Production of oil in microorganisms
Abstract
The invention provides methods and compositions useful for the production of oils, fuels, oleochemicals and other compounds in microorganisms. In particular, the invention provides oil-bearing microorganisms and low-cost culture methods of such microorganisms. The invention also provides microbial cells containing exogenous genes encoding, for example a lipase, sucrose transporter, sucrose invertase, fructokinase, a polysaccharide-degrading enzyme, fatty acyl-ACP thioesterase, acyl-CoA / aldehyde reductase fat, acyl-CoA fat reductase, aldehyde fatty reductase, fatty aldehyde decarbonylase and / or an acyl carrier protein. The invention also includes methods for the manufacture of transportation fuel such as renewable diesel, biodiesel and renewable jet fuel.

Term
No projected expiry on record.
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402 claims: 30 independent, 372 dependent
- 1REIVINDICACIONES 1. Un método para producir diesel renovable, caracterizado porque comprende:(a) cultivar una población de microorganismos en presencia de una fuente fija de carbono, en donde: (i) los microorganismos acumulan por lo menos 10% de su peso seco de células como lípidos y (¡i) la fuente fija de carbono es seleccionada del grupo que consiste de glicerol, material celulósico despolimerizado, sacarosa, melazas, glucosa, arabinosa, galactosa, xilosa, fructosa, arabinosa, mañosa, acetato y cualquier combinación de las anteriores;(b) aislar los componentes lípidos de los microorganismos cultivados y (c) someter los componentes de lípido aislados a una o más reacciones químicas para generar alcanos de cadena recta, mediante el cual se produce el diesel renovable.
- 2El método de conformidad con la reivindicación 1, caracterizado porque el microorganismo es una microalga.
- 3El método de conformidad con la reivindicación 2, caracterizado porque el microorganismo es seleccionado del grupo que consiste de las microalgas enlistadas en la Tabla 1.
- 4El método de conformidad con la reivindicación 3, caracterizado porque el microorganismo es una especie del género Chlorella.
- 5El método de conformidad con la reivindicación 4, caracterizado porque el microorganismo es seleccionado del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella, ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusorium var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris var. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella cnthella y Chlorella zofigiensis.
- 6El método de conformidad con la reivindicación 1, caracterizado porque el microorganismo es una levadura oleaginosa.
- 7El método de conformidad con la reivindicación 6, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis y Yarrowia lipolytica.
- 8El método de conformidad con la reivindicación 1, caracterizado porque el microorganismo es un hongo.
- 9El método de conformidad con la reivindicación 8, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Penniliccium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cadosporium, una especie del gémero Malbranchea, una especie del género Rhizopus y una especie del género Pythium.
- 10El método de conformidad con la reivindicación 1, caracterizado porque el microorganismo contiene por lo menos un gen de utilización de sacarosa exógeno.
- 11El método de conformidad con la reivindicación 10, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa o una fructocinasa.
- 12El método de conformidad con la reivindicación 1, caracterizado porque el microorganismo contiene por lo menos un gen exógeno que codifica una enzima de la ruta de lípido.
- 13El método de conformidad con la reivindicación 12, caracterizado porque la ruta de enzima de lípidos es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenasa, una acetil-CoA carboxilasa, una proteína portadora de acilo y una glicerol-3 fosfato aciltransferasa.
- 14Un método para producir combustible de jet, caracterizado porque comprende:(a) cultivar una población de microorganismos en presencia de una fuente fija de carbono, en donde: (i) los microorganismos acumulan por lo menos 10% de su peso seco de célula como lípido y (¡i) la fuente fija de carbono es seleccionada del grupo que consiste de glicerol, material celulósico despolimerizado, sacarosa, glucosa, arabinosa, galactosa, xilosa, fructosa, arabinosa, mañosa, acetato y cualquier combinación de los anteriores: (b) aislar componentes de lípidos a partir de los microorganismos cultivados y (c) someter los componentes de lípido aislados a una o más reacciones químicas para generar alcanos de cadena recta, mediante el cual se produce el combustible de jet.
- 15El método de conformidad con la reivindicación 14, caracterizado porque el microorganismo es una microalga.
- 16El método de conformidad con la reivindicación 15, caracterizado porque el microorganismo es seleccionado del grupo que consiste de las microalgas enlistadas en la Tabla 1.
- 17El método de conformidad con la reivindicación 16, caracterizado porque el microorganismo es una especie del género Chlorella.
- 18El método de conformidad con la reivindicación 17, caracterizado porque el microorganismo es seleccionado del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella, ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusorium var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris var. tedia, Chlorella vulgaris var. airtáis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tedia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella cnthella y Chlorella zofigiensis.
- 19El método de conformidad con la reivindicación 14, caracterizado porque el microorganismo es una levadura oleaginosa.
- 20El método de conformidad con la reivindicación 19, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, fíhodotorula glutinis, fíhodotorula gracilis y Yarrowia lipolytica.
- 21El método de conformidad con la reivindicación 14, caracterizado porque el microorganismo es un hongo.
- 22El método de conformidad con la reivindicación 21, caracterizado porque el hongo es seleccionado a partir del grupo que consiste de una especie del género Modierella, Modierella vinacea, Modierella alpine, Pythium debaryanum, Mucor circinellotáes, Aspergillus ochraceus, Aspergillus terreus, Penniliccium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cadosporíum, una especie del gémero Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 23El método de conformidad con la reivindicación 14, caracterizado porque el microorganismo contiene por lo menos un gen de utilización de sacarosa exógeno.
- 24El método de conformidad con la reivindicación 23, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa o una fructocinasa.
- 25El método de conformidad con la reivindicación 14, caracterizado porque el microorganismo contiene por lo menos un gen exógeno que codifica una enzima de la ruta de lípidos.
- 26El método de conformidad con la reivindicación 25, caracterizado porque la enzima de la ruta de lípido es seleccionada a partir del grupo que consiste de estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenasa, una acetil-CoA carboxilasa, una proteína portadora de acilo y una glicerol-3 fosfato aciltransferasa.
- 27Una composición de hidrocarburos líquidos, caracterizada porque es elaborada de acuerdo con el método de la reivindicación 1, en donde la composición se conforma a las especificaciones de ASTM D975.
- 28La composición de conformidad con la reivindicación 27, caracterizada porqueel microorganismo es una microalga.
- 29La composición de conformidad con la reivindicación 28, caracterizada porqueel microorganismo es seleccionado a partir del grupo que consiste de las microalgas enlistadas en la Tabla 1.
- 30La composición de conformidad con la reivindicación 29, caracterizada porqueel microorganismo es una especia del género Chlorella.
- 31La composición de conformidad con la reivindicación 30, caracterizada porqueel microorganismo es seleccionado del grupo que consiste de Chlorella anitrata, Chlorella antartica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella dessicata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuolata, Chlorella glucorotropha, Chlorella infosium, Chlorella infosium var actophila, Chlorella infosium var auxeophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var aureoviridis, Chlorella luteoviridis var lutescens, Chlorellaminiata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var mínima, Chlorella regularis var umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharofila var ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris var tedia, Chlorella vulgaris var airidis, Chlorellavulgaris var vulgarís, Chlorella vulgaris var vulgarías f. tedia, Chlorella vulgarias var vulgarias f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 32La composición de conformidad con la reivindicación 27, caracterizada porque el microorganismo es una levadura oleaginosa.
- 33La composición de conformidad con la reivindicación 32, caracterizada porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terrícolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomyces vernalis, Flhodotorula glutinis, fíhodotorula gracilis y Yarrowia lipolytica.
- 34La composición de conformidad con la reivindicación 27, caracterizada porque el microorganismo es un hongo.
- 35La composición de conformidad con la reivindicación 34, caracterizada porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicilium iilacinum, una especie del género Hansenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus y una especie del género Pythium.
- 36La composición de conformidad con la reivindicación 27, caracterizada porque el microorganismo contiene por lo menos un gen de utilización de sacarosa exógeno.
- 37La composición de conformidad con la reivindicación 36, caracterizada porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa o una fructocinasa.
- 38La composición de conformidad con la reivindicación 27, caracterizada porque el microorganismo contiene por lo menos un gen exógeno que codifica a una enzima de ruta de lípido.
- 39La composición de conformidad con la reivindicación 38, caracterizada porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una priruvato deshidrogenasa, acil-CoA carboxilasa, una proteína portadora de acilo y una glicerol-3-fosfato aciltransferasa.
- 40Una composición de hidrocarburos líquidos caracterizada porque es producida de acuerdo con el método de la reivindicación 14, en donde la composición se conforma a las especificaciones de D1655 de ASTM.
- 41La composición de conformidad con la reivindicación 40, caracterizada porque el microorganismo es una microalga.
- 42La composición de conformidad con la reivindicación 41, caracterizada porque el microorganismo es seleccionado del grupo que consiste de las microalgas enlistadas en la tabla 1.
- 43La composición de conformidad con la reivindicación 42, caracterizada porque el microorganismo es una especie del género Chlorella.
- 44La composición de conformidad con la reivindicación 43, caracterizada porque el microorganismo es seleccionado del grupo que consiste de Chlorella anitrata, Chlorella antartica, Chlorella aureoviridis, Chlorella candida, Chlorella capsúlala, Chlorella dessicata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuoiata, Chlorella glucorotropha, Chlorella infosium, Chlorella infosium var actophila, Chlorella infosium var auxeophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var aureoviridis, Chlorella luteoviridis var lutescens, Chlorellaminiata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var mínima, Chlorella regularis var umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharofila var ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris var tertia, Chlorella vulgaris var airidis, Chlorellavulgaris var vulgaris, Chlorella vulgaris var vulgarias f. tertia, Chlorella vulgarias var vulgarias f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 45La composición de conformidad con la reivindicación 40, caracterizada porque el microorganismo es una levadura oleaginosa.
- 46La composición de conformidad con la reivindicación 45, caracterizada porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomyces vernalis, Rhodotorula glutinis, Rhodotorula gracilis y Yarrowia lipolytica.
- 47La composición de conformidad con la reivindicación 40, caracterizada porque el microorganismo es un hongo.
- 48La composición de conformidad con la reivindicación 47, caracterizada porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicilium iilacinum, una especie del género Hansenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus y una especie del género Pythium.
- 49La composición de conformidad con la reivindicación 40, caracterizada porque el microorganismo contiene por lo menos un gen de utilización de sacarosa exógeno.
- 50La composición de conformidad con la reivindicación 49, caracterizada porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa o una fructocinasa.
- 51La composición de conformidad con la reivindicación 40, caracterizada porque el microorganismo contiene por lo menos un gen exógeno que codifica a una enzima de ruta de lípido.
- 52La composición de conformidad con la reivindicación 51, caracterizada porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una priruvato deshidrogenasa, acil-CoA carboxilasa, una proteína portadora de acilo y una glicerol-3-fosfato aciltransferasa.
- 53Una microalga o células de levadura caracterizada porque ha sido diseñada genéticamente y/o seleccionada para expresar una enzima de la ruta de lípido a un nivel alterado en comparación con una célula tipo silvestre de la misma especia.
- 54La célula de conformidad con la reivindicación 53, caracterizada porque la célula produce más lípido en comparación con la célula tipo silvestre cuando ambas células son cultivadas bajo las mismas condiciones.
- 55La célula de conformidad con la reivindicación 53, caracterizada porque la célula ha sido diseñada genéticamente y/o seleccionada para expresar una enzima de ruta de lípido a un nivel más alto que las células tipo silvestre.
- 56La célula de conformidad con la reivindicación 55, caracterizada porque la enzima de ruta de lípido es seleccionada del grupo que consiste de piruvato deshidrogenasa, acil-CoA carboxilasa, una proteína portadora de acilo y una glicerol-3-fosfato aciltransferasa.
- 57La célula de conformidad con la reivindicación 53, caracterizada porque la célula ha sido diseñada genéticamente y/o seleccionada para expresar una enzima de ruta de lípido a un nivel más bajo que las células tipo silvestre.
- 58La célula de conformidad con la reivindicación 57, caracterizada porque la enzima de ruta de lípido comprende citrato cintasa.
- 59La célula de conformidad con la reivindicación 53, caracterizada porque la célula ha sido diseñada genéticamente y/o seleccionada para expresar un regulador global de síntesis de ácido graso a un nivel más alterado en comparación con la célula tipo silvestre, mediante lo cual los niveles de expresión de una prioridad de genes sintéticos de acido graso son alterados en comparación con la célula tipo silvestre.
- 60La célula de conformidad con la reivindicación 57, caracterizada porque la enzima de ruta de lípido comprende una enzima que modifica un acido graso.
- 61La célula de conformidad con la reivindicación 60, caracterizada porque la enzima de ruta de lípido es seleccionada de estearoil-ACP desaturasa y una glicerolípido desaturasa.
- 62La célula de conformidad con la reivindicación 53, caracterizada porque la célula es una especie de microalga seleccionada de la tabla 1.
- 63La célula de microalga de conformidad con la reivindicación 62, caracterizada porque la microalga es del género Chlorella.
- 64La microalga de de conformidad con la reivindicación 63, caracterizada porque la microalga es de la especie seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antartica, Chlorella aureoviridis, Chlorella candida, Chlorella capsúlala, Chlorella dessicata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuoiata, Chlorella glucorotropha, Chlorella infosium, Chlorella infosium var actophila, Chlorella infosium var auxeophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var aureoviridis, Chlorella luteoviridis var lutescens, Chlorellaminiata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var mínima, Chlorella regularis var umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharofila var ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris var tertia, Chlorella vulgaris var airidis, Chlorellavulgaris var vulgaris, Chlorella vulgaris var vulgarias f. tertia, Chlorella vulgarias var vulgarias f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 65Un microbio productor de aceite que contiene una o más genes exógenos caracterizados, porque los genes exógenos codifican proteínas seleccionadas del grupo que consiste de una acil-ACP tioesterasa grasa, acil-CoA reductasa grasa, una aldehido reductasa grasa, una acil-CoA/aldehído reductasa grasa, una aldehido descarbonilasa grasa y una proteína portadora de acilo.
- 66El microbio de conformidad con la reivindicación 65, caracterizada porque el microbio es una microalga.
- 67El microbio de conformidad con la reivindicación 65, caracterizado porque el microorganismo es una levadura oleaginosa.
- 68El microbio de conformidad con la reivindicación 65, caracterizada porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomyces vernalis, Rhodotorula glutinis, Flhodotorula gracilis y Yarrowia lipolytica.
- 69El microbio de conformidad con la reivindicación 65, caracterizada porque el microorganismo es un hongo.
- 70El microbio de conformidad con la reivindicación 69, caracterizada porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicilium iilacinum, una especie del género Hansenulo, una especie del género Chaetomium, una especie del género Cladosporíum, una especie del género Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 71El microbio de conformidad con la reivindicación 65, caracterizada porque el microbio es seleccionado del grupo que consiste de los microorganismos enlistados en la tabla 1.
- 72El microbio de conformidad con la reivindicación 71, caracterizada porque el microbio es una especia del género Chlorella.
- 73El microbio de conformidad con la reivindicación 72, caracterizada porque la microalga es de la especie seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antartica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella dessicata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuolata, Chlorella glucorotropha, Chlorella infosium, Chlorella infosium var actophila, Chlorella infosium var auxeophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var aureoviridis, Chlorella luteoviridis var lutescens, Chlorellaminiata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var mínima, Chlorella regularis var umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharofila var ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris var tedia, Chlorella vulgaris var airidis, Chlorellavulgaris var vulgaris, Chlorella vulgaris var vulgarías f. tedia, Chlorella vulgarias var vulgarias f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 74El microbio de conformidad con la reivindicación 73, caracterizada porque la especie seleccionada es Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea o Chlorella sp.
- 75El microbio de conformidad con la reivindicación 65, caracterizada el gen exógeno está en enlace operable con un promotor, que es inducible o represible en respuesta a un estímulo.
- 76El microbio de conformidad con la reivindicación 75, caracterizada porque el estimulo es seleccionado del grupo que consiste de una molécula pequeña provista exógenamente, calor, frío y luz.
- 77El microbio de conformidad con la reivindicación 65, caracterizada porque el gen exógeno es expresado en un compartimento celular.
- 78El microbio de conformidad con la reivindicación 77, caracterizada porque el compartimento celular es seleccionado del grupo que consiste de un cloroplasto y una mitocondña.
- 79El microbio de conformidad con la reivindicación 65, caracterizada porque el gen exógeno codifica a una acil-ACP tioesterasa de ácido graso.
- 80El microbio de conformidad con la reivindicación 79, caracterizada porque la tioesterasa codificada por el gen exógeno cataliza le escisión de un ácido graso de 8 a 18 átomos de carbono de una proteína portadora de acilo (ACP).
- 81El microbio de conformidad con la reivindicación 80, caracterizada porque la tioesterasa codificada por el gen exógeno cataliza la escisión de un ácido graso de 10 a 14 átomos de carbono de una ACP.
- 82El microbio de conformidad con la reivindicación 81, caracterizada porque la tioesterasa codificada por el gen exógeno cataliza la escisión de un ácido graso de 12 átomos de carbono de una ACP.
- 83El microbio de conformidad con la reivindicación 65, caracterizada porque el gen exógeno codifica a una acil-CoA/aldehído reductasa grasa.
- 84El microbio de conformidad con la reivindicación 83, caracterizada porque la reductasa codificada por el gen exógeno cataliza la reducción de una acil-CoA grasa de 20 a 30 átomos de carbono a un alcohol primario correspondiente.
- 85El microbio de conformidad con la reivindicación 83, caracterizada porque la reductasa codificada por el gen exógeno cataliza la reducción de una acil-CoA grasa de 8 a 18 átomos de carbono a un alcohol primario correspondiente.
- 86El microbio de conformidad con la reivindicación 85, caracterizada porque la reductasa codificada por el gen exógeno cataliza la reducción de una acil-CoA grasa de 10 a 14 átomos de carbono a un alcohol primario correspondiente.
- 87El microbio de conformidad con la reivindicación 86, caracterizado porque la reductasa codificada por el gen exógeno cataliza la reducción de una acil-CoA grasa de 12 átomos de carbono a un dodecanol.
- 88El microbio de conformidad con la reivindicación 65, caracterizado porque el gen exógeno codifica una acil-CoA reductasa grasa.
- 89El microbio de conformidad con la reivindicación 88, caracterizado porque la reductasa codificada por el gen exógeno cataliza la reducción de una acil-CoA grasa de 8 a 18 átomos de carbono a un aldehido correspondiente.
- 90El microbio de conformidad con la reivindicación 89, caracterizado porque la reductasa codificada por el gen exógeno cataliza la reducción de una acil-CoA grasa de 12 átomos de carbono a dodecanal.
- 91El microbio de conformidad con la reivindicación 65, caracterizado porque el microbio contiene además uno o más genes de utilización de sacarosa exógenos.
- 92Un microbio que contiene dos genes exógenos, caracterizado porque un primer gen exógeno codifica una acil-ACP tioesterasa grasa y un segundo gen exógeno codifica una proteína seleccionada del grupo que consiste de una acil-CoA reductasa grasa, una acil-CoA/aldehído reductasa grasa y una proteína portadora de acilo.
- 93El microbio de conformidad con la reivindicación 92, caracterizado porque el microbio es una microalga.
- 94El microbio de conformidad con la reivindicación 92, caracterizado porque el microorganismo es una levadura oleaginosa.
- 95El microbio de conformidad con la reivindicación 94, caracterizada porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomyces vernalis, Flhodotorula glutinis, Rhodotorula gracilts y Yarrowia lipolytica.
- 96El microbio de conformidad con la reivindicación 92, caracterizada porque el microorganismo es un hongo.
- 97El microbio de conformidad con la reivindicación 96, caracterizada porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicilium iilacinum, una especie del género Hansenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus y una especie del género Pythium.
- 98El microbio de conformidad con la reivindicación 92, caracterizada porque el microbio es seleccionado del grupo que consiste de los microorganismos enlistados en la tabla 1.
- 99El microbio de conformidad con la reivindicación 98, caracterizada porque el microbio es una especia del género Chlorella.
- 100El microbio de conformidad con la reivindicación 99, caracterizada porque la microalga es de la especie seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antartica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella dessicata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuolata, Chlorella glucorotropha, Chlorella infosium, Chlorella infosium var actophila, Chlorella infosium var auxeophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var aureoviridis, Chlorella luteoviridis var lutescens, Chlorellaminiata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var mínima, Chlorella regularis var umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharofila var ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris var tedia, Chlorella vulgaris var airidis, Chlorellavulgaris var vulgaris, Chlorella vulgaris var vulgarias f. tedia, Chlorella vulgarias var vulgarias f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 101El microbio de conformidad con la reivindicación 100, caracterizada porque la especie seleccionada es Chlorella minutlssima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea o Chlorella sp.
- 102El microbio de conformidad con la reivindicación 92, caracterizada porque cada uno de los dos genes exógenos están en enlace operable con un promotor, que es Inducible en respuesta a un estímulo.
- 103El microbio de conformidad con la reivindicación 102, caracterizado porque cada promotor es inducible en respuesta a un estímulo idéntico.
- 104El microbio de conformidad con la reivindicación 92, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 8 a 18 átomos de carbono de la ACP.
- 105El microbio de conformidad con la reivindicación 104, caracterizado porque el segundo gen exógeno codificada una acil-CoA/aldehpido reductasa grasa que cataliza la reducción de un acil-CoA graso de 8 a 18 átomos de carbono a un alcohol primario correspondiente.
- 106El microbio de conformidad con la reivindicación 105, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 10 a 14 átomos de carbono de una ACP y la reductasa codificada por el segundo gen exógeno cataliza la reducción de una acil-CoA grasa de 10 a 14 átomos de carbono al alcohol primario correspondiente, en donde la tioesterasa y la reductasa actúan sobre la misma longitud de cadena de carbonos.
- 107El microbio de conformidad con la reivindicación 106, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 12 átomos de carbono de una ACP y la reductasa codificada por el segundo gen exógeno cataliza la reducción de una acil-CoA grasa de 12 átomos de carbono a dodecanol.
- 108El microbio de conformidad con la reivindicación 104, caracterizado porque el segundo gen exógeno codifica una acil-CoA reductasa grasa que cataliza la reducción de una acil-CoA grasa de 8 a 18 átomos de carbono a un aldehido correspondiente.
- 109El microbio de conformidad con la reivindicación 92, caracterizado porque el segundo gen exógeno codifica una acil-CoA reductasa grasa y el microbio contiene además un tercer gen exógeno que codifica una aldehido descarbonilasa grasa.
- 110El microbio de conformidad con la reivindicación 109, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 8 a 18 átomos de carbono de una ACP, la reductasa codificada por el segundo gen exógeno cataliza la reducción de una acil-CoA grasa de 8 a 18 átomos de carbono a un aldehido graso correspondiente y la descarbonilasa codificada por el tercer gen exógeno cataliza la conversión de un aldehido graso de 8 a 18 átomos de carbono a un alcano correspondiente, en donde la tioesterasa, la reductasa y descarbonilasa actúan sobre la misma longitud de cadena de carbonos.
- 111El microbio de conformidad con la reivindicación 92, caracterizado porque el microbio contiene además uno o más genes de utilización de sacarosa exógenos.
- 112El microbio de conformidad con la reivindicación 92, caracterizado porque el segundo gen exógeno codifica una proteína portadora de acilo que es co- expresada naturalmente con al acil-ACP tioesterasa grasa.
- 113El microbio de conformidad con la reivindicación 92, caracterizado porque el segundo gen exógeno codifica una proteína portadora de acilo y el microbio contiene además un tercer gen exógeno que codifica una proteína seleccionada del grupo que consiste de una acil-CoA reductasa grasa y una acil-CoA/aldehído reductasa.
- 114El microbio de conformidad con la reivindicación 113, caracterizado porque el tercer gen exógeno codifica una acil-CoA reductasa grasa y el microbio contiene además un cuarto gen exógeno que codifica una aldehido desacarbonilasa grasa.
- 115Un método para producir una molécula en una población de microbios, el método comprende cultivar una población de microbios en un medio de cultivo, caracterizado porque los microbios contienen:i) un primer gen exógeno que codifica una acil-ACP tioesterasa grasa y ¡i) un segundo gen exógeno que codifica una acil-CoA/aldehído reductasa grasa y en donde los microbios sintetizan un ácido graso enlazado a una proteína portadora de acilo (ACP), la acil-ACP tioesterasa grasa cataliza la escisión del ácido graso de ACP para producir, por medio de procesamiento adicional, una acil-CoA reductasa grasa y la acilCoA/aldehído reductasa grasa cataliza la reducción del acil-CoA a un alcohol.
- 116El método de conformidad con la reivindicación 115, caracterizado porque el microbio es una microalga.
- 117El método de conformidad con la reivindicación 115, caracterizado porque el microorganismo es una levadura oleaginosa.
- 118El método de conformidad con la reivindicación 117, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomyces vernalis, Rhodotorula glutinis, Rhodotorula gracilis y Yarrowia lipolytica.
- 119El método de conformidad con la reivindicación 115, caracterizado porque el microorganismo es un hongo.
- 120El método de conformidad con la reivindicación 119, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicilium iilacinum, una especie del género Hansenulo, una especie del género Chaetomium, una especie del género Cladosporíum, una especie del género Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 121El método de conformidad con la reivindicación 115, caracterizado porque el microbio es seleccionado del grupo que consiste de los microorganismos enlistados en la tabla 1.
- 122El método de conformidad con la reivindicación 121, caracterizado porque el microbio es una especia del género Chlorella.
- 123El método de conformidad con la reivindicación 122, caracterizado porque la especie seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antartica, Chlorella aureoviridis, Chlorella candida, Chlorella capsúlala, Chlorella dessicata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuolata, Chlorella glucorotropha, Chlorella infosium, Chlorella infosium var actophila, Chlorella infosium var auxeophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var aureoviridis, Chlorella luteoviridis var lutescens, Chlorellaminiata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var mínima, Chlorella regularis var umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharofila var ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris var tedia, Chlorella vulgaris var airidis, Chlorellavulgaris var vulgaris, Chlorella vulgaris var vulgarias f. tedia, Chlorella vulgarias var vulgarias f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 124El método de conformidad con la reivindicación 123, caracterizado porque la especie seleccionada es Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea o Chlorella sp.
- 125El método de conformidad con la reivindicación 115, caracterizada porque el medio de cultivo contiene glicerol.
- 126El método de conformidad con la reivindicación 125, caracterizada porque el glicerol es un producto secundario de un proceso de transesterificación.
- 127El método de conformidad con la reivindicación 125, caracterizado porque el medio de cultivo contiene glicerol y por lo menos otra fuente de carbono fija.
- 128El método de conformidad con la reivindicación 127, caracterizado porque la por lo menos otra fuente de carbono fija es sacarosa.
- 129El método de conformidad con la reivindicación 127, caracterizado porque todo del glicerol y todo de la por lo menos otra fuente de carbono fija son provistos a los microbios al comienzo de la fermentación.
- 130El método de conformidad con la reivindicación 127, caracterizado porque el glicerol y por lo menos otra fuente fija de carbono son alimentados a los microbios a una velocidad predeterminada en el curso de la fermentación.
- 131El método de conformidad con la reivindicación 127, caracterizado porque:(a) el glicerol es provisto a los microbios en ausencia de la por lo menos otra fuente fija de carbono por un primer período de tiempo;(b) la por lo menos otra fuente fija de carbono es provista al final del primer período de tiempo y (c) los microbios son cultivados por un segundo período de tiempo en presencia de la por lo menos otra fuente fija de carbono.
- 132El método de conformidad con la reivindicación 115, caracterizado porque los genes exógenos están en enlace operable sobre un promotor que es inducible en respuesta a un primer estímulo y en donde el método comprende además:proveer el primer estímulo y incubar la población de microbios por un primer período de tiempo en presencia del primer estímulo para producir un alcohol.
- 133El método de conformidad con la reivindicación 132, caracterizado porque comprende además extraer el alcohol de la biomasa acuosa.
- 134El método de conformidad con la reivindicación 115, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 8 a 18 átomos de carbono de la ACP y la reductasa codificada por el segundo gen exógeno cataliza la reducción de una acil-CoA grasa de 8 a 18 átomos de carbono a un alcohol primario correspondiente, en donde la tioesterasa y la reductasa actúan sobre la misma longitud de cadena de carbonos.
- 135El método de conformidad con la reivindicación 134, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 10 a 14 átomos de carbono de la ACP y la reductasa codificada por el segundo gen exógeno cataliza la escisión de una acil-CoA grasa de 10 a 14 átomos de carbono a un alcohol primario correspondiente, en donde la tioesterasa y la reductasa actúan sobre la misma longitud de cadena de carbonos.
- 136El método de conformidad con la reivindicación 135, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 12 átomos de carbono de la ACP y la reductasa codificada por el segundo gen exógeno cataliza la reducción de una acil-Coa grasa de 12 átomos de carbono a dodecanol.
- 137El método de conformidad con la reivindicación 115, caracterizado porque los microbios contienen además un tercer gen exógeno que codifica una proteína portadora de acilo.
- 138El método de conformidad con la reivindicación 137, caracterizado porque el tercer gen exógeno codifica una proteína portadora de acilo que es co-expresada naturalmente con la acil-ACP tioesterasa grasa.
- 139Un método para producir una molécula de lípido en una población de microbios, el método está caracterizado porque comprende cultivar una población de microbios en un medio de cultivo, en donde los microbios contienen:(i) un primer gen exógeno que codifica una acil-ACP tioesterasa grasa y (¡i) un segundo gen exógeno que codifica una acil-CoA reductasa grasa y en donde los microbios sintetizan un ácido graso enlazado a una proteína portadora de acilo (ACP), la acil-ACP tioesterasa grasa cataliza la escisión del ácido graso de la ACP para producir, por medio de procesamiento adicional, una acil-CoA grasa y la acil-CoA reductasa grasa cataliza la reducción de la acil-CoA a un aldehido.
- 140El método de conformidad con la reivindicación 139, caracterizado porque el microbio es una microalga.
- 141El método de conformidad con la reivindicación 139, caracterizado porque el microbio es una levadura oleaginosa.
- 142El método de conformidad con la reivindicación 139, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, fíhodotorula glutinis, Flhodotorula gracilis y Yarrowia lipolytica.
- 143El método de conformidad con la reivindicación 139, caracterizado porque el microbio es un hongo.
- 144El método de conformidad con la reivindicación 143, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Penniliccium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cadosporium, una especie del gémero Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 145El método de conformidad con la reivindicación 139, caracterizado porque el microbio es seleccionado del grupo que consiste de los microorganismos enlistados en la tabla 1.
- 146El método de conformidad con la reivindicación 145, caracterizado porque el microbio es una especie del género Chlorella.
- 147El método de conformidad con la reivindicación 146, caracterizado porque la especie es seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis. Chlorella candida, Chlorella capsúlala, Chlorella desiccata, Chlorella, ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusorium var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris var. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella cnthella y Chlorella zofigiensis.
- 148El método de conformidad con la reivindicación 147, caracterizado porque la especie es Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. o Chlorella protothecoides.
- 149El método de conformidad con la reivindicación 139, caracterizado porque los genes exógenos están en enlace operable con un promotor que es inducible en respuesta a un primer estímulo y en donde el método comprende además:proveer el primer estímulo e incubar la población de microbios por un primer período de tiempo en presencia del primer estímulo para producir un aldehido.
- 150El método de conformidad con la reivindicación 149, caracterizado porque comprende además extraer el aldehido de biomasa acuosa.
- 151El método de conformidad con la reivindicación 139, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 8 a 18 átomos de carbono de la ACP y la reductasa codificada por el segundo gen exógeno cataliza la reducción de una acil-Coa grasa a un aldehido correspondiente, en donde la tioesterasa y la reductasa actúan sobre la misma longitud de cadena de carbonos.
- 152El método de conformidad con la reivindicación 139, caracterizado porque los microbios contienen además n tercer gen exógeno que codifica una aldehido descarbonilasa grasa que cataliza la conversión del aldehido a un alcano.
- 153El método de conformidad con la reivindicación 152, caracterizado porque los genes exógenos están en enlace operable con un promotor que es inducible en respuesta a un primer estímulo y en donde el método comprende además:proveer el primer estímulo y incubar la población de microbios por un primer período de tiempo en presencia del primer estímulo para producir un alcano.
- 154El método de conformidad con la reivindicación 153, caracterizado porque comprende además extraer el alcano de biomasa acuosa.
- 155El método de conformidad con la reivindicación 152, caracterizado porque la tioesterasa codificada por el primer gen exógeno cataliza la escisión de un ácido graso de 8 a 18 átomos de carbono de la ACP, la reductasa codificada por el segundo gen exógeno catalizan la reducción de una acil-CoA grasa de 8 a 18 átomos de carbono a un aldehido correspondiene y la descarbonilasa codificada por el tercer gen exógeno cataliza la conversión de un aldehido de 8 a 18 átomos de carbono a un alcano correspondiente, en donde la tioesterasa, la reductasa y la descarbonilasa actúan sobre la misma longitud de cadena de carbonos.
- 156El método de conformidad con la reivindicación 139, caracterizado porque los microbios contienen además un tercer gen exógeno que codifica una proteína portadora de acilo.
- 157El método de conformidad con la reivindicación 156, caracterizado porque el tercer gen exógeno codifica una proteína portadora de acilo que es co-expresada naturalmente con la acil-ACP tioesterasa grasa.
- 158El método de conformidad con la reivindicación 156, caracterizado porque los microbios contienen además un cuarto gen exógeno que codifica una aldehido descarbonilasa grasa que cataliza la conversión del aldehido a un alcano.
- 159El método de conformidad con la reivindicación 139, caracterizado porque el medio de cultivo contiene glicerol.
- 160El método de conformidad con la reivindicación 159, caracterizado porque el glicerol es un producto secundario de un proceso de transesterificación.
- 161El método de conformidad con la reivindicación 159, caracterizado porque el medio de cultivo contiene glicerol y por lo menos otra fuente fija de carbono.
- 162El método de conformidad con la reivindicación 161, caracterizado porque la por lo menos otra fuente fija es sacarosa.
- 163El método de conformidad con la reivindicación 161, caracterizado porque todo de glicerol y toda de la por lo menos otra fuente fija de carbono son provistos a los microbios al comienzo de la fermentación.
- 164El método de conformidad con la reivindicación 161, caracterizado porque el glicerol y la por lo menos otra de la fuente fija de carbono son alimentados a los microbios a una velocidad predeterminada en el curso de la fermentación.
- 165El método de conformidad con la reivindicación 161, caracterizado porque:(a) el glicerol es provisto a los microbios en ausencia de por lo menos otra fuente fija de carbono por un primer período de tiempo;(b) la por lo menos otra fuente fija de carbono es provista al final del primer período de tiempo y (c) los microbios son cultivados por un segundo período de tiempo en presencia de la por lo menos otra fuente de carbono fija.
- 166Un método para producir una molécula de ácido graso que tiene una longitud de cadena de carbonos especificada en una población de microbios, el método está caracterizado porque comprende cultivar una población de microbios que produce lípidos en un medio de cultivo, en donde los microbios contienen un gen exógeno que codifica una acil-ACP bioesterasa grasa que tiene una actividad específica a la longitud de cadena del carbono y en donde los microbios sintetizan un ácido enlazado a una proteína portadora de acilo (ACP) y la tioesterasa cataliza la escisión del ácido graso a partir de la ACP cuando el ácido graso ha sido sintetizado a la longitud de cadena de carbonos específica.
- 167El método de conformidad con la reivindicación 166, caracterizado porque el microbio es una microalga.
- 168El método de conformidad con la reivindicación 166, caracterizado porque el microbio es una levadura oleaginosa.
- 169El método de conformidad con la reivindicación 168, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, fíhodotorula glutinis, fíhodotorula gracilis y Yarrowia lipolytica.
- 170El método de conformidad con la reivindicación 166, caracterizado porque el microbio es un hongo.
- 171El método de conformidad con la reivindicación 170, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Modierella, Modierella vinacea, Modierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Penniliccium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cadosporium, una especie del gémero Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 172El método de conformidad con la reivindicación 170, caracterizado porque el microbio es seleccionado del grupo que consiste de los microorganismos enlistados en la Tabla 1.
- 173El método de conformidad con la reivindicación 172, caracterizado porque el microbio es una especie del género Chlorella.
- 174El método de conformidad con la reivindicación 173, caracterizado porque la especie es seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella, ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusorium var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris var. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella cnthella y Chlorella zofigiensis.
- 175El método de conformidad con la reivindicación 174, caracterizado porque la especie es Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. o Chlorella protothecoides.
- 176El método de conformidad con la reivindicación 166, caracterizado porque el gen exógeno está en enlace operable con un promotor que es inducible en respuesta a un primer estímulo y en donde el método comprende además:proveer el primer estímulo e incubar la población de microbios por un período de tiempo en presencia del primer estímulo.
- 177El método de conformidad con la reivindicación 176, caracterizado porque comprende además extraer el ácido graso de la biomasa acuosa.
- 178El método de conformidad con la reivindicación 166, caracterizado porque los microbios contienen además un segundo gen exógeno que codifica una proteína portadora de acilo.
- 179El método de conformidad con la reivindicación 178, caracterizado porque el segundo gen exógeno codifica una proteína portadora de acilo que es co-expresada naturalmente con la acil-ACP tioesterasa grasa.
- 180El método de conformidad con la reivindicación 166, caracterizado porque la acil-ACP tioesterasa cataliza la escisión de un ácido graso de 8 a 18 átomos de carbono de la ACP.
- 181El método de conformidad con la reivindicación 166, caracterizado porque el medio de cultivo contiene glicerol.
- 182El método de conformidad con la reivindicación 181, caracterizado porque el glicerol es un producto secundario es un proceso de transesterificación.
- 183El método de conformidad con la reivindicación 181, caracterizado porque el medio de cultivo contiene glicerol y por lo menos otra fuente fija de carbono.
- 184El método de conformidad con la reivindicación 183, caracterizado porque la por lo menos otra fuente de carbono es sacarosa.
- 185El método de conformidad con la reivindicación 183, caracterizado porque todo del glicerol y todo de la por lo menos otra fuente fija de carbono son provistos a los microbios al comienzo de la fermentación.
- 186El método de conformidad con la reivindicación 173, caracterizado porque el glicerol y la por lo menos otra fuente fija de carbono son alimentados a los microbios a una velocidad predeterminada en el curso de la fermentación.
- 187El método de conformidad con la reivindicación 183, caracterizado porque:(a) el glicerol es provisto a los microbios en ausencia de la por lo menos otra fuente fija de carbono por un primer período de tiempo;(b) la por lo menos otra fuente fija de carbono es provista al final del primer período de tiempo y (c) los microbios son cultivados por un segundo período de tiempo en presencia de la por lo menos otra fuente fija de carbono.
- 188Una célula de microalga que contiene un gen exógeno, caracterizada porque el gen exógeno codifica una proteína seleccionada del grupo que consiste de una lipasa, un transportador de sacarosa, una invertasa de sacarosa, una fructocinasa o una enzima polisacárido no degradante.
- 189La célula de microalga de conformidad con la reivindicación 188, caracterizada porque la célula es seleccionada de los microorganismos de la Tabla 1.
- 190La célula de microalga de conformidad con la reivindicación 189, caracterizada porque la célula es una especie del género Chlorella.
- 191La célula de microalga de conformidad con la reivindicación 190, caracterizada porque la especie es seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella, ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusorium var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaeríca, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris var. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgarís, Chlorella vulgaris var. vulgaris f. tedia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella cnthella y Chlorella zofigiensis.
- 192La célula de microalga de conformidad con la reivindicación 191, caracterizada porque la célula es Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. o Chlorella protothecoides.
- 193La célula de microalga de conformidad con la reivindicación 188, caracterizada porque el gen exógeno está en enlace operable con un promotor.
- 194La célula de microalga de conformidad con la reivindicación 193, caracterizada porque el promotor es inducible o represible en respuesta aun estímulo.
- 195La célula de microalga de conformidad con la reivindicación 194, caracterizada porque el estímulo es seleccionado del grupo que consiste de una molécula pequeña provista exógenamente, calor, frío y luz.
- 196La célula de microalga de conformidad con la reivindicación 188, caracterizada porque el gen exógeno es expresado en un compartimiento celular.
- 197La célula de microalga de conformidad con la reivindicación 196, caracterizada porque el compartimiento celular es seleccionado del grupo que consiste de un cloroplasto y una mitocondria.
- 198La célula de microalga de conformidad con la reivindicación 188, caracterizada porque el gen codifica una lipasa que tiene por lo menos 70% de identidad de aminoácidos con una lipasa seleccioanda de la Tabla 9.
- 199La célula de microalga de conformidad con la reivindicación 198, caracterizada porque la lipasa es novozym-435.
- 200La célula de microalga de conformidad con la reivindicación 188, caracterizada porque el gen codifica una enzima polisacáñdo-degradante.
- 201La célula de microalga de conformidad con la reivindicación 200, caracterizada porque la enzima polisacárido-degradante es endógena a un virus de Chlorella.
- 202Una célula de microalga que contiene dos genes exógenos, caracterizada porque un primer gen exógeno codifica una lipasa y un segundo gen exógeno codifica una enzima polisacárido-degradante.
- 203La célula de microalga de conformidad con la reivindicación 202, caracterizada porque los genes exógenos están cada uno en enlace operable con un promotor.
- 204La célula de microalga de conformidad con la reivindicación 203, caracterizada porque los genes exógenos están cada uno en enlace operable con promotores que son inducibles en respuesta a un estímulo.
- 205La célula de microalga de conformidad con la reivindicación 204, caracterizada porque los genes exógenos están cada uno en enlace operable con promotores que son inducibles en respuesta al mismo estímulo.
- 206La célula de microalga de conformidad con la reivindicación 204, caracterizada porque los genes exógenos están cada uno en enlace operable con un promotor que es inducible en respuesta por lo menos un estímulo que no induce el otro promotor.
- 207Un método para la manufactura de una molécula de lípido en un microbio, el método está caracterizado porque comprende:(a) cultivar el microbio por un primer período de tiempo suficiente para incrementar la densidad celular, en donde el microbio contiene: (i) un gen exógeno que codifica una lipasa y/o (¡i) un gen exógeno que codifica una enzima polisacárido-degradante;en donde los genes exógenos están en enlace operable con un promotor que es inducible en respuesta a un estímulo;(b) proveer el estímulo y (c) incubar el microbio por un segundo período de tiempo en presencia del estímulo.
- 208Un método para la manufactura de una molécula de lípido en un microbio, el método está caracterizado porque comprende:(a) cultivar un microbio que produce lípidos durante un primer período de tiempo suficiente para incrementar la densidad celular;(b) proveer un virus capaz de infectar y someter a lisis el microbio cuando está en cotnacto directo con el microbio y (c) incubar el microbio por un segundo período de tiempo para producir biomasa acuosa sometida a lisis.
- 209El método de conformidad con la reivindicación 208, caracterizado porque comprende además la etapa de extraer moléculas de lípido de la biomasa acuosa sometida a lisis.
- 210Una célula de microalga que contiene un gen exógeno, caracterizada porque el gen exógeno codifica un co-factor para una enzima de ruta de lípido o codifica una proteína que participa en la síntesis del co-factor.
- 211Un método para cultivar un microbio que produce lípidos, el método está caracterizado porque comprende cultivar el microbio en presencia de una cantidad suficiente de uno o más co-factores para un enzima de ruta de lípido para incrementar el rendimiento de lípido microbiano con respecto al rendimiento de lípido microbiano en ausencia del uno o más cofactores.
- 212El método de conformidad con la reivindicación 211, caracterizado porque el uno o más co factores consisten de una vitamina requerida por una o más enzimas de ruta de lípido.
- 213El método de conformidad con la reivindicación 211, caracterizado porque el uno o más cofactores es biotina.
- 214El método de conformidad con la reivindicación 211, caracterizado porque el uno o más cofactores es/son provistos al incluir en el cultivo un microbio que ha sido diseñado genéticamente para producir el uno o más co-factores.
- 215Un método para la fermentación de un microorganismo caracterizado porque comprende proveer una mezcla que comprende glucosa y xilosa como fuente de energía al microorganismo.
- 216El método de conformidad con la reivindicación 215, caracterizado porque la mezcla comprende además lignina.
- 217El método de conformidad con la reivindicación 215, caracterizado porque la mezcla comprende además por lo menos una especie de furfural.
- 218El método de conformidad con la reivindicación 215, caracterizado porque la mezcla es material celulósico despolimerizado.
- 219El método de conformidad con la reivindicación 215, caracterizado porque el microorganismo es un microalga.
- 220El método de conformidad con la reivindicación 219, caracterizado porque la microalga es seleccionada de la Tabla 1.
- 221El método de conformidad con la reivindicación 220, caracterizado porque la microalga es una especie del género Chlorella.
- 222El método de conformidad con la reivindicación 221, caracterizado porque la microalga es seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsúlala, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. Umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tedia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. Vulgaris f. tedia, Chlorella vulgaris var. Vulgaris f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 223El método de conformidad con la reivindicación 220, caracterizado porque la microalga es seleccionada del grupo que consiste de Bracteococcus minor, Chlorella ellipsoidea. Chlorella kesslerí, Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorella minutissima, Chlorella ovalis, Chlorella protothecoides, Chlorella saccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris, ParaChlorella kesslerí, Prototheca moriformis y Pseudochlorella aquatica.
- 224El método de conformidad con la reivindicación 215, caracterizado porque el microorganismo es una levadura oleaginosa.
- 225El método de conformidad con la reivindicación 224, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, fíhodotorula glutinis, fíhodotorula gracilis y Yarrowia lipolytica.
- 226El método de conformidad con la reivindicación 215, caracterizado porque el microorganismo es un hongo.
- 227El método de conformidad con la reivindicación 226, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 228El método de conformidad con la reivindicación 215, caracterizado porque la mezcla comprende además por lo menos una enzima de utilización de sacarosa.
- 229El método de conformidad con la reivindicación 228, caracterizado porque el microorganismo es del género Chlorella.
- 230El método de conformidad con la reivindicación 215, caracterizado porque el microorganismo ha sido diseñado genéticamente para expresar un gen exógeno que codifica por lo menos una enzima de modificación de lípido, enzima de modificación de hidrocarburo o enzima de utilización de sacarosa.
- 231El método de conformidad con la reivindicación 228, caracterizado porque la mezcla comprende una invertasa de sacarosa.
- 232Un método para cultivar una microalga, caracterizado porque comprende cultivar la microalga en un medio de cultivo que incluye una materia prima de alimentación que comprende por lo menos un sustrato de carbono seleccionado del grupo que consiste de un material celulósico, un azúcar de 5 átomos de carbono, un azúcar de 6 átomos de carbono y acetato.
- 233El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es glucosa y la microalga es de un género seleccionado del grupo que consiste de Chlorella, Parachlorella, Pseudochlorella, Bracteococcus, Prototheca y Scenedesmus.
- 234El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es xilosa y la microalga es de un género seleccionado del grupo que consiste de Chlorella, Pseudochlorella y Prototheca.
- 235El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es sacarosa y la microalga es de un género seleccionado del grupo que consiste de Chlorella y Bracteococcus.
- 236El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es fructosa y la microalga es de un género seleccionado del grupo que consiste de Chlorella, Parachlorella, Prototheca y Scenedesmus.
- 237El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es arabinosa y la microalga es Chlorella sp.
- 238El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es mañosa y la microalga es de un género seleccionado del grupo que consiste de Chlorella, Parachlorella, Bracteococcus, Prototheca y Scenedesmus.
- 239El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es galactosa y la microalga es de un género seleccionado del grupo que consiste de Bracteococcus, Parachlorella, Chlorella, Pseudochlorella, Bracteococcus y Prototheca.
- 240El método de conformidad con la reivindicación 232, caracterizado porque el sustrato de carbono es acetato y la microalga es de un género seleccionado del grupo que consiste de Chlorella, Parachlorella y Prototheca.
- 241El método de conformidad con la reivindicación 232, caracterizado porque el medio de cultivo incluye además por lo menos una enzima de utilización de sacarosa.
- 242El método de conformidad con la reivindicación 241, caracterizado porque la microalga es del género Chlorella.
- 243El método de conformidad con la reivindicación 242, caracterizado porque la microalga ha sido diseñada genéticamente para expresar un gen exógeno que codifica por lo menos una enzima de modificación de lípido, enzima de modificación de hidrocarburo o enzima de utilización de sacarosa.
- 244El método de conformidad con la reivindicación 241, caracterizado porque el medio de cultivo incluye una invertasa de sacarosa.
- 245El método de conformidad con la reivindicación 232, caracterizado porque la microalga es seleccionada de la Tabla 1.
- 246Un método para cultivar microalgas caracterizado porque comprende colocar una pluralidad de células de microalgas en presencia de material celulósico despolimerizado.
- 247El método de conformidad con la reivindicación 246, caracterizado porque las microalgas son cultivadas en presencia de una fuente de carbono fija adicional seleccionada del grupo que consiste de glicerol, sacarosa, glucosa, arabinosa, galactosa, xilosa, fructosa, arabinosa, mañosa, acetato y cualquier combinación de los anteriores.
- 248El método de conformidad con la reivindicación 246, caracterizado porque la microalga es seleccionada de una especie del género Bracteococcus, una especie del género Chlorella, una especie del género Parachlorella, una especie del género Prototheca o una especie del género Pseudochlorella.
- 249El método de conformidad con la reivindicación 248, caracterizado porque la microalga es seleccionada de Bracteococcus minor, Chlorella ellipsoidea, Chlorella kessleri, Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorella minutissima, Chlorella ovalis, Chlorella protothecoides, Chlorella saccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris, Parachlorella kessleri, Prototheca moriformis y Pseudochlorella aquatica.
- 250El método de conformidad con la reivindicación 246, caracterizado porque las microalgas son cultivadas en presencia de por lo menos una enzima de utilización de sacarosa.
- 251El método de conformidad con la reivindicación 250, caracterizado porque la microalga es del género Chlorella.
- 252El método de conformidad con la reivindicación 246, caracterizado porque la microalga ha sido diseñada genéticamente para expresar un gen exógeno que codifica por lo menos una enzima de modificación de lípido, enzima de modificación de hidrocarburo o enzima de utilización de sacarosa.
- 253El método de conformidad con la reivindicación 250, caracterizado porque la por lo menos una enzima de utilización de sacarosa es una invertasa de sacarosa.
- 254El método de conformidad con la reivindicación 252, caracterizado porque la por lo menos una enzima de modificación de lípido es seleccionada de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenase, una acetil-CoA carboxilasa y una glicerol-3 fosfato aciltransferasa.
- 255El método de conformidad con la reivindicación 252, caracterizado porque la por lo menos una enzima de modificación de hidrocarburo es seleccionada de una acil-ACP tioesterasa grasa, una acil-CoA reductasa grasa, una aldehido reductasa grasa, una acil-CoA/aldehído reductasa grasa, una aldehido decarboxilasa grasa y una proteína portadora de acilo.
- 256Un método para cultivar un microbio que produce lípidos, el método está caracterizado porque comprende cultivar el microbio en presencia de ácido acético y en ausencia de una fuente de nitrógeno fija.
- 257El método de conformidad con la reivindicación 256, caracterizado porque el microbio es cultivado en presencia de una cantidad suficiente de ácido acético para incrementar el rendimiento de lípido microbiano con respecto al rendimiento de lípido microbiano en ausencia de ácido acético, en donde las condiciones de cultivo son de otra manera las mismas entre los dos cultivos.
- 258Un cultivo microbiano caracterizado porque contiene:(a) una población de microorganismos;y (b) un medio de cultivo que comprende glucosa, xilosa y una molécula seleccionada del grupo que consiste de lignina y una especie de furfural.
- 259El cultivo microbiano de conformidad con la reivindicación 258, caracterizado porque los microorganismos son microalgas.
- 260El cultivo microbiano de conformidad con la reivindicación 259, caracterizado porque los microorganismos son seleccionados del grupo que consiste de las microalgas enlistadas en la Tabla 1.
- 261El cultivo microbiano de conformidad con la reivindicación 260, caracterizado porque los microorganismos son una especie del género Chlorella.
- 262El cultivo microbiano de conformidad con la reivindicación 261, caracterizado porque los microorganismos son seleccionados del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. Umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. Vulgaris f. tertia, Chlorella vulgaris var. Vulgaris f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 263El cultivo microbiano de conformidad con la reivindicación 258, caracterizado porque los microorganismos son seleccionados de Bracteococcus minor, Chlorella ellipsoidea, Chlorella kessleri, Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorella minutissima, Chlorella ovalis, Chlorella protothecoides, Chlorella saccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris, Parachlorella kessleri, Prototheca moriformis y Pseudochlorella aquatica.
- 264El cultivo microbiano de conformidad con la reivindicación 258, caracterizado porque los microorganismos son levadura oleaginosa.
- 265El cultivo microbiano de conformidad con la reivindicación 264, caracterizado porque la levadura oleaginosa son seleccionadas del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis y Yarrowia lipolytica.
- 266El cultivo microbiano de conformidad con la reivindicación 258, caracterizado porque los microorganismos son hongos.
- 267El cultivo microbiano de conformidad con la reivindicación 266, caracterizado porque los hongos son seleccionados del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopusy una especie del género Pythium.
- 268El cultivo microbiano de conformidad con la reivindicación 258, caracterizado porque los microorganismos contienen por lo menos un gen de utilización de sacarosa exógeno.
- 269El cultivo microbiano de conformidad con la reivindicación 268, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa o una fructocinasa.
- 270El cultivo microbiano de conformidad con la reivindicación 258, caracterizado porque los microorganismos contienen por lo menos un gen exógeno que codifica un enzima de ruta de lípido.
- 271El cultivo microbiano de conformidad con la reivindicación 270, caracterizado porque la enzima de ruta de lípido es seleccionado del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenasa, una acetil-CoA carboxilasa, una proteína portadora de acilo y una glicerol-3 fosfato aciltransferasa.
- 272Un método para la manufactura de biodiesel, el método está caracterizado porque comprende:(a) cultivar un microorganismo que produce lípidos en un primer cultivo microbiano;(b) recuperar el lípido de la biomasa producida por el primer cultivo microbiano;(c) someter el lípido a transesterificación para producir ésteres de ácido graso y glicerol;y (d) agregar el glicerol a un segundo cultivo microbiano.
- 273El método de conformidad con la reivindicación 272, caracterizado porque los primeros y segundos cultivos microbianos son cultivos de la misma especie de microorganismo.
- 274El método de conformidad con la reivindicación 272, caracterizado porque el microorganismo es una microalga.
- 275El método de conformidad con la reivindicación 274, caracterizado porque el microorganismo es seleccionado del grupo que consiste de las microalgas enlistadas en la Tabla 1.
- 276El método de conformidad con la reivindicación 275, caracterizado porque el microorganismo es una especie del género Chlorella.
- 277El método de conformidad con la reivindicación 276, caracterizado porque el microorganismo es seleccionado del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. Umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 278El método de conformidad con la reivindicación 272, caracterizado porque el segundo cultivo microbiano comprende microorganismos seleccionados del grupo que consiste de Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca moriformis, Chlorella minutissima, Chlorella sp. y Chlorella sorokiniana.
- 279El método de conformidad con la reivindicación 272, caracterizado porque el microorganismo es una levadura oleaginosa.
- 280El método de conformidad con la reivindicación 279, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, fíhodotorula glutinis, fíhodotorula gracilis y Yarrowia lipolytica.
- 281El método de conformidad con la reivindicación 272, caracterizado porque el microorganismo es un hongo.
- 282El método de conformidad con la reivindicación 281, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 283El método de conformidad con la reivindicación 272, caracterizado porque el microorganismo contiene por lo menos un gen de utilización de sacarosa exógeno.
- 284El método de conformidad con la reivindicación 283, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa o una fructocinasa.
- 285El método de conformidad con la reivindicación 272, caracterizado porque el microorganismo contiene por lo menos un gen exógeno que codifica una enzima de ruta de lípido.
- 286El método de conformidad con la reivindicación 285, caracterizado porque la enzima de ruta de lípido es seleccionado del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenase, una acetil-CoA carboxilasa, una proteína portadora de acilo y una glicerol-3 fosfato aciltransferasa.
- 287Un método de fermentación caracterizado porque comprende cultivar un microorganismo en presencia de glicerol y por lo menos otra fuente de carbono fija.
- 288El método de conformidad con la reivindicación 287, caracterizado porque el glicerol y la por lo menos otra fuente de carbono fija son provistos al microorganismo simultáneamente a una proporción predeterminada.
- 289El método de conformidad con la reivindicación 288, caracterizado porque todo el glicerol y la por lo menos otra fuente de carbono fija son provistos al microorganismo al comienzo de la fermentación.
- 290El método de conformidad con la reivindicación 288, caracterizado porque todo del glicerol y la por lo menos otra fuente de carbono fija son alimentados al microorganismo a una velocidad predeterminada durante el curso de la fermentación.
- 291El método de conformidad con la reivindicación 287, caracterizado porque (a) el glicerol es provisto al microorganismo en ausencia de la por lo menos otra fuente de carbono fija por un primer período de tiempo;(b) la por lo menos otra fuente de carbono fija es provista al final del primer período de tiempo;y (c) el microorganismo es cultivado por un segundo período de tiempo en presencia de la por lo menos otra fuente de carbono fija.
- 292El método de conformidad con la reivindicación 291, caracterizado porque la por lo menos otra fuente de carbono fija es alimentada al microorganismo a una velocidad predeterminada durante el segundo período de tiempo.
- 293El método de conformidad con la reivindicación 291, caracterizado porque todo de la por lo menos otra fuente de carbono fija es provista al microorganismo al final del primer período de tiempo.
- 294El método de conformidad con la reivindicación 287, caracterizado porque:(a) la por lo menos otra fuente de carbono fija es provista al microorganismo en ausencia de glicerol por un primer período de tiempo;(b) el glicerol es provisto al final del primer período de tiempo;y (c) el microorganismo es cultivado por un segundo período de tiempo en presencia de glicerol.
- 295El método de conformidad con la reivindicación 287, caracterizado porque el microorganismo es una microalga.
- 296El método de conformidad con la reivindicación 295, caracterizado porque la microalga es seleccionada de la Tabla 1.
- 297El método de conformidad con la reivindicación 295, caracterizado porque la microalga es del género Chlorella.
- 298El método de conformidad con la reivindicación 297, caracterizado porque la microalga es seleccionada del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. Umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella y Chlorella zofingiensis.
- 299El método de conformidad con la reivindicación 287, caracterizado porque el microorganismo es una levadura oleaginosa.
- 300El método de conformidad con la reivindicación 299, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis y Yarrowia lipolytica.
- 301El método de conformidad con la reivindicación 287, caracterizado porque el microorganismo es un hongo.
- 302El método de conformidad con la reivindicación 301, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Modierella, Modierrla vinacea, Modierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género fíhizopus y una especie del género Pythium.
- 303El método de conformidad con la reivindicación 287, caracterizado porque el microorganismo contiene por lo menos un gen de utilización de sacarosa exógeno.
- 304El método de conformidad con la reivindicación 303, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, unaglucocinasa, o una fructocinasa.
- 305El método de conformidad con la reivindicación 287, caracterizado porque el microorganismo contiene por lo menos un gen exógeno que codifica una enzima de ruta de lípido.
- 306El método de conformidad con la reivindicación 305, caracterizado porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenase, una acetil-CoA carboxilasa, una proteína portadora de acilo, y una glicerol-3 fosfato aciltransferasa.
- 307El método de conformidad con la reivindicación 287, caracterizado porque el glicerol es un producto secundario de un proceso de transesterificación.
- 308El método de conformidad con la reivindicación 307, caracterizado porque el glicerol es acidulado.
- 309El método de conformidad con la reivindicación 307, caracterizado porque el glicerol es no acidulado.
- 310El método de conformidad con la reivindicación 287, caracterizado porque la por lo menos otra fuente de carbono fija es glucosa.
- 311El método de conformidad con la reivindicación 287, caracterizado porque la por lo menos otra fuente de carbono fija es material celulósico despolimerizado.
- 312El método de conformidad con la reivindicación 287, caracterizado porque la por lo menos otra fuente de carbono fija es sacarosa.
- 313Un termentador caracterizado porque comprende:(a) una población de microorganismos;(b) glicerol y (c) por lo menos un azúcar seleccionado del grupo que consiste de xilosa, glucosa y sacarosa.
- 314El termentador de conformidad con la reivindicación 313, caracterizado porque el glicerol es un producto secundario de un proceso de transesterificación de lípido.
- 315El termentador de conformidad con la reivindicación 313, caracterizado porquelos microorganismos son microalgas.
- 316El termentador de conformidad con la reivindicación 315, caracterizado porquelos microorganismos son seleccionados del grupo que consiste de las microalgas enlistados en la Tabla 1.
- 317El termentador de conformidad con la reivindicación 316, caracterizado porquelos microorganismos son una especie del género Chlorella.
- 318El termentador de conformidad con la reivindicación 317, caracterizado porquelos microorganismos son seleccionados del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kesslerí, Chlorella luteovirídis, Chlorella luteovirídis var. aureoviridis, Chlorella luteovirídis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris/, tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris/, tertia, Chlorella vulgaris var. vulgaris/, viridis, Chlorella xanthella, y Chlorella zofingiensis.
- 319El termentador de conformidad con la reivindicación 313, caracterizado porque los microorganismos son seleccionados del grupo que consiste de Parachlorella kesslerí, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca mori/ormis, Chlorella minutissima, Chlorella sp., y Chlorella sorokiniana.
- 320El termentador de conformidad con la reivindicación 313, caracterizado porque los microorganismos son levadura oleaginosa.
- 321El termentador de conformidad con la reivindicación 320, caracterizado porque la levadura oleaginosa son seleccionadas del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipo/er, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, y Yarrowia lipolytica.
- 322El termentador de conformidad con la reivindicación 313, caracterizado porque los microorganismos son hongos.
- 323El termentador de conformidad con la reivindicación 322, caracterizado porque los hongos son seleccionados del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus, y una especie del género Pythium.
- 324El termentador de conformidad con la reivindicación 313, caracterizado porque los microorganismos contienen por lo menos un gen de utilización de sacarosa exógeno.
- 325El termentador de conformidad con la reivindicación 324, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, unaglucocinasa, o una fructocinasa.
- 326El termentador de conformidad con la reivindicación 313, caracterizado porque los microorganismos contienen por lo menos un gen exógeno que codifica una enzima de ruta de lípido.
- 327El termentador de conformidad con la reivindicación 326, caracterizado porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenasa, una acetil-CoA carboxilasa, una proteína portadora de acilo, y una glicerol-3 fosfato aciltransferasa.
- 328Un método para la fermentación de un microorganismo caracterizado porque comprende proveer glicerol producto secundario de un proceso de transesterificación como una sola fuente de energía de carbono fijo.
- 329El método de conformidad con la reivindicación 328, caracterizado porque no se proporciona energía de luz al microorganismo.
- 330El método de conformidad con la reivindicación 328, caracterizado porque se proporciona energía de luz al microorganismo.
- 331El método de conformidad con la reivindicación 328, caracterizado porque el microorganismo es una microalga.
- 332El método de conformidad con la reivindicación 331, caracterizado porque el microorganismo es seleccionado del grupo que consiste de las microalgas enlistados en la Tabla 1.
- 333El método de conformidad con la reivindicación 332, caracterizado porque el microorganismo es una especie del género Chlorella.
- 334El método de conformidad con la reivindicación 333, caracterizado porque el microorganismo es seleccionado del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureov^dis, Chlorella Candida , Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris/, tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris/, tertia, Chlorella vulgaris var. vulgaris/, viridis, Chlorella xanthella, y Chlorella zofingiensis.
- 335El método de conformidad con la reivindicación 328, caracterizado porque el microorganismo es seleccionado de Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca mori/ormis, Chlorella minutissima, Chlorella sp., y Chlorella sorokiniana.
- 336El método de conformidad con la reivindicación 328, caracterizado porque el microorganismo es una levadura oleaginosa.
- 337El método de conformidad con la reivindicación 336, caracterizado porque la levadura oleaginosa es seleccionada del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipo/er, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, y Yarrowia lipolytica.
- 338El método de conformidad con la reivindicación 328, caracterizado porque el microorganismo es un hongo.
- 339El método de conformidad con la reivindicación 338, caracterizado porque el hongo es seleccionado del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus, y una especie del género Pythium.
- 340El método de conformidad con la reivindicación 328, caracterizado porque el microorganismo contiene por lo menos un gen de utilización de sacarosa exógeno.
- 341El método de conformidad con la reivindicación 340, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, unaglucocinasa, o una fructocinasa.
- 342El método de conformidad con la reivindicación 328, caracterizado porque el microorganismo contiene por lo menos un gen exógeno que codifica una enzima de ruta de lípido.
- 343El método de conformidad con la reivindicación 342, caracterizado porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenasa, una acetil-CoA carboxilasa, una proteína portadora de acilo, y una glicerol-3 fosfato aciltransferasa.
- 344Un microorganismo caracterizado porque contiene un gen de utilización de sacarosa exógeno.
- 345El microorganismo de conformidad con la reivindicación 344, caracterizado porque el gen codifica un transportador de sacarosa.
- 346El microorganismo de conformidad con la reivindicación 344, caracterizado porque el gen codifica una invertasa de sacarosa.
- 347El microorganismo de conformidad con la reivindicación 344, caracterizado porque el gen codifica una fructocinasa.
- 348El microorganismo de conformidad con la reivindicación 344, caracterizado porqueel microorganismo es una microalga.
- 349El microorganismo de conformidad con la reivindicación 348, caracterizado porqueel microorganismo es seleccionado del grupo que consiste de las microalgas enlistadas enla Tabla 1.
- 350El microorganismo de conformidad con la reivindicación 349, caracterizado porque el microorganismo es una especie del género Chlorella.
- 351El microorganismo de conformidad con la reivindicación 350, caracterizado porque el microorganismo es una especie seleccionada del grupo que consiste de Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., o Chlorella protothecoides.
- 352Una célula de la especie Chlorella protothecoides, Chlorella emersonii, o Chlorella minutissima caracterizado porque la célula contiene un gen exógeno.
- 353La célula de conformidad con la reivindicación 352, caracterizada porque el gen exógeno codifica una proteína seleccionado del grupo que consiste de un transportador de sacarosa, una invertasa de sacarosa, una enzima de modificación de lípido, una enzima de modificación de hidrocarburo y una fructocinasa.
- 354La célula de conformidad con la reivindicación 353, caracterizada porque la proteína es una invertasa de sacarosa secretada al espacio extracelular.
- 355La célula de conformidad con la reivindicación 353, caracterizada porque la proteína es una invertasa de sacarosa apuntada al citoplasma.
- 356Un cultivo microbiano caracterizado porque contiene:(a) una población de microorganismos y (b) un medio de cultivo que comprende (i) sacarosa, y (¡i) una enzima de invertasa de sacarosa.
- 357El cultivo microbiano de conformidad con la reivindicación 356, caracterizado porque los microorganismos son microalgas.
- 358El cultivo microbiano de conformidad con la reivindicación 357, caracterizado porque los microorganismos son seleccionados del grupo que consiste de las microalgas enlistadas en la Tabla 1.
- 359El cultivo microbiano de conformidad con la reivindicación 358, caracterizado porque los microorganismos son una especie del género Chlorella.
- 360El cultivo microbiano de conformidad con la reivindicación 359, caracterizado porque los microorganismos son seleccionados del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida , Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris/, tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris/, tertia, Chlorella vulgaris var. vulgaris/, viridis, Chlorella xanthella, y Chlorella zofingiensis.
- 361El cultivo microbiano de conformidad con la reivindicación 356, caracterizado porque los microorganismos son levadura oleaginosa.
- 362El cultivo microbiano de conformidad con la reivindicación 361, caracterizado porque la levadura oleaginosa son seleccionados del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, y Yarrowia lipolytica.
- 363El cultivo microbiano de conformidad con la reivindicación 356, caracterizado porque los microorganismos son hongos.
- 364El cultivo microbiano de conformidad con la reivindicación 363, caracterizado porque los hongos son seleccionado del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Miicor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus y una especie del género Pythium.
- 365El cultivo microbiano de conformidad con la reivindicación 356, caracterizado porque los microorganismos contienen por lo menos un gen de utilización de sacarosa exógeno.
- 366El cultivo microbiano de conformidad con la reivindicación 365, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa, o una fructocinasa.
- 367El cultivo microbiano de conformidad con la reivindicación 366, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa.
- 368El cultivo microbiano de conformidad con la reivindicación 356, caracterizado porque la enzima de sacarosa invertasa es una enzima de sacarosa invertasa secretable codificada por un gen de sacarosa invertasa exógeno expresado por la población de microorganismos.
- 369El cultivo microbiano de conformidad con la reivindicación 356, caracterizado porque el microorganismo contiene por lo menos un gen exógeno que codifica una enzima de ruta de lípido.
- 370El cultivo microbiano de conformidad con la reivindicación 369, caracterizado porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenasa, una acetil-CoA carboxilasa, una proteína portadora de acilo, y una glicerol-3 fosfato aciltransferasa.
- 371Un cultivo microbiano caracterizado porque contiene:(a) una población de microorganismos y (b) un medio de cultivo que comprende (i) melasas y (¡i) una enzima de sacarosa invertasa.
- 372El cultivo microbiano de conformidad con la reivindicación 371, caracterizado porque los microorganismos son microalgas.
- 373El cultivo microbiano de conformidad con la reivindicación 372, caracterizado porque los microorganismos son seleccionados del grupo que consiste de las microalgas enlistados en la Tabla 1.
- 374El cultivo microbiano de conformidad con la reivindicación 373, caracterizado porque los microorganismos son una especie del género Chlorella.
- 375El cultivo microbiano de conformidad con la reivindicación 374, caracterizado porque los microorganismos son seleccionados del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pnngsheimii, Chlorella protothecoides, Chlorella regular is, Chlorella regularis var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris/, tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris/, tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, y Chlorella zofingiensis.
- 376El cultivo microbiano de conformidad con la reivindicación 371 , caracterizado porque los microorganismos son levadura oleaginosa.
- 377El cultivo microbiano de conformidad con la reivindicación 376, caracterizado porque las levaduras oleaginosas son seleccionadas del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, y Yarrowia lipolytica.
- 378El cultivo microbiano de conformidad con la reivindicación 371, caracterizado porque los microorganismos son hongos.
- 379El cultivo microbiano de conformidad con la reivindicación 378, caracterizado porque los hongos son seleccionados del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium ¡ilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus, y una especie del género Pythium.
- 380El cultivo microbiano de conformidad con la reivindicación 371, caracterizado porque el microorganismo contienen por lo menos un gen de utilización de sacarosa exógeno.
- 381El cultivo microbiano de conformidad con la reivindicación 380, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una invertasa de sacarosa, una hexocinasa, una glucocinasa, o una fructocinasa.
- 382El cultivo microbiano de conformidad con la reivindicación 381, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa.
- 383El cultivo microbiano de conformidad con la reivindicación 371, caracterizado porque la enzima de sacarosa invertasa es una enzima de sacarosa invertasa secretable codificada por un gen de sacarosa invertasa exógeno expresado por la población de microorganismos.
- 384El cultivo microbiano de conformidad con la reivindicación 371, caracterizado porque los microorganismos contienen por lo menos un gen exógeno que codifica una enzima de ruta de lípido.
- 385El cultivo microbiano de conformidad con la reivindicación 384, caracterizado porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una esteaoril-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenase, una acetil-CoA carboxilasa, una proteína portadora de acilo, y una glicerol-3 fosfato aciltransferasa.
- 386Un cultivo microbiano caracterizado porque contiene:(a) una población de microorganismos y (b) un medio de cultivo que comprende (i) sacarosa;(¡i) lignina y (¡ii) una enzima de sacarosa invertasa.
- 387El cultivo microbiano de conformidad con la reivindicación 386, caracterizado porque los microorganismos son microalgas.
- 388El cultivo microbiano de conformidad con la reivindicación 387, caracterizado porque los microorganismos son seleccionados del grupo que consiste de las microalgas enlistadas en la Tablal.
- 389El cultivo microbiano de conformidad con la reivindicación 388, caracterizado porque los microorganismos son una especie del género Chlorella.
- 390El cultivo microbiano de conformidad con la reivindicación 389, caracterizado porque los microorganismos son seleccionados del grupo que consiste de Chlorella anitrata, Chlorella antárctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regular is var. mínima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris/, tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris/, tertia, Chlorella vulgaris var. vulgaris/, viridis, Chlorella xanthella, y Chlorella zofingiensis.
- 391El cultivo microbiano de conformidad con la reivindicación 386, caracterizado porque los microorganismos son levadura oleaginosa.
- 392El cultivo microbiano de conformidad con la reivindicación 391, caracterizado porque la levadura oleaginosa son seleccionadas del grupo que consiste de Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipo/er, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, y Yarrowia lipolytica.
- 393El cultivo microbiano de conformidad con la reivindicación 386, caracterizado porque los microorganismos son hongos.
- 394El cultivo microbiano de conformidad con la reivindicación 393, caracterizado porque los hongos son seleccionados del grupo que consiste de una especie del género Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium ¡ilacinum, una especie del género Hensenulo, una especie del género Chaetomium, una especie del género Cladosporium, una especie del género Malbranchea, una especie del género Rhizopus, y una especie del género Pythium.
- 395El cultivo microbiano de conformidad con la reivindicación 386, caracterizado porque los microorganismos contienen por lo menos un gen de utilización de sacarosa exógeno.
- 396El cultivo microbiano de conformidad con la reivindicación 395, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa, una sacarosa invertasa, una hexocinasa, unaglucocinasa, o una fructocinasa.
- 397El cultivo microbiano de conformidad con la reivindicación 396, caracterizado porque el gen de utilización de sacarosa codifica un transportador de sacarosa.
- 398El cultivo microbiano de conformidad con la reivindicación 386, caracterizado porque la enzima de sacarosa invertasa es una enzima de sacarosa invertasa secretable codificada por un gen de sacarosa invertasa exógeno expresado por la población de microorganismos.
- 399El cultivo microbiano de conformidad con la reivindicación 386, caracterizado porque los microorganismos contienen por lo menos un gen exógeno que codifica una enzima de ruta de lípido.
- 400El cultivo microbiano de conformidad con la reivindicación 399, caracterizado porque la enzima de ruta de lípido es seleccionada del grupo que consiste de una estearoil-ACP desaturasa, una glicerolípido desaturasa, una piruvato deshidrogenasa, una acetil-CoA carboxilasa, una proteína portadora de acilo, y una glicerol-3 fosfato aciltransferasa.
- 401Un ácido nucleico caracterizado porque comprende:(a) un cADN que codifica un gen de utilización de sacarosa y (b) un cADN que codifica una proteína que confiere resistencia al antibiótico higromicina o el antibiótico G418.
- 402Un método para cultivar microalgas, caracterizado porque comprende:(a) proveer una célula de microalga apta de efectuar crecimiento heterotrópico;(b) colocar la célula de microalga en un medio de cultivo, en donde el medio de cultivo comprende material celulósico despolimerizado y (c) incubar las microalgas por un periodo de tiempo suficiente para permitir que las células crezcan.
Independent claims402
1,049 paragraphs in 70 sections, as filed
The present disclosure is concerned with the production of oils, fuels and oleochemicals made from microorganisms. In particular, the disclosure is concerned with oil-bearing microorganisms, including microalgae, yeast, and fungi, and with methods of cultivating such microorganisms for the production of useful compounds, including lipids, fatty acid esters, fatty acids, fatty acids, aldehydes, alcohols and alkanes, for use in industry or as a source of energy or food.
Microorganisms of the invention may be selected or genetically engineered for use in the methods or other aspects of the invention described herein.
BACKGROUND OF THE INVENTION
Fossil fuel is a general term for buried combustible geological deposits of organic materials formed from decaying plants and animals that have been converted to acceptable crude oil, coal, natural gas, or heavy oils by exposure to heat and pressure in the earth's crust during hundreds of millions of years.
In common dialogue, fossil fuel, also known as mineral fuel, is used synonymously with other hydrocarbon-containing natural resources such as coal, oil, and natural gas. The use of fossil fuels has enabled large-scale industrial development and has largely supplanted water-powered mills, as well as burning wood or ++++ turba for heating. Fossil fuels are a finite nonrenewable resource.
When electricity is generated, the energy from burning fossil fuels is often used to drive a turbine. Older generations often used steam generated from burning fuel to turn the turbine, but in newer power plants, the gases produced by burning fuel directly turn a gas turbine. With global modernization in the 20th and 21st centuries, the thirst for energy from fossil fuels, especially petroleum-derived gasoline, is one of the causes of major regional and global conflicts.
The burning of fossil fuels by humans is the largest source of carbon dioxide emissions, which is one of the greenhouse gases that enable radioactive forcing and contribute to global warming. In the United States of America, more than 90% of greenhouse gas emissions come from the combustion of fossil fuels. In addition, other air pollutants, such as nitrogen oxides, sulfur dioxide, volatile organic compounds (VOCs), and heavy metals are produced.
Human activity raises greenhouse gas levels mainly through the release of carbon dioxide from fossil fuel combustion, but other gases, for example methane, are not negligible. Concentrations of various greenhouse gases have increased over time due to human activities such as the burning of fossil fuels and deforestation leading to higher concentrations of carbon dioxide. According to the global warming hypothesis, greenhouse gases from industry and agriculture have played a major role in the recently observed global warming.
The increased demand for energy by the global economy has also placed increased pressure on the cost of hydrocarbons. In addition to energy, many industries, including plastics and chemical manufacturers, are heavily dependent on the availability of hydrocarbons as raw materials for their manufacturing processes. Cost-effective alternatives to current sources of supply could help mitigate the upward pressure on energy and these raw material costs.
BRIEF DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a microbe, which in various embodiments may comprise a microalgal cell, an oleaginous yeast or a fungus that contains an exogenous gene encoding a protein selected from the group consisting of a lipase, sucrose transporter, sucrose invertase, fructokinase, polysaccharide-degrading enzyme, a fatty acyl-ACP thioesterase, a fatty acyl-CoA aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase and an acyl carrier protein (ACP). The microbe (for example, microalga cell) can be selected for example from Table 1. In particular embodiments, the cell is a species of the genus Chlorella, such as for example Chlorella fusca, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella kessleri, Chlorella vulgaris, Chlorella saccharophila, Chlorella sorokiniana or Chlorella ellipsoidea. In other embodiments, the microbe is an oily yeast selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica. In still other embodiments, the microbe is a fungus selected from the group consisting of a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor clrcinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicilllum iilacinum, a species of the genus Hensenulo, a species from the genus Chaetomium, one species from the genus Cladosporium, one species from the genus Malbranchea, one species from the genus Rhlzopus, and one species from the genus Pythium. In other embodiments, the invention includes the expression of hydrocarbon modification enzymes in bacterial hosts such as E. coli and Badila method for producing renewable diesel. In one embodiment, the method comprises: (a) culturing a population of microorganisms in the presence of a fixed carbon source, where (i) the microorganisms accumulate at least 10% of their dry cell weight as lipid and (ii) the fixed carbon source is selected from group consisting of glycerol, depolymerized cellulosic material, sucrose, molasses, glucose, arabnose, galactose, xylose, fructose, arabnose, mannose, acetate, and any combination of the above, (b) isolating the lipid components from the cultured microorganisms and (c) subjecting the isolated lipid components to one or more chemical reactions to generate straight chain alkanes, whereby the renewable diesel is produced.
In another aspect, the present invention is concerned with a liquid hydrocarbon composition made in accordance with the method described directly above, wherein the composition conforms to the specifications of ASTM D975.
In another aspect, the present invention is concerned with a method for producing jet fuel. In one embodiment, the method comprises (a) culturing a population of microorganisms in the presence of a fixed carbon source, wherein (i) the microorganisms accumulate at least 10% of their dry cell weight as lipid and (ii) the fixed carbon source is selected from the group consisting of glycerol, depolymerized cellulosic material, sucrose, glucose, arabinose, galactose, xylose, fructose, arabinose, mannose, acetate, and any combination of the above, (b) isolating the lipid components from the cultured microorganisms, (c) subjecting the isolated lipid components to one or more chemical reactions to generate straight chain alkanes, (d) subjecting the straight chain alkanes to catalytic pyrolysis or cracking , whereby jet fuel is produced.
In another aspect, the present invention is concerned with a composition of liquid hydrocarbons produced in accordance with the method described directly above, wherein the composition conforms to the specifications of ASTM D 1655.
In another aspect, the present invention is concerned with a microalgae or yeast cell that has been genetically engineered and/or selected to express a lipid pathway enzyme at an altered level compared to a wild-type cell of the same species. In some cases, the cell produces more lipid compared to the wild-type cell when both cells are grown under the same conditions. In some cases, the cell has been genetically engineered and/or selected to express a lipid pathway enzyme at a higher level than the wild-type cell. In some cases, the lipid pathway enzyme is selected from the group consisting of pyruvate dehydrogenase, acetyl-CoA carboxylase, acyl carrier protein, and glycerol-3 phosphate acyltransferase. In some cases, the cell has been genetically engineered and/or selected to express a lipid pathway enzyme at a lower level than the wild-type cell. In at least one embodiment in which the cell expresses the lipid pathway enzyme at a lower level, the lipid pathway enzyme comprises citrate synthase.
In some embodiments, the microalga or yeast cell described above has been genetically engineered and/or selected to express a global regulator of fatty acid synthesis at an altered level compared to the wild-type cell, whereby expression levels of a plurality of synthetic fatty acid genes are altered compared to the wild-type cell. In some cases, the lipid pathway enzyme comprises an enzyme that modifies a fatty acid. In some cases, the lipid pathway enzyme is selected from a stearoyl-ACP desaturase and a glycerolipid desaturase.
In another aspect, the present invention is concerned with an oil-producing microbe containing one or more foreign genes, wherein the foreign genes encode protein(s) selected from the group consisting of a fatty acyl-ACP thioesterase, a a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty acyl-CoA/aldehyde reductase, a fatty aldehyde decarboxylase, and an acyl carrier protein. In some cases, the microbe is Chlorella protothecoides, Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, or Chlorella sp. In other cases, the microbe is of another species as described herein. In one embodiment, the foreign gene is in operable linkage to a promoter, which is inducible or repressible in response to a stimulus. In some cases, the stimulus is selected from the group consisting of an exogenously supplied small molecule, heat, cold, and light. In some cases, the foreign gene is expressed in a cellular compartment. In some embodiments, the cellular compartment is selected from the group consisting of a chloroplast and a mitochondria.
In one embodiment, the exogenous gene encodes a fatty acid acyl-ACP thioesterase. In some cases, the thioesterase encoded by the exogenous gene catalyzes the cleavage of a C8-C18 fatty acid from an acyl carrier protein (ACP). In some cases, the thioesterase encoded by the exogenous gene catalyzes the cleavage of a 10- to 14-carbon fatty acid from a PCA. In one embodiment, the thioesterase encoded by the exogenous gene catalyzes the cleavage of a C12 fatty acid from a PCA.
In one embodiment, the exogenous gene encodes a fatty acyl-CoA/aldehyde reductase. In some cases, the reductase encoded by the exogenous gene catalyzes the reduction of a fatty acyl-CoA of 20 to 30 carbon atoms to the corresponding primary alcohol. In some cases, the reductase encoded by the exogenous gene catalyzes the reduction of a fatty acyl-CoA of 8 to 18 carbon atoms to a corresponding primary alcohol. In some cases, the reductase encoded by the exogenous gene catalyzes the reduction of a fatty acyl-CoA of 10 to 14 carbon atoms to a corresponding primary alcohol. In one embodiment, the reductase encoded by the exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol.
In one embodiment, the exogenous gene encodes a fatty acyl-CoA reductase. In some cases, the reductase encoded by the foreign gene catalyzes the reduction of a fatty acyl-CoA of 8 to 18 carbon atoms to a corresponding aldehyde. In one embodiment, the reductase encoded by the exogenous gene catalyzes the reduction of a C 12 fatty acyl-CoA to dodecanal.
In at least one embodiment, the microbe of the invention further contains one or more exogenous sucrose utilization genes.
In another aspect, the present invention is concerned with a microbe containing two foreign genes, wherein a first foreign gene codes for a fatty acyl-ACP thioesterase and a second foreign gene codes for a protein selected from the group consisting of an acyl-CoA reductase. a fatty acyl-CoA/fatty aldehyde reductase and an acyl carrier protein. In some cases, the microbe is Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. or Chlorella protothecoides. In other cases, the microbe is another species as described herein. In some cases, the two foreign genes are each in operable linkage to a promoter, which is inducible in response to a stimulus. In some cases, each promoter is inducible in response to an identical stimulus.
In one embodiment, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a C8 to C18 fatty acid from a PCA. In some embodiments, the second exogenous gene encodes a fatty acyl-CoA/aldehyde reductase that catalyzes the reduction of a fatty acyl-CoA of 8 to 18 carbon atoms to a corresponding primary alcohol. In some cases, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 10- to 14-carbon fatty acid from a PCA, and the reductase encoded by the second exogenous gene catalyzes the reduction of a 10-to fatty acyl-CoA. 14 carbon atoms to the corresponding primary alcohol, where thioesterase and reductase act on the same carbon chain length. In one embodiment, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 12-carbon fatty acid from a PCA and the reductase encoded by the second exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA. to dodecanol. In some embodiments, the second exogenous gene encodes a fatty acyl-CoA reductase that catalyzes the reduction of a fatty acyl-CoA of 8 to 18 carbon atoms to a corresponding aldehyde.
In some embodiments, the second exogenous gene encodes a fatty acyl-CoA reductase and the microbe further contains a third exogenous gene encoding a fatty aldehyde decarbonylase. In some cases, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18 carbon fatty acid from a PCA, the reductase encoded by the second exogenous gene catalyzes the reduction of an 8 to 18 fatty acyl-CoA. where the thioesterase, reductase and decarbonylase act on the same carbon chain length.
In some embodiments, the second exogenous gene encodes an acyl carrier protein that is naturally co-expressed with fatty acyl-ACP thioesterase.
In some embodiments, the second exogenous gene encodes an acyl carrier protein and the microbe further contains a third exogenous gene encoding a protein selected from the group consisting of a fatty acyl-CoA reductase and a fatty acyl-CoA/aldehyde reductase. In some cases, the third exogenous gene encodes a fatty acyl-CoA reductase and the microbe additionally contains a fourth exogenous gene encoding a fatty aldehyde decarbonylase.
In another aspect, the present invention is concerned with a method for producing a molecule in a population of microbes. In one embodiment, the method comprises culturing a population of microbes in a culture medium, wherein the microbes contain (i) a first foreign gene encoding a fatty acyl-ACP thioesterase and (ii) a second foreign gene encoding a fatty acyl-ACP thioesterase. acyl-CoA/fatty aldehyde reductase and microbes synthesize a fatty acid bound to an acyl carrier protein (ACP), fatty acyl-ACP thioesterase catalyzes the cleavage of fatty acid from ACP to produce, Through further processing, a fatty acyl-CoA and fatty acyl-CoA/aldehyde reductase catalyzes the reduction of acyl-CoA to an alcohol.
In one embodiment the method for producing a molecule in a population of microbes, the microbe is Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. or Chlorella protothecoides. In other cases, the microbe is another species of microorganism as described herein. In some cases, the culture medium contains glycerol. In one embodiment, glycerol is a by-product of a transesterification process. In some cases, the culture medium contains glycerol and at least one other source of fixed carbon. In one embodiment, the at least one other fixed carbon source is sucrose. In some cases, all of the glycerol and all of the other fixed carbon sources are supplied to the microbes at the start of fermentation. In some cases, glycerol and the at least one other fixed carbon source are fed to the microbes at a predetermined rate in the course of fermentation. In some culture methods of the invention, glycerol is provided to microbes in the absence of the at least one other fixed carbon source for a first period of time, the at least one other fixed carbon source is provided at the end of the first period of time. period of time and the microbes are cultured for a second period of time in the presence of the at least one other source of fixed carbon.
In some embodiments, the foreign genes are in operable linkage to a promoter that is inducible in response to a first stimulus. In some cases, the method further comprises providing the first stimulus and incubating the population of microbes for a first period of time in the presence of the first stimulus to produce an alcohol. In some cases, the method further comprises extracting alcohol from aqueous biomass comprising the culture medium and microbes.
In some embodiments, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18 carbon fatty acid from ACP and the reductase encoded by the second exogenous gene catalyzes the reduction of an 8 to 18 fatty acyl-CoA. 18 carbon atoms to a corresponding primary alcohol, where thioesterase and reductase act on the same carbon chain length. In some cases, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 10- to 14-carbon fatty acid from ACP and the reductase encoded by the second exogenous gene catalyzes the reduction of a 10 to 10-carbon fatty acyl-CoA. 14 carbon atoms to a corresponding primary alcohol, where thioesterase and reductase act on the same carbon chain length. In one embodiment, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 12-carbon fatty acid from ACP and the reductase encoded by the second exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA. to dodecanol. In some cases, the microbes also contain a third exogenous gene that encodes an acyl carrier protein. In some embodiments, the third exogenous gene encodes an acyl carrier protein that is naturally co-expressed with fatty acyl-ACP thioesterase.
In another aspect, the present invention is concerned with a method for producing a lipid molecule in a population of microbes. In one embodiment, the method comprises culturing a population of microbes in a culture medium, wherein the microbes contain (i) a first foreign gene encoding a fatty acyl-ACP thioesterase and (ii) a second foreign gene encoding a fatty acyl-ACP thioesterase. Fatty acyl-CoA reductase and where microbes synthesize a fatty acid bound to an acyl carrier protein (ACP), fatty acyl-ACP thioesterase catalyzes the cleavage of fatty acid from ACP to produce, Through further processing, a fatty acyl-CoA and fatty acyl-CoA reductase catalyze the reduction of acyl-CoA to an aldehyde. In some cases, the microbe is Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides. In other cases, the microbe is another species of microorganism as described herein.
In some embodiments, the foreign genes are in operable linkage to a promoter that is inducible in response to a first stimulus, and the method further comprises providing the first stimulus and incubating the population of microbes for a first period of time in the presence of the first stimulus to produce an aldehyde. In one embodiment, the method further comprises extracting the aldehyde from aqueous biomass comprising the culture medium and the microbe population.
In some embodiments, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18 carbon fatty acid from ACP and the reductase encoded by the second exogenous gene catalyzes the reduction of an 8 to 18 fatty acyl-CoA. 18 carbon atoms to a corresponding aldehyde, where thioesterase and reductase act on the same carbon chain length. In some cases, the microbes also contain a third exogenous gene that encodes a fatty aldehyde decarbonylase that catalyzes the conversion of the aldehyde to an alkane.
In some cases, the foreign genes are in operable linkage to a promoter that is inducible in response to a first stimulus, and the method further comprises providing the first stimulus and incubating the population of microbes for a first period of time in the presence of the first stimulus to produce an alkane. In some cases, the method further comprises extracting the alkane from aqueous biomass comprising the culture medium and the microbe population.
In some cases, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18 carbon fatty acid from ACP, reductase encoded by the second exogenous gene catalyzes the reduction of an 8 to 18 fatty acyl-CoA carbon atoms to a corresponding aldehyde and the decarbonylase encoded by the third exogenous gene catalyzes the conversion of an aldehyde of 8 to 18 carbon atoms to a corresponding alkane, wherein the thioesterase, reductase and decarbonylase act on the same carbon chain length. In some embodiments, the microbes further contain a third exogenous gene encoding an acyl carrier protein. In some cases, the third exogenous gene encodes an acyl carrier protein that is naturally co-expressed with the fatty acyl-ACP thioesterase. In some cases, the microbes also contain a fourth exogenous gene that encodes a fatty aldehyde decarbonylase that catalyzes the conversion of the aldehyde to an alkane.
In some methods, the culture medium contains glycerol. In one embodiment, glycerol is a by-product of a transesterification process. In some cases, the culture medium contains glycerol and at least one other source of fixed carbon. In one embodiment, the at least one other fixed carbon source is sucrose. In some cases, all of the glycerol and all of the at least one other fixed carbon source are supplied to the microbes at the start of fermentation. In some cases, glycerol and the at least one other fixed carbon source are fed to the microbes at a predetermined rate in the course of fermentation. In one embodiment, glycerol is provided to the microbes in the absence of the at least one other fixed carbon source for a first time period, the at least one other fixed carbon source is provided at the end of the first time period, and the microbes are grown for a second period of time in the presence of the at least one other fixed carbon source.
In another aspect, the present invention is concerned with a method of producing a fatty acid molecule having a specific carbon chain length in a population of microbes. In one embodiment, the method comprises culturing a population of lipid-producing microbes in a culture medium, wherein the microbes contain an exogenous gene encoding a fatty acyl-ACP thioesterase having a specific activity at a carbon chain length and wherein microbes synthesize a fatty acid bound to an acyl carrier protein (ACP) and thioesterase catalyzes the cleavage of the fatty acid from the ACP, when the fatty acid has been synthesized to the specific carbon chain length. In some cases, the microbe is Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides. In other cases, the microbe is another species of microorganism as described herein.
In some embodiments, the foreign gene is operably linked to a promoter that is induced in response to a first stimulus, and the method further comprises providing the first stimulus and incubating the population of microbes for a period of time in the presence of the first stimulus. In some cases, the method further comprises extracting the fatty acid from aqueous biomass comprising the culture medium and the microbe population.
In some cases, the microbes also contain a second foreign gene that encodes an acyl carrier protein. In some embodiments, the second exogenous gene encodes an acyl carrier protein that is naturally co-expressed with fatty acyl-ACP thioesterase. In one embodiment, acyl-ACP thioesterase catalyzes the cleavage of a C8 to C18 fatty acid from ACP.
In some cases, the culture medium contains glycerol. In one embodiment, glycerol is a by-product of a transesterification process. In some embodiments, the culture medium contains glycerol and at least one other fixed carbon source. In one embodiment, the at least one other carbon source is sucrose. In some cases, all of the glycerol and all of the at least one other fixed carbon source are supplied to the microbes at the start of fermentation. In some cases, glycerol and the at least one other fixed carbon source are fed to the microbes at a predetermined rate in the course of fermentation. In one embodiment, glycerol is provided to the microbes in the absence of the at least one other fixed carbon source for a first time period, the at least one other fixed carbon source is provided at the end of the first time period, and the microbes are grown for a second period of time in the presence of the at least one other fixed carbon source.
In another aspect, the present invention is concerned with a microalgal cell containing a foreign gene, wherein the foreign gene encodes a protein selected from the group of a lipase, a sucrose transporter, a sucrose invertase, a fructosinase or a polysaccharide-degrading enzyme. . In some cases, the cell is Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides. In other cases, the cell is another species of microalgae as described herein.
In some cases, the foreign gene is in operable linkage to a promoter. In some cases, the promoter is inducible or repressible in response to a stimulus. In various modalities, the stimulus is selected from the group consisting of an exogenously supplied small molecule, heat, cold, and light. In some cases, the foreign gene is expressed in a cellular compartment. In some embodiments, the cellular compartment is selected from the group consisting of a chloroplast and a mitochondria.
In some cases, the gene encodes a lipase that has at least 70% amino acid identity to a lipase selected from Table 9. In one embodiment, the lipase is novozim-435. In one embodiment, the polysaccharide-degrading enzyme is endogenous to a Chlorella virus.
In another aspect, the present invention is concerned with a microalgal cell containing two foreign genes, wherein a first foreign gene codes for a lipase and a second foreign gene codes for a polysaccharide-degrading enzyme. In some cases, the foreign genes are each in operable linkage to a promoter. In some cases, the foreign genes are each in operable linkage to promoters that are inducible in response to a stimulus. In some cases, the foreign genes are each in operable linkage to promoters that are inducible in response to the same stimulus. In some cases, the foreign genes are each in operable linkage to a promoter that is inducible in response to at least one stimulus that does not induce the other promoter.
In another aspect, the present invention is concerned with a method for manufacturing a lipid molecule in a microbe. In one embodiment, the method comprises (a) culturing the microbe for a first period of time sufficient to increase cell density, wherein the microbe contains (i) an exogenous gene encoding a lipase and/or (ii) a gene exogenous encoding a polysaccharide-degrading enzyme, wherein the exogenous gene(s) is(are) in operable linkage to a promoter that is inducible in response to a stimulus, (b) providing the stimulus and (c) incubating the microbe for a second period of time in the presence of the stimulus.
In another aspect, the present invention is concerned with a method for manufacturing a lipid molecule in a microbe. In one embodiment, the method comprises (a) culturing a lipid-producing microbe for a first period of time sufficient to increase cell density, (b) providing a virus capable of infecting and lysing the microbe when in direct contact with the microbe and (c) incubating the microbe for a second period of time to produce lysed aqueous biomass. In one embodiment, the method further comprises extracting lipid molecules from the lysed aqueous biomass.
In another aspect, the present invention is concerned with a microalgal cell containing a foreign gene, wherein the foreign gene encodes a co-factor for a lipid pathway enzyme or encodes a protein that participates in the synthesis of the co-factor. .
In another aspect, the present invention is concerned with a method for culturing a lipid-producing microbe. In one embodiment, the method comprises culturing the microbe in the presence of a sufficient amount of one or more co-factors for a lipid pathway enzyme to increase the microbial lipid yield relative to the microbial lipid yield in the absence of the one or more. co-factors. In some cases, the one or more co-factors consist of a vitamin required by one or more lipid pathway enzymes. In one embodiment, the one or more co-factors is biotin. In some cases, the one or more co-factors are provided by including in the culture a microbe that has been genetically engineered to produce the one or more co-factors.
In another aspect, the present invention is concerned with a method for fermenting a microorganism, which comprises providing a mixture comprising glucose and xylose as an energy source to the microorganism. In one embodiment, the mixture further comprises lignin. In one embodiment, the mixture further comprises at least one furfural species. In some cases, the blend is depolymerized cellulosic material. In some cases, the mixture further comprises at least one sucrose utilization enzyme. In one embodiment, the mixture comprises a sucrose invertase.
In some cases, the microorganism is selected from the group consisting of Bracteococcus minor, Chlorella ellipsoidea, Chlorella kessleri, Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorella minutissima, Chlorella ovalis, Chlorella protothecoides, Chlorella saccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris, Parachlorella kessleri, Prototheca moriformis, and Pseudochlorella aquatica. In other cases, the microorganism is another species of microorganism as described herein. In some cases, the microorganism has been genetically engineered to express an exogenous gene encoding at least one lipid-modifying enzyme, hydrocarbon-modifying enzyme, or sucrose-utilizing enzyme.
In another aspect, the present invention is concerned with a method for culturing a microalgae, which comprises culturing the microalgae in a culture medium that includes a feedstock comprising at least one carbon substrate selected from the group consisting of a cellulosic material, a 5 carbon atom sugar, a 6 carbon atom sugar and an acetate. In some cases, the carbon substrate is glucose and the microalgae is of a genus selected from the group consisting of Chlorella, Parachlorella, Pseudochlorella, Bracteococcus, Prototheca, and Scenedesmus. In some cases, the carbon substrate is xylose and the microalgae is from a genus selected from the group consisting of Chlorella, Pseudochlorella, and Prototheca. In some cases, the carbon substrate is sucrose and the microalgae is from a genus selected from the group consisting of Chlorella, and Bracteococcus. In some cases, the carbon substrate is fructose and the microalgae is from a genus selected from the group consisting of Chlorella, Parachlorella, Prototheca, and Scenedesmus. In some cases, the carbon substrate is arabinose and the microalgae is Chlorella sp. In some cases, the carbon substrate is crafty and the microalgae is of a genus selected from the group consisting of Chlorella, Parachlorella, Bracteococcus, Prototheca, and Scenedesmus. In some cases, the carbon substrate is galactose and the microalgae is from a genus selected from the group consisting of Bracteococcus, Parachlorella, Chlorella, Pseudochlorella, Bracteococcus, and Prototheca. In some cases, the carbon substrate is acetate and the microalgae is of a genus selected from the group consisting of Chlorella, Parachlorella, and Prototheca.
In one embodiment, the culture medium further includes at least one sucrose utilization enzyme. In some cases, the microalga has been genetically engineered to express an exogenous gene encoding at least one lipid-modifying enzyme, hydrocarbon-modifying enzyme, or sucrose-modifying enzyme. In some cases, the culture medium includes a sucrose invertase.
In another aspect, the present invention is concerned with a method for culturing microalgae comprising placing a plurality of microalgae cells in the presence of depolymerized cellulosic material. In some cases, the microalgae are grown in the presence of an additional fixed carbon source selected from the group consisting of glycerol, sucrose, glucose, arabinose, galactose, xylose, fructose, arabinose, mannose, acetate, and any combination of the above. In one embodiment, the microalgae are grown in the presence of at least one sucrose utilization enzyme.
In some cases, the microalgae are selected from a Bracteococcus species, a Chlorella species, a Parachlorella species, a Prototheca species, or a Pseudochlorella species. In some cases, the microalgae is selected from Bracteococcus minor, Chlorella ellipsoidea, Chlorella kessleq, Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorella minutissima, Chlorella ovalis, Chlorella protothecoides, Chlorella saccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris, Parachlorella kestoscalaris. moriformis, and Pseudochlorella aquatica. In other cases, the microalga is another species of microalga as described herein.
In some embodiments, the microalga has been genetically engineered to express an exogenous gene encoding at least one lipid-modifying enzyme, hydrocarbon-modifying enzyme, or sucrose-modifying enzyme. In one embodiment, the at least one sucrose utilization enzyme is a sucrose invertase. In some cases, the at least one lipid-modifying enzyme is selected from a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, and a glycerol-3 phosphate acyltransferase. In some cases, the at least one hydrocarbon-modifying enzyme is selected from a fatty acylACP thioesterase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, an acylCoA/fatty aldehyde reductase, a fatty aldehyde decarboxylase, and a carrier protein. acyl.
In another aspect, the present invention is concerned with a method of culturing a lipid-producing microbe, the method comprising culturing the microbe in the presence of acetic acid and in the absence of a fixed nitrogen source. In some cases, the microbe is cultured in the presence of a sufficient amount of acetic acid to increase the microbial lipid yield relative to the microbial lipid yield in the absence of acetic acid, where the culture conditions are otherwise the same between the two crops.
In another aspect, the present invention is concerned with a microbial culture containing a population of microorganisms and a culture medium comprising glucose, xylose and a molecule selected from the group consisting of lignin and a furfural species. In some cases, the microorganisms are selected from Bracteococcus minor, Chlorella ellipsoidea, Chlorella kessleri, Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorella minutissima, Chlorella ovalis, Chlorella protothecoides, Chlorella saccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgari, Protheescalori moriformis and Pseudochlorella aquatica. In other cases, the microorganisms are another species of microorganism as described herein.
In another aspect, the present invention is concerned with a method for culturing microalgae. In one embodiment, the method comprises a) providing a microalga cell capable of heterotrophic growth, b) placing the microalga cell in culture media, wherein the culture media comprise depolymerized cellulosic material, and c) incubating the microalga for a period of time. long enough to allow the cell to grow.
In another aspect, the present invention is concerned with a biodiesel manufacturing method. In one embodiment, the method comprises a) culturing a lipid-producing microorganism in a first microbial culture, b) recovering the lipid from the biomass produced by the first microbial culture, c) subjecting the lipid to transesterification to produce lipid ester(s). fatty acid and glycerol and d) adding the glycerol to a second microbial culture. In some cases, the first and second microbial cultures are cultures of the same species of microorganism. In some cases, the second microbial culture comprises microorganism selected from the group consisting of Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca moriformis, Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana. In other cases, the second microbial culture comprises another species of microorganism as described herein.
In another aspect, the present invention is concerned with a fermentation method comprising culturing a microorganism in the presence of glycerol and at least one other fixed carbon source. In some cases the glycerol and the at least one other fixed carbon source are produced to the microorganism simultaneously at a predetermined ratio. In some cases, all of the glycerol and at least one other fixed carbon source are supplied to the microorganism at the start of fermentation. In some cases, all of the glycerol and the at least one other fixed carbon source are fed to the microorganism at a predetermined rate during the course of fermentation. In one embodiment of the method, glycerol is provided to the microorganism in the absence of the at least one other fixed carbon source for a first time period, the at least one other fixed carbon source is provided at the end of the first time period, and the microorganism is cultured for a second period of time in the presence of at least one other fixed carbon source. In one embodiment, the at least one other fixed carbon source is fed to the microorganism at a predetermined rate during the second time period. In some cases, all of the at least one other fixed carbon source is provided to the microorganism at the end of the first time period. In one embodiment of the method, the at least one other fixed carbon source is provided to the microorganism in the absence of glycerol for a first period of time, the glycerol is provided at the end of the first time period, and the microorganism is cultured for a second period. of time in the presence of glycerol. In one embodiment, glycerol is a by-product of a transesterification process. In one embodiment, the glycerol is acidified. In another embodiment, the glycerol is not acidified. In some cases, the at least one other fixed carbon source is glucose. In some cases, the at least one other fixed carbon source is depolymerized cellulosic material. In one embodiment, the at least one other fixed carbon source is sucrose.
In another aspect, the present invention is concerned with a fermentor comprising a population of microorganisms, glycerol and at least one sugar selected from the group consisting of xylose, glucose and sucrose. In one embodiment, glycerol is a by-product of a lipid transesterification process. In some cases, the microorganisms are selected from the group consisting of Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca moriformis, Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana. In other cases, the microorganisms are another species as described herein.
In another aspect, the present invention is concerned with a method for the fermentation of a microorganism. In one embodiment, the method comprises providing glycerol by-product of a transesterification process as a single fixed carbon energy source. In one embodiment, no light energy is provided to the microorganism. In another embodiment, light energy is provided to the microorganism. In some cases, the microorganism is selected from Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca moriformis, Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana. In other cases, the microorganism is another species as described herein.
In another aspect, the present invention is concerned with a microorganism containing an exogenous sucrose utilization gene. In one embodiment, the gene encodes a sucrose transporter. In one embodiment, the gene encodes a sucrose invertase. In one embodiment, the gene encodes a fructokinase. In some cases, the microorganism is a species selected from the group consisting of Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellepsoidea, Chlorella sp. and Chlorella protothecoides. In other cases, the microorganism is another species as described herein.
In another aspect, the present invention is concerned with a cell of the species Chlorella protothecoides, Chlorella emersonii or Chlorella minutissima, wherein the cell contains an exogenous gene. In some cases, the exogenous gene encodes a protein selected from the group consisting of a sucrose transporter, a sucrose invertase, a lipid-modifying enzyme, a hydrocarbon-modifying enzyme, and a fructokinase. In some embodiments, the protein is a sucrose invertase secreted into the extracellular space. In some embodiments, the protein is a cytoplasmic targeted sucrose invertase.
In another aspect, the present invention is concerned with a microbial culture containing a population of microorganisms and a culture medium comprising i) sucrose and ii) a sucrose invertase enzyme.
In another aspect, the present inversion is concerned with a microbial culture containing a population of microorganisms and a culture medium comprising i) melases and ii) a sucrose invertase enzyme.
In another aspect, the present invention is concerned with a microbial culture containing a population of microorganisms and a culture medium comprising i) sucrose, ii) lignin and iii) a sucrose invertase enzyme.
In the various microbial cultures described above, the microorganisms contain at least one exogenous sucrose utilization gene. In some embodiments, the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, and a fructokinase. In one embodiment, the sucrose invertase enzyme is a secreted sucrose invertase enzyme encoded by an exogenous sucrose invertase gene expressed by the microorganism population. In some cases, the microorganisms contain at least one exogenous gene encoding a lipid pathway enzyme or a hydrocarbon modification enzyme.
In another aspect, the present invention is concerned with a nucleic acid comprising a cDNA encoding a sucrose utilization gene and a cDNA encoding a protein that confers resistance to the antibiotic hygromycin or the antibiotic G418.
In embodiments of the various methods, compositions, cells, microorganisms, microbes, microbial cultures, terminators, and the like, described above, the microorganism or microbe may be a microalgae, an oily yeast, a fungus, or a bacterium, unless otherwise specified. specify otherwise. In some cases, the microorganism is selected from the group consisting of the microalgae listed in Table 1. In some cases, the microorganism is a species of the genus Chlorella. In some cases, the microorganism is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. Vacuolata, Chlorella glocotropha, Chlorella infusionum, Chlorella infusioum var. Actophila, Chlorella infusioum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothercoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella f. tedia, Chlorella vulgaris var airidis, Chlorella vulgaris var vulgaris, Chlorella vulgaris var vulgaris f. tedia, Chlorella vulgaris var vulgaris f. viridis, Chlorella xantella and Chlorella zofingiensis. In some cases, the microorganism is an oily yeast selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces satarkeyi lipofer, Endomycopsis vernalis, Flhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica. In some cases, the microorganism is a fungus selected from the group consisting of a species of the genus Mortierella, Mortierella vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus achraceus, Aspergillus terreus, Pennicillium iilacium, a species of the genus Hansenulo, a species from the genus Chaetomium, one species from the genus Cladosporium, one species from the genus Malbranchea, one species from the genus Rhlzopus and one species from the genus Pythium.
In the various embodiments described above, the microorganism may contain at least one exogenous sucrose utilization gene. In some cases, the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.
In the various embodiments described above, the microorganism may contain at least one exogenous gene encoding a lipid pathway enzyme. In some cases, the lipid pathway enzyme is selected from the group consisting of a stearyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3-phosphate acyltransferase.
In the various embodiments described above, the microorganism may contain at least one exogenous gene encoding a hydrocarbon modification enzyme. In some cases, the hydrocarbon modifying enzyme is selected from the group consisting of a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and /or an acyl carrier protein.
Any two or more of the various embodiments described above may be combined together to produce additional embodiments encompassed within the scope of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the dry cell weight per liter of multiple Chlorella species and strains when grown in the presence of various types of glycerol with and without additional glucose.
Figure 2 shows the cell dry weight per liter of multiple Chlorella species and strains when grown in the presence of various types of glycerol with added glucose.
Figure 3 shows the relative culture lipid concentrations of multiple Chlorella species and strains when grown in the presence of various types of glycerol with added glucose.
Figure 4 shows the culture lipid concentration of multiple Chlorella species and strains when grown in the presence of various types of glycerol with and without additional glucose.
Figure 5 shows lipid as percent dry cell weight of two Chlorella species and strain when grown in the presence of various types of glycerol with additional glucose, where glycerol is added sequentially after glucose.
Figure 6 shows lipid as percent dry cell weight of two Chlorella species and strains when grown in the presence of various types of glycerol with additional glucose.
Figure 7 shows the relative lipid concentration of cultures of multiple species and strains of
Chlorella when grown in the presence of 2% glucose and 1% glucose plus 1% reagent grade glycerol.
Figure 8 shows lipid as percent dry cell weight of multiple Chlorella species and strains when grown in the presence of glucose with and without reagent grade glycerol, where glycerol is added sequentially or in combination with glucose.
Figure 9 shows the relative lipid concentration of cultures of multiple Chlorella species and strains when grown in the presence of various types of glycerol with additional glucose, where glycerol is added sequentially or in combination with glucose.
Figure 10 shows the dry cell weight per liter of multiple Chlorella species and strains when grown in the presence of various types of glycerol with additional glucose, where glycerol is added sequentially or in combination with glucose.
Figure 11(a) shows lipid as percent dry cell weight of Spirulina platensis when grown in the presence of glucose, reagent grade glycerol, unacidulated biodiesel glycerol by-product, and a combination of glycerol and glucose.
Figure 11(b) shows lipid as dry cell weight percent of Navicula pelliculosa when cultured in the presence of various types of glycerol in the presence of combinations of glycerol and glucose.
Figure 12(a) shows lipid as dry cell weight percent of Scenedesmus armatus when grown in the presence of various types of glycerol and in the presence of a combination of glycerol and glucose.
Figure 12(b) shows the dry cell weight per liter of Scenedesmus armatus when grown in the presence of various types of glycerol and in the presence of a combination of glycerol, biodiesel by-product, and glucose.
Figure 13 shows the cell dry weight per liter of Navicula pelliculosa when grown in the presence of various types of glycerol and in the presence of a combination of glycerol, non-acidulating biodiesel by-product, and glucose.
Figure 14 shows the dry cell weight per liter of Scenedesmus armatus and Navícula pelliculosa when grown in the presence of glycerol, a by-product of acidified and non-acidulated biodiesel with additional glucose, where glycerol is added sequentially or in combination with glucose.
Figure 15 shows a synergistic effect with the combination of xylose and glucose on Chlorella growth compared to xylose or glucose alone.
Figure 16 shows the genotype of Chlorella protothecoides transformant containing an exogenous gene.
Figure 17 shows the codon usage of Chlorella protothecoides.
Figure 18 shows the codon usage of D. salina and Chlorella pyrenoidosa.
Figure 19 shows a) reagent grade glycerol; b) glycerol by-product of unacidulated biodiesel and c) glycerol by-product of acidulated diesel, all of which were used in the experiments described in the examples.
Figure 20 shows the cultivation of Chlorella protothecoides on glucose and fructose.
Figure 21 shows the cultivation of Chlorella fusca on 1% sucrose.
Figure 22 shows the cultivation of Chlorella kesslerí on 1% sucrose.
Figure 23 shows the cell dry weight per liter of Chlorella protothecoides when grown in the presence of glucose, sucrose, or one of several honeydew samples (designated BS1, BS2, and HTM) in the presence or absence of a sucrose invertase.
Figure 24 shows the culture of Chlorella protothecoides when grown in the presence of glucose, sucrose, or one of several honeydew samples (designated BS1, BS2, and HTM) in the presence or absence of a sucrose invertase as measured by cell density. relative.
Figure 25 shows a mimic of various yeast invertase (SUC2) plasmid concepts with three different promoters (designated CMV, CV and HUP1) also as useful restriction sites for sub-cloning.
Figure 26 shows the genotype of Chlorella protothecoides transformants selected on sucrose in the dark containing an exogenous sucrose invertase gene.
Figure 27 shows the genotype of Chlorella protothecoides cells transformed with a gene encoding a secreted sucrose invertase from S. cerevisiae.
Figure 28 shows the genotype of Chlorella minutissima and Chlorella emersonii cells transformed with a gene encoding a secreted sucrose invertase from S. cerevisiae.
Figure 29 illustrates a gas chromatograph generated by analysis of a renewable diesel product produced in accordance with the methods of the present invention as described in Example 27.
Figure 30 illustrates a plot of the boiling point distribution for a renewable diesel product produced in accordance with the methods of the present invention and described in Example 27.
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
Unless otherwise defined, all technical and scientific terms shown herein have the meaning commonly understood by a person skilled in the art with which the invention is concerned. The following references provide one of ordinary skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genectics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); Y
Hale & Marham, The HarperCollins Dictionary of Biology (1991). As used herein the following terms have the meanings ascribed to them unless otherwise specified.
As used with reference to a nucleic acid, "active in microalgae" refers to a nucleic acid that is functional in microalgae. For example, a promoter that has been used to drive an antibiotic resistance gene to impart antibiotic resistance to transgenic microalgae is active in microalgae. Examples of promoters active in microalgae are promoters endogenous to certain species of algae and promoters found in plant viruses.
An "acyl carrier protein" or "ACP" is a protein that binds to a growing acyl chain during fatty acid synthesis as a thiolester at the thiol distant from the 4'phosphopantetheine moiety and comprises a component of the complex. of fatty acid synthase. The phrase "naturally co-expressed" with reference to an acyl carrier protein in conjunction with a synthesis - fatty ACP thioesterase means that the ACP and thioesterase are co-expressed naturally in a tissue or organism from which they are derived, for example because the genes encoding the two enzymes are either under the control of a common regulatory sequence or because they are expressed in response to the same stimuli.
An “acyl-CoA molecule” or “acyl-CoA” is a molecule comprising an acyl moiety covalently attached to Co-enzyme A via a thiolester bond at the thiol distant from the 4'-phosphopantetheine moiety. of Co-enzyme A.
"Axenic" means a culture of an organism that is free from recombination by other living organisms.
"Biodiesel" is a biologically produced fatty acid alkyl ester suitable for use as a fuel in a diesel engine.
The term "biomass" refers to material produced by culturing and/or propagating cells. The biomass may contain cells and/or intracellular content, as well as extracellular material. Extracellular material includes but is not limited to compounds secreted by a cell.
"Bioreactor" means an envelope or partial envelope in which cells are grown, optionally in suspension.
As used herein, "catalyst" refers to an agent, such as a macromolecular molecule or complex, capable of facilitating or promoting a chemical reaction from reactant to product without becoming part of the product. Thus, a catalyst increases the rate of a reaction, after which the catalyst can act on another reactant to form the product. A catalyst generally lowers the overall activation energy required for the reaction, so that it proceeds faster or at a lower temperature. Thus, an equilibrium reaction can be obtained more quickly. Examples of catalysts include enzymes, which are biological catalysts, heat, which is a non-biological catalyst, and metal catalysts used in fossil petroleum refining processes.
"Cellulosic material" means the digestion products of cellulose, including glucose and xylose, and optionally additional compounds such as disaccharides, oligosaccharides, lignin, turtles, and other compounds. Non-limiting examples of sources of cellulosic material include sugarcane bagasse, sugar beet pulp, corn stover, wood chips, sawdust, and switchgrass.
The term "co-cultivation" and variants thereof, such as "co-cultivate", refer to the presence of two or more cell types in the same bioreactor. The two or more cell types may both be microorganisms, such as microalgae, or may be a microalgae cell cultured with a different cell type. Culture conditions may be those that enhance the growth and/or propagation of the two or more cell types or those that facilitate the growth and/or proliferation of one or a subset of the two or more cells, while maintaining cell growth for the rest.
The term "co-factor" is used herein to refer to any molecule, other than a substrate, that is required for an enzyme to carry out its enzymatic activity.
As used herein, "complementary DNA" ("cDNA") is a DNA representation of mRNA, usually obtained by messenger RNA (mRNA) reverse transcription or amplification (for example, via polymerase chain reaction ("PCR"). ”)).
The term "cultured" and variants thereof refer to the unintentional enhancement of growth (increases in cell size, cell content, and/or cell activity) and/or propagation (increases in cell numbers via mitosis) of one or more cells by using proposed culture conditions. The combination of both cultivation and propagation can be termed proliferation. The one or more cells may be those of a microorganism, such as microalgae. Examples of proposed conditions include the use of a defined medium (with known characteristics such as pH, ionic strength, and carbon source), specified temperature, oxygen tension, carbon dioxide levels, and growth in a bioreactor. The term does not refer to the growth or propagation of microorganisms in nature or otherwise without direct human intervention, such as the natural growth of an organism that ultimately becomes a fossil to produce geological crude oil.
As used herein, the term "cytolysis" refers to the lysis of cells in a hypotonic environment. Cytolysis is caused by excessive osmosis, or movement of water, into a cell (hyperhydration). The cell cannot withstand the osmotic pressure of the water inside and so it explodes.
As used herein, the terms "expression vector" or "expression construct" refer to a nucleic acid construct, recombinantly or synthetically generated, with a set of specified nucleic acid elements that allow transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Commonly, the expression vector includes a nucleic acid to be transcribed operatively linked to a promoter.
"Exogenous gene" refers to a nucleic acid transformed into a cell. A transformed cell may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The foreign gene may be from a different species (and thus heterologous) or from the same species (and thus homologous) in relation to the cell that is transformed. In the case of a homologous gene, it occupies a different site in the cell's genome relative to the endogenous copy of the gene. The foreign gene may be present in more than one copy in the cell. The exogenous gene can be maintained in a cell as an insert to the genome or as an episomal molecule.
"Exogenously provided" describes a molecule provided to the culture medium of a cell culture.
As used herein, a "fatty acyl-ACP thioesterase" is an enzyme that catalyzes the cleavage of a fatty acid from an acyl carrier protein (ACP) during lipid synthesis.
As used herein, an "acyl-CoA/fatty aldehyde reductase" is an enzyme that catalyzes the reduction of an acyl-CoA molecule to a primary alcohol.
As used herein, a "fatty acyl-CoA reductase" is an enzyme that catalyzes the reduction of an acyl-CoA molecule to an aldehyde.
As used herein, a "fatty aldehyde decarbonylase" is an enzyme that catalyzes the conversion of a fatty aldehyde to an alkane.
As used herein, a "fatty aldehyde reductase" is an enzyme that catalyzes the reduction of an aldehyde to a primary alcohol.
"Fixed carbon source" means carbon-containing molecule(s), preferably organic, that is present at ambient temperature and pressure in solid or liquid form.
"Fungus", as used herein, means heterotrophic organisms characterized by a chitinous cell wall of the fungal kingdom.
"Homogenate" means biomass that has been subjected to physical disruption.
As used herein, "hydrocarbon" refers to: (a) a molecule containing only hydrogen and carbon atoms, wherein the carbon atoms are covalently bonded to form a typical or partially typical straight, branched, backbone chain to which hydrogen atoms are attached; or (b) a molecule that contains only primarily hydrogen and carbon atoms and that can be converted to contain only hydrogen and carbon atoms by one to four chemical reactions. Non-limiting examples of the latter include hydrocarbons that contain an oxygen atom between a carbon atom and a hydrogen atom to form an alcohol molecule, as well as aldehydes that contain a single oxygen atom. Methods for the reduction of alcohols to hydrocarbons containing only carbon and hydrogen atoms are well known. Another example of a hydrocarbon is an ester, in which an organic group replaces a hydrogen atom (or more than one) in an acidic oxygen. The molecular structure of hydrocarbon compounds varies from the simplest form, in the form of methane (CH<sub>4</sub>), which is a constituent of natural gas, to the very heavy and complex ones, such as some molecules such as asphaltenes found in crude oil, petroleum and bitumen. Hydrocarbons may be in gas, liquid, or solid form or any combination of these forms and may have one or more double or triple bonds between adjacent carbon atoms in the backbone. Thus, the term includes linear, branched, cyclic or partially cyclic alkanes, alkenes, lipids and paraffin. Examples include propane, butane, pentane, hexane, octane, triolein, and squalene.
"Hydrocarbon modifying enzyme" refers to an enzyme that alters the covalent structure of a hydrocarbon. Examples of hydrocarbon modifying enzymes include a lipase, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase and a fatty aldehyde decarbonylase. Compounds produced by the enzymatic activity of hydrocarbon modifying enzymes, including fatty acids, alcohols, aldehydes, alkanes and other compounds derived therefrom are interchangeably referred to herein as hydrocarbons or lipids.
The term "hydrogen:carbon ratio" refers to the ratio of hydrogen atoms to carbon atoms in a molecule on an atom-to-atom basis. The ratio can be used to refer to the number of carbon and hydrogen atoms in a hydrocarbon molecule. For example, the hydrocarbon with the highest ratio is methane CH<sub>4</sub> (4:1).
"Hydrophobic fraction" refers to that portion, or fraction, of a material that is more soluble in a hydrophobic phase compared to an aqueous phase. A hydrophobic moiety is substantially insoluble in water and usually nonpolar.
As used herein, the phrase "increased lipid yield" refers to an increase in the productivity of a microbial culture by increasing, for example, the dry weight of cells per liter of culture, increasing the percentage of cells constituting the lipid or increase the overall amount of lipid per liter of culture volume per unit time.
An "inducible promoter" is one that moderates the transcription of an operably linked gene in response to a particular stimulus.
As used herein, the phrase "operable link" refers to a functional link between two sequences, such as a control sequence (commonly a promoter) and the linked sequence. A promoter is in operable link to a foreign gene if it can moderate transcription of the gene.
The term “in situ” means “in place” or “in its original position”. For example, a culture may contain a first microalga that secretes a catalyst and a second microorganism that secretes a substrate, with the first and second cell types producing the components necessary for a particular chemical reaction to occur in situ in the co-culture. without requiring additional separation or processing of the materials.
A "limiting concentration of a nutrient" is a concentration in a culture that limits the spread of a cultured organism. A "non-limiting concentration of a nutrient" is a concentration that supports maximum propagation during a given growing period. Thus, the number of cells produced during a given culture period is less in the presence of a limiting concentration of a nutrient than when the nutrient is non-limiting. A nutrient is said to be "in excess" in a crop, when the nutrient is present in a concentration greater than that which supports maximum propagation.
As used herein, a "lipase" is a water-soluble enzyme that catalyzes the hydrolysis of ester bonds in water-insoluble lipid substrates. Lipases catalyze the hydrolysis of lipids to glycerols and fatty acids.
As used herein, a "lipid pathway enzyme" is any enzyme that plays a role in lipid metabolism, that is, either in lipid synthesis, modification, or degradation. This term encompasses proteins that chemically modify lipids, also as carrier proteins.
"Lipids" are a class of hydrocarbons that are soluble in nonpolar solvents (such as ether and chloroform) and are relatively or completely insoluble in water. Lipid molecules have these properties because they consist largely of long hydrocarbon tails that are hydrophobic in nature. Examples of lipids include fatty acids (saturated and unsaturated); glycerides or glycerolipids (such as monoglycerides, diglycerides, triglycerides or neutral fats and phosphoglycerides or glycerophospholipids); nonglycerides (sphingolipids, sterol lipids including cholesterol and spheroid hormones, prenol lipids including terpenoids, fatty alcohols, waxes, and polyketides); and derivatives of complex lipids (sugar-linked lipids or glycolipids and protein-linked lipids). "Fats" are a subgroup of lipids called "triacylglycerides."
As used herein, the term "Spent" refers to a solution containing the contents of lysed cells.
As used herein, the term "lysis" refers to the disruption of the cell membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular contents, often by mechanical, viral, or osmotic mechanisms that compromise its integrity.
As used herein, the term "lysing" refers to the disruption of the cell membrane and optionally the cell wall of a biological organism or cell sufficient to release at least some intracellular contents.
"Microalga" means a eukaryotic microbial organism containing a chloroplast and optionally capable of photosynthesis or a prokaryotic microbial organism capable of photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source for energy, as well as heterotrophs, which can live only on a fixed carbon source. Microalgae can refer to single-celled organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, and can also refer to microbes such as Volvox, which is a single multicellular photosynthetic microbe of two distinct cell types. “Microalgae can also refer to cells such as Chlorella and Dunaliella. "Microalgae" also includes other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum. Anabaena and Pyrobotrys. "Microalgae" also includes obligate heterotrophic microorganisms that have lost the ability to photosynthesize, such as certain species of dinoflagellate algae.
The terms "microorganism" and "microbe" are used interchangeably herein to refer to microscopic single-celled organisms.
"Oleaginous yeast", as used herein, means yeast that can naturally accumulate more than 10% of its dry cell weight as lipid and can do this as a result of genetic engineering. Oily yeasts include organisms such as Yarrowia lipolytica, as well as engineered strains of yeast such as Saccharomyces cerevisiae that have been engineered to accumulate greater than 10% of their cell dry weight as lipids.
As used herein, the term "osmotic shock" refers to the rupture of cells in solution following a sudden reduction in osmotic pressure. Osmotic shock is sometimes induced to release cellular components from such cells into solution.
"Photobioreactor" refers to a container, at least part of which is at least partially transparent or partially open, thereby allowing light to pass through, in which one or more microalgae cells are grown. Photobioreactors can be closed, as in the case of a polyethylene bag or Erlenmeyer flask, or they can be open to the environment, as in the case of an outdoor lagoon.
As used herein, a "polysaccharide-degrading enzyme" refers to any enzyme capable of catalyzing the hydrolysis or depolymerization of any polysaccharide. For example, cellulases catalyze the hydrolysis of cellulose.
"Polysaccharides" (also called "glycans") are carbohydrates made up of monosaccharides linked together by glycosidic bonds. Cellulose is an example of a polysaccharide that makes up certain plant cell walls. Cellulose can be depolymerized by enzymes to produce monosaccharides such as xylose and glucose, as well as larger disaccharides and oligosaccharides.
"Gate", in the context of a bioreactor, refers to an opening in the bioreactor that allows the inflow or outflow of materials such as gases, liquids, and cells. The ports are usually connected to tubing leading from the photobioreactor.
A "promoter" is defined as an arrangement of nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the transcription start site, such as in the case of a polymerase II-type promoter, a TATA element. A promoter also optionally includes distant enhancer or repressor elements, which may be located as much as several thousand base pairs from the transcription start site.
As used herein, the term "recombinant" when used with reference to, for example, a cell or nucleic acid, protein or vector indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a nucleic acid. exogenous or protein or the alteration of a natural nucleic acid or protein or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in the natural (non-recombinant) form of the cell or express natural genes that are otherwise abnormally expressed, under-expressed or not expressed. The term "recombinant nucleic acid" herein means nucleic acid, originally formed in vitro, generally by manipulation of nucleic acid, for example using polymerases and endonucleases in a form not normally found in nature. In this way, the operative linkage of different sequences is obtained. Thus, an isolated nucleic acid, in linear form, or an expression vector formed in vitro by sprinkling of DNA molecules that are not normally spliced, are both considered recombinant for the purposes of this invention. It will be understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, that is using the host cell's in vivo cellular machinery, rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently found to be non-recombinant, are still considered recombinant for purposes of the invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, that is, through expression of a recombinant nucleic acid as illustrated above.
As used herein, the term "renewable diesel" refers to alkanes (such as C:10:0, C12:0, C:14:0, C16:0, and C18:0) produced by means of hydrogenation and lipid deoxygenation.
As used herein, the term "sonification" refers to a process for disrupting biological materials, such as a cell, through the use of sound wave energy.
"Furfural species" refers to 2-furancarboxaldehyde or a derivative thereof that retains the same basic structural features.
As used herein, "stubble" refers to the dry cuttings and leaves of a crop remaining after a grain has been harvested.
A "sucrose utilization gene" is a gene that, when expressed, aids in a cell's ability to utilize sucrose as an energy source. Proteins encoded by a sucrose utilization gene are referred to herein as "sucrose utilization enzymes" and include sucrose transporters, sucrose invertases, and exosinases such as glucosinases and fructosinases.
“Water waste” is aqueous waste that commonly contains wash water, laundry waste, feces, urine, and other liquid or semi-liquid wastes. They include some forms of municipal waste as well as secondarily treated sewage.
For sequence comparison to determine percent nucleotide or amino acid identity, commonly one sequence acts as the reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input to a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal sequence alignment by comparison can be carried out for example by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), using the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), using the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. Madison, Wl), or by visual inspection (see generally Ausubel et al, supra).
Another exemplary algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403410 (1990). Software for performing BLAST analyzes are publicly available through the National Center for Biotechnology Information (at the web address www.ncbi.nlm.nih.gov). This algorithm involves first identifying high-score sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in the same database sequence. T is referred to as the neighbor word score threshold (Altschul et al, supra.). These initial neighbor word hits act as seeds to initiate searches to find longer HSPs containing them. Word hits are spread out in both directions along each sequence by as much as the cumulative alignment score can be increased. Cumulative scores are calculated using for nucleotide sequences, the parameters M (backscore for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The spread of word hits in each direction are stopped when: the cumulative alignment score drops by the amount X of its maximum rolled value; the cumulative score goes to zero or less due to the accumulation of one or more negative score residue alignments or either one or the other of the end of the sequence is reached. To identify whether a nucleic acid or polypeptide is within the scope of the invention, the default parameters of the BLAST programs are appropriate. The BLASTN program (for nucleotide sequences) defaults to a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program defaults to a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. The TBLATN program (using protein sequence for nucleotide sequences) defaults to a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM scoring matrix of 62. (see Henikoff & Henikoff, Proc. Nati Acad ScL USA 89:10915 (1989).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (eg, Karlin & Altschul, Proc. Nati. Acad. Sci. USA 90:5873-5787 (1993)). . One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences can occur per probability. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
II. GENERAL
The premise of the invention lies in part in the concept that certain microorganisms can be used to produce oils, fuels, and other hydrocarbon or lipid compositions economically and in large quantities for use in the transportation fuel, petrochemical, and/or food or cosmetics among other applications. Suitable microorganisms include microalgae, oily yeast, and fungi. A preferred genus of microalgae for use in the invention is the lipid-producing microalgae Chlorella. Lipid transesterification produces long-chain fatty acid esters useful as biodiesel. Other enzymatic and chemical processes can be tailored to produce fatty acids, aldehydes, alcohols, alkanes, and alkenes. The present application describes methods for the genetic modification of multiple species and strains of microorganisms, including Chlorella and similar microbes, to provide organisms that have characteristics that facilitate the production of lipid suitable for conversion to oils, fuels, and oleochemicals. In some applications, renewable diesel, jet fuels, or other hydrocarbon compounds are produced. The present application also describes methods for culturing microalgae for increased productivity and increased lipid yield and/or for more cost-effective production of the compositions described herein.
In particular embodiments, the present application describes genetically engineered strains of microalgae with one or more exogenous genes. For example, microalgae that produce high levels of triacylglycerides (TAGs) suitable for biodiesel can be engineered to express a lipase, which can facilitate the transesterification of TAGs from microalgae. The lipase can optionally be expressed using an inducible promoter, such that cells can first be grown to a desirable density in a fermenter and then harvested, followed by induction of the promoter to express the lipase, optionally in the presence of sufficient alcohol to drive the conversion of TAGs to fatty acid esters.
Some microalgal lipid is sequestered in cell membranes and other non-aqueous parts of the cell. Therefore, to increase the yield of the transesterification reaction, it may be beneficial to lyse cells to increase lipase access to lipid. Cell disruption can be effected, for example mechanically by adding pressurized steam or by using a virus that lyses microalgae cells, expressing a gene to produce a cell lytic protein, or treating the culture with an agent. that lyses microalgae cells. Steam treatment of microalgae for cell disruption is described for example in US Patent 6,750,048.
Also disclosed herein is the genetic engineering of microalgae that produce high levels of TAG to express a gene that lyses microalgae cells, such as, for example, a lytic virus gene. This gene can be expressed using an inducible promoter, such that cells can first be grown to a desirable density in a teapot and then harvested, followed by induction of the promoter to express the gene to lyse the cells. A gene encoding a polysaccharide-degrading enzyme, for example, can be expressed to lyse cells.
Optionally, the lipase can be expressed in an intracellular compartment, where it remains separated from the majority of the microalgal lipid until transesterification. In general, it is preferable to carry out the transesterification after water has been substantially removed from the preparation and/or excess alcohol has been added. Lipases can use water, also like alcohol, as a substrate in transesterification. With water, the lipid is conjugated to a hydroxyl moiety to produce a polar fatty acid, rather than an ester. With an alcohol, such as methanol, the lipid is conjugated to a methyl group, producing a nonpolar fatty acid ester, which is commonly preferable for a transportation fuel. To limit the exposure of the lipase to the microalgal lipid until conditions are appropriate for transesterification to produce fatty acid esters, the lipase can be expressed, for example, in the chloroplast, mitochondrion, or other cell organelle. This compartmentalized expression results in the sequestration of the lipase from most of the cellular lipid until after the cells have been disrupted.
In other particular embodiments, the present application describes genetically engineered strains of microalgae, oily yeasts, bacteria or fungi with one or more exogenous genes to produce various hydrocarbon compounds. For example, microalgae that naturally or through genetic modification would produce high levels of lipids can be engineered (or additionally engineered) to express an exogenous fatty acyl-ACP thioesterase, which can facilitate the cleavage of fatty acids from lipid-carrying protein. acyl (ACP) during lipid synthesis. These fatty acids can be recovered or, through further enzymatic processing within the cell, produce other hydrocarbon compounds. Optionally, the fatty acyl-ACP thioesterase can be expressed from a gene operably linked to an inducible promoter and/or can be expressed in an intracellular compartment.
The fatty acyl-ACP thioesterase can be chosen based on its specificity for a growing fatty acid (during fatty acid synthesis) having a particular carbon chain length. For example, the fatty acyl-ACP thioesterase may have a specificity for a carbon chain length ranging from 8 to 34 carbon atoms, preferably 8 to 18 carbon atoms, and more preferably 10 to 14 carbon atoms. A specificity for a C12 fatty acid is more preferred.
Furthermore, the invention provides genetically engineered strains of microalgae to express two or more exogenous genes, such as, for example, a lipase and a lytic gene, for example, one encoding a polysaccharide-degrading enzyme. One or both genes can be expressed using an inducible promoter, which allows the relative timing of expression of those genes to be controlled to improve lipid yield and conversion to fatty acid esters. The invention also provides vectors and methods for engineering microbes that produce lipid to metabolize sucrose, which is an advantageous trait because it allows the engineered cells to convert sugarcane or other feedstocks into lipids suitable for the production of oils, fuels, oleochemicals and the like.
In other embodiments, the invention provides genetically engineered strains of microbes (eg, microalgae, oily yeast, matter, or fungus) that express two or more exogenous genes, such as, for example, a fatty acyl-ACP thioesterase and a fatty acyl-CoA/aldehyde. fatty reductase, the combined action of which produces an alcohol product. The invention further provides other combinations of exogenous genes, including without limitation, a fatty acyl-ACP thioesterase and a naturally co-expressed acyl carrier protein to generate fatty acids of specific length or a fatty acyl-ACP thioesterase and a Fatty acyl-CoA reductase to generate aldehydes. The invention also provides for the combination of a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and a fatty aldehyde decarbonylase to generate alkanes or alkenes. One or more of the foreign genes can be expressed using an inducible promoter.
The invention provides further modifications of microalgae, for example to provide microalgae with desired growth characteristics and/or to improve the quantity and/or quality of lipids produced. For example, microalgae can be engineered to increase carbon flux to the lipid pathway and/or modify the lipid pathway to beneficially alter the ratios or properties of lipid produced by cells.
The present application discloses genetically engineered strains of microalgae to express two or more foreign genes, one encoding a fixed carbon source transporter (such as sucrose) and a second encoding a sucrose invertase enzyme. The resulting fermentable organisms produce hydrocarbons at a lower manufacturing cost than has been obtainable by previously known methods of biological hydrocarbon production. Insertion of the two exogenous genes described above can be combined with disruption of polysaccharide biosynthesis by site-directed and/or random mutagenesis, which directs even greater carbon flux to hydrocarbon production. Individually and in combination, tropic conversion, design to alter oil production, and treatment with exogenous enzymes alter the composition of oil produced by an organism. The alteration may be a change in the amount of hydrocarbons produced, the amount of one or more hydrocarbon species produced relative to other hydrocarbons, and/or the types of hydrocarbon species produced in the microorganism. For example, microalgae can be engineered to produce a higher amount and/or percentage of TAG.
III. MICROORGANISMS THAT PRODUCE OIL OR LIPID
Any species of organism that produces appropriate lipid or hydrocarbon can be used, although microorganisms that naturally produce high levels of appropriate lipid or hydrocarbon are preferred. Hydrocarbon production by microorganisms is reviewed by Metzger et al. Appl Microbiol Biotechnol (2005) 66: 486-496 and A Look Back at the US Department of Energy's Aquatic Species Program: Biodiesel of Algae, NREL/TP-580-24190, John Sheehan, Terri Dunahay, John Benemann, and Paul Roessler (1998).
Considerations affecting the selection of microorganisms for use in the invention include, in addition to the production of lipids or hydrocarbons suitable for the production of oils, fuels, and oleochemicals: (1) high lipid content as a percentage of cell weight; (2) ease of cultivation; (3) ease of genetic design; and (4) ease of biomass processing. In particular embodiments, the wild type or genetically engineered microorganism produces cells that are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70% or more lipid. Preferred organisms grow heterotrophically (on sugars in the absence of light) or can be engineered to do so using, for example, the methods disclosed herein. The ease of transformation and the availability of constitutive and/or inducible selectable markers and promoters that are functional in the microorganism affect the ease of genetic design. Processing considerations may include, for example, the availability of effective means to lyse the cells.
a. algae
In one embodiment of the present invention, the microorganism is a microalga. Non-limiting examples of microalgae that can be used in accordance with the present invention can be found in Table 1.
Table 1. Examples of microalgae
Achnanthes orientalis, Agmenellum, Amphiprora hyaline, Amphora coffeeformis, Amphora coffeeformis linea, Amphora coffeeformis punctata, Amphora coffeeformis taylorí, Amphora coffeeformis tenuis, Amphora delicatissima, Amphora delicatissima capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus sp falcatus, Boekelovia boekelovia, Boekelovia ., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kesslerí, Chlorella lobophora (strain SAG 37.88), Chlorella luteovirídis, Chlorella luteovirídis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides (including any of UTEX strains 1806, 411, 264, 256, 255,
250, 249, 31, 29, 25), Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regulaos var. minimal, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. Ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris f. tedia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f tedia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella spiana, Cyclotella meneghin ., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granúlate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terrestrial, Dunaliella tediolecta, Dunaliella viridis, Dunaliella tediolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena, Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Hymenomonas sp. , Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEXLB 2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navícula acceptata, Navícula biskanterae, Navícula pseudotenelloides, Navícula pelliculosa, Navícula saprophila ., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria subcillatoria sp. , Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phagus, Phormidium, Platymonas sp., Pleurochrysis hipae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca podoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, fihodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Temistracusel sp., Synetraselchocco. , Tetraselmis suecica, Thalassiosira weissflogii and Viridiella fridericiana
one. Chlorella
In a preferred embodiment of the present invention, the microorganism is of the genus Chlorella, preferably Chlorella protothecoides, Chlorella ellipsoidea, Chlorella minutissima or Chlorella emersonii.
Chlorella is a genus of one-celled green algae, belonging to the phylum Chlorophyta. It is spherical in shape, about 2 to 10 pm in diameter, and is without flagella. Some Chlorella species are naturally heterotrophic.
Chlorella, particularly Chlorella protothecoides, is a preferred microorganism for use in the invention due to its high lipid composition, particularly long chain lipid suitable for biodiesel. Furthermore, this microalga grows heterotrophically and can be genetically engineered as demonstrated in the examples herein.
In a preferred embodiment of the present invention, the microorganism used for the expression of a transgene is from the genus Chlorella, preferably Chlorella protothecoides, Chlorella minutissima or Chlorella emersonii. Examples of transgene expression in, for example, Chlorella, can be found in the literature (see for example Current Microbiology Vol. 35 (1997), pp. 356-362; Sheng Wu Gong Cheng Xue Bao. 2000 Jul; 16(4 ):443-6, Current Microbiology Vol.38 (1999), pp. 335-341; Appl Microbiol Biotechnol (2006) 72: 197-205; Marine Biotechnology 4, 63-73, 2002; Current Genetics 39:5, 365-370 (2001); Plant Cell fieports 18:9, 778-780, (1999); Plantarium Biology 42(2): 209-216, (1999); PlantPathol. J 21(1): 13-20, (2005)). Also see Examples herein. Other lipid-producing microalgae can also be engineered, including procanyontic microalgae (see Kalscheuer st al., Applied Microbiology and Biotechnology, Volume 52, Issue 4 / October, 1999).
2. Chlorella Species Identification
Chlorella species for use in the invention can be identified by amplifying certain target regions of the genome. For example, identification of a specific Chlorella species or strain can be obtained by means of nuclear and/or chloroplast DNA amplification and sequencing using primers and methodology using any region of the genome, for example using the methods described in Wu et al. ., bot. Bull. Acad. Without. (2001) 42:115-121 Identification of Chlorella spp isolates using ribosomal DNA sequences. Well-established methods of phylogenetic analysis, such as amplification and sequencing of ribosomal internal transcribed spacer (ITS1 and ITS2 rDNA), 18S rRNA, and other conserved genomic regions can be used by those skilled in the art to identify not only Chlorella species, but others. hydrocarbon and lipid producing organisms apt to use the methods disclosed herein. For examples of algae identification and classification methods see also eg Genetics, August 2005; 170(4):1601-10 and RNA, April 2005; 11(4):361-4.
B. Oily Yeast
In one embodiment of the present invention, the microorganism is an oily yeast. Non-limiting examples of oleaginous yeast that can be used in accordance with the present invention can be found in Table 2.
Table 2. Examples of oleaginous yeast.
Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Fihodotorula glutinis, Rhodotorula gracilis and Yarrowia lipolytica
C. Other Fungi
In one embodiment of the present invention, the microorganism is a fungus. Non-limiting examples of fungi that can be used in accordance with the present invention can be found in Table 3.
Table 3. Examples of fungi
Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, Hensenulo, Chaetomium, Cladosporium, Malbranchea. fihizopus and Pythium
D. Bacteria
In one embodiment of the present invention, the microorganism is a bacterium.
Examples of exogenous gene expression in bacteria, such as E. coti, are well known; see for example Molecular Cloning: A Laboratory Manual, Sambrook et al. (3rd edition, 2001, Cold Spring Harbor Press).
IV. METHODS OF CULTURE OF MICROORGANISMS
Microorganisms are cultivated both for the purposes of carrying out genetic manipulations and for the subsequent production of hydrocarbons (eg, lipids, fatty acids, aldehydes, alcohols, and alkanes). The first type of cultivation is carried out on a small scale and initially, at least, under conditions in which the starting microorganism can grow. For example, if the starting microorganism is a photoautotroph, the initial culture is carried out in the presence of light. Culture conditions can be changed if the microorganism is developed or designed to grow independently of light. Cultivation for hydrocarbon production purposes is usually carried out on a large scale. Preferably a fixed carbon source is present. The culture can also be exposed to light some or all of the time.
Microalgae can be cultivated in liquid media. The culture may be contained within a bioreactor. Optionally, the bioreactor does not allow light to enter. Alternatively, microalgae can also be grown in photobioreactors that contain a fixed carbon source and allow light to strike the cells. Exposure of microalgae cells to light, even in the presence of a fixed carbon source that the cells transport and use (i.e., mixotrophic growth), nevertheless accelerates growth compared to culturing the cells in the dark. . Culture condition parameters can be manipulated to optimize total hydrocarbon production, the combination of hydrocarbon species produced, and/or production of one hydrocarbon species. In some instances it is preferable to grow cells in the dark, such as when using extremely large tankers (40,000 liters and larger) that do not allow light to hit the culture.
Microalgae culture media commonly contain components such as a fixed nitrogen source, trace elements, optionally a pH buffer solution for pH maintenance, and phosphate. Other components may include a fixed carbon source such as acetate or glucose and salts such as sodium chloride, particularly for seawater microalgae. Examples of trace elements include zinc, boron, cobalt, copper, manganese, and molybdenum in, for example, the respective forms of ZnCI.<sub>2</sub>,H<sub>3</sub>BO<sub>3</sub>, CoCI<sub>2</sub>*60H<sub>2</sub>0, IoC<sub>2</sub>*2H<sub>2</sub>O, MnCI<sub>2</sub>»4H<sub>2</sub>Or and (ΝΗ<sub>4</sub>)<sub>6</sub>Μο<sub>7</sub>0<sub>24</sub>4Η<sub>2</sub>0.
For organisms apt to grow on a fixed carbon source, the fixed carbon source may be, for example, glucose, fructose, sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside, and/or glucuronic acid. The one or more carbon sources may be supplied at a concentration of about 50 μΜ, at least about 100 μΜ, at least about 500 μΜ, at least about 5 mM, at least about 50 mM, and at least about 500 mM, from one or more exogenously supplied fixed carbon sources. Some species of microalgae can be grown using a fixed carbon source such as glucose or acetate in the absence of light. Such a culture is known as a heterotrophic culture. For Chlorella protothecoides, for example, heterotrophic growth results in high biomass production and high lipid accumulation in cells.
Some microorganisms grow naturally on or can be engineered to grow on a fixed carbon source that is a heterogeneous source of compounds such as municipal waste, secondarily treated sewage, wastewater, and other sources of fixed carbon and other nutrients such as sulfates, phosphates and nitrates. The sewage component serves as a nutrient source in hydrocarbon production, and cultivation provides an inexpensive source of hydrocarbons.
Other culture parameters can also be manipulated, such as the pH of the culture medium, the identity and concentration of trace elements and other constituents of the medium.
A. Photosynthetic Growth
Microalgae can be cultivated in the presence of light. The number of photons striking a culture of microalgae cells can be manipulated, as can other parameters such as the wavelength spectrum and ratio of dark:light hours per day. Microalgae can also be grown in natural light, as well as simultaneous and/or alternating combinations of natural light and artificial light. For example, microalgae of the Chlorella genus can be grown under natural light during daylight hours and under artificial light during night hours.
The gas content of a photobioreactor for growing microorganisms such as microalgae can be manipulated. Part of the volume of a photobioreactor may contain gas instead of liquid. Gas inlets can be used to pump gases into the photobioreactor. Any gas can be pumped into a photobioreactor, including air, air/CO mixtures<sub>2</sub>, noble gases such as argon and others. The rate of gas entry into a photobioreactor can also be manipulated. Increasing the gas flow to a photobioreactor increases the turbidity of a microalgae culture. The placement of gates that transport gases into a photobioreactor can also affect the turbidity of a culture at a given gas flow rate. air/CO mixtures<sub>2</sub> can be modulated to generate optimal amounts of CO<sub>2</sub> for maximum growth by a particular organism. Microalgae grow significantly faster in low light, for example 3% CO<sub>2</sub>/97% air than in 100% air. 3% CO<sub>2</sub>/97% air is about 100 times more CO<sub>2</sub> than that found in the air. For example, air:CO mixtures<sub>2</sub> of approximately 99.75% air:0.25% CO<sub>2</sub>, approximately 99.5% air:0.5% CO<sub>2</sub>, approximately 99.0% air: 1.00% CO<sub>2</sub>, approximately 98.0% air:2.0% CO<sub>2</sub>, approximately 97.0% air:3.0% CO<sub>2</sub>, approximately 96.0% air:4.0% CO<sub>2</sub>, and approximately 95.00% air:5.0% CO<sub>2</sub> they can be infused into a bioreactor or photobioreactor.
Microalgae cultures can also be subjected to mixing using devices such as centrifugal blades and impellers, shaking a culture, stir bars, pressurized gas infusion, and other instruments.
Photobioreactors can have inlets that allow gases, solids, semi-solids, and liquids to enter the chamber containing the microalgae. Gates are usually attached to pipes or other means to transport substances. Gas locks, for example, transport gases to the crop. The pumping of gases into a photobioreactor can serve both to feed the cells CO<sub>2</sub> and other gases and to aerate the culture and therefore generate turbidity. The amount of turbidity in a culture varies as the number and position of gas gates is altered. For example, gas gates can be placed along the bottom of a cylindrical polyethylene bag. Microalgae grow faster when CO is added<sub>2</sub> into the air and pumped into a photobioreactor. For example, an air mixture of 5% CO is infused<sub>2</sub>:95% air to a photobioreactor containing Botryococcus cells (see for example J Agrie Food Chem. 2006 Jun 28;54(13):4593-9; J Biosci Bioeng. 1999;87(6):811-5; and J Nat Prod. 2003 Jun;66(6):772-8).
Photobioreactors can be exposed to one or more light sources to provide microalgae with light as an energy source via light directed to the surface of the photobioreactor. Preferably, the light source provides an intensity that is sufficient for cells to grow, but not so intense that it causes oxidative damage or elicits a photoinhibitory response. In some instances, a light source has a wavelength range that mimics or roughly mimics the sun's range. In other instances, a different wavelength range is used. Photobioreactors can be placed outdoors or in a greenhouse or other equipment that allows sunlight to reach the surface. Preferred photon intensities for Botryococcus species are between 25 and 500 μΕ m<sup>2</sup> yes<sup>1</sup> (see for example Fotosynth Res. 2005 Jun; 84(13):21-7).
Photobioreactors preferably have one or more gates that allow entry of media. It is not necessary that only one substance enters or leaves a gate. For example, a gate can be used to flow culture media into the photobioreactor and then later used for sampling, gas inlet, gas outlet or other purposes. In some instances, a photobioreactor is filled with culture media at the start of a culture and no further culture media is infused after the culture is inoculated. In other words, the microalgae biomass is cultivated in an aqueous medium for a period of time during which the microalgae reproduce and increase in number; however, quantities of aqueous culture medium are not flowed through the photobioreactor for the entire period of time. Thus, in some embodiments, the aqueous culture medium is not flowed through the photobioreactor after inoculation.
In other instances, the culture medium can be flowed through the photobioreactor throughout the period of time during which the microalgae reproduce and increase in number. In some embodiments, the medium is infused into the photobioreactor after inoculation but before the cells reach a desired density. In other words, a turbulent flow regime of gas inlet and media inlet is not maintained for microalgae reproduction until a desired increase in microalgae numbers has been obtained.
Photobioreactors preferably have one or more gates that allow gas to enter. The gas can serve both to provide nutrients such as CO<sub>2</sub>, as well as to provide turbulence in the culture medium. Turbulence can be obtained by placing a gas inlet gate below the level of the aqueous culture medium, such that gas entering the photobioreactor bubbles to the culture surface. One or more gas outlet ports allows gas to escape, thereby preventing pressure buildup in the photobioreactor. Preferably a gas outlet port leads to a "one-way" valve that prevents contaminating microorganisms from entering the photobioreactor. In some instances, cells are grown in a photobioreactor for a period of time during which the microalgae reproduce and increase in number, however, a turbulent flow regime with turbulent eddies predominantly throughout the culture medium causes ingress. of gas is not maintained for the entire period of time. In other instances, a turbulent flow regime with turbulent eddies predominantly throughout the culture medium caused by gas ingress may be maintained for the entire period of time during which the microalgae reproduce and increase in numbers. In some instances, a predetermined range of ratios between the photobioreactor scale and the eddy scale is not maintained for the period of time during which the microalgae reproduce and increase in number. In other instances, such an interval may be maintained.
Photobioreactors preferably have at least one gate that can be used for sampling of the culture.
Preferably, a sampling gate can be used repeatedly without altering or compromising the axenic nature of the culture. A sample port can be configured with a valve or other device that allows sample flow to be stopped and started. Alternatively a sampling gate can allow continuous sampling. Photobioreactors preferably have at least one gate that allows inoculation of a culture. Such a gate can also be used for other purposes such as media inlet or gas inlet.
B. Heterotrophic culture
As an alternative to photosynthetic growth of microorganisms, as described above, some microorganisms can be grown under heterotrophic culture conditions in which a fixed carbon source provides energy for growth and lipid accumulation.
In a heterotrophic cultivation method according to the invention, the cost of biodiesel production, crude, partially purified or purified glycerol produced as a by-product of lipid transesterification can be used as a feedstock for fermentation, for example of lipid-producing microbial cultures. Thus, the invention encompasses culturing a microbe (eg, a microalga) in a first culture medium; recovering the microbial lipid from the culture; subjecting the microbial lipid to transesterification to produce glycerol fatty acid ester(s), as described above; and adding the glycerol to a second microbial culture as feedstock. The first and second microbial cultures may be, but need not be, cultures of the same microbe. If desired, a continuous system can be devised whereby glycerol produced from lipid recovered from a culture can be fed back to the same culture.
The invention provides significantly improved culture parameters incorporating the use of glycerol for the fermentation of multiple genera of both eukaryotic and prokaryotic microbes, including microbes from the genera Chlorella, Navícula, Scenedesmus and Spirulina. As the examples demonstrate, microbes of extremely divergent test lineages, including Chlorella, Navícula, Scenedesmus and Spirulina as well as cultures of multiple different Chlorella species and strains grow very well on not only purified reagent grade glycerol, but also on an acidified and non-acidulated glycerol by-product of biodiesel transesterification, in some instances, microalgae, such as Chlorella strains, undergo cell division faster in the presence of glycerol than in the presence of glucose. In these instances, two-step culture processes in which cells are first fed glycerol to rapidly increase heat density and are then fed glucose to accumulate lipids can improve the efficiency with which lipids are produced. The use of the glycerol by-product of the transesterification process provides significant economic advantages when it is put back into the production process. Other feeding methods are also provided, such as glycerol and glucose mixtures. Feeding such mixes also captures the same economic benefits. Furthermore, the invention provides methods of feeding alternative sugars to microalgae such as sucrose in various combinations with glycerol. These benefits provided by the invention have been demonstrated herein in microbes of extremely divergent lineages of evolution, including both prokaryotes and eukaryotes, demonstrating the utility of the Invention for microbial fermentation.
Standard methods for the cultivation and propagation of Chlorella protothecoides are known (see for example Miao and Wu, J. Biotechnology, 2004, 11: 85-93 and Miao and Wu, Biosource Technology (2006) 97:841-846). The invention also provides new culture conditions for Chlorella. For example, multiple Chlorella species and multiple strains within a species can be grown in the presence of glycerol, including glycerol by-products of biodiesel transesterification.
For the production of hydrocarbons, cells, including recombinant cells of the invention described herein, are preferably cultured or fermented in large quantities. Culture can be in large volumes of liquid, such as in suspension culture as an example. Other examples include starting with a small cell culture expanding to a large biomass in combination with cell culture and propagation as well as hydrocarbon production. Bioreactors or steel tankers can be used to accommodate large volumes of culture. A proofer similar to those used in beer and/or wine production is appropriate, as are extremely large proofers used in ethanol production.
Appropriate nutrient sources are provided for growing in a termentador. These include raw materials such as one or more of the following: a fixed carbon source such as glucose, corn starch, depolymerized cellulosic material, sucrose, sugar cane, sugar beet, lactose, whey, or molasses; a source of fat, such as vegetable fats or oils; a nitrogen source, such as protein, soybean meal, corn liquor, ammonia (pure or in salt form), nitrate or nitrate salt, or molecular nitrogen; and a source of phosphorus, such as phosphate salts. Additionally, a fermenter allows control of growing conditions such as temperature, pH, oxygen tension, and carbon dioxide levels. Optionally, gaseous components, such as oxygen or nitrogen, can be bubbled through a liquid culture. Other sources of starch (glucose) such as wheat, potato, rice and sorghum. Other carbon sources include process streams such as technical grade glycerol, black liquor, organic acids such as acetate, and molasses. Carbon sources can also be provided as a mixture, such as a mixture of sucrose and depolymerized sugar beet pulp.
A shaker can be used to allow cells to go through the various phases of their growth cycle. As an example, an inoculum of hydrocarbon-producing cells may be introduced to a medium followed by a lag period (lag phase) before the cells begin to grow. Following the lag period, the growth rate increases steadily and enters the logarithmic or exponential phase. The exponential phase is in turn followed by a slowdown in growth due to decreases in nutrients and/or increases in toxic substances. After this arrest, growth stops and the cells enter a stationary phase or steady state, depending on the particular environment provided to the cells.
Hydrocarbon production by the cells disclosed herein can occur during or after log phase, including the stationary phase where nutrients are supplied or still available, to allow continuation of hydrogen production in the absence of division. cell phone.
Preferably, microorganisms grown using the conditions described herein and known in the art comprise at least about 20% by weight lipid, preferably at least about 40% by weight, more preferably at least about 50% in weight and more preferably at least about 60% by weight.
A surprising discovery is that multiple species and multiple strains within a Chlorella species perform better in the presence of the glycerol by-product of transesterification than in an equivalent amount of reagent grade glycerol. The glycerol by-product of transesterification usually contains residual methanol and other contaminants in addition to glycerol. For example, Figures 1-6 demonstrate that Chlorella protothecoides and Chlorella kessleri strains exhibit better productivity on the acidified and non-acidulated glycerol by-product of lipid transesterification reactions than when grown on pure reagent grade glycerol. Other microbes, such as Scenedesmus and Navícula microalgae may also function better in the presence of the glycerol by-product of transesterification than an equivalent amount of reagent grade glycerol.
Cell Dry Weight per Liter: Figure 1 demonstrates that cell dry weight was higher on the biodiesel glycerol by-product than on neat glycerol and this trend held true when cells were grown on glycerol by itself or in combination with glucose. Figure 2 shows the same trends with additional Chlorella strains. Figure 12(b) demonstrates that cell dry weight per liter of Scenedesmus armatus is higher on acidified and unacidulated biodiesel by-product glycerol than on pure reagent grade glycerol. Figure 13 demonstrates that the cell dry weight per liter of Navicula pelliculosa is higher on unacidulated biodiesel by-product glycerol than on pure reagent grade glycerol.
Lipid Content per Liter: Figures 3 and 4 demonstrate that multiple Chlorella species, and multiple strains within a Chlorella species, lipid levels per liter are higher when cells are grown in the presence of biodiesel glycerol by-product than when grown in the presence of equivalent concentrations of pure reagent grade glycerol.
Lipid as a percentage of cell weight: Figures 5 and 6 demonstrate that multiple Chlorella species and multiple strains within a Chlorella species accumulate a higher percentage of cell dry weight as lipid when cultured in the presence of the glycerol by-product. of biodiesel than when grown in the presence of equivalent concentrations of pure reagent grade glycerol. Figure 11 demonstrates that both Spirulina platensis and Navícula pelliculosa can accumulate a higher percentage of cell dry weight as lipid when grown in the presence of biodiesel glycerol by-product than when grown in the presence of equivalent concentrations of pure reagent grade glycerol. . Figure 12(a) demonstrates that Scenedesmus armatus can accumulate a higher percentage of cell dry weight as lipid when cultured in the presence of biodiesel glycerol by-product than when cultured in the presence of equivalent concentrations of pure reagent grade glycerol.
Another surprising result is that multiple species of microbes, including microalgae such as Chlorella and multiple strains within a species of Chlorella and other microalgae such as Scenedesmus, Navícula and Spirulina exhibit better characteristics as biodiesel producers in the presence of mixtures of glycerol and glucose than in the presence of glucose alone.
Lipid Content per Liter: Figure 7 demonstrates that Chlorella can accumulate higher levels of lipid per liter of culture in the presence of 1% glycerol/1% glucose than in the presence of 2% glucose.
Lipid as percentage of cell weight: Figure 8 demonstrates that Chlorella can accumulate a higher percentage of cell dry weight as lipid when cultured in the presence of an equal concentration (weight percent) mixture of glycerol and glucose than when it is cultivated in the presence of only glucose. Figure 11(a) demonstrates that Sprullna platensis can accumulate a higher percentage of cell dry weight as lipid when cultured in the presence of an equal concentration (weight percent) mixture of glycerol by-product of biodiesel and glucose than when it is cultivated in the presence of only glucose. Figure 11(b) demonstrates that Navicula pelliculosa can accumulate a higher percentage of cell dry weight as lipid when cultured in the presence of a mixture of equal concentration (weight percent) of reagent grade glycerol and glucose, also as glycerol. by-product of biodiesel and glucose, than when grown in the presence of glucose alone. Figure 12(b) demonstrates that Scenedesmus armatus can accumulate a higher percentage of cell dry weight as lipid when cultured in the presence of equal concentration (wt percent) of biodiesel by-product glycerol and glucose than when cultured in presence of only glucose.
An additional and unexpected discovery is that the addition of glycerol and glucose to the microbe, including microalgae such as Chlorella, Scenedesmus and Navícula sequentially rather than as a single batch mixture of glycerol and glucose can generate additional yield gains. This attribute of multiple Chlorella species and multiple strains within a Chlorella species was tested in the presence of both biodiesel glycerol by-product and reagent grade glycerol.
Lipid as percentage of cell weight: Figure 8 demonstrates that Chlorella can accumulate a higher percentage of cell dry weight as lipid when glycerol is added to a culture for an initial period of time, followed by the addition of glucose and continued cultivation. for a second period of time, than when the same amounts of glycerol and glucose are added together at the beginning of the experiment.
Lipid content per liter: Figure 9 shows Chlorella exhibiting higher levels of lipid per liter of culture when glycerol and glucose are added sequentially than when the same amounts of glycerol and glucose are added together at the beginning of the experiment. This trend was observed when glycerol by-product of acidulated biodiesel, glycerol by-product of unacidulated biodiesel, or reagent grade glycerol was used.
Cell Dry Weight per Liter: Figure 10 demonstrates four different Chlorella strains from two different species that accumulate a higher cell dry weight per liter of culture when glycerol and glucose are added sequentially than when the same amounts of glycerol and glucose are added. added together at the beginning of the experiment. This trend was observed when glycerol by-product of acidulated biodiesel, glycerol by-product of unacidulated biodiesel, or reagent grade glycerol was used. Figures 14(a) and (b) demonstrate that both Scenedesmus armatus and Navícula pelliculosa can exhibit increases in cell dry weight per liter when biodiesel by-product glycerol is only added to a culture for an initial period of time, followed by more. late by the addition of glucose, compared to the addition of identical amounts of glycerol and glucose at the beginning of the fermentation.
Three different productivity markers (cell dry weight per liter, grams per liter lipid, and percentage of cell dry weight as lipid) in microbial lipid production are enhanced by the use of biodiesel by-product and temporal separation of fuel sources. carbon. The invention therefore provides new methods for generating higher amounts of lipid per unit time in multiple species of microbes from highly divergent areas of the evolutionary tree, including both prokaryotes and eukaryotes. The methods for manufacturing lipids and hydrocarbons disclosed herein using glycerol are not limited to microalgae, but can be used with any microbe capable of using glycerol as an energy source.
In an alternative heterotrophic growth method according to the present invention, microorganisms can be cultivated using depolymerized cellulosic biomass as feedstock. Cellulosic biomass (eg, stover, such as corn stover) is inexpensive and readily available; however, attempts to use this material as feed stock for yeast have failed. In particular, such feed stock has been found to be inhibitory to yeast growth and yeast cannot use the five carbon sugars produced from cellulosic materials (eg, hemicellulose xylose). In contrast, microalgae can grow on processed cellulosic material. Thus, the invention provides a method for culturing microalgae in the presence of a cellulosic material and/or a five-carbon sugar. Cellulosic materials generally include:
<td>Component</td><td>dry weight percent</td>
<td>Cellulose</td><td> 40-60%</td>
<td>hemicellulose</td><td> 20-40%</td>
<td>lignin</td><td> 10-30%</td>
Suitable cellulosic materials include residues of herbaceous energy crops and wood energy crops, as well as agricultural crops, that is, the parts of plants, mainly stems and leaves, not removed from the primary food or fiber product fields. Examples include agricultural wastes such as sugarcane bagasse, rice husks, corn fiber (including stalks, leaves, husks, and cobs), wheat straw, rice straw, sugar chocolate pulp, citrus pulp, citrus peels. ; forest wastes such as hardwood and softwood thinning and hardwood and softwood residues from log operations; wood waste such as ground mill waste (wood chips, wood dust) and pulp mill waste; urban waste such as municipal solid waste paper fractions; urban wood waste and urban organic waste such as fragments of municipal grass and construction waste with wood. Additional cellulosics include dedicated cellulosic crops such as switchgrass, hybrid cottonwood and miscanthus, sugarcane, and sorghum fiber. The five carbon sugars that are produced from such materials include xylose.
Surprisingly, it has been shown herein that some Chlorella species exhibit higher levels of productivity when grown on a combination of glucose and xylose than when grown on either glucose or xylose alone. This synergistic effect provides a significant advantage in that it allows the cultivation of Chlorella on combinations of xylose and glucose, such as cellulosic material and is shown in Figure 15.
In yet another alternative heterotrophic culture method according to the present invention, which can optionally be used by itself in combination with the methods described above, sucrose, produced for example from sugar cane or sugar beet, is used as feed raw material. As described in more detail in the section entitled Design of Microbes later herein, lipid production can be facilitated or made more efficient by engineering microbes, such as Chlorella, to use sucrose as a carbon source. For example, expression of an ascarase transporter and an agarose invertase allow Chlorella to transport sucrose into the cell from the culture medium and hydrolyze sucrose to produce glucose and fructose. Optionally, a fructokinase can also be expressed in instances where endogenous hexokinase activity is sufficient for maximal fructose phosphorylation. Examples of suitable useful sucrose transporters are GenBank accession numbers CAD91334, CAB92307 and CAA53390. Examples of suitable sucrose invertases are GenBank accession numbers CAB95010, NP012104 and CAA06839. Examples of suitable sucrose invertases are GenBank accession numbers P26984, P26420 and CAA43322. Vectors for transformation of microalgae, including Chlorella, encoding one or more such genes can be designed as described herein.
Secretion of a sucrose invertase may obviate the need for expression of a transporter that can transport sucrose into the cell. This is due to a secreted invertase catalyzing the conversion of a sucrose molecule into a glucose molecule and a fructose molecule, both of which can be transported and used by the microbes disclosed herein. For example, the expression of a sucrose invertase (such as SEQ ID NO: 14) with a secretion signal (such as that of SEQ ID NO: 15 (from yeast), SEQ ID NO: 16 (from higher plants), SEQ ID NO: 17 (eukaryotic consensus secretion signal) and SEQ ID BO: 18 (combination of higher plant and eukaryotic consensus signal sequences) generate invertase activity outside the cell See Hawkins et al., Curren Microbiology Vol. 38 (1999), p. 335-341 for examples of signals of active secretion in Chlorella. Expression of such a protein, such as disabled by the genetic engineering methodology disclosed herein, allows cells to be able to use extracellular glucose as an energy source by utilizing sucrose as an extracellular energy source. In cells such as Chlorella protothecoides, Chlorella minutissima, and Chlorella emersonii such as those demonstrated herein can use both extracellular fructose and extracellular glucose as energy sources, secretion of an invertase may provide the only catalytic activity necessary to use sucrose as a source. energy efficient, not expensive.
For example, as shown in Figure 26, Chlorella protothecoides can be engineered with a sucrose invertase gene under the regulatory control of one of three promoters (cauliflower mosaic virus (CMV) 35S promoter, Chlorella virus promoter (CV) or Chlorella HUP1 promoter (HUP1)). The sucrose invertase gene used in this example comprises a modification to the S. cerevisiae SUC2 gene to optimize C. protothecoides codon usage. The cDNA and amino acid sequences of the optimized gene correspond to SEQ ID NO: 8 and SEQ ID NO: 19, respectively. An illustration of the plasmid constructs used in the transformation is shown in Figure 25. Expression of a secreting sucrose invertase, such as that described herein, allows the use of molasses, sugarcane juice, and other raw materials. feed containing sucrose for cell fermentation.
Similarly, Figures 27 and 28 show the results of transformation of Chlorella protothecoides and Chlorella minutissima and Chlorella emersonii, respectively, with the S. cerevisiae sucrose invertase gene under the control of the CMV promoter.
The growth potential of microorganisms expressing an exogenous secretable sucrose invertase is illustrated by the addition of an invertase to the Chlorella protothecoides culture medium, as described in further detail in the Examples. Figures 23 and 24 illustrate the surprising result that Chlorella cells grow as well on waste molasses from sugarcane processing as they do on reagent grade pure glucose; The use of this low-value waste product from sugarcane processing can provide significant cost savings in the production of hydrocarbons and other oils. Molasses contains lignin and other cellulosic waste products that poison many microorganisms and retard their growth, however Chlorella cells have been found to thrive in the presence of such poisons. Figures 23-24 show growth of cells on three unique sources of molasses (designated BS1, BS2 and HTM), compared to growth on glucose or sucrose in the presence or absence of an extracellular sucrose invertase.
Alternatively, a sucrose invertase can also be expressed intracellularly in cells that express a sucrose transporter, as well as in cells that express any carbohydrate transporter that allows sucrose to enter the cell.
A foreign gene was transformed into and expressed in Chlorella protothecoides, as described in Example 12. Expression of the sucrose utilization gene can be monitored using the same or similar methodology and vector design.
Bioreactors may be employed for use in heterotrophic growth methods. As will be appreciated, precisions made to make light available to cells in photosynthetic culture methods are unnecessary when a fixed carbon source is used in the heterotrophic culture methods described herein.
The specific examples of process conditions and heterotrophic growth methods described herein may be combined in any appropriate manner to improve microbial growth and lipid production efficiencies. Furthermore, the invention includes the selection and/or genetic engineering of microbes, such as microalgae, to produce microbes that are even more suitable for use in the methods described above. For example, microbes that have a greater ability to utilize any of the feedstocks described herein for increased proliferation and/or lipid (eg, fatty acids) production are within the scope of the invention.
C. Mixotrophic growth
Mixotrophic growth is the use of both light and fixed carbon sources as energy sources for the growth of hydrocarbon-producing cells. Mixotrophic growth can be carried out in a photobioreactor. Microalgae can be grown and maintained in closed photobioreactors made of different types of transparent or semi-transparent material. Such material may include Plexiglas® wrappers, glass wrappers, bags made of substances such as polyethylene, transparent or semi-transparent tubing, and other materials. Microalgae can be grown or maintained in open photobioreactors such as channel lagoons, treatment lagoons, and other non-enclosed vessels.
D. Culture media
Microorganisms useful in accordance with the method of the present invention are found in various locations and environments throughout the world. As a consequence of their isolation from other species and their ongoing evolutionary divergence, the particular culture medium for optimal growth and generation of lipid and/or hydrocarbon constituents can be difficult to predict. In some cases, certain strains of microorganisms may be unsuitable for growth on a particular culture medium due to the presence of some inhibitory component or the absence of some essential nutritional requirement required by the particular strain of microorganism.
Solid and liquid culture media are generally available from a wide variety of sources, and instructions for the preparation of particular media that are appropriate for a wide variety of microorganism strains can be found, for example, online at httD://www. iitex.org/, a site maintained by the University of Texas at Austin for its algae culture (UTEX) collection. For example, various freshwater and saltwater media include those shown in Table 4 below.
Table 4. Specimen algal media.
<td>medium sweet aaua</td><td>Salty water medium</td>
<td>Diatom medium 1/2 CHEV</td><td>1%F/2</td>
<td>Diatom medium 1/3 CHEV</td><td>Enriched seawater medium 1/2</td>
<td>Diatom medium 1/5 CHEV</td><td>Medium Erdchreiber 1/2</td>
<td>DYIII/PEA + Gr+ 1:1</td><td>Seawater medium + 1/2 substrate</td>
<td>Diatom medium 2/3 CHEV</td><td>Seawater medium + 1/3 substrate</td>
<td>2X CHEV Diatom Medium</td><td>1/4 erd</td>
<td>Ag diatom medium</td><td>Seawater medium + 1/4 substrate</td>
<td>Half Alien</td><td>Seawater medium + 1/2 substrate</td>
<td>Medium BG11-1</td><td>Enriched seawater medium 2/3</td>
<td>Medium INV Bold</td><td>20% Alien + 80% ERD</td>
<td>Medium 3N Bold</td><td>Erdschreiber medium 2X</td>
<td>Botryococcus medium</td><td>Seawater medium + 2X substrate</td>
<td>middle bristol</td><td>Medium F/2 at 5%</td>
<td>CHEV Diatom Medium</td><td>Agar medium in seawater + substrate 5/3</td>
<td>Middle of Chu</td><td>artificial seawater medium</td>
<td>CR1 Diatom Medium</td><td>BG11 -1 + 36% NaCI Medium</td>
<td>Diatom medium XR1 +</td><td>BG11-1 + 1% NaCI medium</td>
<td>Diatom medium CR1 -S</td><td>INV Bold:Erdshreiber (1:1)</td>
<td>Cyanidium medium</td><td>INV Bold:Erdshreiber (4:1)</td>
<td>Cyanophyte medium</td><td>Bristol-NaCI medium</td>
<td>Medium Desmid</td><td>Medium seawater with Dasycladales</td>
<td>Medium DYIII</td><td>Enriched seawater medium</td>
<td>Middle Euglena</td><td>Middle Erdshreiber</td>
<td>Medium HEPES</td><td>ES/10 Enriched Seawater Medium</td>
<td>Medium J</td><td>ES/2 Enriched Seawater Medium</td>
<td>Medium Malt</td><td>ES/4 Enriched Seawater Medium</td>
<td>Half month</td><td>Medium F/2</td>
<td>Modified Medium 3N Bold</td><td>F/2 + NH4</td>
<td>COMBO Modified Medium</td><td>Medium BOM</td>
<td>Medium N/20</td><td>2 X Modified CHEV</td>
<td>Medium Ochromonas</td><td>2 X modified CHEV + substrate</td>
<td>Medium P49</td><td>Modified artificial seawater medium</td>
<td>Medium Polytomella</td><td>modified CHEV</td>
<td>medium proteose</td><td>Medium Porphridium</td>
<td>Frozen Seaweed Medium</td><td>Seawater medium + substrate</td>
<td>Medium with substrate extract</td><td>SS Diatom Medium</td>
<td>Substrate in water: Medium BAR</td><td></td>
<td>Substrate in water: Medium-GR</td><td></td>
<td>Substrate in water: Medium GR-/NH4</td><td></td>
<td>Substrate in water: Medium GR+</td><td></td>
<td>Substrate in water: Medium GR+/NH4</td><td></td>
<td>Substrate in water: Medium PEA</td><td></td>
<td>Substrate in water: Peat medium</td><td></td>
<td>Substrate in water: Medium VT</td><td></td>
<td>Medium Spirulina</td><td></td>
<td>binding medium</td><td></td>
<td>Middle Trebouxia</td><td></td>
<td>Medium Volvocaceous</td><td></td>
<td>Medium volvocaceous-3N</td><td></td>
<td>Medium Volvox</td><td></td>
<td>Volvox-Dextrose Medium</td><td></td>
<td>Middle Waris</td><td></td>
<td>Waris + medium with substrate extract</td><td></td>
In a particular example, a suitable medium for the cultivation of Chlorella protothecoides (UTEX 31) comprises proteose medium. This medium is suitable for axenic cultures and a one liter volume of the medium (pH approximately 6.8) can be prepared by adding one gram of proteose peptone to one liter of Bristol medium. The Bristol medium comprises NaNO<sub>3</sub> 2.94 mM, CaCI<sub>2</sub>h<sub>2</sub>OR 0.17 mM, MgSO<sub>4</sub>-7H<sub>2</sub>0.3mM, KH<sub>2</sub>PO<sub>4</sub> 0.43 mM, 1.29 mM and 1.43 mM NaCI in an aqueous solution. For a 1.5% agar medium, 15 g of agar can be added to one liter of the solution. The solution is covered and autoclaved and then stored at a refrigerated temperature before use.
Other media suitable for use with the methods of the invention can be readily identified by consulting the URL identified above or by consulting other organizations that maintain cultures of microorganisms, such as SAG, CCAP, or COALA. SAG refers to the Collection of Algal Cultures at the University of Gottingen (Gottingen, Germany), CCAP refers to the collection of algal and protozoan cultures managed by the Scottish Association for Marine Science (Scotland, United Kingdom Greater Brittany) and CCALA refers to the collection of laboratory algae cultures at the Institute of Botany (Treboñ, Czech Republic).
E. Increased lipid yield
Process conditions can be adjusted to increase the yield of lipids appropriate for a particular use and/or to reduce the cost of production. For example, in certain embodiments, a microbe (eg, a microalga) is grown in the presence of a limiting concentration of one or more nutrients, such as, for example, carbon and/or nitrogen, phosphorous, or sulfur, while providing excess carbon energy fixed as glucose. Nitrogen limitation tends to increase microbial lipid yield relative to microbial lipid yield in a culture in which excess nitrogen is provided. In particular embodiments, the increase in lipid yield is at least about: 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500%. The microbe can be cultured in the presence of a limiting amount of a nutrient for a portion of the entire culture period or for the entire culture period. In particular embodiments, the concentration of the nutrient is cycled between a limiting concentration and a non-limiting concentration at least twice during the entire culture period.
To increase lipid yield, acetic acid can be used as feedstock for a lipid-producing microbe (eg, microalgae). Acetic acid is fed directly to the point of metabolism that initiates fatty acid synthesis (ie, acetyl-CoA); thus providing acetic acid in the culture which can increase fatty acid production. In general, the microbe is grown in the presence of a sufficient amount of acetic acid to increase the microbial lipid yield and/or the microbial fatty acid yield, specifically, with respect to the microbial lipid (eg, fatty acid) yield in absence of acetic acid.
In another embodiment, lipid yield is increased by culturing a lipid-producing microbe (eg, microalgae) in the presence of one or more co-factors for a lipid pathway enzyme (eg, a fatty acid synthetic enzyme). . In general, the concentration of the co-factor(s) is sufficient to increase the yield of the microbial lipid (eg fatty acid) relative to the yield of the microbial lipid in the absence of the co-factor(s). In a particular embodiment, the co-factor(s) is (are) provided to the culture by including in the culture a microbe (eg, microalgae) that contains an exogenous gene encoding the co-factor(s). -factor(s). Alternatively, cofactors can be provided to a culture by including a microbe (eg, microalgae) that contains an exogenous gene encoding a protein that participates in cofactor synthesis. In certain embodiments, appropriate co-factors include any vitamin required by a lipid pathway enzyme, such as for example: biotin, pantothenate. Genes encoding co-factors suitable for use in the invention or participating in the synthesis of such co-factors are well known and can be introduced into microbes (eg microalgae) using concepts and techniques such as those described above.
V. LIPID PATH DESIGN
In some embodiments of the present invention, the microorganisms of the present invention are modified to alter known lipid properties and/or ratios and/or to increase carbon flux to lipids. The pathway can additionally or alternatively be modified to alter the properties and/or ratios of various hydrocarbon molecules produced through enzymatic processing of lipids.
A. Alteration of properties or proportion of lipid or hydrocarbons produced
In the case of microalgae, some wild type cells already have good growth characteristics but do not produce the desired types or amounts of lipids. Examples include Pyrobotrys, Phormidium, Agmenellum, Carteria, Lepocinclis, Pyrobotrys, Nitzschia, Lepocinclis, Anabaena, Euglena, Spirogyra, Chlorococcum, Tetrahedron, Oscillatoria, Phagus and Chlorogonium which have the desirable growth characteristic of growing in municipal sewage or waste water. . Such cells, as well as Chlorella species and other microbes, can be engineered to have enhanced lipid production characteristics. Desired characteristics include optimization of lipid yield per unit volume and/or per unit time, carbon chain length (for example, for biodiesel production or for initial applications requiring hydrocarbon feedstock), reducing the number of double or triple bonds, optionally zero, remove or eliminate rings and cyclic structures and increase the proportion of hydrogen: carbon from a particular species of lipid or from a distinct lipid population. In addition, appropriate hydrocarbon-producing microalgae can also be engineered to have even more desirable hydrocarbon outputs. Examples of such microalgae include species of the genus Chlorella.
one. Regulation of enzymes that control branch points in fatty acid synthesis
In particular embodiments, one or more key enzymes that control branch points in metabolism for fatty acid synthesis may be up-regulated or down-regulated to enhance lipid production. Upregulation can be stopped for example by transforming cells with expression concepts in which a gene encoding the enzyme of interest is expressed, for example using a strong promoter and/or transition enhancing enhancer elements. Such concepts may include a selectable marker, such that transformants may be subjected to selection, which may result in application of the concept and an increase in the expression level of the encoded enzyme. Examples of enzymes suitable for upregulation according to the methods of the invention include pyruvate dehydrogenase, which plays a role in converting pyruvate to acetyl-CoA (examples, some from algae, include Genbank accession numbers NP_415392; AAA53047; Q1XDM1 and CAF05587). Upregulation of pyruvate dehydrogenase can increase acetyl-CoA production and thereby increase fatty acid synthesis. Acetyl-CoA carboxylase catalyzes the initial step in fatty acid synthesis. Thus, this enzyme can be up-regulated to increase fatty acid production (eg, some from microalgae, include Genbank accession numbers BAA94752; AAA75528; AAA81471; YP 537052; YP 536879; NP 045833 and BAA57908). Fatty acid production can also be increased by upregulating acyl carrier protein (ACP), which carries the growing acyl chains during fatty acid synthesis (examples, some from microalgae, include Genbank accession numbers AOTOF8 ; P51280; NP_84041; YP_874433). Glycerol-3-phosphateacetyltransferase catalyzes the rate-limiting step of fatty acid synthesis. Upregulation of this enzyme can increase fatty acid production (examples, some from microalgae, include Genbank accession numbers AAA74319; AAA33122; AAA37647; P44857 and ABO94442). The foregoing proteins are candidates for expression in microalgae, including species of the genus Chlorella.
Down-regulation of an enzyme of interest can be obtained using, for example, catalytic antisense RNA/DNA, RNA interference (RNAγ), "knockout", "knockdown" or other mutagenesis techniques. Enzyme expression/function can also be inhibited using intrabodies. Examples of enzymes suitable for down-regulation according to the methods of the invention include citrate synthase, which consumes acetyl-CoA as part of the tricarboxylic acid (TCA) cycle. Down-regulation of citrate synthase can force more acetyl-CoA into the fatty acid synthesis pathway.
2. Modulation of global regulators of fatty acid synthesis genes
Global regulators modulate the expression of fatty acid biosynthetic pathway genes. Thus, one or more global regulators of fatty acid synthesis must be up-regulated or down-regulated, as appropriate, to inhibit or enhance, respectively, the expression of a priority fatty acid synthetic gene and finally, to increase production. of lipid. Examples include sterol regulatory element binding proteins (SREBPs), such as SREBP-1a and SREBP-1c (for examples see Genbank accession numbers NP 035610 and Q9WTN3). Global regulators can be up-regulated or down-regulated, eg, as described above with respect to checkpoint enzyme regulation.
3. Regulation of hydrocarbon modifying enzyme
The methods of the invention also include transformant cells, with one or more genes encoding hydrocarbon modifying enzymes such as for example a fatty acyl-ACP thioesterase (see examples in Table 5 with accession numbers), a fatty acyl-CoA/ fatty aldehyde reductase (see examples in Table 6 with accession numbers), a fatty acyl-CoA reductase (see examples in Table 7 with accession numbers), a fatty aldehyde decarbonylase (see examples in Table 8 with accession numbers), a fatty aldehyde reductase or a squalene synthase gene (see Genbank accession number AF205791). In some embodiments, genes encoding a fatty acyl-ACP thioesterase and a naturally co-expressed acyl carrier protein can be transformed into a cell, optionally with one or more genes encoding other hydrocarbon modification enzymes. In other embodiments, ACP and fatty acyl-ACP thioesterase may have an affinity for each other that imparts an advantage when the two are used together in the microbes and methods of the present invention, regardless of whether they are naturally co-expressed in a tissue. or private body. Thus, the present invention contemplates both naturally co-expressed pairs of these enzymes, as well as those that share an affinity for interacting with one another to facilitate carbon chain cleavage from ACP-specific sites.
In still other embodiments, an exogenous gene encoding a desaturase can be transformed into the cell in conjunction with one or more genes encoding other hydrocarbon modification enzymes in order to provide hydrocarbon saturation modifications. Stearoyl-ACP desaturase (see, for example, Genbank accession numbers AAF15308; ABM45911 and AAY86086), for example, catalyzes the conversion of stearoyl-ACP to oloyl-ACP. Upregulation of this gene can increase the proportion of monounsaturated fatty acids produced by a cell; while down-regulation can reduce the proportion of monounsaturates. Similarly, the expression of one or more glycerolipid desaturases can be controlled to alter the ratio of unsaturated to saturated fatty acids, such as omega-6 fatty acid desaturase, omega-3 fatty acid desaturase, or omega-6-oleate desaturase. . In some embodiments, the desaturase can be selected with reference to a desired carbon chain length such that the desaturase is capable of making site-specific modifications within a substrate of specified carbon length or substrates having a length of carbon within a specified interval.
In particular embodiments, the microbes of the present invention are genetically engineered to express one or more exogenous genes selected from a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase fat or a naturally co-expressed acyl carrier protein. Appropriate expression methods are described above with respect to expression of a lipase gene, including, among other methods, inducible expression and combative expression.
Without intending to be limited by any particular theory or cellular mechanism, a fatty acyl-ACP thioesterase cleaves a fatty acid from an acyl carrier protein (ACP) during lipid synthesis. Through further enzymatic treatment, the cleaved fatty acid is then combined with a co-enzyme to produce an acyl-CoA molecule. This acyl-CoA is the substrate for the enzymatic activity of a fatty acyl-CoA reductase to produce an aldehyde, as well as for a fatty acyl-CoA/aldehyde reductase to produce an alcohol. The aldehyde produced by the action of fatty acyl-CoA reductase identified above is the substrate for further enzyme activity by either a fatty aldehyde reductase to produce an alcohol or a fatty aldehyde deacaboxylase to produce an alkane or alkene.
The enzymes described directly above have a specificity for acting on a substrate that includes a specific number of carbon atoms. For example, a fatty acyl-ACP thioesterase may have a specificity for cleaving a fatty acid having twelve carbon atoms from ACP. In some embodiments, ACP and length-specific thioesterase may have an affinity for each other that makes them particularly useful as a combination (for example, exogenous ACP and thioesterase genes may be co-expressed naturally in a particular tissue or organism). from which they are derived). Thus, in various embodiments, the microbe may contain an exogenous gene encoding a protein with specificity to catalyze an enzymatic activity (for example, cleavage of a fatty acid from ACP, reduction of acyl-CoA to an aldehyde or alcohol). or conversion of an aldehyde to an alkane) with respect to the number of carbon atoms contained in the substrate. Enzyme specificity can be in various ways, for a substrate having 8 to 34 carbon atoms, preferably 8 to 18 carbon atoms and more preferably 10 to 14 carbon atoms. The most preferred specificity is for a substrate having twelve carbon atoms. In other embodiments, the specificity may be for 20 to 30 carbon atoms.
Fatty acyl-ACP thioesterase suitable for use with the microbes and methods of the invention include without limitation those listed in Table 5.
Table 5. Fatty acyl-ACP thioesterases and Genbank accession numbers.
Umellularia califomica fatty acyl-ACP thioesterase (GenBank # AAC49001) Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank # Q39473) Fatty acyl-ACP thioesterase (GenBank # AAB71730) Elaeis guineensis fatty acyl-ACP thioesterase (GenBank # ABD83939) Elaeis guineensis fatty acyl-ACP thioesterase (GenBank # AAD42220) Populus tormentosa Fatty acyl-ACP thioesterase (GenBank # ABC47311) Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank # NP_172327) Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank # CAA85387) Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank8 # CAA8538)
Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank #O9SOI3)
Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank #CAA54060)
Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank # AAC72882)
Cuphea calophylla subsp. mesostemon fatty acyl-ACP thioesterase (GenBank # ABB71581)
Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank # CAC19933)
Elaeis guineensis fatty acyl-ACP thioesterase (GenBank # AAL15645)
Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank #Q39513)
Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank # AAD01982)
Vitis vinifera fatty acyl-ACP thioesterase (GenBank #CAN81819)
Garcinia mangostana fatty acyl-ACP thioesterase (GenBank # AAB51525)
Brassica júncea fatty acyl-ACP thioesterase (GenBank # AB118986)
Madhuca longifolia fatty acyl-ACP thioesterase (GenBank # AAX51637)
Brassica napus fatty acyl-ACP thioesterase (GenBank #ABH11710)
Oryza sativa (indica crop group) fatty acyl-ACP thioesterase (GenBank # EAY86877)
Oryza sativa (japonica cultivar group) fatty acyl-ACP thioesterase (GenBank # NP_001068400)
Oryza sativa (indica crop group) fatty acyl-ACP thioesterase (GenBank # EAY99617)
Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank # AAC49269)
Fatty acyl-CoA/fatty aldehyde reductase suitable for use with microbes and methods of the invention include without limitation those listed in Table 6.
Table 6. Fatty acyl-CoA/aldehyde reductase listed by Gen Bank accession number.
AAC45217, YP_047869, BAB85476, YP_001086217, YP_001280274, YP_264583, YP_436109, YP_959769, ZP_01736962, ZP_01900335, ZP_01892096, ZP_01103974, ZP 01915077, YP 924106, YP_130411, ZP_01222731, YP 550815, YP 983712, YP_001019688, YP_524762, YP_856798, ZP_011155000, YP_001141848, NP_336047, NP_216059, YP 882409, YP_706156, YP_001136150, YP_952365, ZP_01221833, YP_130076, NP_567936, AAR88762, ABK28586, NP_197634,
CAD30694, NP_001063962, BAD46254, NP_001030809, EAZ10132, EAZ43639, EAZ07989, NP_001062488, CAB88537, NP_001052541, CAH66597, CAE02214,
CAH66590, CAB88538, EAZ39844, AAZ06658, CAA68190, CAA52019, and BAC84377
Fatty acyl-CoA reductases suitable for use with the microbes and methods of the invention include without limitation those listed in Table 7.
Table 7. Fatty acyl-CoA reductases listed by Gen Bank accession numbers
NP 187805, ABO14927, NP_001049083, CAN83375, NPJ91229, EAZ42242, EAZ06453, CAD30696, BAD31814, NP_190040, AAD38039, CAD30692, CAN81280, NP_197642, NP_1900, NP_1900.
Fatty aldehyde decarbonylases suitable for use with the microbes in methods of the invention include without limitation those listed in Table 8.
Table 8. Fatty aldehyde decarbonylases listed by Gen Bank accession numbers.
NP_850932, ABN07985, CAN60676, AAC23640, CAA65199, AAC24373,
CAE03390, ABD28319, NPJ81306, EAZ31322, CAN 63491, EAY94825, EAY86731,
CAL55686, XP_001420263, EAZ23849, NP_20058, NP_001063227, CAN83072, AAR90847, and AAR97643
Combinations of naturally co-expressed fatty acyl-ACP thioesterases and acyl carrier proteins are suitable for use with the microbes and methods of the invention.
Additional examples of hydrocarbon modifying enzymes include amino acid sequences contained in or referenced in or encoded by nucleic acid sequences contained or referenced in any of the following US patents: 6,610,527; 6,451,576; 6,429,014; 6,342,380; 6,265,639; 6,194,185; 6,114,160; 6,083,731; 6,043,072; 5,994,114; 5,891,697; 5,871,988; 6,265,639 and further described in Gen Bank Accession Numbers: AAO18435; ZP 00513891; Q38710; AAK60613, AA60610; AAK60611; NP 113747; CAB75874; AAK60612; AAF20201; BAA11024; AF20591 and CAA03710.
Other enzymes suitable for use with the microbes and methods of the invention include those that have at least 70% amino acid identity to one of the proteins listed in Table 5-8 and that exhibit the corresponding desired enzyme activity (for example , cleavage of a fatty acid from an acyl carrier protein, reduction of an acyl-CoA to an aldehyde or an alcohol, or conversion of an aldehyde to an alkane). In additional embodiments, the enzyme activity is present in a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or by at least about 99% identity to one of the sequences described above, all of which are incorporated herein by reference as if fully summarized.
The above-described hydrocarbon-modifying enzymes are useful in the production of various hydrocarbons from a microbe (for example, a microalga, oily yeast, or fungus) or population of microbes, whereby a fatty acyl-ACP thioesterase cleaves a fatty acyl-ACP thioesterase. fatty acid from an acyl carrier protein (ACP) during lipid synthesis. Through further enzymatic treatment, the cleaved fatty acid is then combined with a co-enzyme to produce an acyl-CoA molecule. This acyl-CoA is the substrate for the enzymatic activity of a fatty acyl-CoA reductase to produce an aldehyde, as well as for a fatty acyl-CoA/aldehyde reductase to produce an alcohol. The aldehyde produced by the action of fatty acyl-CoA reductase identified above is the substrate for further enzyme activity by either a fatty aldehyde reductase to produce an alcohol or a fatty aldehyde decarbonylase to produce an alkane or alkene.
Hydrocarbon modification enzymes have specificity to act on a substrate that includes a specific number of carbon atoms. For example, a fatty acyl-ACP thioesterase may have specificity for cleaving a fatty acid having 12 carbon atoms from ACP. Thus, in various embodiments, the microbe may contain an exogenous gene encoding a protein with specificity for satellite enzymatic activity (for example, cleavage of a fatty acid from ACP, reduction of acyl-CoA to an aldehyde or alcohol). or conversion of an aldehyde to an alkane) with respect to the number of carbon atoms contained in the substrate. The enzyme specificity may be in various embodiments for a substrate having 8 to 34 carbon atoms, preferably 8 to 14 carbon atoms and more preferably 10 to 14 carbon atoms. The most preferred specificity is for a substrate having 12 carbon atoms. In other embodiments, the specificity may be for 20 to 30 carbon atoms.
In some embodiments, fatty acids or the corresponding primary alcohols, aldehydes, alkanes, or alkenes generated by the methods described herein contain at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 32, or at least about 34 or more carbon atoms. Preferred fatty acids for the production of biodiesel, renewable diesel or jet fuel or the corresponding primary alcohols, aldehydes, alkanes and alkenes for industrial applications contain at least about 8 carbon atoms or more. In certain embodiments, the above fatty acids, as well as the other corresponding hydrocarbon molecules are saturated (without any carbon-carbon double or triple bonds); monounsaturated (single double bond); polyunsaturated (two or more double bonds); they are linear (non-cyclic) and/or have little or no branching in their structures.
By selecting the desired combination of foreign genes to be expressed, the product generated by the microbe can be made, which can then be extracted from the aqueous biomass. For example, the microbe may contain: i) an exogenous gene encoding a fatty acyl-ACP thioesterase and optionally ii) a naturally co-expressed acyl carrier protein or an acyl carrier protein that otherwise has fatty acyl-ACP thioesterase activity (or vice versa) and optionally iii) an exogenous gene encoding a fatty acyl-CoA/aldehyde reductase or fatty acyl-CoA reductase and optionally iv) an exogenous gene encoding a fatty aldehyde reductase or fatty aldehyde decarbonylase. The microbe, when cultured as described hereinafter, synthesizes a fatty acid bound to an ACP and fatty acyl-ACP thioesterase catalyzes the cleavage of the fatty acid from the ACP to produce, through further enzymatic processing, a molecule of fatty acyl-CoA. When present, acyl-CoA/fatty aldehyde reductase catalyzes the reduction of acyl-CoA to an alcohol. Similarly, fatty acyl-CoA reductase, when present, catalyzes the reduction of acyl-CoA to an aldehyde. In those embodiments in which an exogenous gene encoding a fatty acyl-CoA reductase is present and is expressed to produce an aldehyde product, a fatty aldehyde reductase, encoded by the third exogenous gene, catalyzes the reduction of the aldehyde to an alcohol. Similarly, a fatty aldehyde decarbonylase catalyzes the compression of the aldehyde to an alkane or an alkene, when present.
Genes encoding such enzymes can be obtained from cells already known to exhibit significant lipid production such as Chlorella protothecoides. Genes already known to have a role in lipid production, eg a gene encoding an enzyme that saturates double bonds, can be individually transformed into recipient cells. However, for the practice of the invention it is not necessary to make a priori assumptions as to which genes are required. A DNA library containing different genes, such as cDNA from a good lipid-producing organism, can be transformed into recipient cells. The cDNA is preferably in operable linkage with a promoter active in microalgae. Different recipient microalgae cells transformed by a library receive different genes from the library. Transformants having enhanced lipid production are identified by means of selection methods known in the art, such as for example HPLC acid, gas chromatography and hydrocarbon analysis mass spectrometry methods (for examples of such analyses, see Biomass and Bioenergy Vol 6. No. 4. pp. 269-274 (1994), Experientia 38, 47-49 (1982), and Phytochemistry 65 (2004) 3159-3165). These transformants are then subjected to further transformation with the original library and/or optionally cross-crossed to generate a further round of organisms having enhanced lipid production. General procedures for growing whole organisms to acquire a desired property are described in for example US Patent 6,716,631. Such methods encompass, for example, introducing a library of DNA fragments into a plurality of cells, whereby at least one of the fragments undergoes recombination with a segment in the cells' genome or episome to produce modified cells. The modified cells are then selected for modified cells that have evolved to acquire the desired function. Vectors and methods for transformation are analogous to those discussed in connection with expression of lipase genes.
In addition, subtractive libraries can be used to identify genes whose transcription is induced under different conditions, especially conditions employed in the cultivation of microorganisms for the production of biodiesel or for the production of hydrocarbons useful as a feedstock for industrial applications. Subtractive libraries contain nucleotide sequences that reflect the difference between two different samples. Such libraries are prepared by procedures that include the steps of denaturing and hybridizing populations of nucleotides (eg mRNA, cDNA, amplified sequences) from each sample. Sequences common to both samples are hybridized and removed, leaving sequences that differ between samples. In this way, sequences that are induced under particular conditions can be identified. This technique can be used, for example, to identify genes useful for increasing lipid (eg fatty acid) production and in particular lipid production under any desired culture conditions. The subtractive hybridization technique can also be used to identify promoters, eg, inducible promoters useful in the expression of constructs according to the invention.
Thus, for example, subtractive libraries can be prepared from cultures of microorganisms grown autotropically (in the light without a fixed carbon source) or heterotropically (in the dark in the presence of a fixed carbon source). In particular, heterotrophic genes can be induced during dark culture in the presence of a fixed carbon source and can therefore be present in an accelerated library by subtracting autotrophic cell sequences from dark heterotrophic cell sequences. Subtractive libraries can also be prepared from cultures to which a particular carbon substrate, such as glucose, has been added to identify genes that play a role in metabolizing the substrate. Subtractive libraries prepared from cultures grown in the presence of counterlimited excess nitrogen can be used to identify genes that control cell division as opposed to producing hydrocarbon accumulation. Preparation of a subtractive library from a culture to which lipids (eg, fatty acids) have been added can help identify genes whose overexpression implements fatty acid production. More specifically, the addition of fatty acids to a culture of cells that can use the added fatty acids will lead to the down-regulation of fatty acid synthetic genes to down-regulate fatty acid production. Overexpression of one or more of such genes will have the opposite effect.
B. Increased Carbon Flux to the Lipid Pathway.
Some microalgae produce significant amounts of non-lipid metabolites, such as, for example, polysaccharides. Because polysaccharide biosynthesis can use a significant proportion of the total metabolic energy available to cells, mutagenesis of lipid-producing cells followed by selection for reduced or eliminated polysaccharide production generates new strains that are capable of producing high yields. higher in lipids.
The phenol:sulfuric acid assay detects carbohydrates (see, Hellebust, Handbook of Phycological Methods, Cambridge University Press, 1978; and Cuesta G., et al., J Microbiol Methods. 2003 Jan;52(l):69-73) . The 1,6-dimethylmethylene blue assay detects anionic polysaccharides, (see for example Braz J Med Biol Res. 1999 May;32(5):545-50; Clin Chem. 1986 Nov;32(l 1):2073 -6).
Polysaccharides can also be analyzed by methods such as HPLC, size exclusion chromatography and anion exchange chromatography (see, for example Prosky L, Asp N, Schweizer TF, DeVries JW & Furda I (1988). Determination of insoluble , soluble and total dietary fiber in food and food products: Interlaboratory study. Journal of the Association of Official Analytical Chemists 71, 1017+1023; Int J Biol Macromol. 2003 Nov;33(l-3):9-18). Polysaccharides can also be detected using gel electrophoresis (see, for example Anal Biochem. 2003 Oct 15;321(2):174-82; Anal Biochem. 2002 Jan 1;300(l) :53-68).
SAW. LIPID AND HYDROCARBON RECOVERY METHODS
Hydrocarbons (eg, lipids, fatty acids, aldehydes, alcohols, and alkanes) produced by the cell of the invention may be harvested or otherwise collected by any convenient means. For example, hydrocarbons secreted from cells can be centrifuged to separate hydrocarbons in a hydrophobic layer from contaminants in an aqueous layer and optionally from any solid materials as a precipitate after centrifugation. Material containing cells or cell fractions can be treated with proteases to degrade contaminating proteins before or after centrifugation. In some instances the contaminating proteins are associated, possibly covalently, with hydrocarbons or hydrocarbon precursors that form hydrocarbons after protein removal. In other instances, the hydrocarbon molecules are in a preparation that also contains protein. Proteases can be added to protein-containing hydrocarbon preparations to degrade proteins (for example, the protease from Streptomyces griseus can be used (SigmaAldrich catalog number P5147)). After digestion, the hydrocarbons are preferably purified from residual proteins, peptide fragments and amino acids. This purification can be carried out, for example by methods listed above, such as centrifugation and filtration.
Extracellular hydrocarbons can also be extracted in vivo from living microalgae cells which are then returned to a bioreactor by exposing the cells in an otherwise sterile environment to a non-toxic extraction solvent, followed by separation of the living cells. and the hydrophobic fraction of the extraction solvent and hydrocarbons, where the separated living cells are then returned to a culture vessel, such as a stainless steel proofer or photobioreactor (see, Biotechnol Bioeng. 2004 Dec 5;88(5):593-600 and Biotechnol Bioeng. 2004 Mar 5;85(5):475-81).
Hydrocarbons can also be isolated by whole cell extraction. Cells are first subjected to disruption, as described in the section entitled "Cell Lysis" and then intracellular cell membrane/cell wall associated hydrocarbons as well as extracellular hydrocarbons can be harvested from the entire cell mass such as by using centrifugation as described above.
Several methods are available to separate hydrocarbons and lipids from cell lysates produced by the above methods. For example, the hydrocarbons can be extracted with a hydrophobic solvent such as hexane (see, for example, Frenz et al. 1989, Enzyme Microb. Technol., 11 :717). Hydrocarbons can also be extracted using liquefaction (see, for example, Sawayama et al. 1999, Biomass and Bioenergy 17:33-39 and Inoue et al. 1993, Biomass Bioenergy 6(4):269274); oil liquefaction (see, for example, Minowa et al. 1995, Fuel 74(12):1735-1738); and CO removal<sub>2</sub> supercritical (see, for example Mendes et al. 2003, Inorganica Chimica Acta 356:328-334).
Miao and Wu describe a protocol for microalgal lipid recovery from a Chlorella prototheocoides culture in which cells were harvested by centrifugation, washed with distilled water, and freeze-dried. The resulting cell powder was pulverized in a mortar and then extracted with n-hexane. Miao and Wu, Biosource Technology (2006) 97:841-846.
A. Lysis of Cells
Intracellular lipids and hydrocarbons produced in microorganisms are in some embodiments extracted after lysis of the cells of the microorganism. Once extracted, the lipids and/or hydrocarbons can be further refined to produce oils, fuels, or oleochemicals.
After the completion of the culture, the microorganisms can be separated from the fermentation broth. Optionally, the separation is effected by centrifugation to generate a concentrated paste. Centrifugation does not remove significant amounts of intracellular water from microorganisms and is not a drying step. The biomass can then be washed with a washing solution (eg DI water) to get rid of fermentation broth and debris. Optionally, the washed microbial biomass can also be dried (oven-dried, freeze-dried, etc.) prior to cell disruption. Alternatively, cells can be lysed without removal of some or all of the fermentation broth when fermentation is complete. For example, the cells may be at less than a 1:1 v:v ratio of cells to extracellular fluid when the cells are lysed.
Microorganisms containing a lipid and/or hydrocarbon can be lysed to produce a lysate. As detailed herein, the step of lysing a microorganism (also referred to as cell lysis) can be accomplished by any convenient means, including heat-induced lysis, addition of base, addition of acid, using enzymes such as proteases and polysaccharide degradation enzymes such as amylases, using ultrasound, mechanical lysis, using osmotic shock, infection with a lytic virus and/or expression of one or more Utic genes. Lysis is performed to release intracellular molecules that have been produced by the microorganism. Each of these methods for the lysis of a microorganism can be used as a single method or in combination simultaneously or sequentially.
The extent of cell disruption can be observed by microscopic analysis. Using one or more of the methods described herein, commonly greater than 70% cell disruption is observed. Preferably cell disruption is more than 80%, more preferably more than 90% and most preferably around 100%.
In particular embodiments, the microorganism is lysed after cultivation, for example to increase exposure of the cellular lipid and/or hydrocarbon for extraction or further processing. Timing of lipase expression (eg via an inducible promoter) or cell lysis can be adjusted to optimize lipid and/or hydrocarbon yield. A number of lysis techniques are described below. These techniques can be used individually or in combination.
one. Thermally induced lysis
In a preferred embodiment of the present invention, the step of lysing a microorganism comprises heating a cell suspension containing the microorganism. In this mode, the fermentation broth containing the microorganisms (or a suspension of microorganisms isolated from the fermentation broth) is heated until the microorganisms, that is, the cell membranes and cell walls of the microorganisms are degraded or ruptured. Commonly, applied temperatures are at least 50°C. Higher temperatures, such as, at least 30°C, at least 60°C, at least 70°C. at least 80°C, at least 90°C, at least 100°C, at least 110°C, at least 120°C, at least 130°C or higher are used for the most efficient cell lysis .
Cell lysis by heat treatment can be effected by boiling the microorganism. Alternatively, heat treatment (without boiling) can be carried out in an autoclave. The heat treated lysate can then be cooled for further treatment.
Cell disruption can also be effected by saturated steam treatment, that is, by means of the addition of pressurized steam. Saturated steam treatment of microalgae for cell disruption is described for example in US Patent 6,750,048.
2. Lysis Using a Base
In another preferred embodiment of the present invention, the step of lysing a microorganism comprises adding a base to a cell suspension containing the microorganism.
The base must be strong enough to hydrolyze at least a portion of the proteinaceous compounds of the microorganisms used. Bases that are useful for solubilizing proteins are known in the art of chemistry. Exemplary bases that are useful in the methods of the present invention include but are not limited to hydroxides, carbonates and bicarbonates of lithium, sodium, potassium, calcium and mixtures thereof. A preferred base is KOH. Microalgae-based treatment for cell disruption is described for example in US Patent 6,750,048.
3. Acid Lysis
In another preferred embodiment of the present invention, the step of lysing a microorganism comprises adding an acid to a cell suspension containing the microorganism. Acid lysis can be performed using an acid at a concentration of 10-500 nN or preferably 40-160 nM. Acid lysis is preferably carried out at a temperature higher than room temperature (for example, at 40-160Ό and preferably at a temperature of 50-130Ό). For moderate temperatures (eg, room temperature to 100C and particularly room temperature to 65°C), acid treatment can usefully be combined with sonication or other methods of cell disruption.
Four. Cell Lysis Using Enzymes
In another preferred embodiment of the present invention, the step of lysing a microorganism comprises lysing the microorganism using an enzyme. Preferred enzymes for lysing a microorganism are proteases and polysaccharide-degrading enzymes such as hemicellulase (eg, hemicellulase from Aspergillus niger; Sigma Aldrich, St. Louis, MO; #H2125), pectinase, (eg, pectinase from Rhizopus sp. .; Sigma Aldrich, St. Louis, MO; #P2401), Mannaway 4.0 L (Novozymes), cellulase (for example, cellulose from Trichoderma viride; Sigma Aldrich, St. Louis, MO; #09422), and driselase (for example, driselase from Basidiomycetes sp.; Sigma Aldrich, St. Louis, MO; #D9515.
a) Cellulase
In a preferred embodiment of the present invention, a cellulase for lysis of a microorganism is a polysaccharide-degrading enzyme, optionally from Chlorella or a Chlorella virus.
b) Proteases
Proteases such as Streptomysis grisis protease, chymotrypsin, proteinase K, proteases listed in Degradation of Polylactide by Commercial Proteases, Oda Yet al., Journal of Polymers and the Environment, Volume 8, Number 1, January 2000, pp. 29-32(4), and other proteases can be used to lyse microorganisms. Other proteases that can be used include Alcalase 2.4 FG (Novozymes) and Flavourzyme 100 L (Novozymes).
c) Combinations
Any combination of a protease and a polysaccharide-degrading enzyme may also be used, including any combination of the foregoing proteases and polysaccharide-degrading enzymes.
5. Cell Lysis Using Ultrasound
In another preferred embodiment of the present invention, the step of lysing a microorganism is carried out using ultrasound, that is, sonification. Thus, cells can also be lysed with high frequency sound. Sound can be produced electronically and transported through a metal junction to an appropriately concentrated cell suspension. This sonication (or ultrasonication) disrupts cell integrity based on the creation of cavities in the cell suspension.
6. mechanical lysis
In another preferred embodiment of the present invention, the microorganism lysis step is performed by mechanical lysis. Cells can be mechanically lysed and optionally homogenized to facilitate collection of hydrocarbons (eg, lipids). For example, a pressure disruptor device can be used to pump a cell-containing suspension through a restricted orifice valve. High pressure (up to 1500 bar) is applied followed by instant expansion through an outlet nozzle. Cell disruption is accomplished by three different mechanisms: shock on the valve, high liquid shear in the orifice, and sudden pressure drop at the discharge, causing cell explosion. The method releases intracellular molecules.
Alternatively, a ball mill can be used. In a Ball Mill, cells are agitated into suspension with small abrasive particles, such as beads. Cells rupture due to shear forces, grinding between beads, and collisions with beads. The beads disrupt cells to release cell contents. Cells can also be subjected to disruption by shear forces, such as with the use of mixing (such as with a high-speed mixer or Waring mixer as examples), the French press, or even centrifugation in the case of weak cell walls to subdue to disruption the cells.
7. Cell lysis by osmotic shock (cytolysis)
In another preferred embodiment of the present invention, the microorganism lysis step is carried out by applying an osmotic shock.
8. Infection with a lytic virus
In a preferred embodiment of the present invention, the step of lysing a microorganism comprises infection of the microorganism with a lytic virus. A wide variety of viruses are known to lyse microorganisms suitable for use in the present invention and the selection and use of a particular lytic virus for a particular microorganism is within the skill level of those skilled in the art.
For example, Paramecium bursaria chlorella virus (PBCV-1) is the prototype of a group of large, eicosahedral, plaque-forming, double-stranded DNA viruses (Family Phyconaviridae, genus Chlorovirus) that replicate in and lyse certain Unicellular eukaryotic Chlorella-like green algae. Thus, any susceptible microalgae can be lysed by infecting the culture with an appropriate Chlorella virus. Methods for infecting Chlorella species with a chlorella virus are known. See, for example, Adv. Trusted viruses 2006: 293-336; Virology, 1999 Apr 25, 257(1): 15-23; Virology, 2004 Jan 5, 318(1): 214-23; Nucleic Acids Symp. Ser. 2000(44): 161-2; J. Virol., March 2006, 80(5): 2437-44 and Annu. fiev. Microbiol. 1999, 53:447-94.
9. Autolysis (expression of a lytic gene)
In another preferred embodiment of the present invention, the step of lysing a microorganism comprises autolysis. In this embodiment, a microorganism according to the invention is genetically engineered to produce a lytic protein that will lyse the microorganism. This lytic gene can be expressed using an inducible promoter, such that cells can first be grown to a desirable density in a fermenter, followed by induction of the promoter to express the lytic gene to lyse the cells. In one embodiment, the lytic gene encodes a polysaccharide-degrading enzyme.
In certain other embodiments, the lytic gene is a gene from a lytic virus. Thus, for example, a lytic gene from a Chlorella virus can be expressed in a cell of algae of the genus Chlorella such as C. protothecoides.
Appropriate expression methods are described herein with respect to expression of a lipase gene. Expression of lytic genes is preferably done using an inducible promoter, such as a promoter active in microalgae that is induced by a stimulus such as the presence of a small molecule, light, heat, and other stimuli. Chlorella virus lytic genes are known. For example, see Virology 263, 376-387 (1999) and Virology 230, 361-368 (1997).
B. Extraction of lipids and hydrocarbons
The lipids and hydrocarbons generated by the microorganisms of the present invention can be recovered by extraction with an organic solvent. In some cases, the preferred organic solvent is hexane. Commonly, the organic solvent is added directly to the lysate without prior separation of the lysate components. In one embodiment the lysate generated by one or more of the methods described above is contacted with an organic solvent for a period of time sufficient to allow the lipid and/or hydrocarbon components to form a solution with the organic solvent. In some cases, the solution can then be further refined to recover specific desired hydrocarbon or lipid components. Hexane extraction methods are well known in the art.
Vile. METHODS FOR THE PROCESSING OF LIPIDS AND HYDROCARBONS
A. Enzyme modification
Hydrocarbons (for example, lipids, fatty acids, aldehydes, alcohols, and alkanes) produced by cells as described herein can be modified through the use of one or more enzymes, including a lipase, as described previously. When hydrocarbons are in the extracellular environment of the cell, the one or more enzymes can be added to that environment under conditions in which the enzyme modifies the hydrocarbon or completes its synthesis of a hydrocarbon precursor. Alternatively, the hydrocarbons may be partially or completely isolated from the cellular material prior to the addition of one or more catalysts such as enzymes. Such catalysts are added exogenously and their activity occurs outside the cell or in vitro.
B. Thermal Modification and Other Catalytic Modification
Hydrocarbons produced by cells in vivo or enzymatically modified in vitro, as described herein, can optionally be further processed by conventional means. Processing may include "catalytic pyrolysis" to reduce the size and thus increase the hydrogen:carbon composition of hydrocarbon molecules. Catalytic and thermal pyrolysis methods are systematically used in the processing of hydrocarbons and tungsten oil. Catalytic methods involve the use of a catalyst, such as a solid acid catalyst. The catalyst can be silicaalumina or a zeolite, which results in heterolytic or asymmetric cleavage of a carbon-carbon bond to result in a carbocation and a hydride anion. These active intermediates then undergo either rearrangement or hydride transfer with another hydrocarbon. The reactions can thus regenerate the intermediates to result in a self-propagating chain mechanism. The hydrocarbons can also be processed to reduce, optionally to zero, the number of carbon-carbon double, triple bonds therein. Hydrocarbons can also be processed to remove or eliminate a ring or cyclic structure in them. Hydrocarbons can also be processed to increase the hydrogen:carbon ratio. This may include the addition of hydrogen (“hydrogenation”) and/or the “catalytic pyrolysis” of hydrocarbons to smaller hydrocarbons.
Thermal methods involve the use of elevated temperature and pressure to reduce the size of hydrocarbons. An elevated temperature of about 800 can be used.<sup>and</sup> C and a pressure of approximately 700 kPa. These conditions generate "light," a term that is sometimes used to refer to hydrogen-rich hydrocarbon molecules (as distinguished from photon flux), while they also generate, through condensation, heavy hydrocarbon molecules that are relatively depleted of hydrogen. The methodology provides for homolytic or symmetrical cleavage and produces alkenes that can optionally be enzymatically saturated as described above.
The catalytic and thermal methods are conventional in plants for the processing of hydrocarbons and oil refining. Thus, hydrocarbons produced by cells as described herein can be collected and processed for refining through conventional means. See Hillen et al. (Biotechnology and Bioengineering, Vol. XXIV: 193-205 (1982)) for a report on hydrocracking of hydrocarbons produced by microalgae. In alternative embodiments, the fraction is treated with another catalyst, such as an organic compound, heat, and/or an inorganic compound. For the processing of lipids to biodiesel, a transesterification process is used as described in Section IV herein.
The hydrocarbons produced via the methods of the present invention are useful in a variety of industrial applications. For example, the production of linear alkyl benzene sulfonate (LAS), an anionic surfactant used in almost all types of detergents and cleaning preparations, uses hydrocarbons generally comprising a chain of 10-14 carbon atoms. See, for example, United States Patent Nos. 6,496,430; 5,506,201; 6,692,730; 6,268,517; 6,020,509; 6,140,302; 5,080,848 and 5,567,359. Surfactants, such as LAS, can be used in the manufacture of personal care compositions and detergents, such as those described in US Patent Nos. 5,942,479; 6,086,903; 5,833,955 and 6,407,044.
VIII. METHODS FOR PRODUCING FUELS SUITABLE FOR USE IN DIESEL VEHICLES AND JET ENGINES
Increased interest is directed at the use of bio-based hydrocarbon components in fuel, such as biodiesel, renewable diesel, and jet fuel, since renewable bio-based starting materials that can replace fossil fuel-derived starting materials are available and their use is desirable. There is an urgent need for methods to produce hydrocarbon components from biological materials. The present invention satisfies this need by providing methods for compounding biodiesel, renewable diesel, and jet fuel using the lipids generated by the methods described herein as biological material for producing biodiesel, renewable diesel, and jet fuel.
Traditional diesel fuels are petroleum distillates rich in paraffinic hydrocarbons. They have boiling ranges as wide as 188-C (370<sup>5</sup> F) to 415.5<sup>5</sup> C(780<sup>5</sup> F), which are suitable for combustion in a compression inhibited engine, such as a diesel motor vehicle. The American Society for Testing and Materials (ASTM) establishes diesel grade according to boiling range, along with allowable ranges for other fuel properties, such as cetane number, cloud point, boiling temperature, etc. flash, viscosity, aniline point, sulfur content, water content, ash content, copper band corrosion, and carbon residues. Technically, any hydrocarbon distillate material derived from biomass or otherwise meeting the appropriate ASTM specification can be defined as diesel fuel (ASTM D975), jet fuel (ASTM D1655), or biodiesel (ASTM D6751).
After extraction, the lipid and/or hydrocarbon components recovered from the microbial biomass described herein can be subjected to chemical treatment for the manufacture of a fuel for use in diesel vehicles and jet engines.
A. Biodiesel
Biodiesel is a liquid that varies in color - between golden and dark brown - depending on the production feedstock. It is practically immiscible with water, has a high boiling point and a low vapor pressure. Biodiesel refers to a processed fuel equivalent to diesel for use in diesel motor vehicles. Biodiesel is biodegradable and non-toxic. An additional benefit of biodiesel over conventional diesel fuel is lower engine wear.
Biodiesel commonly comprises alkyl esters of C<sub>14</sub>-C<sub>18</sub>. Such processes convert biomass or a lipid produced and isolated as described herein to diesel fuels. A preferred method of producing biodiesel is by transesterification of a lipid as described herein. A preferred alkyl ester for use as biodiesel is a methyl ester or ethyl ester.
Biodiesel produced by a method described herein can be used alone or combined with conventional diesel fuel at any concentration in most modern diesel motor vehicles. When combined with conventional diesel fuel (petroleum diesel), biodiesel can be present from about 0.1% to about 99.9%. Worldwide, a system known as the “B” factor is used to state the amount of biodiesel in any fuel blend. For example, fuel that contains 20% biodiesel is labeled D20. Pure biodiesel is called B100.
Biodiesel can also be used as a heating fuel in home and commercial heaters. Existing oil heaters or boilers may contain rubber or rubber parts and may require conversion to run on biodiesel. The conversion process is usually relatively simple, involving exchanging rubber parts for synthetic parts because biodiesel is a strong solvent. Due to its strong solvent power, burning biodiesel will increase the efficiency of boilers or heaters.
Biodiesel can be used as an additive in diesel buildups to increase the lubricity of pure ultra-low sulfur diesel (ULSD) fuel, which is advantageous because it has virtually no sulfur content.
Biodiesel is a better solvent than petrodiesel and can be used to break up residue deposits in fuel lines of vehicles that have previously been put into operation with petrodiesel.
one. biodiesel production
Biodiesel can be produced by transesterification of triglycerides contained in oil-rich biomass. Thus, in another aspect of the present invention, a method for producing biodiesel is provided. In a preferred embodiment, the method for producing biodiesel comprises the steps of: (a) culturing a lipid-containing microorganism using the methods disclosed herein; (b) lysing a lipid-containing microorganism to produce a lysate; (c) isolating the lipid from the lysed microorganism and (d) transesterifying the lipid composition, whereby biodiesel is produced.
Methods for culturing a microorganism, lysing a microorganism to produce a Used, treating the Used in a medium comprising an organic solvent to form a heterogeneous mixture, and recovering the treated lysate to a lipid composition have been described above and can be also be used in the method to produce biodiesel.
The lipid compositions can be subjected to transesterification to produce long chain fatty acid esters useful as biodiesel. Preferred transesterification reactions are outlined below and include base-catalyzed transesterification and transesterification using recombinant lipases.
In a base-catalyzed transesterification process, triacylglycerides are reacted with an alcohol, such as methanol or ethanol, in the presence of an alkaline catalyst, commonly potassium hydroxide. This reaction forms methyl or ethyl esters or glycerin (glycerol) as a by-product.
a) General chemical process
Animal and plant oils are commonly composed of triglycerides which are esters of free fatty acids with vinyl alcohol, glycerol. In transesterification, the glycerol in a triacylglyceride (TAG) is replaced with a short chain alcohol such as methanol or ethanol. A typical reaction scheme is as follows:
—0---OCR-i base catalyst
-----------fyCOOEt + R<sub>2</sub>COOEt + R<sub>3</sub>COOEt + C<sub>3</sub>h<sub>5</sub>(oh)<sub>3</sub>
EtOH<sub>r</sub>.„
Fatty acid ethyl esters Glycerol —o—ocr<sub>2</sub> —o—ocr<sub>3 </sub>triglyceride
In this reaction scheme, the alcohol is deprotonated with a base to make it a stronger nucleophile. Commonly, ethanol or methanol is used in vast excess (up to 50 times). Normally, this reaction will proceed either excessively slowly or not. Heat, as well as an acid or base, can be used to help the reaction proceed more quickly. The acid or base are not consumed by the transesterification reaction, thus they are not reactants but catalysts. Almost all biodiesel has been produced using the base-catalyzed technique since it requires only lowering temperatures and pressures and produces more than 98% conversion yield (provided the starting agent is low in moisture and free fatty acids).
b. Use of recombinant lipases
Transesterification can also be carried out experimentally using an enzyme, such as a lipase instead of a base. The lipase-catalyzed transesterification can be carried out, for example, at a temperature between room temperature and 80<sup>yes</sup> C and a molar ratio of TAG to lower alcohol greater than 1:1, preferably around 3:1.
Suitable lipases for use in transesterification include but are not limited to those listed in Table 9. Other examples of useful lipases for transesterification are found, for example, in US Patent Nos. 4,798,793; 4,940,845; 5,156,963; 5,342,768; 5,776,741 and WO 89/01032.
Table 9. Lipases suitable for use in transesterification.
Lipase. ABG73614 from Aspergillus niger, lipase B CAA83122 from
Candida antarctica (novozym-435), AAR24090 lipase from Candida cylindracea, lipase from Candida lipolytica (Lipasa L; Amano Pharmaceutical Co., Ltd.), lipase from Candida rugosa (e.g., Lipase-OF; Meito Sangyo Co., Ltd. ), Mucor mieheik lipase (Lipozyme IM 20), Pseudomonas fluorescens lipase AAA25882, Rhizopus japonicus lipase AAAF32408 (Lipo
F), lipase ABI13521 from Serratia marcescens (Enzyme SM), lipase CAB58509 from Thermomyces lanuginosa, Lipase P (Nagase
ChemteX Corporation) and Lipase QLM (Meito Sangyo Co., Ltd., Nagoya, Japan)
A challenge to using a lipase for the production of suitable fatty acid esters for biodiesel is that the price of lipase is much higher than the price of sodium hydroxide (NaOH) used by the strong base process. This challenge has been addressed by using an immobilized lipase, which can be recycled. However, the immobilized lipase activity must be maintained after recycling for a minimum of cycles to allow a lipase-based process to compete with the strong-base process in terms of production cost. Immobilized lipases are subjected to poisoning by the lower alcohols commonly used in transesterification. US Patent No. 6,398,707 (issued June 4, 2002 to Wu et al. ) describes methods for enhancing the activity of immobilized lipases and regenerating immobilized lipases having reduced activity.
In particular embodiments, a recombinant lipase is expressed in the same lipid-producing microorganisms on which the lipase acts. Appropriate recombinant lipases include those listed above in Table 9 and/or having GenBank accession numbers listed above in Table 9 or a polypeptide that has at least 70% amino acid identity to one of the lipases listed above and in Table 9 and exhibiting lipase activity. In further embodiments, the enzyme activity is present in a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, for at least about 99% identity to one of the sequences described above, all of which are incorporated by reference herein as if fully summarized. The DNA encoding the lipase and selectable marker is preferably codon-optimized cDNA. Methods for recoding genes for expression in microalgae are described in US Patent No. 7,135,290.
2. standards
The common international standard for biodiesel is EN 14214. ASTM D6751 is the most common biodiesel standard referenced in the United States of America and Canada.
Germany uses DIN EN 14214 and the United Kingdom of Great Britain requires compliance with BS EN 14214.
Basic industry tests to determine if products conform to these standards commonly include gas chromatography, HPLC, and others. Biodiesel that meets quality standards is highly non-toxic, with a Nominal Toxicity or Toxicity Classification (LD<sub>50</sub>) greater than 50 mL/Kg.
B. Renewable diesel
Renewable diesel comprises alkanes, such as C16:0 and C18:0, and thus are distinguishable from biodiesel. The high-quality renewable diesel conforms to the standard of ASTM D975.
The lipids produced by the methods of the present invention can serve as the raw material of the invention to produce renewable diesel. Thus, in another aspect of the present invention, a method for producing renewable diesel is provided. Renewable diesel can be produced by at least three processes: hydrothermal processing (hydrotreating); hydroprocessing and indirect liquefaction. These processes produce distillates without ester. During these processes, the triacylglycerides produced and isolated as described herein are converted to alkanes.
In a preferred embodiment, the method for producing renewable diesel comprises a) culturing a lipid-containing microorganism using methods recommended herein; b) lysing the microorganism to produce a lysate; c) isolating the lipid from the lysed microorganism and d) deoxygenating and hydrotreating the lipid to produce an alkane, whereby renewable diesel is produced. Lipids suitable for the manufacture of renewable diesel can be obtained via extraction of microbial biomass using an organic solvent such as hexane or via other methods, such as those described in US patent 5,928,696.
In some methods the microbial lipid is first subjected to catalytic pyrolysis in conjunction with hydrotreating to reduce the carbon chain length and saturate the double bonds, respectively. The material is then isomerized, also in conjunction with hydrotreating. The naphtha fraction can then be removed by distillation, followed by further distillation to vaporize and distill desired components in the diesel fuel to meet the D975 standard while leaving components that are heavier than desired to meet the standard. D975 standard. Methods of hydrotreating, hydrocracking, deoxygenation and isomerization of chemically modified oils, including triglyceride oils, are well known in the art. See, for example, European patent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1); EP1640437 (A1); EP1681337 (A1); EP1795576 (A1) and US Patents 7, 238, 277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746; 5,885,440; 6,881,873.
one. hydrotreating
In a preferred embodiment of the method for producing renewable diesel, the lipid treatment to produce an alkane is effected by hydrotreating the lipid composition. In hydrothermal processing, biomass is commonly reacted in water at elevated temperature and pressure to form oils and residual solids. Conversion temperatures are commonly 300°F (149Ό) to 660°F (349°C), with sufficient pressure to hold water primarily as a liquid, 100 to 170 standard atmospheres (atm). Reaction times are less than 15 to 30 minutes. After the reaction is complete, the organics are separated from the water. By this, a distillate suitable for diesel is produced.
2. hydroprocessing
A renewable diesel, referred to as "green diesel", can be produced from fatty acids using traditional hydroprocessing technology. Oils containing triglyceride can be hydroprocessed either as a co-feed with oil or as a dedicated feed. The product is a diesel fuel that conforms to ASTM specification D975. Thus, in another preferred embodiment of the method for producing renewable diesel, treatment of the lipid composition to produce an alkane is effected by hydroprocessing of the lipid composition.
In some methods for making renewable diesel, the first stage of treatment of a triglyceride is hydroprocessing to saturate double bonds, followed by deoxygenation at elevated temperature in the presence of hydrogen and a catalyst. In some methods, hydrogenation and deoxygenation occur in the same reaction. In other methods, deoxygenation occurs before hydrogenation. The isomerization is then optionally carried out, also in the presence of hydrogen and a catalyst. The naphtha components are preferably separated by means of distillation. For example, see US Patents 5,475,160 (hydrogenation of triglycerides); 5,091,116 (deoxygenation, hydrogenation and gas removal); 6,391,815 (hydrogenation) and 5,888,947 (isomerization).
Petroleum refiners use old processing to remove impurities by treating feeds with hydrogen. Hydroprocessing conversion temperatures are commonly 300°F (149°C) to 700°F (361°C). Pressures are commonly 40 to 100 atmospheres. Reaction times are commonly on the order of 10 to 60 minutes.
Solid catalysts are used to increase certain reaction rates, improve effectiveness for certain products, and optimize hydrogen consumption.
Hydrotreating and hydroprocessing ultimately lead to a reduction in the molecular weight of the feed. In the case of triglyceride-containing oils, the triglyceride molecule is reduced to four hydrocarbon molecules under hydroprocessing conditions: one propane molecule and three heavier hydrocarbon molecules, commonly in the C range.<sub>8</sub> to Ci<sub>8</sub>.
3. indirect liquefaction
A traditional ultra-low sulfur diesel can be produced from any form of biomass using a two-stage process. First, the biomass is converted to a synthetic gas, a gaseous mixture rich in hydrogen and carbon monoxide. The synthetic gas or synthesis gas is then catalytically converted to liquids. Commonly, the production of liquids is carried out using Fischer-Tropsch (FT) synthesis. This technology applies to carbon, natural gas and heavy oils. Thus, in yet another preferred embodiment of the method for producing renewable diesel, the treatment of the lipid composition to produce an alkane is effected by indirect liquefaction of the lipid composition.
C. Jet fuel
Annual US jet fuel use in 2006 was approximately 80 billion liters (21 billion gallons). Airplane fuel is light to straw in color. The most common fuel is an A-1 airplane grade unleaded/paraffin oil-based fuel, which is produced to a set of specifications that are internationally indexed. Airplane fuel is a mixture of a large number of different hydrocarbons, possibly as many as a thousand or more. The range of their sizes (molecular weights or carbon numbers) is restricted by product requirements, eg freezing point or smoke point. Kerosene type airplane fuel (including Jet A and Jet A-1) has a carbon number distribution between about 8 and 16 carbon numbers. Wide-cut or naphtha-type airplane fuel (including Jet B) commonly has a carbon number distribution of between about 5 and 15 carbons.
Both airplane fuels (Jet A and Jet B) can contain a number of additives. Useful additives include but are not limited to antioxidants, antistatic agents, corrosion inhibitors, and fuel system icing inhibition (FSII) agents. Antioxidants prevent gumming, and are usually based on alkylated phenols, for example AO-30, AO-31 or AO-37. Antistatic agents dissipate static electricity and prevent the formation of sparks. Stadis 450 with dinonylnaphthylsulfonic acid (DINNSA) as the active ingredient, is an example. Corrosion inhibitors, for example DCI-4A is used for civil and military fuels and DCI-6A is used for military fuels. FSII agents include, for example, Di-EGME.
One solution is to combine algae fuels with existing jet fuels. The present invention provides such a solution. The lipids produced by the methods of the present invention can serve as a limiting feedstock for producing jet fuel. Thus, in another aspect of the present invention, a method for producing jet fuel is provided. Here two methods for producing jet fuel from the lipids produced by the methods of the present invention are provided: fluid catalytic cracking (FCC) and hydrodeoxygenation (HDO).
one. fluid catalytic cracking
Fluid catalytic cracking (FCC) is a method that is used to produce olefins, especially propylene, from heavy crude fractions. There are reports in the literature that vegetable oils such as canola oil could be processed using FCC to give a hydrocarbon stream useful as a gasoline fuel.
The lipids produced by the method of the present invention can be converted to olefins. The process involves flowing the produced lipids through an FCC and locating a product stream consisting of olefins, which is useful as jet fuel. The produced lipids are contacted with a cracking catalyst at cracking conditions to provide a product stream comprising definis and hydrocarbons useful as jet fuel.
In a preferred embodiment, the method for producing jet fuel comprises: a) culturing a lipid-containing microorganism using methods disclosed herein, b) lysing the lipid-containing microorganism to produce a lysate, c) isolating the lipid from the lysate and djtreat lipid composition, whereby jet fuel is produced.
In a preferred embodiment of the method for producing a jet fuel, the lipid composition may be flown through a fluid catalytic cracking zone, in one embodiment, may comprise contacting the lipid composition with a cracking catalyst. to cracking conditions to provide a product stream comprising C<sub>2</sub>-C<sub>5</sub> olefins.
In certain embodiments of this method it may be desirable to remove any contaminants that may be present in the lipid composition. Thus, before flowing the lipid composition through a fluid catalytic cracking zone, the lipid composition is pre-treated. The pretreatment may involve contacting the lipid composition with an ion exchange resin. The ion exchange resin is an acidic ion exchange resin, such as Amberlyst™-15 and can be used as a bed in a reactor through which the lipid composition is flowed, either flow up or flow down. Other pre-treatments can influence mild acid washes by contacting the lipid composition with an acid, such as sulfuric, acetic, nitric or hydrochloric acid. Contact is made with a dilute acid solution, usually at room temperature and atmospheric pressure.
The lipid composition, optionally pre-treated, is flowed to an FCC zone, where the hydrocarbonaceous components are cracked to olefins. Catalytic cracking is carried out by contacting the lipid composition in a reaction zone with a catalyst composed of finely divided particulate material. The reaction is catalytic cracking, as opposed to hydrocracking, and is carried out in the absence of added hydrogen or the consumption of hydrogen. As the cracking reaction proceeds, substantial amounts of coke are deposited on the catalyst. The catalyst is regenerated at high temperature by burning the coked catalyst in a regeneration zone. The coked-containing catalyst, referred to herein as "coked catalyst", is continuously transported from the reaction zone to the regeneration zone to be regenerated and replaced by essentially coked-free regenerated catalyst from the regeneration zone. Fluidization of the catalyst particles by various gas streams allows catalyst transport between the reaction zone and the regeneration zone. Methods for cracking hydrocarbons, such as those of the lipid composition described herein, in a fluidized stream of catalyst, transporting the catalyst between reaction and regeneration zones, and subjecting coked fuel to the regenerator are well known for those experienced in the art of FCC processes. Exemplary FCC applications and useful catalysts for cracking the lipid composition to produce C<sub>2</sub> -C<sub>5</sub> olefins are described in US Pat. Nos. 6,538,169; 7, 288, 685, which are incorporated in their capacity by reference.
In one embodiment, the cracking of the lipid composition of the present invention is matured in the elevator section or alternatively, the elevated section of the FCC zone. The lipid composition is introduced to the elevator via a nozzle resulting in rapid vaporization of the lipid composition. Prior to contacting the catalyst, the lipid composition will ordinarily have a temperature of from about 149°C (300°F) to about 316°C (600°F). The catalyst is flowed from a mixing vessel to the riser, where it is contacted with the lipid composition for a time of approximately two seconds or less.
The mixed catalyst and reacted lipid composition vapors are then discharged from the top of the riser through an outlet and separated into a cracked product vapor stream that includes olefins and a bond of catalyst particles coated with substantial amounts of coked. and generally referred to as "coked catalyst". In an effort to minimize the contact time of the lipid composition and the catalyst that can promote further composition of the desired products to other undesirable products, any arrangement of separators such as a swirl arm arrangement can be used to remove the catalyst with coked from the product stream quickly. The spacer, eg, vortex arm spacer, is located in an upper portion of a chamber with a zone of separation located in the lower portion of the chamber. The catalyst separated by the eddy arm arrangement falls into the separation zone. The cracked product vapor stream comprising cracked hydrocarbons including light olefins and some catalyst exits the chamber via a conduit which is in communication with cyclones. Cyclones remove remaining catalyst particles from the product vapor stream to reduce particle concentrations to very low levels. The product vapor stream then exits from the top of the separation vessel. The catalyst separated by the cyclones is returned to the stripper vessel and then to the stripper zone. The stripping zone removes adsorbed hydrocarbons from the catalyst surface by countercurrent contact with saturated steam.
The low hydrocarbon partial pressure operates to favor the production of light olefin. Thus, the lifter pressure is set to approximately 172 to 241 KPa (25 to 35 pound-force/inch).<sup>2</sup>) with a hydrocarbon partial pressure of approximately 35 to 172 KPa (5 to 25 pound-force/inch<sup>2</sup>), with a preferred partial pressure of about 69 to 138 KPa (10 to 20 pound-force/in<sup>2</sup>). This relatively low partial pressure for hydrocarbons is obtained by using steam as the diluent to the extent that the diluent is from 10 to 55% by weight of the lipid composition and preferably about 15% by weight of the lipid composition. Other diluents such as dry gas can be used to arrive at equivalent hydrocarbon partial pressures.
The temperature of the cracked stream at the riser exit will be approximately 510°C to 621°C (950°F to 1150°F). However, riser exit temperatures greater than 566°C (1050°F) make more dry gas and more olefins. While riser exit temperatures less than 566Ό (1050°F) make less ethylene and propylene. Thus, it is preferred to operate the FCC process at a preferred temperature of about 566Ό to about 630°C, a preferred pressure of about 138 kPa to about 240 kPa (20 to 35 pound-force/in<sup>2</sup> absolute). Another condition for the process is that the ratio of catalyst to lipid composition can vary from about 5 to about 20 and preferably from about 10 to about 15.
In one embodiment of the method for producing a jet fuel, the lipid composition is introduced into the riser section of an FCC reactor. The temperature in the riser section will be very hot and will range from about 700°C (1292°F) to about 760°C (1400°F) with a catalyst to lipid composition ratio of about 100 to about 150. It is anticipated that introducing the lipid composition to the riser section will produce considerable amounts of propylene and ethylene.
The produced liquid hydrocarbon and gas products can be analyzed by gas chromatography, HPLC, etc.
2. Hydrodeoxygenation
In another embodiment of the method for producing a jet fuel using the lipid composition(s) produced as described herein, the structure of the lipid composition(s) is decomposed by a process called hydrodeoxygenation (HDO).
HDO stands for removal of oxygen by means of hydrogen, that is, oxygen is removed while the structure of the material is broken. The olefinic double bonds are hydrogenated and any sulfur and nitrogen compounds are removed. The removal of sulfur is called hydrodesulfurization (HDS). The pre-treatment and purity of the raw materials (lipid composition or lipids) contribute to the service life of the catalyst.
In general in the HDO/HDS stage, hydrogen is mixed with the feedstock (lipid composition or lipids) and then the mixture is passed through a catalytic bed as a co-current flow, since either as a single phase or as a two phase feedstock. After the HDO/MDS step, the product fraction is separated and passed to a separate isomerization reactor. An isomerization reactor for the biological starting material is described in the literature (Fl 100 248) as a cocurrent reactor.
The process for producing a fuel by hydrogenation of a hydrocarbon feed, eg, the lipid composition or lipids herein, can also be effected by passing the lipid composition or lipids as a co-current flow with hydrogen gas through a first hydrogenation zone and thereafter, The hydrocarbon influent is further hydrogenated in a second hydrogenation zone by passing hydrogen gas into the second hydrogenation zone as a counter-current flow relative to the hydrocarbon effluent. Exemplary HDO applications and catalysts useful for cracking the lipid composition to produce C<sub>2</sub>-C<sub>5</sub> olefins are described in US Patent No. 7,232,935, which is incorporated by reference in its entirety.
Commonly, in the hydrodeoxygenation step, the structure of the biological component, such as the composition of lipid(s) herein, is decomposed, oxygen, nitrogen, phosphorous and sulfur compounds and gas-like light hydrocarbons are removed, and olefinic bonds are removed. they are hydrogenated. In the second stage of the process, that is, in the so-called isomerization stage, the isomerization is carried out for the branching of the hydrocarbon chain and to improve the performance of the paraffin at low temperatures.
In the first stage, that is, the HDO stage of the cracking process, hydrogen gas and the lipid(s) composition herein to be hydrogenated are passed to an HDO catalyst bed system either as co-current or counter-current flows, the catalyst bed system comprises one or more catalyst beds, preferably 1-3 catalyst beds. The HDO stage is commonly operated in a co-current fashion. In the case of an HDO catalyst bed system comprising two or more catalyst beds, one or more of the beds can be put into operation using the countercurrent flow principle.
In the HDO stage, the pressure varies between 20 and 150 bars, preferably between 50 and 100 bars and the temperature varies between 200 and 500 Ό, preferably in the range of 300-400°C.
In the HDO step, known hydrogenation catalysts containing metals from Group VI and/or VIB of the periodic table can be used. Preferably, the hydrogenation catalysts are supported Pd, Pt, Ni, NIMo or CoMo catalysts, the support consisting of alumina and/or silica. NIMo/AI catalysts are commonly used<sub>2</sub>EITHER<sub>3</sub> and HOW/AI<sub>2</sub>0<sub>3</sub>.
Prior to the HDO step, the lipid composition(s) herein may optionally be treated by pre-hydrogenation under milder conditions, thus avoiding double bond side reactions. Such pre-hydrogenation is carried out in the presence of a pre-hydrogenation catalyst at temperatures of 50-400'C and hydrogen pressures of 1-200 bars, preferably at a temperature of between 150 and 250Ό and a hydrogen pressure of between 10 and 100 bars. The catalyst may contain metals from Group VIII and/or VIB of the periodic table. Preferably, the pre-hydrogenation catalyst is a supported Pd, Pt, Ni, NiMo and CoMo catalyst, the support consisting of alumina and/or silica.
A gaseous stream from the HDO stage containing hydrogen is cooled and then carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulfur compounds, gaseous light hydrocarbons and other impurities are removed therein. After compression, the purified hydrogen or recycle hydrogen is returned to the first catalyst bed and/or between catalyst beds to compensate for the extraction of the gas stream. Water is removed from the condensed liquid. The liquid is passed to the first catalyst bed or between catalyst beds.
After the HDO stage, the product is subjected to an isomerization stage. It is essential to the process that impurities are removed as completely as possible before the hydrocarbons come into contact with the isomerization catalyst. The isomerization step comprises an optional separation step, wherein the reaction product of the HDO step can be purified by stripping or distillation with steam or an appropriate gas such as light hydrocarbon, nitrogen or hydrogen. The optional distillation or separation step is carried out in a counter-current manner in a unit upstream of the isomerization catalyst, where the gas and liquid are brought into contact with each other or before the actual isomerization reactor in a Separate separation unit using the counter current principle.
After the hydrogen gas stripping step and the hydrogenated lipid composition or lipids herein and optionally a mixture of n-paraffin, are passed to a reactive isomerization unit comprising one or more catalyst beds. The catalyst beds of the isomerization stage can operate in either a co-current or counter-current manner.
It is important for the process that the countercurrent flow principle is applied in the isomerization stage. In the isomerization stage. This is done by carrying out either the optional separation step or the isomerization reaction step or both in a counter-current manner.
The isomerization step and the HDO step can be carried out in the same pressure vessel or in separate pressure vessels. The optional pre-hydrogenation can be carried out in a separate pressure vessel or in the same pressure vessel as the HDO and isomerization steps.
In the isomerization stage, the pressure varies in the range of 20-150 bars, preferably in the range of 20-100 bars, the temperature is between 200 and 500°C, preferably between 300 and 400°C.
In the isomerization step, isomerization catalysts known in the art can be used. Suitable isomerization catalysts contain molecular sieves and/or a Group VII metal and/or a carrier. Preferably the isomerization catalyst contains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrierite and Pt, Pd or Ni and AI<sub>2</sub>EITHER<sub>3</sub> i YES<sub>2</sub>. Typical isomerization catalysts are for example Pt/SAPO-11/AI<sub>2</sub>EITHER<sub>3</sub>, Pt/ZSM-22/AI<sub>2</sub>EITHER<sub>3</sub>, Pt/ZSM-23/AI<sub>2</sub>EITHER<sub>3</sub>and Pt/SAPO-11 l/S¡0<sub>2</sub>.
As for the product, a high-quality hydrocarbon component of biological origin, useful as a diesel fuel or a component thereof, is obtained, the density, cetane number and low-temperature performance of said hydrogen component are excellent.
IX. MICROBE DESIGN
As indicated above, in certain embodiments of the present invention, it is desirable to genetically modify a microorganism to improve lipid production, modify the properties or ratios of components generated by the microorganism, or improve or provide de novo growth characteristics on a variety. of raw food materials. Chlorella, particularly Chlorella protothecoides, Chlorella minutissima, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. and Chlorella emersonii are preferred microorganisms for use in the genetic engineering methods described herein, although other Chlorella species as well as other varieties of microorganisms can be used.
Promoters, cDNA and 3'UTR, as well as other vector elements, can be generated by cloning techniques using fragments isolated from natural sources (see for example Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; and US Patent 4,683,202.) Alternatively, elements can be generated synthetically using known methods (see for example Gene. 1995 Oct 16;164(1):49-53).
A. Codon Optimization for Expression
DNA encoding a polypeptide to be expressed in a microorganism, eg, a lipase and selectable marker are preferably codon-optimized cDNAs. Methods for recoding genes for expression in microalgae are described in US Patent 7,135,290. Additional information for codon optimization is available, for example, in the GenBank codon usage database. As non-limiting examples, the codon usage in Chlorella pyrenoidosa, Dunaliella salina and Chlorella protothecoides are shown in Tables 10, 11 and 12, respectively.
Table 10. Chlorella pyrenoidosa codon usage.
<td>Phe</td><td>uuuu UUC</td><td> 39 (0.82) 56(1.18)</td><td>To be</td><td>UCU 50 (1.04) UCC 60(1.25)</td>
<td>read</td><td>USA</td><td> 10(0.20)</td><td></td><td>ACU 6 (0.96)</td>
<td></td><td>OUGG</td><td> 46 (0.91)</td><td></td><td>UCG 43 (0.89)</td>
<td>tyr</td><td>wow</td><td> 15(0.59)</td><td>Cys</td><td>UGU 46 (0.77)</td>
<td></td><td>UAC</td><td> 36(1.41)</td><td></td><td>UGC 73 (1.23)</td>
<td>ter</td><td>uaa</td><td> 9 (0.00)</td><td>ter</td><td>AGU 43 (0.00)</td>
<td>ter</td><td>UAG</td><td> 15(0.00)</td><td>trp</td><td>UGG 69 (1.00)</td>
<td>read</td><td>cuu</td><td> 49 (0.97)</td><td>Pro</td><td>UCC 80 (0.98)</td>
<td></td><td>CUC</td><td> 73 (1.45)</td><td></td><td>CCC 88(1.08)</td>
<td></td><td>WHICH</td><td> 22 (0.44)</td><td></td><td>CAC 93(1.14)</td>
<td></td><td>CUG</td><td> 103 (2.04)</td><td></td><td>CCG 65 (0.80)</td>
<td>his</td><td>CAU</td><td> 50 (0.88)</td><td>arg</td><td>UGC 39 (0.76)</td>
<td></td><td>ACC</td><td> 3(1.12)</td><td></td><td>GCC 63 (1.23)</td>
<td>gln</td><td>CAA</td><td> 59 (0.84)</td><td></td><td>AGC 46 (0.90)</td>
<td></td><td>AGC</td><td> 2(1.16)</td><td></td><td>GCG 47 (0.92)</td>
<td>lies</td><td>wow</td><td> 24 (0.69)</td><td>Thr</td><td>ACCU 32 (0.67)</td>
<td></td><td>abc</td><td> 61 (1.76)</td><td></td><td>ACC 76(1.60)</td>
<td></td><td>wow</td><td> 19(0.55)</td><td></td><td>ACA 41 (0.86)</td>
<td>met</td><td>AUG</td><td> 42 (1.00)</td><td></td><td>GCA 41 (0.86)</td>
<td>asn</td><td>AAU</td><td> 26 (0.75)</td><td>To be</td><td>AGU 23 (0.48)</td>
<td></td><td>CAA</td><td> 3(1.25)</td><td></td><td>AGC 67(1.39)</td>
<td>Lys</td><td>AAA</td><td> 32 (0.54)</td><td>arg</td><td>AGA51 (1.00)</td>
<td></td><td>AAG</td><td> 86(1.46)</td><td></td><td>AGG61 (1.19)</td>
<td>Val</td><td>wow</td><td> 36 (0.75)</td><td>To the</td><td>CUG 57 (0.79)</td>
<td></td><td>GUC</td><td> 54(1.13)</td><td></td><td>GCC 97(1.34)</td>
<td></td><td>GUA</td><td> 30 (0.63)</td><td></td><td>GCA 89(1.23)</td>
<td></td><td>GUG</td><td> 71 (1.49)</td><td></td><td>GCG 47 (0.65)</td>
<td>Asp</td><td>GAU</td><td> 60 (0.95)</td><td>gly</td><td>GGU 35 (0.60)</td>
<td></td><td>GAC</td><td> 66(1.05)</td><td></td><td>GGC 78(1.33)</td>
<td>glu</td><td>GAA</td><td> 41 (0.68)</td><td></td><td>GGA 54 (0.92)</td>
<td></td><td>GAG</td><td> 80 (1.32)</td><td></td><td>GGG 67(1.15)</td>
Table 11. Preferred codon usage in Dunaliella salina.
<td>TTC (Phe)</td><td>CT (Tyr)</td><td>TGC (Cys)</td><td>TAA (Stop)</td>
<td>TGG (Trp)</td><td>CCC (Pro)</td><td>ACC (His)</td><td>CGC (Arg)</td>
<td>CTG (Leu)</td><td>GAC (gn)</td><td>ATC (lie)</td><td>ACC(Thr)</td>
<td>AAC (Asn)</td><td>AGC (Being)</td><td>ATG (Met)</td><td>AAG (Lys)</td>
<td>GCC (Wing)</td><td>GAC (Asp)</td><td>GGC (Gly)</td><td>GTG (Val)</td>
<td>GAG (Glu)</td><td></td><td></td><td></td>
Table 12. Preferred codon usage in Chlorella protothecoides.
<td>TTC (Phe)</td><td>CT (Tyr)</td><td>TGC (Cys)</td><td>TGA (Stop)</td>
<td>TGG (Trp)</td><td>CCC (Pro)</td><td>ACC (His)</td><td>CGC (Arg)</td>
<td>CTG (Leu)</td><td>GAC (gn)</td><td>ATC (lie)</td><td>ACC(Thr)</td>
<td>GAC (Asp)</td><td>CBT (Being)</td><td>ATG (Met)</td><td>AAG (Lys)</td>
<td>GCC (Wing)</td><td>AAC (Asn)</td><td>GGC (Gly)</td><td>GTG (Val)</td>
GAG (Glu)
B. Promoters
Many promoters are active in microalgae, including promoters that are endogenous to the algae being transformed, as well as promoters that are not endogenous to the algae being transformed (i.e., promoters from other algae, promoters from higher plants and plant virus or algae virus promoters). Exogenous and/or endogenous promoters that are active in microalgae and antibiotic resistance genes functional in microalgae are described, eg, Curr Microbiol. 1997 Dec; 35(6):356-62 (Chlorella vulgaris); Mar Biotechnol (NY). 2002 Jan; 4(1):63-73 (Chlorella ellipsoidea); Mol Gene Genet. 1996 Oct 16; 252(5):572-9 (Phaeodactylum tricornutum); Plant Mol Biol. 1996 Apr; 31(1):1-12 (Volvox carteri); Proc Nati Acad Sci USA. 1994 Nov 22;91(24):11562-6 (Volvox carteri); Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C, PMID: 10383998, 1999 May; 1(3):239-251 (Laboratory of Molecular Plant Biology, Stazione Zoológica, Villa Comunale, 1-80121 Naples, Italy) (Phaeodactylum tricornutum and Thalassiosira weissflogii); Plant Physiol. 2002 May; 129(1):7-12. (Porphyridium sp.); Proc Nati Acad Sci USA. 2003 Jan 21 ;100(2):438-42. (Chlamydomonas reinhardtii); Proc Nati Acad Sci USA. 1990 Feb; 87(3): 1228-32. (Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jan 25;20(12):2959-65; Mar Biotechnol (NY). 2002 Jan; 4(1):63-73 (Chlorella); Biochem Mol Biol Int. 1995 Aug; 36(5): 1025-35 (Chlamydomonas reinhardtii); J Microbiol. 2005 Aug; 43(4):361-5 (Dunaliella); Yi Chuan Xue Bao. 2005 Apr; 32(4):424-33 (Dunaliella); Mar Biotechnol (NY). 1999 May; 1(3):239-251. (Thalassiosira and Phaedactylum); Koksharova, Appl Microbiol Biotechnol 2002 Feb; 58(2): 123-37 (various species); Mol Genet Genomics. 2004 Feb; 271(1):50-9 (Thermosynechococcus elongates); J. Bacterid. (2000), 182, 211-215; FEMS Microbiol Lett. 2003 Apr 25; 221(2):155-9; Plant physiol. 1994 Jun; 105(2):635-41; Plant Mol Biol. 1995 Dec; 29(5):897-907 (Synechococcus PCC 7942); Sea Pollut Bull. 2002; 45(1-12):163-7 (Anabaena PCC 7120); Proc Nati Acad Sci USA. 1984 Mar; 81(5):1561-5 (Anabaena (various strains)); Proc Nati Acad Sci USA. 2001 Mar 27; 98(7):4243-8 (Synechocystis); Wirth, Mol Gene Genet 1989 Mar; 216(1):175-7 (various species); Mol Microbiol, 2002 Jun; 44(6): 1517-31 and Plasmid, 1993 Sep; 30(2):90-105 (Fremyella diplosiphon); Hall et al. (1993) Gene 124: 75-81 (Chlamydomonas reinhardtii); Gruber et al. (1991). Current Micro. 22:15-20; Jarvis et al. (1991) Current Genet. 19: 317-322 (Chlorella); for additional promoters see also Table 1 of US Patent 6,027,900).
The promoter used to express a foreign gene may be the promoter naturally linked to that gene or may be a heterologous gene. Some promoters are active in more than one species of microalgae. Other promoters are species specific. Preferred promoters include promoters such as RBCS2 from Chlamydomonas reinhardtik and viral promoters, such as cauliflower mosaic virus (CMV) and Chlorella virus, which have been shown to be active in multiple species of microalgae (see for example Plant Cell Rep. 2005 Mar; 23(10-11):727-35; J Microbiol. 2005 Aug; 43(4):361-5; Mar Biotechnol (NY). 2002 Jan; 4(1):63-73). In other embodiments, the Botryococcus malate dehydrogenase promoter, such as a nucleic acid comprising any part of SEQ ID NO: 3 or the RBCS2 promoter from Chlamydomonas reinhardtik (SEQ ID NO: 4) can be used. Optionally, at least 10, 20, 30, 40, 50 or 60 nucleotides or more of these promoter-containing sequences are used. Preferred promoters endogenous to species of the genus Chlorella are SEQ ID NO: 1 and SEQ ID NO: 2.
Preferred promoters useful for expression of exogenous genes in Chlorella are listed in the sequence listing of this application, such as the Chlorella HUP1 gene promoter (SEQ ID NO: 1) and the Chlorella ellipsoidea nitrate reductase promoter (SEQ ID NO : 2). Chlorella virus promoters can also be used to express genes in Chlorella, such as SEQ ID NO: 1-7 of US Patent 6,395,965. Additional promoters active in Chlorella can be found, for example in Biochem Biophys Res Commun. 1994 Oct 14; 204(l): 187-94; Plant Mol Biol. 1994 Oct; 26(l):85-93; Virology. 2004 Aug 15; 326(1):150-9; and Virology. 2004 Jan 5; 318(1):214-23.
C. Select Markers
Any of a wide variety of selectable markers can be used in a transgene construct useful for transforming Chlorella. Examples of suitable selectable markers include the nitrate reductase gene, the hygromycin phosphotransferase (HPT) gene, the neomycin phosphotransferase gene, and the ble gene, which confers resistance to phleomycin. Methods to determine the susceptibility of microalgae to antibiotics are well known. For example, Mol Gene Genet. nineteen ninety six Oct 16; 252(5):572-9.
More specifically, Dawson et al. (1997), Current Microbiology 35:356-362 (incorporated by reference herein in its entirety), describe the use of the Chlorella vulgaris nitrate reductase (NR) gene as a selectable marker for NR-deficient Chlorella sorokiniana mutants. Kim et al. (2002), Mar. Biotechnol. 4:63-73 (incorporated by reference herein in its entirety), disclosed the use of the HPT gene as a selectable marker to transform Chorella ellipsoidea. Huang et al. (2007), Appl. Microbiol. Biotechnol. 72: 197-205 (incorporated by reference herein in its entirety), reported the use of Sh ble as a selectable marker for Chlorella sp. DT.
D. Inducible Expression
The present invention also provides the use of an inducible promoter to express a gene of interest. In particular, the use of an inducible promoter to express a lipase gene allows lipase production after growth of the microorganism when conditions have been adjusted, if necessary, to enhance transesterification, for example, after disruption of the lipases. cells, reduction of the water content of the reaction mixture, and/or addition of sufficient alcohol to drive the conversion of TAG to fatty acid esters.
Inducible promoters useful in the invention include those that moderate the transcription of an operably linked gene in response to a stimulus, such as an exogenously supplied small molecule (for example, glucose, as in SEQ ID NO: 1), temperature (heat or cold), light, etc. Appropriate promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level. In the latter case, the transcription level of the lipase preferably does not significantly interfere with the growth of the microorganism in which it is expressed.
Expression of transgenes in Chlorella can be undecidably effected by promoters such as the promoter that drives the Chlorella hexose transporter gene (SEQ ID NO:
one). This promoter is strongly activated by the presence of glucose in the culture medium.
E. Expression of two or more foreign genes
In addition, a genetically engineered microorganism, such as a microalga, may comprise and express two or more exogenous genes, such as, for example, a lipase and a lytic gene, for example one encoding a polysaccharide-degrading enzyme. One or both genes can be expressed using an inducible promoter, which allows the relative expression timing of these genes to be controlled to improve lipid yield and conversion to fatty acid esters. The expression of the two or more foreign genes may be under the control of the same inducible promoter or under the control of different inducible promoters. In the latter situation, the expression of a first foreign gene may be induced for a first period of time (during which the expression of a second foreign gene may or may not be induced) and the expression of a second foreign gene may be induced. for a second period of time (during which the expression of a first exogenous gene may or may not be induced). Provided herein are vectors and methods for engineering microbes that produce lipids to metabolize sucrose, which is an advantageous trait because it allows the engineered cells to convert sugarcane feedstock to lipids.
Also provided herein are genetically engineered strains of microbes (eg, microalgae, oily yeast, bacteria, or fungi) that express two or more exogenous genes, such as, for example, a fatty acyl-ACP thioesterase and a fatty acyl-CoA/aldehyde. fatty reductase, the combined action of which produces an alcohol product. Further provided are other combinations of exogenous genes, including without limitation, a fatty acyl-ACP thioesterase and a fatty acyl-CoA reductase to generate aldehydes. Furthermore, the present application provides the combination of a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase and a fatty aldehyde decarboxylase to generate alkanes. One or more of the foreign genes can be expressed using an inducible promoter.
Examples of modifications further suitable for use in the present invention include genetically engineered strains of microalgae to express two or more foreign genes, one encoding a fixed carbon source transporter (such as sucrose) and a second encoding an invertase enzyme. saccharose. The resulting fermentable organisms produce hydrocarbons at a lower manufacturing cost than has been obtainable by previously known methods of biological hydrocarbon production. Insertion of the two exogenous genes described herein can be combined with disruption of polysaccharide biosynthesis by direct and/or random mutagenesis, which directs even greater carbon flux to hydrogen production. Individually and in combination, trophic conversion, design to alter oil production, and treatment with exogenous enzymes alter the oil composition produced by a microorganism. The alteration may be a change in the amount of hydrogens produced, the amount of one or more hydrocarbon species produced relative to other hydrocarbons, and/or the types of hydrocarbon species produced in the microorganism. For example, microalgae can be engineered to produce a higher amount and/or higher percentage of TAG.
F. Expression by Compartments
The present invention also provides for compartmentalized expression of a gene of interest. In particular, it may be advantageous in particular embodiments to target lipase expression to one or more cellular compartments, where it is sequestered from most cellular lipids until initiation of the transesterification reaction. Preferred organelles for targeting are chloroplasts, mitochondria, and endoplasmic reticulum.
one. Expression in Chloroplasts
In one embodiment of the present invention, expression of a polypeptide in a microorganism is targeted to chloroplasts. Methods for targeting the expression of a heterologous gene to the chloroplast are known and can be employed in the present invention. Methods for targeting foreign gene products to chloroplasts are described in Shrier et al., EMBO J. (1985) 4:25-32. See also Tomai et al. Gen. Biol. Chem. (1988) 263:15104-15109 and US Pat. No. 4,940,835 for the use of transit peptides to translocate nuclear gene products into the chloroplast. Methods for directing the transport of proteins to the chloroplast are also reviewed in Kenauf TIBTECH (1987) 5:40-47. Endogenous chloroplast targeting sequences to Chlorella are known, such as genes in the nuclear genome of Chlorella that encode proteins that are targeted to the chloroplast. chloroplast; see for example GenBank accession number AY646197 and AF499684.
Wageningen UR - Plant Research International sells a vector IMPACTVECTOR1.4, which uses the Chrysanthemum morifolium small subunit protein secretion signal to deliver a heterologous protein into the chloroplast stromal (cytoplasmic) environment, by delivery via a system double membrane. The protein is fused to the first 11 amino acids of the mature rubisco protein in order to allow proper processing of the signal peptide (Wong et al., Plant Molecular Biology 20: 81-93 (1992)). The signal peptide contains a natural intron of the RbcS ge.
In another procedure, the chloroplast genome is genetically engineered to express the heterologous protein. Stable transformation of Chlamydomonas reinhardtik (a green algae) chloroplasts has been described using foreign DNA-coated high velocity tungsten micro-projectile bombardment of recipient cells. See, for example, Boynton et al., Science (1988) 240: 1534-1538; Blowers et al. Plant Cell (1989) 1:123-132 and Debuchy et al., EMBO J. (1989) 8:2803-2809. The transformation technique using tungsten micro-projectiles is described by Klein et al., Nature (London) (1987) 7:70-73. Other chloroplast transformation methods for both plants and microalgae are known. See, for example, US Patents 5,693,507; 6,680,426; and Plant Physiol. 2002 May; 129(1):7-12; and Plant Biotechnol J. 2007 May; 5(3):402-12.
As described in US Patent No. 6,320,101 (issued November 20, 2001 to Kaplan et al.; which is incorporated herein by reference), cells can be chemically treated to reduce the number of chloroplasts per cell to approximately one. . The heterologous nucleic acid can then be introduced into the cells via particle bombardment with the goal of introducing at least one heterologous nucleic acid molecule into the chloroplasts. The heterologous nucleic acid is selected such that it is integrable into the chloroplast genome via homologous recombination, which is readily effected by chloroplast-inherent enzymes. For this purpose, the heterologous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid sequence that is derived from the chloroplast genome. In addition, the heterologous nucleic acid includes a selectable marker. Additional details regarding this technique are found in the US patents. Nos. 4,945,050 and 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the chloroplast protein expression system.
US Patent No. 7,135,620 (issued November 14, 2006 to Daniell et al.; incorporated herein by reference) describes chloroplast expression vectors and related methods. Expression cassettes are DNA constructs that include a coding sequence and appropriate control sequences to provide for proper expression of the coding sequence in the chloroplast. Typical expression cassettes include the following components: the 5' untranslated region of a microorganism gene or chloroplast gene such as psbA that will provide for transcription and translation of a DNA sequence encoding a polypeptide of interest in the chloroplast; a DNA sequence encoding a polypeptide of interest; and a translation and transcription termination region, such as a 3' inverted repeat region of a chloroplast gene that can stabilize RNA of introduced genes, thereby enhancing expression of the foreign gene. The cassette can optionally include an antibiotic resistance gene.
Commonly, the expression cassette is flanked by convenient restriction sites for insertion into an appropriate genome. The expression cassette may be flanked by DNA sequences from chloroplast DNA to facilitate stable integration of the expression cassette into the chloroplast genome, particularly by homologous recombination. Alternatively, the expression cassette may remain unintegrated, in which case, the expression cassette commonly includes a chloroplast origin of replication, which is capable of providing for replication of heterologous DNA in the chloroplast.
The expression cassette generally includes a promoter region of a gene capable of expression in the chloroplast. The promoter region may include promoters obtainable from chloroplast genes, such as the spinach or chickpea psbA gene or the maize rbcL and atpB promoter region and Rrna promoters. Examples of promoters are described in Hanley-Bowdoin and Chua, TIBS (1987) 12:67-70; Mullet et al., Plant Molec Biol. (1985) 4: 39-54; Hanley-Bowdoin (1986) PhD. Dissertation, the Rockefeller University; Krebbers et al., Nucleic Acids Res. (1982) 10: 4985-5002; Zurawaki et al., Nucleic Acids Res. (1981) 9:3251-3270 ; and Zurawskl et al., Proc. Nat'l Acad Sel. USA (1982) 79: 7699-7703. Other promoters can be identified and the relative strength of promoters so identified assessed, by placing a promoter of interest 5' to a promoterless marker gene and observing its effectiveness relative to transcription obtained from e.g. the promoter of the psbA gene, a promoter of relatively strong chloroplast. The efficiency of expression of the heterologous gene can be further improved by any of a variety of techniques. These include the use of multiple promoters inserted in tandem 5' to the heterologous gene, eg a double psbA promoter, the addition of enhancer sequences and the like.
Numerous promoters active in the Chlorella chloroplast can be used for expression of exogenous genes in the Chlorella chloroplast, such as those found in Genbank accession number NC_001865 (Chlorella vulgaris chloroplast, whole genome).
Where it is desired to provide inducible expression of the heterologous gene, an inducible promoter and/or a 5' untranslated region containing sequences that provide for regulation at the transcriptional and/or translational level (at the 3' end) can be included in the expression cassette. For example, the 5' untranslated region may be from a gene where expression is light regulatable. Similarly, the 3' inverted repeat regions could be used to stabilize RNA from heterologous genes. Inducible genes can be identified by enhanced expression in response to a stimulus of particular interest and low expression or absent expression in the absence of the stimulus. For example, a light-inducible gene can be identified, where enhanced expression occurs during light irradiation, while substantially reduced expression or no expression occurs in low or no light. Light-regulating promoters from green microalgae are known (see for example Mol Genet Genomics. 2005 Dec; 274(6):625-36).
The termination region that is employed will primarily be one of convenience, since the termination region appears to be relatively interchangeable between proplasts and bacteria. The termination region may be native to the transcription initiation region, may be native to the DNA sequence of interest, or may be obtainable from another source. See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.
Expression cassettes can be transformed into a plant cell of interest by any of a variety of methods. These methods include, for example, biolistic methods (see, for example, Sanford, Trends In Biotech. (1988) 6:299-302, US Patent 4,945,050 ; electroporation (Fromm et al, Proc. Nati. Acad. Sci. (USA) (1985 ) 82:5824 5828); the use of a laser, microinjection or any other method capable of introducing DNA into a chloroplast.
Additional descriptions of chloroplast expression vectors suitable for use in microorganisms such as microalgae are found in US Patent 7,081,567 (issued July 25, 2006 to Xue et al.); 6,680,426 (issued Jan. 20, 2004 to Daniell et al.); and 5,693,507 (issued December 2, 1997 to Daniell et al.).
Proteins expressed in the nuclear chlorella genome can be targeted to the chloroplast using chloroplast targeting signals. Endogenous chloroplast targeting sequences to Chlorella are known, such as genes in the Chlorella nuclear genome that encode proteins that are targeted to the chloroplast; see, for example, GenBank accession numbers AY646197 and AF499684. Proteins can also be expressed in the Chlorella chloroplast by inserting genes directly into the chloroplast genome. Chloroplast transformation commonly occurs via homologous recombination and can be effected if chloroplast genome sequences are known for creation of targeting vectors (see, for example, the complete genome sequence of a Chlorella chloroplast; Genbank accession number NC 001865). See previous sections herein for details of chloroplast transformation.
2. Expression in mitochondria
In another embodiment of the present invention, expression of a polypeptide in a microorganism is targeted to mitochondria. Methods for targeting foreign gene products to mitochondria Boutry et al. Nature (London) (1987) 328:340-342) have been described, where they are included in green microalgae (see, for example, Mol Gen Genet. 1993 Jan;236(2-3):235-44).
For example, an expression vector encoding an appropriate secretion signal can target a heterologous protein to mitochondria. The vector IMP ACTVECTOR 1.5 vector, from Wageningen URPlant Research International, uses the yeast CoxIV secretion signal, which has been shown to deliver proteins into the mitochondrial matrix. The protein is fused to the first four amino acids of the yeast CoxIV protein in order to allow proper processing of the signal peptide (Kohler et al. Plant J 11:613-621 (1997)). Other mitochondrial targeting sequences are known, including those functional in green microalgae. For example, see FEBS Lett. 1990 Jan 29;260(2): 165-8; and J Biol Chem. 2002 Feb 22;277(8):6051-8.
Proteins expressed in the Chlorella nuclear genome can be targeted to mitochondria using mitochondrial targeting signals. See previous sections herein for details of mitochondrial protein targeting and transformation.
3. Expression in Endoplasmic Reticulum
In another embodiment of the present invention, expression of a polypeptide in a microorganism is targeted to the endoplasmic reticulum. Inclusion of an appropriate sorting or retention signal in an expression vector ensures that proteins are required in the endoplasmic reticulum (ER) and do not proceed downstream to the Golgi apparatus. For example, the vector IMPACTVECTOR1.3, from Wageningen UR-Plant Research International, includes the well known KDEL sort or retention signal. With this vector, ER retention has a practical advantage in that it has been reported to improve expression levels 5-fold or more. The main reason for this seems to be that the ER contains lower concentrations and/or different proteases responsible for post-production degradation of expressed proteins that are present in the cytoplasm. Functional ER retention signals in green microalgae are known. For example, see Proc Nati Acad Sci USA. 2005 Apr 26;102(17):6225-30.
G. Transformation
Cells can be transformed by any appropriate technique including, for example, biolistics, electroporation, bed glass transformation, and silicon carbide whisker transformation. See, for example, Examples herein.
Any convenient technique for introducing a transgene into Chlorella can be used in the present invention. Dawson et al. (1997) (supra) described the use of microprojectile bombardment to introduce the Chlorella vulgaris nitrate reductase (NR) gene into Chlorella sorokiniana NR deficient mutants, resulting in stable transformants. Briefly, 0.4 micron tungsten beads were coated with plasmid; 3X10<sup>7</sup> c.sorokiniana cells were spread in the central third of a non-selective agar plate and bombarded with the PDS-1000/He Biolistic Particle Delivery© (Bio-Rad) biolistic particle delivery system.
A preferred method for introducing a gene into Chlorella is the method described by Kim et al. (2002), Mar. Biotechnol. 4:63-73. Kim reports the transformation of Chorella ellipsoidea protoplasts using CaCI<sub>2</sub> and polyethylene glycol (PEG). In particular, protoplasts were prepared by culturing cells from
C.ellipsoidea at a density of 1-2 X 10<sup>8</sup>/M1. Cells were recovered and washed by centrifugation for 5 minutes at 1600 g and resuspended in 5 M1 phosphate pH buffer solution (Ph 6.0) containing 0.6 MI sorbitol, 0.6 M mannitol, 4% (w/v) cellulose (Calbiochem ), 2% (w/v) macerase (Calbiochem) and 50 units of pectinase (Sigma). The cell suspension was incubated at 25°C for 16 hours in the dark with moderate shaking. The resulting protoplasts were recovered by centrifugation at 400 g for 5 minutes. The pellet was gently resuspended in 5 ml f/2 medium containing 0.6 M sorbitol and 0.6 M mannitol and centrifuged at 400 g for 5 minutes. This pellet was resuspended in 1 MI of 0.6 M sorbitol/mannitol solution containing mMCaC2. 50 mL. Then, 5 mg of transgene DNA, along with 25 pg of calf thymus DNA (Sigma), was added to 10<sup>7</sup>-10<sup>8</sup> protoplasts in 0.4 mi. After 15 min at room temperature, 200 pL of PNC (polyethylene glycol 4000, 40%, 0.8 M NaCl, 50 mM CaCl2) was added and mixed gently for 30 min at room temperature. After this, 0.6 ml of f/2 medium supplemented with 0.6 M sorbitol/mannitol solution, 1% yeast extract and 1% glucose were added and the transformed cells were incubated at 25°C for 12 hours in the dark. for cell wall regeneration. A similar method was used by Huang et al. (2007) (supra) to introduce a transgene encoding mercuric reductase into Chlorella sp. DT.
Electroporation has also been used to transform Chorella. As reported by Maruyama et al. (2004), Biotechnology Techniques 8:821-826 (incorporated by reference herein in its entirety), this technique was used to introduce a transgene into Chlorella saccharophila c-211-1 protoplasts prepared from cells in the stationary phase. Transient expression of the introduced plasmid was observed under a field strength between 600 and 900 V/cm, and a pulse duration of around 400 ms, where high membrane permeability to 70-kDa FITC-dextran was determined.
Examples of transgene expression in Chlorella can be found in the literature (see, for example Current Microbiology Vol. 35 (1997), pp. 356-362; Sheng Wu Gong Cheng Xue Bao. 2000 Jul;16(4):443- 6;Current Microbiology Vol. ); Plant Cell Reports 18:9, 778-780, (1999); Plantarium Biology 42(2): 209-216, (1999); Plant Pathol. J 21(1): 13-20, (2005)). See also examples herein.
Examples of transgene expression in oilseed yeast (eg Yarrowia lipolytica) can be found in the literature (see eg Bordes et al., J Microbiol Methods, Jun 27 (2007)).
Examples of transgene expression in fungi (for example, Mortierella alpine, Mucor circinelloides, and Aspergillus ochraceus) can also be found in the literature (see, for example, Microbiology, Jul; 153(Pt. 7):2013-25 (2007); Mol Genet Genomics, Jun; 271(5):595-602 (2004); Curr Genet, Mar;21(3):21523 (1992); Current Microbiology, 30(2):83-86 (1995); Sakuradani, NISR Research Grant, Studies of Metabolic Engineering of Useful Lipid-producing Microorganisms (2004); and PCT/JP2004/012021). Examples of exogenous gene expression in bacteria such as E. coli are well known; see, for example, Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press.
Vectors for transformation of microorganisms in accordance with the present invention may be prepared by known techniques familiar to those skilled in the art. The nucleotide sequence of the construct used for transformation of multiple Chlorella species corresponds to SEQ ID NO:25. In one embodiment, an exemplary vector design for expression of a lipase gene in a microorganism such as a microalga contains a gene encoding a lipase in operable linkage to a promoter active in microalgae. Alternatively, if the vector does not contain a promoter operably linked to the gene of interest, the gene can be transformed into the cell such that it is operably linked to an endogenous promoter at the point of integration of the vector. The promoterless transformation method has been shown to work in microalgae (see, for example, Plant Journal 14:4, (1998), pp.441-447). The vector may also contain a second gene encoding a protein that imparts resistance to an antibiotic or herbicide for example, ie a selectable marker. Optionally, one or both genes are followed by a 3' untranslated sequence containing a polyadenylation signal. Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Microalgal co-transformation in which different vector molecules are used simultaneously to transform cells can also be used (see, for example, Protist 2004 Dec; 155(4):381-93). Transformed cells can optionally be selected based on the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.
All references cited herein, including Patents, Patent Applications, and Publications, are incorporated by reference herein in their entirety, whether or not previously specifically incorporated. The publications mentioned herein are cited for the purpose of describing and disclosing reagents, methodologies, and concepts that may be used in connection with the present invention. Nothing herein shall be construed as an admission that these references are prior art in connection with the invention described herein.
Although this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification. The present application is intended to cover any variations, uses or adaptations of the invention which generally follow the principles of the invention and which include such departures from the present disclosure as they come from the known or customary practice in the art with which the invention is performed. concerning and how they may be applied to the essential elements summarized above herein.
X. EXAMPLES
The following examples are offered to illustrate but not limit the claimed invention.
EXAMPLE 1
Chlorella strains from the University of Texas culture collection were tested for growth on glycerol and glucose. The following Chlorella species and strains were cultivated: Chlorella kessleri (strains 263, 397, 398, 2228); Chlorella sorokiniana (strains 1663, 1665, 1669, 1671, 1810); Chlorella saccharophila (2911; 2469); Chlorella protothecoides (31, 249, 250, 264). Each strain was inoculated from the solid medium to a 25 ml liquid-based medium (2g/L yeast extract, NaNO<sub>3</sub> 2.94mM, CaCI<sub>2</sub>'2H<sub>2</sub>OR 0.17mM, MgSO<sub>4</sub>«7H<sub>2</sub>0.3mM, K<sub>2</sub>HPO<sub>4</sub>0.4mM, KH<sub>2</sub>PO<sub>4</sub> 1.28mM, NaCI 0.43mM) and cultured with shaking at 27°C for 72 hours under a light intensity of 75 75pEm~V<sup>1</sup>. These cultures were used to inoculate each strain at a final density of 1x10<sup>5</sup> cells/mL in 24-well plates containing 2 mL of (a) base media only (b) base media plus 0.1% glucose and (c) base media plus 0.5% reagent grade glycerol (EM Science, catalog #GX0185-6). The plates were placed in the dark and cultured for 72 hours with shaking at 27°C. Samples of each strain grown in the three conditions were diluted 1.9:1 in H<sub>2</sub>O distilled and absorbance was read at 600 nm on a Molecular Devices SpectraMax 340PC. All strains exhibited growth in the presence of glucose and glycerol compared to basal media alone.
EXAMPLE 2
Strains and media: Chlorella protothecoides #1 (CEPA 250), #2 (CEPA 264) and Chlorella kessleri #1 (CEPA 398) were obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified Proteose medium. The modified Proteose medium consisted (in g/L) of 0.25 g NaNO<sub>3</sub>, 0.09 g of K<sub>2</sub>HPO<sub>4</sub>, 0.175 g of KH<sub>2</sub>PO<sub>4</sub>, 0.025 g of CaCl<sub>2</sub>-2H<sub>2</sub>Or, 0.075 g MgSO<sub>4</sub>-7H<sub>2</sub>Or and 2 g of yeast extract per liter. Glycerol waste from biodiesel production (acidulated glycerol (AG)) and non-acidulated glycerol (NAG) were obtained from Imperial Western Productions (Selma, CA, USA). "Pure or reagent grade" glycerol was from EM Science (a division of Merck KGA), catalog number #GX0185-6.
Experimental design and growth measurement: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Protease + 1% pure glycerol
2. Protease + 1% acidulated glycerol
3. Protease + glycerol without acidifying at 1%
Four. Protease + 1% pure glycerol + 1% glucose (added after 72 hours).
5. Protease + 1% acidified glycerol + 1% glucose (added after 72 hours)
6. Protease + 1% non-acidulating glycerol + 1% glucose (added after 72 hours).
Each strain was inoculated to different media at a concentration of 5 x 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken by orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hours of initial culture, 1% (w/v) glucose was added to samples #4, 5, and 6 and cultured another 24 hours. To measure cell dry weight, 1 ml of each culture was pelleted by centrifugation at 5000 rpm for 5 minutes in an Eppendorf 5415C centrifuge. After removing the supernatant, the cell pellets were frozen at -80°C and lyophilized in a laboratory scale freeze dryer (Labconco, MO, United States of America). The results are shown in Figure 1.
EXAMPLE 3
Strains and media: Chlorella protothecoides #1 (STRAIN 250), #3 (STRAIN 249) and Chlorella kessleri #2 (strain 397) were obtained from the Algal Culture Collection at the University of Texas (Austin, TX, United States of America). America). The concentrated cultures were maintained on a modified proteose medium (see EXAMPLE 2).
Experimental design and growth measurement: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Proteose + 1% pure glycerol + 1% glucose
2. Proteose + 1% acidulated glycerol + 1% glucose
3. Proteose + 1% non-acidulating glycerol + 1% glucose.
Each strain was inoculated to different media at a concentration of 5 x 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken on a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 96 hours, cell growth was measured by cell dry weight (see EXAMPLE 2). The results are shown in Figure 2.
EXAMPLE 4
Strains and Media: Chlorella protothecoides #3 (CEPA 249), #4 (CEPA 31) and Chlorella kessleri #2 (CEPA 397) were obtained from the algal culture collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained in a modified Proteose medium (see EXAMPLE 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Proteose + 1% pure glycerol + 1% glucose
2. Proteose + 1% acidulated glycerol + 1% glucose
3. Proteose + 1% non-acidulating glycerol + 1% glucose
Each strain was inoculated to media containing different glycerols (pure, acidified or non-acidulated) at a concentration of 5 χ 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken by the Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 96 hours, the lipid content was measured. To measure the amount of lipid content in cells, 100 μΙ of cultures were harvested and washed once with the same volume of media. To each tube, 5 μΙ of washed cells and 200 μΙ of 18 M sulfuric acid were added. The tubes were incubated at 90°C in a water bath for 30 minutes and 1 ml of phosphoric acid-vanillin reagent was added to the tubes and incubated at 37°C for 15 minutes. To prepare the phosphoric acid-vanillin reagent, 0.12 g of vanillin was added to 20 mL of water and the volume was adjusted to 100 mL with 85% phosphoric acid. The optical density at 530 nm was measured in a glass cuvette against a reference tube with 5 μΙ of water as a sample. The results are shown in Figure 3.
EXAMPLE 5
Strains and Media: Chlorella protothecoides #2 (CEPA 264) and Chlorella kessleri #1 (CEPA 398) were obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified proteose medium (see EXAMPLE 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Proteose + 1% pure glycerol
2. Proteose + glycerol without acidifying at 1%
3. Proteose + 1% pure glycerol + 1% glucose (added after 72 hours)
Four. Proteose + 1% non-acidulating glycerol + 1% glucose (added after 72 hours).
Each strain was inoculated to media containing different glycerols (pure or without acidifying) at a concentration of 5 χ 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 72 hours of initial growth, 1% glucose was added to sample #3 and #4 and cultured another 24 hours. The lipid content was measured in all samples (see EXAMPLE 4). The optical density at 600 nm was also measured to check for non-specific absorbance and subtracted from OD 530 nm to calculate the amount of lipid. The reference curve is composed of tnolein dissolved in chloroform ranging from 1 to 10 pg. The results are shown in Figure 4.
EXAMPLE 6
Strains v Media: Chlorella protothecoides #3 (CEPA 249) and Chlorella kessleri #2 (CEPA 397) were obtained from the algal culture collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified proteose medium (see EXAMPLE 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Proteose + 1% pure glycerol + 1% glucose (added after 72 hours)
2. Proteose + 1% acidulated glycerol + 1% glucose (added after 72 hours)
3. Proteose + 1% non-acidulating glycerol + 1% glucose (added after 72 hours)
Each strain was inoculated to media containing different glycerols (pure, acidified or non-acidulated) at a concentration of 5 x 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 72 hours of initial growth, 1% glucose was added and cultured for another 24 hours. Cell dry weight and lipid content were measured in all samples (see EXAMPLES 2 and 5). The lipid percentage was calculated from the amount of total lipid divided by the cell dry weight. The results are shown in Figure 5.
EXAMPLE 7
Strains v Media: Chlorella protothecoides #2 (CEPA 264) and Chlorella kessleri #1 (CEPA 398) were obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified proteose medium (see EXAMPLE 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Proteose + 1% pure glycerol + 1% glucose (added after 72 hours)
2. Proteose + 1% non-acidulating glycerol + 1% glucose (added after 72 hours)
Each strain was inoculated with media containing either 1% neat glycerol or 1% unacidulated glycerol at a concentration of 5 x 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 72 hours of initial growth, 1% glucose was added and cultured for another 24 hours. Cell dry weight and lipid content were measured in all samples (see EXAMPLES 1 and 4). The lipid percentage was calculated from the amount of total lipid divided by the cell dry weight. The results are shown in Figure 6.
EXAMPLE 8
Strains and Media: Chlorella protothecoides #1 (CEPA 250), #4 (CEPA 31) and Chlorella kessleri #2 (CEPA 397) were obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified proteose medium (see EXAMPLE 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following different media was prepared in the 24-well plates.
one. Proteose + 2% glucose
2. Proteose + 1% glycerol + 1% glucose
Each strain was inoculated to different media at a concentration of 5 x 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken on a Labnet (Berkshire, UK) orbital shaker at
430 rpm. After 96 hours of initial growth, the lipid content was measured (see EXAMPLE 5). The results are shown in Figure 7.
EXAMPLE 9
Strains and media: Chlorella protothecoides #3 (strain 249), #4 (strain 31) and Chlorella kesslerí #1 (strain 398) were obtained from the algal culture collection at the University of Texas (Austin, Texas, United States of America). America). The concentrated cultures were maintained on a modified proteose medium (see example 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following media was prepared in 24-well plates.
one. Proteose + 2% glucose
2. Proteose + 1% glycerol + 1% glucose
3. Proteose + 1% glycerol + 1% glucose (added after 72 hours)
Each strain was inoculated to different media at a concentration of 5X10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430rpm. After 72 hrs of initial growth, 1% (w/v) glucose was added to medium number 3 and cultured another 24 hrs. Cell dry weight and lipid content were measured in all samples (see examples 2 and 5). The lipid percentage was calculated from the total amount divided by the cell dry weight. The results are shown in figure 8.
EXAMPLE 10
Strains and media: Chlorella protothecoides #1 (strain 250), #3 (strain 249) and Chlorella kessleri #2 (strain 397) were obtained from the algal culture collection at the University of Texas (Austin, Texas, United States of America). America). The concentrated cultures were maintained on a modified proteose medium (see example 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Proteose + 1% pure glycerol + 1% glucose
2. Proteose + 1% pure glycerol + 1% glucose (added after 72 hours)
3. Proteose + 1% acidulated glycerol + 1% glucose
Four. Proteose + 1% acidulated glycerol + 1% glucose (added after 72 hours)
5. Proteose + 1% non-acidulating glycerol + 1% glucose
6. Proteose + 1% non-acidulating glycerol + 1% glucose (added after 72 hours)
Each strain was inoculated to different media at a concentration of 5X10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430rpm. After 72 hrs of initial growth, 1% (w/v) glucose was added to medium number 2, number 4 and number 6 and cultured another 24 hrs. The lipid content was measured in all samples (see examples 4). The results are shown in figure 9.
EXAMPLE 11
Strains and media: Chlorella protothecoides #1 (strain 250), #3 (strain 249), #4 (strain 31), and Chlorella kessleri #2 (strain 397) were obtained from the algal culture collection at the University of Texas ( Austin, Texas, United States of America). The concentrated cultures were maintained on a modified proteose medium (see example 2).
Experimental design and lipid analysis: For each strain, 1 ml of the following different media was prepared in 24-well plates.
one. Proteose + 1% pure glycerol + 1% glucose
2. Proteose + 1% pure glycerol + 1% glucose (added after 72 hours)
3. Proteose + 1% acidulated glycerol + 1% glucose
Four. Proteose + 1% acidulated glycerol + 1% glucose (added after 72 hours)
5. Proteose + 1% non-acidulating glycerol + 1% glucose
6. Proteose + 1% non-acidulating glycerol + 1% glucose (added after 72 hours)
Each strain was inoculated to different media at a concentration of 5X10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430rpm. After 72 hrs of initial growth, 1% (w/v) glucose was added to media number 2, number 4 and number 6 and cultured another 24 hrs. Cell dry weight was measured in all samples (see example 2). The results are shown in figure 10.
EXAMPLE 12
vector construction
A BaMHI-SacII fragment containing the CMV promoter, a hygromycin resistant cDNA and CMV 3' UTR (SEQ ID NO:5, a subsequence of the vector pCAMBIA1380, Cambia, Canberra, Australia) was cloned into the BamHI and SacII sites of pBluescnpt and is referred to herein as pHyg.
Biolistic transformation of Chlorella
Seashell Technology's S550d gold carriers were prepared according to the manufacturer's protocol. The linearized pHyg plasmid (20 ug) was mixed with 50 ul of binding buffer solution and 60 ul (30 mg) of S550d gold carriers and incubated on ice for 1 min. Precipitation buffer solution (100 ul) was added and the mixture was incubated on ice for another minute. After being vortexed, the DNA-coated particles were agglomerated by centrifugation at 10,000rpm in an Eppendo microfuge. rf5415C microfuge for 10sec. The gold pellet was washed once with 500 µl cold 100% ethanol, pelleted by brief centrifugation in the microfuge and resuspended with 50 µl ice-cold ethanol. After brief sonication (1-2 sec), 10 µl of DNA-coated particles were immediately transferred to the carrier's membrane.
The Chlorella protothecoides culture (University of Texas, Culture Collection 250) was grown in Proteose medium (2gr/L yeast extract, 2.94mM NaNO3, 0.17mM CaCl2*2H2O, 0.3mM MgSO4«7H2O, 0.4mM K2HPO4 , 1.28mM KH2PO4, 0.43mM NaCI) on a rotary shaker under continuous light at 75 Jmol ni photons<sup>2</sup> yes<sup>1</sup> until it reached a cell density of 2X10<sup>6</sup> cells/ml. Cells were harvested, washed once with sterile distilled water, and resuspended in 50 Jl of medium. 1X10<sup>7</sup> cells were spread in the central third of a non-selective Proteose media plate. Cells were bombarded with the PDS1000/He Biolistic Particle Delivery System (Bio-Rad). Rupture discs (1100 and 1350 psi) were used and the plates were placed 9 and 12 cm below the sieve/macrocarrier assembly. Cells are allowed to recover at 25°C for 12-24 hrs. After recovery, the cells were scraped from the plates with a rubber or rubber spatula, mixed with 100 Jl of the medium and spread on plates containing hygromycin (200 Jg/ml). After 7-10 days of incubation at 25°C, colonies representing transformed cells were visible on the 1100 and 1350 inch-pound rupture disc plates.<sup>2</sup> and at a distance of 9 and 12 cm. Colonies were picked and plated on selective agar plates for a second round of selection.
Chlorella transformation by Electroporation
The Chlorella protothecoides culture was grown in Proteose medium on a rotary shaker under continuous light at 75 Jmol photons m'<sup>2</sup> yes<sup>1 </sup>until they reached a cell density of 2X10<sup>6</sup> cells/ml. Cells were harvested, washed once with sterile distilled water, and resuspended in Tris-phosphate buffer solution (20mM Tris-HCl, pH 7.0; 1mM potassium phosphate) containing 50mM sucrose at a density of 1X10.<sup>8 </sup>cells/ml. A suspension of approximately 250 Jl of cells (1X10<sup>8</sup> cells) was placed in a 4mm gap disposable electroporation cuvette. To the cell suspension, 5 μg of linearized pHgy plasmid DNA and 200 μg of carrier DNA (salmon sperm DNA under constant stress) were added. Then, the electroporation cuvette was incubated in a water bath at 16°C for 10min. An electrical pulse (1100 V/cm) was then applied to the cuvette at a capacitance of 25 JF (no shunt receptor used for electroporation) using a Gene Pulser II electroporation apparatus (Bio-Rad Labs, Hercules, CA ). The cuvette was then incubated at room temperature for 5 min, after which the cell suspension was transferred to 50 ml Proteose medium and shaken on a rotary shaker for 2 days. Following recovery, cells were harvested by low speed centrifugation, resuspended in Proteose media, and plated at low density on plates supplemented with 200 μg/ml hygromycin. The plates were incubated under continuous light at 75 Jmol photons m'.<sup>2</sup> yes<sup>1</sup>. The transformants appeared as colonies in 1-2 weeks. Colonies were picked and plated on selective agar plates for a second round of selection.
Genotype
A subset of colonies that survived a second round of selection were grown in a small volume and harvested. Pellets of about 5 — 10 □! volume were resuspended in 50ZII of 10mM NaEDTA by vortexing and then incubated at 100*0 for 10. Tubes were then vortexed briefly and sonicated for 10sec, then centrifuged at 12,000 xg for 1min. 2 TI of supernatant were used as template in a 507I PCR reaction. the primers used for genotyping were SEQ ID NO:6 and SEQ ID NO:7. PCR conditions were as follows: 95°C 5min x 1 cycle; 95°C 30 sec - 58Ό 30 sec - 72°C, 1 min, 30 sec x 35 cycles; 72Ό 10min x 1 cycle. The expected 992 bp fragment was found in 6 of 10 colonies from the biolistic method and from a single colony from electroporation. A smaller, non-specific band was present in all lanes. The results are shown in figure 16. To confirm the identity of the 992bp amplified fragments, two biolistic bands and the electroporation band were excised from the gel and individually sequenced. The sequence of all three bands corresponded to the expected 992bp fragment. (DNA ladder:Bionexus® All Purpose Hi-Lo® DNA ladder catalog #BN2050).
EXAMPLE 13
Strains and media: a) Spirulina platensis (UTEX 2340) and b) Navícula pelliculosa (UTEX 667) were obtained from the algae culture collection at the University of Texas (Austin, Texas, United States of America). The concentrated Spirulina culture was maintained in Spirulina medium and Navícula was maintained in soil extract medium (SEM). The Spirulina medium consisted of NaHCO<sub>3</sub>162mM, Na<sub>2</sub>CO<sub>3</sub> 38mM, K<sub>2</sub>HPO<sub>4</sub> 1.9mM, NaNO<sub>3</sub> 29mM, K<sub>2</sub>SW<sub>4</sub> 5.75mM, 17.1mM NaCI, MgSO<sub>4</sub>*7H<sub>2</sub>OR 0.8mM, CaCI<sub>2</sub>*2H<sub>2</sub>0.25mM O, Na<sub>2</sub>2mM EDTA, FeCI<sub>3</sub>*6H<sub>2</sub>OR 0.36mM, MnCI<sub>2</sub>*4H<sub>2</sub>OR 0.21 mM, ZnCI<sub>2</sub> 0.037mM, CoCI<sub>2</sub>*6H<sub>2</sub>0 0.0085mM, NaMoO<sub>4</sub>«2H<sub>2</sub>OR 0.017, CuSO<sub>4</sub>«5H<sub>2</sub>OR 0.787M, ZnSO<sub>4</sub>«7H<sub>2</sub>OR 0.15ΞΙΜ, H<sub>3</sub>BO<sub>3</sub>10DM and vitamin B<sub>12</sub> 0.001mM. The soil extract medium consists of NaNO<sub>3</sub> 2.94mm, CaCI<sub>2</sub>*2H<sub>2</sub>OR 0.17mM, MgSO<sub>4</sub>*7H<sub>2</sub>0.3mM, K<sub>2</sub>HPO<sub>4</sub> 0.43mM, KH<sub>2</sub>PO<sub>4</sub> 1.29mM, NaCI 0.43mM and soil extract. Glycerol waste from biodiesel production (acidulated glycerol (AG) and non-acidulated glycerol (NAG)) were obtained from Imperial Western Products (Selma, CA, USA).
Experimental design and growth measurement: For each strain, 1 ml of the following different media was prepared in 24-well plates.
a)
7. Spirulina medium + 2% glucose
8. Spirulina medium + 2% reagent grade glycerol
9. Spirulina medium + 2% non-acidulating glycerol
10. Spirulina medium + 1% unacidulated glycerol + 1% glucose
b)
one. SEM + 2% glucose
2. SEM + 2% reagent grade glycerol
3. SEM + 1% reagent grade glycerol + 1% glucose
Four. SEM + 2% acidulated glycerol
5. SEM + 1% acidulated glycerol + 1% glucose
6. SEM + 2% unacidulated glycerol
7. SEM + 1% unacidulated glycerol + 1% glucose
Each strain was inoculated to different media at a concentration of 5X10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430rpm. After 96 hrs the lipid content was measured. To measure the amount of lipid content in cells, 10011 cultures washed once with the same volume of media were harvested. To each tube, 511 washed cells and 200Jl of 18M sulfuric acid were added. the tubes were incubated at 90°C in a water bath for 10min and 1ml of vanillin phosphoric acid reagent was added to the tubes and incubated at 37°C for 15min. To prepare the phosphoric acid-vanillin reagent, 0.12 g of vanillin was added to 20 ml of water and the volume was adjusted to 100 ml with 85% phosphoric acid. The optical density at 530nm was read in a glass cuvette against a reference tube with 51I water as sample. The reference curve is composed of Triolein dissolved in chloroform that fluctuates from 1 to 10ig.
To measure cell dry weight, 0.5ml of each culture was pelleted by centrifugation at 5000rpm for 5min. After removing the supernatant, the cell pellets were frozen at -80°C and dried overnight in a freeze-drying system (Labconco, MO, United States of America). The lipid percentage was calculated from the amount of total lipid divided by the cell dry weight. The results are shown in figure 11.
EXAMPLE 14
Strains and media: a) Scenedesmus armandus (UTEX 2552) was obtained from the algal culture collection at the University of Texas (Austin, Texas, United States of America). The concentrated cultures were maintained on modified Proteose medium. The modified Proteose medium consisted (g/L) of 0.25g NaNO<sub>3</sub>, 0.09g of K<sub>2</sub>HPO<sub>4</sub>, KH<sub>2</sub>PO<sub>4</sub>, 0.025g of CaCI<sub>2</sub>-2H<sub>2</sub>Or, 0.075g of MgSO<sub>4</sub>'7H<sub>2</sub>Or and 2g of yeast extract per liter.
Experimental Design and Measurement of Growth and Lipid: For each culture condition, 1 ml of the following different media was prepared in 24-well plates.
a),b)
one. Proteose + 2% glucose
2. Proteose + glycerol 2%
3. Proteose + 2% acidulated glycerol
Four. Proteose + glycerol without acidifying at 2%
5. Proteose + 1% non-acidulating glycerol + 1% glucose
Scenedesmus armandus (UTEX 2552) was inoculated to different media at a concentration of 5X10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken on a Labnet (Berkshire, UK) orbital shaker at 430rpm. After 96 hrs cell growth was measured by cell dry weight and lipid content was measured by phospho-vanillin assay (see example 13). The lipid percentage was calculated from the total amount of lipid divided by the cell dry weight. The results are shown in figure 12.
EXAMPLE 15
Strains v Media: Navícula pelliculosa (UTEX 667) was obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a soil extract medium (see EXAMPLE 13).
Experimental design and growth measurement: For each culture condition, 1 ml of the following different media was prepared in 24-well plates.
one. SEM + 2% glucose
2. SEM + 2% glycerol
3. SEM + 2% acidulated glycerol
Four. SEM + 1% acidulated glycerol + 1% glucose
5. SEM + 2% unacidulated glycerol
6. SEM + 1% unacidulated glycerol + 1% glucose.
Navícula pelliculosa (UTEX 667) was inoculated to media containing glucose or different glycerols (pure, acidified or without acidifying) at a concentration of 5 χ 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken on a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 96 hours, cell growth was measured by cell dry weight (see EXAMPLE 13). The results are shown in Figure 13.
EXAMPLE 16
Strains and Media: Scenedesmus armatus (UTEX 2552) and Navícula pelliculosa (UTEX 667) were obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified proteose medium for Scenedesmus armatus and a soil extract medium for Navícula pelliculosa (see EXAMPLE 1).
Experimental design and growth measurement: For each strain, 1 ml of the following different media was prepared in 24-well plates.
scenedesmus armatus
5. Proteose + 1% acidulated glycerol + 1% glucose
6. Proteose + 1% acidulated glycerol + 1% glucose (added after 72 hours)
navicula pellicularus
one. SEM + 1% acidulated glycerol + 1% glucose
2. SEM + 1% acidified glycerol + 1% glucose (added after 72 hours)
Each strain was inoculated into media at a concentration of 5 x 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 72 hours of initial growth, 1% glucose was added to sample #2 and cultured for another 24 hours. Cell growth was measured by cell dry weight (see EXAMPLE 13). The results are shown in Figure 14.
EXAMPLE 17
Strains and Media: Chlorella protothecoides (UTEX 31) was obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified proteose medium (see EXAMPLE 1).
Experimental design: For each condition, 1 ml of the following different media was prepared in 24-well plates.
Four. proteose
5. Proteose + 0.5% glucose
6. Proteose + xylose 0.5%
7. Proteose + 0.25% glucose + 0.25% xylose.
Chlorella protothecoides #4 (UTEX 31) was inoculated to media containing different sugars (glucose or xylose) at a concentration of 3 x 10<sup>5</sup> cells/ml. Cultures were kept in the dark and shaken on a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 72 hours of growth, cell growth was measured by counting cell numbers in each culture. The results are shown in Figure 15.
EXAMPLE 18
Chlorella protothecoides strains #1, #3, and #4 were obtained from the Algal Culture Collection at the University of Texas (Austin, TX, USA). The concentrated cultures were maintained on a modified proteose medium (see EXAMPLE 1). For each condition, 1 ml of the following different media was prepared in 24-well plates.
one. proteose
2. Proteose + 1% glucose
3. Proteose + fructose 1%
Each strain was inoculated to media containing different sugars (glucose or fructose) at a concentration of 1 x 10<sup>6</sup> cells/ml. Cultures were kept in the dark and shaken using a Labnet (Berkshire, UK) orbital shaker at 430 rpm. After 96 hours of growth, cell density was measured by counting the cell numbers of each culture. The results are shown in Figure 20.
EXAMPLE 19
Chlorella on Sucrose
Materials and methods: Chlorella protothecoides (UTEX 249) was inoculated into three 50-ml flasks of proteose medium with 1% sucrose (2.94 mM NaNO<sub>3</sub>, 0.428 mM K<sub>2</sub>HPO<sub>4</sub>, 1.28 mM KH<sub>2</sub>PO<sub>4</sub>, 0.427 mM NaCI, 0.17 mM CaCI<sub>2</sub>-2H<sub>2</sub>OR, 0.3 mM MgSO<sub>4</sub>-7H<sub>2</sub>O, Proteose peptone 1 g/L) at a final cell density of 4 x 10<sup>5</sup> cells for me Invertase (Sigma #14504) was added to two of the cultures at 0.01 U/ml and 0.05 U/ml. All three cultures were grown in the dark for -60 hrs shaking at 150rpm.
Results: Final cell counts were made in all three cultures after -60 hrs of shaking in the dark. The control or witness flask reached 4.4x10<sup>5</sup> cells per ml while the 0.01 U/ml and 0.05 U/ml flasks reached cell densities of 1x10<sup>8</sup> and 3x10<sup>8</sup> respectively. Each flask was checked for contamination at the end of the experiment by microscopic analysis and all were clean.
EXAMPLE 20
Chlorella strains that grow on sucrose
Cultures of Chlorella kessleri ((a) UTEX 397 and (b) UTEX 398) and Chlorella fusca ((a) UTEX 251 and (b) UTEX 1801) were inoculated from autotropic liquid cultures to 10 ml of proteose + 1 sucrose medium. % in 50 ml flasks at a concentration of 1 x 10<sup>6</sup> cells/ml. Control cultures were also inoculated at the same density with only proteose media. Cultures were grown at 200 in the dark shaking at 250 rpm for 7 days, at which point cell density was measured by hemocytometer. As shown in Figures 21-22, all four strains grew on sucrose compared to the initial cell density and the proteose only control.
EXAMPLE 21
Growth of Chlorella protothecoides on honeydew with a sucrose invertase.
Preparation of Chlorella cells for inoculation: A 10 ml liquid culture of Chlorella was started by taking the inoculum from a solid proteose plate. Cultures were grown in the light for approximately 2 days at 26°C. Growth was measured using an optical densitometer (OD) at 750 nm and by determining cell dry weights.
Preparation of molasses and concentrated sugar solutions: A 5% concentrated solution was prepared with glucose, sucrose, and 3 different molasses samples (marked BSI, BS2, and HTM) obtained from the commercial sugarcane-to-sugar process, as shown. in Table 13 below. The pH of all stock solutions was checked to be in the range of 6-6.6 and then the stock solutions were autoclaved.
Table 13. Molasses and sugar solutions
<td>molasses</td><td>% of sugar</td><td>5% sugar diluted in 100 ml</td>
<td></td><td></td><td>grams or my</td>
<td></td><td></td><td></td>
<td>htm</td><td> 78.72</td><td> 6.4</td>
<td>BSI (FL)</td><td> 44.25</td><td> 11.3</td>
<td>BS2 (AU)</td><td> 51.55</td><td> 9.7</td>
<td>Saccharose</td><td>too</td><td> 5</td>
<td>Glucose</td><td>too</td><td> 5</td>
Preparation of Invertase Solution: A stock solution of 40 units/mL of invertase was prepared by reconstituting 1 mg of a 400 units/mg invertase (Sigma) in 10 mL of distilled water.
Experimental conditions and setups: 10 ml cultures were prepared, each consisting of a final concentration of 1% molasses/sugar, 0.05 units/ml invertase and 1.0x10<sup>6</sup> cells per ml of Chlorella protothecoides in a protease-based medium. Cultures were numbered as follows: (1) control media only; (2) 1% HTM; (3) 1% BSI ; (4) 1% BS2; (5) 1% glucose; and (6) 1% sucrose. A similar control set was also prepared without the addition of invertase. Cultures were grown in the dark for 5 days shaking at 250 rpm at 28°C.
Results: The growth of Chlorella protothecoides cells was evaluated immediately after 5 days of incubation on the respective feeding raw material in the dark. As shown in Figures 23-24, cells can be grown on molasses in the presence of a sucrose invertase with yields comparable to those on pure reagent grade glucose.
EXAMPLE 22
Genetic design of Chlorella protothecoides to express an Exogenous Sucrose Invertase.
Strains and Media: Chlorella protothecoides (UTEX 250) was obtained from the Algal Culture Collection at the University of Texas (Austin, TX, United States of America). Concentrated cultures were maintained on modified proteose medium. The modified proteose medium consists of 0.25 g NaNO<sub>3</sub>, 0.09 g of K2HPO<sub>4</sub>, 0.175 g of KH2PO<sub>4</sub>, 0.025 g of CaCl<sub>2</sub>-2H<sub>2</sub>Or, 0.075 g MgSO<sub>4</sub>-7H<sub>2</sub>Or, and 2 g of yeast extract per liter (g/L).
Plasmid construction: To express the secreted form of invertase in Chlorella protothecoides, a Saccharomyces cerevisiae SUC2 gene was placed under the control of three different promoters: Cauliflower Mosaic Virus (CMV) 35S promoter, Chlorella virus (NC) promoter -IA) and Chlorella HUPI promoter. A yeast SUC2 gene was synthesized to accommodate the optimized codon usage of C. protothecoides and includes a signal sequence required to direct extracellular secretion of invertase. Each construct was integrated into pBluescript KS+, and EcoRI/AscI, Ascl/Xhol, and Xhol/BamHI sites were introduced to each promoter, invertase gene, and
CMV 3'UTR, respectively, by PCR amplification using specific primers. Purified PCR products were sequentially cloned. An illustration of the final constructs is shown in Figure 25.
Chlorella protothecoides transformation: A culture of Chlorella protothecoides was grown in modified protease medium on a rotary shaker under continuous light at 75 pmol m'photons.<sup>2</sup> Yes<sup>1</sup> until it reached a cell density of 6xl0<sup>6</sup>cells/ml.
For biolistic transformation, Seashell Technology S550d gold carriers were prepared according to the manufacturer's protocol. Briefly, a construct linearized (20 pg) by Bsal was mixed with 50 µl of binding buffer and 60 µl (3 mg) of S550d gold carriers and incubated on ice for 1 minute. Precipitation buffer (100 µL) was added and the mixture was incubated on ice for another 1 minute. After moderate vortexing, the DNA-coated particles were agglomerated by centrifugation at 10,000 rpm in an Eppendorf microfuge for 10 seconds. The gold pellet was washed once with 500 µl cold 100% ethanol, pelleted by brief centrifugation in the microfuge and resuspended with 50 µl ice-cold ethanol. After a brief solidification (1-2 seconds), 10 µl of DNA-coated particles were immediately transferred to the carrier membrane. Cells were harvested, washed once with sterile distilled water, resuspended in 50 µl of medium (1 x 10<sup>7</sup> cells) and were spread in the central third of a nonselective proteinaceous plate. Cells were bombarded with the PDS-1000/He Biolistic Particle Delivery System (Bio-Rad). Rupture disks (1100 and 1350 psi) were used and the plates were placed 9-12 cm below the screen/macrocarrier assembly. Cells are allowed to recover at 25°C for 12-24 hours. After recovery, cells were scraped from the plates with a rubber spatula, mixed with 100 µl of medium, and spread on 1% sucrose-modified proteose plates. After 7-10 days of incubation at 25 Ό in the dark, colonies representing transformed cells were visible on the plates.
For transformation with electroporation, cells were harvested, washed once with sterile distilled water, and resuspended in Tris-phosphate buffer solution (20 mM Tris-HCl pH 7.0; 1 mM potassium phosphate) containing 50 mM sucrose at a density of 4x10<sup>8</sup> cells/ml. Approximately 250 pl of cell suspension (1x10<sup>8</sup> cells) was placed in a 4 mm gap disposable electroporation cuvette. To the cell suspension, 5 pg of linearized plasmid DNA and 200 pg of carrier DNA (sheared salmon sperm DNA) were added. Then the electroporation cuvette was incubated in an ice water bath at 16°C for 10 minutes. An electrical pulse (1100 V/cm) was then applied to the cuvette at a capacitance of 25 pF (no shunt resistor used for electroporation) using a Gene Pulser II electroporation apparatus (Bio-Rad Labs, Hercules, CA ). The cuvette was then incubated at room temperature for 5 minutes, after which the cell suspension was transferred to 50 ml of modified proteose medium and shaken on a rotary shaker for 2 days. Following recovery, cells were harvested at low speed (4000 rpm), resuspended in modified proteose medium, and plated at low density on 1% sucrose modified proteose plates. After 7-10 days of incubation at 25°C in the dark, colonies representing transformed cells were visible on the plates.
Selection of Transformants and Genotypes: Colonies were projected from modified proteose plates grown in the dark with 1% sucrose and approximately the same number of cells were transferred to 24-well plates containing 1 ml liquid medium of modified proteose with 1% sucrose. Cultures were kept in the dark and shaken using the Labnet orbital shaker (Berkshire, UK) at 430 rpm for 5 days.
To verify the presence of the introduced invertase gene in Chlorella transformants, DNA from each transformant was isolated and amplified with a gene-specific primer set (CMV construct: forward primer (CAACCACGTCTTCAAAGCAA) (SEQ ID NO:6)/reverse primer (TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO:9), CV constructs: forward primer (TTGTCGGAATGTCATATCAA) (SEQ ID NO: 10)/reverse primer (TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 11), and HUPI construct: forward primer (AACGCCTTTGTACAACTGCA) (SEQ ID NO: 12)/ reverse primer (TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO: 13)). For rapid DNA isolation, a volume of cells (approximately 5-10 uL in size) were resuspended in 50 uL of 10 mM NaEDTA. The cell suspension was incubated at 100°C for 10 minutes and sonicated for 10 seconds. After centrifugation at 12,000 g for 1 minute, 3 uL of supernatant was used for the PCR reaction. PCR amplification was performed in a DNA thermal cycler (Perkin-Elmer GeneAmp 9600). The reaction mix (50 uL) contained 3 uL of extracted DNA, 100 pmol each of the respective primers described above, 200 uM dNTP, 0.5 unit of Taq DNA polymerase (NEB), and Taq DNA polymerase buffer. according to manufacturer's instructions. DNA denaturation was carried out at 95C for 5 minutes during the first cycle and then for 30 seconds. Primer annealing and extension reactions were carried out at 58°C for 30 seconds and 72°C for 1 minute, respectively. PCR products were then visualized on 1% agarose genes stained with ethidium bromide. Figure 26 shows the results of PCR genotyping of C. protothecoides transformants using the gene-specific primers identified above. The arrows show the expected size of the PCR product and the stars represent DNA samples from each transformant that show the PCR product matching the expected size ((V: Vector only, WT: wild type).
Growth in Liquid Culture: After five days of growth in the dark, genotype-positive transformants showed growth on liquid minimal proteose medium + 1% sucrose in the dark, while wild-type cells showed no growth on the same medium. in the dark.
EXAMPLE 23
Transformation of algal strains with a S. cerevisiae invertase
Secreted Invertase: A gene encoding a secreted sucrose invertase (Saccharomyces cerevisiae Gen Bank accession number NP 012104) was synthesized de novo as a 1599 bp Ase I-Xho fragment that was subsequently subcloned into a pUC19 derivative that it possesses the Cauliflower Mosaic Virus 35s promoter and 3' UTR as EcoR 1/Asc I and Xho/Sac I cassettes, respectively.
Algal Cell Growth: The media used in these experiments consisted of liquid base media (see EXAMPLE 1) and solid base media (+1.5% agarose) containing fixed carbon in the form of sucrose or glucose (as designated) at a final concentration of 1%. The strains used in this experiment did not grow in the dark on medium base in the absence of an additional fixed carbon source. The species were deposited on the plates and cultivated in the dark at 28°C. Individual colonies were screened and used to inoculate 500 mL of liquid base medium containing 1% glucose and allowed to grow in the dark to mid-log phase, measuring cell counts each day. Each of the following strains had previously been tested for growth on sucrose in the dark as a single carbon source and exhibited no growth and were thus excluded for transformation with a secreted invertase: (1) Chlorella protothecoides (UTEX 31); (2) Chlorella minutissima (UTEX 2341); and (3) Chlorella emersonii (CCAP 211/15).
Algal Cell Transformation via Particle Bombardment: A sufficient culture was centrifuged to give approximately 1-5x10<sup>8</sup> total cells. The resulting pellet was washed with base medium without any fixed carbon sources added. The cells were spun down again and the pellet was resuspended in a volume of base medium sufficient to give 5x10<sup>7</sup> to 2x10<sup>8</sup> cells/ml. 2501000 µl of cells were then plated onto the solid base medium supplemented with 1% sucrose and allowed to dry on the plate in a sterile hood. Plasmid DNA was precipitated onto gold particles according to the manufacturer's recommendations (Seashell Technology, La Jolla, CA). Transformations were carried out using a BioRad PDS He-1000 Particle Delivery System using 1350 pound-force/inch rupture disks.<sup>2</sup> (psi) with the macrocarrier assembly set 9 cm from the rupture disc carrier. Following the transformations, the plates were incubated in the dark at 28°C. All strains generated multiple transformant colonies. Control plates transformed without any invertase insert, but otherwise identically prepared, contained no colonies.
Analysis of Chlorella protothecoides transformants: Genomic DNA was extracted from wild-type Chlorella protothecoides cells and transformant colonies as follows: cells were resuspended in 100 ul of extraction buffer (87.5 mM Tris Cl, pH 8.0, NaCl 50 mM, 5 mM EDTA, pH 8.0, 0.25% SDS) and incubated at 60°C, with occasional mixing via inversion for 30 min. For PCR, samples were diluted 1:100 in 20 mM Tris Cl, pH 8.0.
Genotyping was performed on genomic DNA extracted from WT, transformants, and plasmid DNA. Samples were genotyped for the marker gene. Primers 2383 (5' CTGACCCGACCTATGGGAGCGCTCTTGGC 3') (SEQ ID NO:20) and 2279 (5'
CTTGACTTCCCTCACCTGGAATTTGTCG 3') (SEQ ID NO:21) were used in this genotyping PCR. The PCR profile used was as follows: denaturation at 94°C for 5 minutes; 35 cycles of 94°C-30 seconds, 60°C-30 seconds, 72°C-3 minutes; 72°C-5 minutes. A band of identical size was amplified from the positive controls (plasmids) and two Chlorella protothecoides transformants (UTEX 31), as shown in Figure 27.
Chlorella minutissima and Chlorella emersonii transformant analysis: Genomic DNA was extracted from Chlorella WT and WT transformants as follows: cells were resuspended in 100 ul of extraction buffer (87.5 mM Tris Cl, pH 8.0, NaCI 50 mM, 5 mM EDTA, pH 8.0, 0.25% SDS) and incubated at 60C with occasional mixing via inversion for 30 min. For PCR, samples were diluted 1:100 in 20 mM Tris Cl, pH 8.0. Genotyping was performed on genomic DNA extracted from WT, transformants and plasmid DNA. The samples were subjected to genotyping regarding the marker gene. Primers 2336 (5' GTGGCCATATGGACTTACAA 3') (SEQ ID NO:22) and 2279 (5' CTTGACTTCCCTCACCTGGAATTTGTCG 3') (SEQ ID NO:21) were designated as primer set 2 (1215 bp expected product), in so that primers 2465 (5' CAAGGGCTGGATGAATGACCCCCAATGGACTGTGGTACGACG 3') (SEQ ID NO:23) and 2470 (5' CACCCGTCGTCATGTTCACGGAGCCCAGTGCG 3') (SEQ ID NO:24) were designated primer set 4 (expected product of 1442 bp). The PCR profile used was as follows: denaturation at 94°C for two minutes; 29 cycles of 94°C-30 seconds, 60°C-30 seconds, 72°C-1 minute, 30 seconds; 72°C-5 minutes. A control plasmid containing the secreted invertase was used as a PCR control. Figure 28 shows the transformation of Chlorella minutissima (UTEX 2341) and Chlorella emersonii (CCAP 21 1/15) species of microalgae with the gene encoding a secreted invertase.
The sequence of the invertase construct corresponds to SEQ ID NO:25.
EXAMPLE 24
Growths of algae strains compared to S. cerevisiae on a cellulosic feed stock prepared with Celluclast
Strains and culture conditions: The algal strains used in this study are listed in Table 14 below and were cultured in Proteose medium with exogenously supplied cellulosic material and in some cases additional fixed carbon in the form of glucose. Twenty-four algae strains were used in this study, including 5 different genera encompassing 11 different Chlorella species, two each of Parachlorella and Prototheca, and one each of Bracteococcus and Pseudochlorella. Saccharomyces serevisiae (strain PJ-69-4A) was cultured in YPD medium (per liter, 10g Bacto-yeast extract, 20g Bacto-peptone and 20g glucose). Both algae and yeast were grown at 28°C in the dark. Growth of this strain on Proteose medium in the dark in the absence of cellulosic material or other additional fixed carbon either did not occur or was extremely minimal.
Glucose release from cellulosic material via enzymatic depolymerization treatment: Wet exploited corn stover material was prepared by the National Renewable Energy Laboratory (Golden, CO) by cooking corn stover in a 1.4% sulfuric acid solution and dewatering the resulting suspension. Using a Mettler Toledo Moisture Analyzer the dry solids in the wet corn stover was determined to be 24%. A 10Og wet sample was resuspended in deionized water to a final volume of 420ml and the pH was adjusted to 4.8 using
10N NaOH. Celluclast™ (Novozymas) (a cellulase) was added to a final concentration of 4% and the resulting suspension incubated with shaking at 50°C for 72hrs. The pH of this material was then adjusted to 7.5 with NaOH (negligible volume change), filter sterilized through a 0.221m filter, and stored at -20°C. One sample was reserved for glucose concentration determination using a hexokinase-based kit from Sigma, as described herein.
Determination of Glucose Concentration Released by Celluclast Treatment of Wet Corn Stubble: Glucose concentrations were determined using sigma #G3293 Glucose Assay Reagent. Samples, treated as outlined above, were diluted 400-fold and 40 I added to the reaction. It was determined that the cellulosic preparation of corn stover contains approximately 23g/l of glucose.
Table 14. Strains of algae grown on cellulosic feed prime material
<td>genus/species</td><td>Source/designation</td>
<td>Bracteoccus minor</td><td>UTEX 66</td>
<td>Chlorella ellipsoidea</td><td>SAG 2141</td>
<td>Chlorella kessleri</td><td>UTEX 1808</td>
<td>Chlorella kessleri</td><td>UTEX 397</td>
<td>Chlorella emersonii</td><td>CCAP 211/15</td>
<td>Chlorella luteoviridis</td><td>SAG 2133</td>
<td>Chlorella luteoviridis</td><td>SAG 2198</td>
<td>Chlorella luteoviridis</td><td>SAG 2214</td>
<td>Chlorella luteoviridis</td><td>UTEX 22</td>
<td>Bracteococcus medionucleatus</td><td>UTEX 1244</td>
<td>Chlorella minutissima</td><td>CCALA 20024</td>
<td>Chlorella minutissima</td><td>UTEX 2341</td>
<td>Chlorella ovalis</td><td>CCAP 211/21A</td>
<td>Chlorella protothecoides</td><td>CCAP211/8d</td>
<td>Chlorella protothecoides</td><td>UTEX 250</td>
<td>Chlorella saccharophila</td><td>UTEX 2911</td>
<td>Chlorella sorokiniana</td><td>UTEX 1230</td>
<td>Chlorella sp.</td><td>SAG 241.80</td>
<td>Chlorella vulgaris</td><td>CCAP 211/11C</td>
<td>Parachlorella kessleri</td><td>SAG 12.80</td>
<td>Parachlorella kessleri</td><td>SAG 27.87</td>
<td>prototheca moroformis</td><td>UTEX 1441</td>
<td>prototheca moriformis</td><td>UTEX 1434</td>
<td>Pseudochlorella aquatica</td><td>SAG 2149</td>
In Table 14 and as used herein, refers to the algae culture collection at the University of Texas (Austin, Texas, United States of America), SAG refers to the algae culture collection at the University of Gottingen (Goettingen, Germany), CCAP refers to the algae and protozoan culture collection managed by the Scottish Association for Marine Science (Scotland, United Kingdom of Great Britain) and CCALA refers to the collection of algal laboratory cultures at the Institute of Botany (Trebon, Czech Republic).
Determination of growth on cellulosic material: after enzymatic treatment and saccharification of cellulose to glucose, xylose and other monosaccharide sugars, the previously prepared material was evaluated as experimental raw material for the growth of 24 strains of algae or S. cerevisiae in a Proteose medium or YPD medium respectively. The Proteose medium was made up to a final glucose concentration of 23 g/l (the final glucose concentration generated via cellulolytic treatment of corn stover), as was YPD for the S. cerevisiae culture, by adding varying amounts of glucose. pure and/or depolymerized cellulosic material. Varying concentrations of cellulosic material were included, providing 0, 12.5, 25, 50, and 100% of the 23 g/L glucose in each medium, the components of which are shown in Table 15 below. One ml of the appropriate media was added to the wells of a 24-well plate. S. cerevisiae grown heterotrophically at 28°C in YPD served as the inoculum (201I) for the yeast cavities. twenty Microliters of inoculum for the 24 algal strains were provided by the algal cells grown mixotrophically in a Proteose medium containing 20 g/l glucose.
Table 15. Cellulosic feedstock preparations.
<td>Vol. of YPD 23.1 g/l glucose (ml)</td><td>Vol. Proteose medium 23.1 g/l glucose (ml)</td><td>Vol. Of 100% cellulosics compensated to the medium of Proteose (my)</td><td>Vol. of 100% of cellulosics compensated to Y.P.D. (my)</td><td>Vol. Final (me)</td><td>cellulosic percentage</td>
<td> 0</td><td> 1</td><td> 0</td><td> 0</td><td> 1</td><td> 0</td>
<td> 0</td><td> 0.875</td><td> 0.125</td><td> 0</td><td> 1</td><td> 12.5</td>
<td> 0</td><td> 0.75</td><td> 0.25</td><td> 0</td><td> 1</td><td> 25</td>
<td> 0</td><td> 0.5</td><td> 0.5</td><td> 0</td><td> 1</td><td> 50</td>
<td> 0</td><td> 0</td><td> 1</td><td> 0</td><td> 1</td><td> 100</td>
<td> 1</td><td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 0</td>
<td> 0.875</td><td> 0</td><td> 0</td><td> 0.125</td><td> 1</td><td> 12.5</td>
<td> 0.75</td><td> 0</td><td> 0</td><td> 0.25</td><td> 1</td><td> 25</td>
<td> 0.5</td><td> 0</td><td> 0</td><td> 0.5</td><td> 1</td><td> 50</td>
<td> 0</td><td> 0</td><td> 0</td><td> 1</td><td> 1</td><td> 100</td>
Table 15 shows the volumes of depolymerized corn stover modified to contain Proteose or YPD media components that were added to the Proteose or YPD media, respectively to produce media containing the indicated percentage of cellulosics. The media were prepared in order to obtain a final glucose concentration of 23 g/l in all cases. The volume of media before the addition of either 2011 yeast or algal cells grown in mid-log phase was 1 ml. Cells were incubated two days in the dark over varying concentrations of cellulosic feed stocks at 28°C with shaking (300rpm). Growth was determined by absorbance measurement at 750nm in a UV spectrophotometer. Surprisingly, all strains were grown on cellulosic material prepared with Celluclast, including media conditions in which 100% of the fermentable sugar was cellulosic fermentable.
EXAMPLE 25
Growth of 24 strains of algae v S. cerevisiae on various cellulosic feedstocks prepared with Accellerase 1000™ and Celluclast™
Strains and Culture Conditions: The algal strains used in this example are listed in Table 14 (above) and were grown in a Proteose medium plus additional fixed carbon in the form of depolymerized cellulosic material and/or pure glucose. Saccharomyces cerevisiae (strain pJ69-4a) was grown on YPD medium plus additional fixed carbon in the form of depolymerized cellulosic material and/or pure glucose. Both algae and yeast were cultured at 28<sup>,J</sup>C in the dark
Glucose release from cellulosic material via enzymatic depolymerization treatment: Wet exploited corn stover material was prepared by the National Renewable Energy Laboratory (Golden, CO) by cooking corn stover in a 1.4% sulfuric acid solution and dewatering the resulting suspension. Switchgrass was also prepared by the National Renewable Energy Laboratory (Golden, CO) using the same method as for corn stover. Sugar beet pulp, generated via pectinase treatment, was supplied by Atlantic Biomass, Inc. Of Frederick, MD. Using a Mettler Toledo moisture analyzer, dry solids were 24% in wet corn stover, 26% in switchgrass, and 3.5% in sugar beet pulp. A 10Og wet sample of corn stover or switchgrass was resuspended in deionized water to a final volume of 420ml and the pH adjusted to 4.8 using 10N NaOH. For beet pulp, 8.8g of dry solids were brought to 350ml with deionized water and the pH adjusted to 4.8 10N NaOH. For all feedstocks, Accellerase 1000™ (Genencor) (a cellulase enzyme complex) was used at a ratio of 0.25ml enzyme per gram of dry biomass. The samples were incubated with shaking (110rpm) at 50°C for 72hrs. The pH of this material was then adjusted to 7.5 with NaOH (negligible volume change), filter sterilized through a 0.22µm filter, and used in culture experiments summarized below. One sample was set aside for glucose concentration determination using a hexokinase-based kit from Sigma, as described hereinafter. The same set of cellulosic feedstocks was also prepared using Celluclast™ (Novozymes) (a cellulase) as described in the previous example.
Determination of Glucose Concentrations in Various Cellulosic Feedstocks Treated with Accellerase 1000: Glucose concentrations were determined using Sigma's Assay Reagent #G3293. Samples treated as outlined above were diluted 400-fold and 401I was added to the reaction. Cellulosic preparations of corn stover, switchgrass, and beet pulp were determined to contain approximately 23.6, 17.1, and 13.7 g/l of glucose, respectively.
Determination of growths on cellulosic material: after the enzymatic depolymerization of sources of glucose, xylose and other monosaccharides, the previously prepared materials were evaluated as feed raw materials for the growth of the 24 strains of algae listed in table 14 and S cerevisiae on Proteose or YPD medium respectively. The media were designed to contain a consistent concentration of glucose while varying the amount of cellulosic material derived from corn stover, switchgrass, or beet pulp. A first set of Proteose and YPD medium containing 23.6 gg/L pure glucose, while a second set of media contained depolymerized corn stover, switchgrass, and beet pulp, each containing 23.6 g/L. of glucose. Switchgrass and beet pulp media were supplemented with 6.5 to 9.9 g/l of pure glucose to normalize glucose in all cellulosic media to 23.6 g/l. One ml of the appropriate medium was added to the wells of a 24-well plate. S. cerevisiae grown heterotrophically at 28°C in YPD served as the inoculum (2011) for the yeast cavities. Twenty microliters of inoculum for the 24 algal strains were provided by the algal cells grown mixotropically in Proteose medium containing 20 g/l glucose. All cells were incubated two days in the dark in varying concentrations of cellulosic feed raw materials at 28°C with shaking (300rpm). Growth was determined by absorbance measurement at 750nm in a UV spectrophotometer. Surprisingly, all algal strains grew on corn stover, switchgrass, and beet pulp material prepared with Accellerase 1000™ or Celluclast™, including media conditions in which 100% fermentable sugar was cellulosic-derived. Under no combination of cellulosic feedstock and depolymerization enzyme S. cerevisiae outgrew on an equivalent amount of pure glucose, indicating that inhibitors to yeast growth on cellulosic material make a major impact on fermentation productivity. Combinations of algae strains, depolymerization enzymes, and 100% cellulosic-derived monosaccharide feedstocks that outperformed at 100% pure glucose are shown in Table 16.
Table 16. Combinations of algae, enzymes and feed raw materials
<td>Feed raw material and depolymerization enzyme</td><td>genus/species</td><td>Font /designation</td>
<td>Corn stubble / Celluclast™</td><td>Bracteococcus minor</td><td>UTEX 66</td>
<td>Beet Pulp/Accellerase™</td><td>Chlorella ellipsoidea</td><td>SAG 2141</td>
<td>Switchgrass/ Accellerase™</td><td>Chlorella kessleri</td><td>UTEX 252</td>
<td>beet pulp/ Accellerase™</td><td>Chlorella kessleri</td><td>UTEX 397</td>
<td>Switchgrass/ Accellerase™</td><td>Chlorella luteoviridis</td><td>SAG 2133</td>
<td>Beet pulp/ Accellerase™</td><td>Chlorella luteoviridis</td><td>SAG 2133</td>
<td>Switchgrass/ Accellerase™</td><td>Chlorella luteoviridis</td><td>UTEX 22</td>
<td>beet pulp/ Accellerase™</td><td>Chlorella luteoviridis</td><td>UTEX 22</td>
<td>Corn Stubble/ Accellerase™</td><td>Chlorella luteoviridis</td><td>UTEX 22</td>
<td>beetroot pulp/ Accellerase™</td><td>Chlorella protothecoides</td><td>UTEX 250</td>
<td>beet pulp/ Accellerase™</td><td>Chlorella sp.</td><td>SAG 241.80</td>
<td>beet pulp/ Accellerase™</td><td>Parachlorella kessleri</td><td>SAG 12.80</td>
<td>Switchgrass/ Accellerase™</td><td>prototheca moriformis</td><td>UTEX 1441</td>
<td>beet pulp/ Accellerase™</td><td>prototheca moriformis</td><td>UTEX 1441</td>
<td>Corn Stubble/ Accellerase™</td><td>prototheca moriformis</td><td>UTEX 1441</td>
<td>Corn Stubble/Celluclast™</td><td>prototheca moriformis</td><td>UTEX 1441</td>
<td>Corn Stubble/ Accellerase™</td><td>prototheca moriformis</td><td>UTEX 1434</td>
<td>beet pulp/ Accellerase™</td><td>Pseudochlorella aquatica</td><td>SAG2149</td>
EXAMPLE 26
Carbon Utilization Sieves
Mines v Culture Conditions: Seed cultures of the various microalgae strains identified below were started as 1 ml liquid cultures in 24-well plates and grown autotrophically for 48hrs in light, shaking ~350rpm. Plates were set with a 1.5% agarose-based solid Proteose medium containing 1% glucose, glycerol, xylose, sucrose, fructose, arabinose, mannose, galactose, or acetate as the only source of fixed carbon. For each strain, 5JI of autotropic 24-well plate culture were plated on solid media. The plates were incubated for 7 days in the dark at 28°C and examined for growth in comparison to a control plate containing no additional fixed carbon. Growth was observed for each of the species tested with each respective feed stock as shown in Table 17 below. Growth of these strains on Proteose medium in the dark in the absence of additional fixed carbon did not occur or was extremely minimal.
Table 17. Species of algae cultured on various fixed carbon feedstocks
<td>fixed carbon source</td><td>genus/species</td><td>Font /designation</td>
<td>Glucose</td><td>Chlorella protothecoides</td><td>UTEX 250</td>
<td>Glucose</td><td>Chlorella kessleri</td><td>UTEX 397</td>
<td>Glucose</td><td>Chlorella sorokiniana</td><td>UTEX 2805</td>
<td>Glucose</td><td>Parachlorella kessleri</td><td>SAG 12.80</td>
<td>Glucose</td><td>Pseudochlorella aquatica</td><td>SAG2149</td>
<td>Glucose</td><td>Chlorella reisiglii</td><td>CCAP 8/11</td>
<td>Glucose</td><td>Bracteococcus medionucleatus</td><td>UTEX 1244</td>
<td>Glucose</td><td>Prototheca stagnora</td><td>UTEX 1442</td>
<td>Glucose</td><td>prototheca moriformis</td><td>UTEX 1434</td>
<td>Glucose</td><td>prototheca moriformis</td><td>UTEX 1435</td>
<td>Glucose</td><td>scenedesmus rubescens</td><td>CCAP 232/1</td>
<td>Glycerol</td><td>Parachlorella kessleri</td><td>SAG 12.80</td>
<td>Glycerol</td><td>Chlorella protothecoides</td><td>CCAP211/8d</td>
<td>Glycerol</td><td>Bracteococcus medianucletus</td><td>UTEX 1244</td>
<td>Glycerol</td><td>prototheca moriformis</td><td>UTEX 288</td>
<td>Glycerol</td><td>prototheca moriformis</td><td>UTEX 1435</td>
<td>Glycerol</td><td>Chlorella minutissima</td><td>UTEX 2341</td>
<td>Glycerol</td><td>Chlorella sp.</td><td>CCAP 211/61</td>
<td>Glycerol</td><td>Chlorella sorokiniana</td><td>UTEX 1663</td>
<td>xylose</td><td>Chlorella luteoviridis</td><td>SAG 2133</td>
<td>xylose</td><td>Chlorella ellipsoidea</td><td>SAG 2141</td>
<td>xylose</td><td>Pseudochlorella aquatica</td><td>SAG 2149</td>
<td>xylose</td><td>Chlorella sp.</td><td>CCAP 211/75</td>
<td>xylose</td><td>prototheca moriformis</td><td>UTEX 1441</td>
<td>xylose</td><td>prototheca moriformis</td><td>UTEX 1435</td>
<td>Saccharose</td><td>Chlorella saccharophila</td><td>UTEX 2469</td>
<td>Saccharose</td><td>Chlorella luteoviridis</td><td>UTEX 22</td>
<td>Saccharose</td><td>Chlorella sp.</td><td>UTEX-EE102</td>
<td>Saccharose</td><td>Chlorella luteoviridis</td><td>SAG 2198</td>
<td>Saccharose</td><td>Bracteococcus medionucleatus</td><td>UTEX 1244</td>
<td>Saccharose</td><td>Chlorella minutissima</td><td>CCALA 20024</td>
<td>Fructose</td><td>Chlorella kessleri</td><td>UTEX 398</td>
<td>Fructose</td><td>Chlorella trebouxiodes</td><td>SAG 3.95</td>
<td>Fructose</td><td>Parachlorella kessleri</td><td>SAG 27.87</td>
<td>Fructose</td><td>Chlorella luteoviridis</td><td>SAG 2214</td>
<td>Fructose</td><td>Chlorella protothecoides</td><td>UTEX 31</td>
<td>Fructose</td><td>Chlorella protothecoides</td><td>UTEX 250</td>
<td>Fructose</td><td>Chlorella reisiglii</td><td>CCAP 8/11</td>
<td>Fructose</td><td>Chlorella protothecoides</td><td>CCAP211/8d</td>
<td>Fructose</td><td>prototheca moriformis</td><td>UTEX 1435</td>
<td>Fructose</td><td>scenedesmus rubescens</td><td>CCAP 232/1</td>
<td>arabinose</td><td>Chlorella sp.</td><td>CCAP 211/75</td>
<td>crafty</td><td>Chlorella kessleri</td><td>UTEX 263</td>
<td>crafty</td><td>Chlorella saccharophila</td><td>UTEX 2911</td>
<td>crafty</td><td>Parachlorella kessleri</td><td>SAG 12.80</td>
<td>crafty</td><td>Chlorella sp.</td><td>SAG 241.80</td>
<td>crafty</td><td>Chlorella angustoellipsoidea</td><td>SAG 265</td>
<td>crafty</td><td>Chlorella ellipsoidea</td><td>SAG 2141</td>
<td>crafty</td><td>Chlorella protothecoides</td><td>UTEX 250</td>
<td>crafty</td><td>Chlorella emersonii</td><td>CCAP 211/15</td>
<td>crafty</td><td>Bracteococcus minor</td><td>UTEX 66</td>
<td>crafty</td><td>Prototheca stagnora</td><td>UTEX 1442</td>
<td>crafty</td><td>prototheca moriformis</td><td>UTEX 1439</td>
<td>crafty</td><td>Chlorella cf. minute</td><td>CCALA 20024</td>
<td>crafty</td><td>scenedesmus rubescens</td><td>CCAP 232/1</td>
<td>Galactose</td><td>Bracteococcus minor</td><td>UTEX 66</td>
<td>Galactose</td><td>Parachlorella kessleri</td><td>SAG 14.82</td>
<td>Galactose</td><td>Parachlorella beijerinckii</td><td>SAG 2046</td>
<td>Galactose</td><td>Chlorella protothecoides</td><td>UTEX 25</td>
<td>Galactose</td><td>Chlorella sorokiniana</td><td>UTEX 1602</td>
<td>Galactose</td><td>Parachlorella kessleri</td><td>SAG 12.80</td>
<td>Galactose</td><td>Pseudochlorella aquatica</td><td>SAG 2149</td>
<td>Galactose</td><td>Chlorella luteoviridis</td><td>SAG 2214</td>
<td>Galactose</td><td>Chlorella ellipsoidea</td><td>CCAP 211/42</td>
<td>Galactose</td><td>Chlorella ellipsoidea</td><td>CCAP 211/50</td>
<td>Galactose</td><td>Chlorella protothecoides</td><td>UTEX 250</td>
<td>Galactose</td><td>Chlorella protothecoides</td><td>UTEX 264</td>
<td>Galactose</td><td>Bracteococcus medionucleatus</td><td>UTEX 1244</td>
<td>Galactose</td><td>prototheca moriformis</td><td>UTEX 1439</td>
<td>Galactose</td><td>prototheca moriformis</td><td>UTEX 1441</td>
<td>Galactose</td><td>Chlorella kessleri</td><td>KOALA 252</td>
<td>Acetate</td><td>Chlorella sorokiniana</td><td>UTEX 1230</td>
<td>Acetate</td><td>Chlorella sorokiniana</td><td>UTEX 1810</td>
<td>Acetate</td><td>Chlorella luteoviridis</td><td>UTEX 22</td>
<td>Acetate</td><td>Parachlorella kessleri</td><td>SAG 12.80</td>
<td>Acetate</td><td>Parachlorella kessleri</td><td>SAG 27.87</td>
<td>Acetate</td><td>Chlorella sp.</td><td>SAG 241.80</td>
<td>Acetate</td><td>Chlorella luteoviridis</td><td>SAG 2214</td>
<td>Acetate</td><td>Chlorella protothecoides</td><td>UTEX 31</td>
<td>Acetate</td><td>Chlorella protothecoides</td><td>UTEX 411</td>
<td>Acetate</td><td>Chlorella ellipsoidea</td><td>CCAP211/42</td>
<td>Acetate</td><td>Chlorella ovalis</td><td>CCAP211/21A</td>
<td>Acetate</td><td>Chlorella protothecoides</td><td>CCAP211/8d</td>
<td>Acetate</td><td>Prototheca stagnora</td><td>UTEX 1442</td>
<td>Acetate</td><td>Chlorella protothecoides</td><td>UTEX 250</td>
<td>Acetate</td><td>Chlorella sorokiniana</td><td>KOALA 260</td>
<td>Acetate</td><td>Chlorella vulgaris</td><td>CCAP211/79</td>
<td>Acetate</td><td>Parachlorella kessleri</td><td>SAG 14.82</td>
EXAMPLE 27
Renewable diesel production
Cell Production: One F tank batch of Chlorella protethecoides (UTEX 250) (approximately 1200 gallons) was used. The batch was allowed to run (#ZA07126) for 10 Ohrs, while glucose levels were controlled at 16 g/ l after which the corn syrup feed is terminated. Residual glucose levels dropped to <0 g/L two hours later. This resulted in a final age of 102hrs. The final volume of the broth was 4542 L (1120 gallons). Both ongoing contamination checks and a full analysis of a final broth sample failed to show any signs of contamination. The fermentation broth was centrifuged and drum dried. The drum-dried cells were resuspended in hexane and homogenized at approximately 100 bar. Hexane extraction was then performed using standard methods and the resulting algal triglyceride oil was determined to be free of residual hexane.
Renewable diesel production: The algal triglyceride oil had a lipid profile of approximately (3% C18:0, 71% C18:1,15% C18:2, 1% C18:3, 8% C16:0 and 2 % of other components The oil was first subjected to hydrocracking resulting in a loss of yield of approximately 20% to water and gases, then hydroisomerization was carried out with an approximate loss of 10% in yield to gases. Then a first distillation was carried out to remove the naptha fraction, leaving the desired product. Approximately 20% of the material was lost to naptha in this first distillation. A second distillation was then carried out at a temperature sufficient to remove fractions necessary to meet ASTM specification D975 but leaving a bottom fraction that did not meet the 90% point for the D975 distillation. Approximately 30% of the material was left in the bottom fraction in the second distillation. The resulting material was then tested to all ASTM D975 specifications.
Figures 29 and 30 illustrate a gas chromatograph and boiling point distribution plot, respectively, of the final renewable diesel product produced by the method of the invention. Table 18 shows the boiling point distribution of the resulting renewable diesel product and Table 19 shows the results of an analysis of the final product for compliance with ASTM D975 specifications.
Table 18. Renewable diesel product boiling point distribution. BP = boiling point
<td>% dough</td><td>BP °C</td><td>% dough</td><td>bp</td><td>% dough</td><td>bp</td><td>% dough</td><td>bp</td>
<td>recovered</td><td></td><td>recovered</td><td>°C</td><td>recovered</td><td>°C</td><td>recovered</td><td>°C</td>
<td>PPI</td><td> 150.4</td><td> 26.0</td><td> 261.8</td><td> 52.0</td><td> 303.8</td><td> 78.0</td><td> 315.8</td>
<td> 1.0</td><td> 163.6</td><td> 27.0</td><td> 264.6</td><td> 53.0</td><td> 304.4</td><td> 79.0</td><td> 316.4</td>
<td> 2.0</td><td> 173.4</td><td> 28.0</td><td> 266.2</td><td> 54.0</td><td> 304.8</td><td> 80.0</td><td> 317.0</td>
<td> 3.0</td><td> 175.2</td><td> 29.0</td><td> 268.2</td><td> 55.0</td><td> 305.2</td><td> 81.0</td><td> 317.6</td>
<td> 4.0</td><td> 188.0</td><td> 30.0</td><td> 271.0</td><td> 56.0</td><td> 305.6</td><td> 82.0</td><td> 318.4</td>
<td> 5.0</td><td> 194.8</td><td> 31.0</td><td> 272.4</td><td> 57.0</td><td> 306.0</td><td> 83.0</td><td> 319.0</td>
<td> 6.0</td><td> 196.6</td><td> 32.0</td><td> 273.4</td><td> 58.0</td><td> 306.4</td><td> 84.0</td><td> 319.6</td>
<td> 7.0</td><td> 197.8</td><td> 33.0</td><td> 276.2</td><td> 59.0</td><td> 306.6</td><td> 85.0</td><td> 320.2</td>
<td> 8.0</td><td> 207.4</td><td> 34.0</td><td> 280.0</td><td> 60.0</td><td> 307.0</td><td> 86.0</td><td> 320.8</td>
<td> 9.0</td><td> 210.0</td><td> 35.0</td><td> 282.4</td><td> 61.0</td><td> 307.4</td><td> 87.0</td><td> 321.2</td>
<td> 10.0</td><td> 214.4</td><td> 36.0</td><td> 285.2</td><td> 62.0</td><td> 307.6</td><td> 88.0</td><td> 321.8</td>
<td> 11.0</td><td> 216.6</td><td> 37.0</td><td> 287.8</td><td> 63.0</td><td> 308.0</td><td> 89.0</td><td> 322.2</td>
<td> 12.0</td><td> 217.6</td><td> 38.0</td><td> 289.6</td><td> 64.0</td><td> 308.4</td><td> 90.0</td><td> 322.4</td>
<td> 13.0</td><td> 221.4</td><td> 39.0</td><td> 291.8</td><td> 65.0</td><td> 308.8</td><td> 91.0</td><td> 322.8</td>
<td> 14.0</td><td> 227.6</td><td> 40.0</td><td> 294.2</td><td> 66.0</td><td> 309.2</td><td> 92.0</td><td> 323.2</td>
<td> 15.0</td><td> 229.8</td><td> 41.0</td><td> 295.8</td><td> 67.0</td><td> 309.6</td><td> 93.0</td><td> 324.4</td>
<td> 16.0</td><td> 233.2</td><td> 42.0</td><td> 296.8</td><td> 68.0</td><td> 310.2</td><td> 94.0</td><td> 326.8</td>
<td> 17.0</td><td> 235.8</td><td> 43.0</td><td> 297.6</td><td> 69.0</td><td> 310.6</td><td> 95.0</td><td> 329.4</td>
<td> 18.0</td><td> 236.8</td><td> 44.0</td><td> 298.4</td><td> 70.0</td><td> 311.2</td><td> 96.0</td><td> 333.6</td>
<td> 19.0</td><td> 240.2</td><td> 45.0</td><td> 299.2</td><td> 71.0</td><td> 311.6</td><td> 97.0</td><td> 339.4</td>
<td> 20.0</td><td> 245.6</td><td> 46.0</td><td> 299.8</td><td> 72.0</td><td> 312.2</td><td> 98.0</td><td> 346.2</td>
<td> 21.0</td><td> 248.0</td><td> 47.0</td><td> 300.6</td><td> 73.0</td><td> 313.0</td><td> 99.0</td><td> 362.8</td>
<td> 22.0</td><td> 250.2</td><td> 48.0</td><td> 301.2</td><td> 74.0</td><td> 313.6</td><td>FBP</td><td> 401.4</td>
<td> 23.0</td><td> 253.6</td><td> 49.0</td><td> 302.0</td><td> 75.0</td><td> 314.2</td><td></td><td></td>
<td> 24.0</td><td> 255.2</td><td> 50.0</td><td> 302.6</td><td> 76.0</td><td> 314.8</td><td></td><td></td>
<td> 25.0</td><td> 256.8</td><td> 51.0</td><td> 303.2</td><td> 77.0</td><td> 315.4</td><td></td><td></td>
Table 19. Analytical report for renewable diesel product using D975 specifications.
<td>number method</td><td>test description</td><td>Results</td><td>units</td>
<td>D93A</td><td>Flash point (PMCC)</td><td> 70</td><td>°C</td>
<td>D2709</td><td>water and sediment</td><td> 0</td><td>Vol %</td>
<td>D86</td><td>90% distillation (recovered)</td><td> 301.0/573.9</td><td>°C/°F</td>
<td>D445</td><td>Kinematic viscosity @ 40.0°C (104.0°F)</td><td> 2.868</td><td>mm2/sec</td>
<td>D482</td><td>Sulfur</td><td> <0.001</td><td>Weight %</td>
<td>D5453</td><td>Sulfide</td><td> 2.4</td><td>ppm</td>
<td>D130</td><td>Copper Corrosion 3hrs @ 50°C</td><td>lbs</td><td></td>
<td>D613</td><td>Cetane number***</td><td> >65</td><td></td>
<td>D976</td><td>calculated cetane index</td><td> 71.2</td><td></td>
<td>D2500</td><td>cloud point</td><td> -3</td><td>°C</td>
<td>D524</td><td>Carbon residue of 10% of Ramsbotton</td><td> 0.02</td><td>Weight%</td>
<td>D97</td><td>Pour point</td><td> -3</td><td>°C</td>
<td>D2274</td><td>Total insolubles (oxidation stability) 40hr test</td><td> 4.0</td><td>mg/100ml</td>
<td>D4052</td><td>Density @15.0 °C (59.0°F)</td><td> 793.8</td><td>kg/mt3</td>
<td>D4176-1</td><td>Appearance by visual inspection (lab)</td><td>clear and bright/ happens</td><td>Visual</td>
<td>D4176-1</td><td>Appearance by visual inspection (lab)</td><td>water free /happens</td><td>Visual</td>
<td>D4176-1</td><td>Appearance by visual inspection (lab)</td><td>particles / passes</td><td>Visual</td>
<td>D1500</td><td>ASTM color</td><td>L0.5</td><td></td>
<td>D664</td><td>acid number</td><td> <0.10</td><td>mg KOH/g</td>
<td>D6079</td><td>Lubricity (wear mark)</td><td> 405</td><td>zm</td>
SEQUENCES
SEQIDNOil
Chlorella HUP promoter (subsequence GenBank accession number X55349); gaieagacgggcctgacctgcgagataatcaagtgctcgtaggcaaccaactcagcagctgcttggtgttgggtctgcaggatagtgtt gcagggccccaaggacagcaggggaacttacacctlgtccccgacccagttttatggagtgcattgcctcaagagoctagccggagc gctaggctacatacttgccgcaccggtatgaggggatatagtactcgcactgcgctgtctagtgagatgggcagtgctgcccataaac aactggclgclcagccatógttggcggaccattctgggggggccagcaatgcctgactttcg^tagggtgaaaactgaacaaagac taccaaaacagaatttcttcctccttggaggtaagcgcaggccggcccgcctgcgcccacatggcgctccgaacacctccatagctgt aagggcgcaaacatggccggactgttgtcagcactctttcatggccatacaaggtcatgtcgagattagtgctgagtaagácacUtca ccccaigUcgaltgaagcc^gacttcalgccaacctgccc^gggc^agcagacgtatgccalcatgaccactagccgacatgcg ctgtcttttgccaccaaaacaactggtacaccgctcgaagtcgtgccgcacacctccgggagtgagtccggcgactcctccccggcg ggccgcggccctacctgggtagggtcgccatacgcccacgaccaaacgacgcaggaggggallggggtagggaatccGaaccag cctaaccaagacggcacctataataataggtggggggactaacagccctatatcgcaagctttgggtgcctatcttgagaagcacgag ttggagtggctgtgtacggtcgaccctaaggtgggtgtgccgcagcctgaaacaaagcgtctagcagctgcttctataatgtgtcagcc gttgtgtttcagttatattgtatgclattgtttgttcgtgclaggglggí;gcaggcccacctactgtggcgggccattggttggtgcttgaaú gcctcaccMctaaggtctgaacgctcactcaaacgcctttgtacaactgcagaactttccttggcgctgcaactacagtgtgcaaacca gcacaíagcactcccttacatcacccagcagtacaaca cgccggcagcgRcatgtccgggcccagggcagcggtggtgecataaatgtcggtgatggtggggaggggggecgtcgccacacc attgccgttgctggcigacgcatgcacatgtggcctggctggcaccggcagcaotggtctccagccagccagcaagtggctgttcag gaaagcggccatgtt^tggtccetgcgcatgtaattococagí4caaaggagggaacagcttggatttgatgtag1.gcccaaccg^ ctgaaigtgcgatggcaggtccctttgagtolccc^aattactagcagggcactgtgacctaacgcagcatgccaaccgcaaaaaaat gattgacagaaaalgaageggtgt^caaiatttgctgtíd.ttattc gttttaatcagcaaccaagttcgaaacgcaactatcgtggtgatca agtgaacctcatcagacttacctcgttcggcaaggaaacggaggcaccaaattccaatttgatatiaícgcttgccaagctagagctgat aaccctcagtgaagggaccaícagaccagaaagaccagatctcctcctcgacaccgagagagtgttgcggcagtaggacgacaag
SEQ ID NO:3
b. braunii malate dehydrogenase 5'UTR: aattggaaaccccgcgcaagaccgg^tgtttggccgcctgaccggaaagggggggcctgfcccgaagggggtciatctcttgggg gatgtcgggcgcggmgtcgatgttgatggacctcttcttcgaccatgtcggggtcgaggccaagagccgcgtccatttcgccgagt tcatgatggaggtgaatgaccgcatogccaccgaacgcgccaagaagcgggcgaccgatogcccccgtcgctgcagcccttgccg aggaagtccggctgctggcgttcgacgagatgatggtgacgaacagcccggacgcgatgatcctgtcgcggctgjtcaccgcgctg atcgaggcgggggtgacgatcgtcaccaectccaaccggccgcccagggatetetataagaacgggctcaaccgcgagcatttcct gcccttcaícgcgctgaicgaggcgcggctggacgtgctggcgctgaacggcccgaccgactatcggcgcgaccggctggggcg gctggacacgtggttggtgcccaatggccccaaggcgacgattaccttgtcggcggcgttcttccgcctgaccgactatccggtogag gatgccgcgcatgtgccctctgaggacctgaaggtgggcgggcgcgtgctgaatgtccccaaggcgctgaagggcgtcgcggtctt cicgttcaagcggttgtgcggcgaagcgcggggggcggcggactatctggcggtegcgcggg^ttccacaccgtcatcctggtcg gaatccccaagctgggggcggagaaccgcaacgaggcggggcgcttc^ccagclgatcgacgegctctacgaacataaggtcaa gctgctcgccgcagccgatgccagcccgccgaactctatgaaaccggcga.cggccggttcgagtttgagcgcagatcagccggttg gaagagatgcgctccgaggattatctggcccaaggccatggcteggag^gccttgatcaggccttaatgcacttcgcaaccattatc gtttaaaatcttaaactctgtggaataacggttccccgacgccgcaatacacgtacgtccactacggagtaggattgga
SEQIDN0:4
Promotor de Chlamydomonas RBCS2: cgcttagaag3tttegata3ggcgccaga3ggagcgcagccaaaccaggatgatgtttgatggggtatttgagcacttgcaacccttat ccggaagccccctggcccacaaaggcíaggcgccaatgcaagcagtÉcgcatgcagcccctggagcggtgccctcctgataaacc ggccagggggccialgUclltaclUUíacaagagaaglcacloaacatcllaaacggícliaagaagtotatocgg
SEQ ID NO:5
CMV-Hyg-CMV BamHI-SacII pCAMBIA cassette:
ggatccccgggaaftcggcgcgccgggcccaacatggtggagcacgíicactctcgtctactccaagaatatcaaagatacagtctca gaagaccaaagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccaltgcccagctatctgtcacttcatca aaaggacagtagaa&aggaaggtggcacctacaaatgccatcawgcgataaaggaaaggctatcgttcaagatgectctgccgaca glggtcccaaagatggacccccacccacgaggagcalcgtggaaaaaga^acgttccaacc&cgtcttcaaagcaagt^attgat gtgataacaiggtggagcacgacactctcgtctactccaagaaiatcaaagatacagtctcagaagaccaaagggctattgagacttttc aacmggglaatot^gmaaAcctcMattccatígcocagctetctgteacttcídcaaaaggacagtamaaggaaggtgg cacctacaaatgccaícattgcgataaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaagatggacccccaccc acgcacaatcccactatccttcgcaagaccttectctatataaggaagttcatttcatttggagaggacacgctgaaatcaccagtctctct cíacaaalctatclctcfcgagcttlcgcagatcccggggggcaatgagatatgaaaaagcctgaactcaccgcgacgtctgtcgagaa gtitctgatcgaaaagitcgacagcgtctecgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtag^g ggcgtggautgtcctgcgg^aaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcstcggccgcgctccc gattccggaagtgcttgacattggggagtttagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacc tgcctgaaaccgaactgcccgctgítctacaaccggtcgcggaggctatggatgcgatcgctgcggccgatcttagccagacgagcg ggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcact ggcaaacígtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaa gtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcaltgactggagcgag gcgatgttoggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcsgacgcgctactt cgagcggaggcatccggagcttgcaggatcgccacgactocgggcgtatatgctccgcattggtcttgaccaactctatcagagcttg gttgacggcaatttegatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtaca caaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactc gtccgagggcaaagaaatagagtagatgccgaceggatctgtcgategacaagctcgagtttctccataataatgtgtgagtagttccc agataaggga^tagggttectatagg^ttcgctcatgtgttgagcataiaagaaacccttagtatgtatttgtatttgtaaaatacttctatc a.ataaaatttctaattcctaaaaccaaaatccagtactaaaatccagatcocccgaattaattcggcgttaattcagtacattaaaaacgtcc gcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgccaccagccagccaacagctccccgaccggcagct cggcacaaa8tcaccactcgatacaggcagcccatcagtccgggacggcgtcagcgggag8gcegttgtaaggcggcagactttg ctc^gttaccgatgctattcggaagaacggcaactaagctgccgggtttgaaacacggatgatctcgcggagggtagcatgttgattg taacgatgacagagcgttgctgcctgtgiitcaccgcgg
SEQ ID N0:6 caaccacgtcttcaaagcaa
SEQ ID ΝΌ-.7 agcaatcgcgcatatgaaat
SEQ ID X0:8
ArGCTTCTTCAGGCCTTTCrrTTTCTTCTTGCTOGTTTTGCTGCCAAGATCAGCGCC
TCTATGACGAACGAAAACCTCGGATAGACCACTTGTGCACTTTACACCAAACAAG
GGCTGGATGAATGACCCCAATGGACTGTGGTACGACGAAAAAGATGCCAAGTGG
CATCTGTACTTTCAATAC.AACCCAGAACGATACTGTCTGGGGGACGCCA'nOTm'
GGGGCCACGCCACGTCCGACGACCTGACCAATTGGGAGGACCAACCAATAGCTA
TCGCTCCGAAGAGGAACGACTCCGüAGCATTCTCGGGTTCCATGGTGGTTGACTA
CAACAATACTrCCGGCTTTTTCAACGATACCATTGACCCGAGACAACGCTGCGTG
CTGGACGGTGGATACACTTTTACAGAGTATCAGAAGAACCCTGTGCTTGCTGCAA ATTCGACTCAGTTCCGAGATCCGAAGGTCFTTTGGTACGAGCCCTCGCAGAAGTG GATCATGACAGCGGCAAAGTCACAGGACTACAAGATCGAAATTTACTCGTCTGA CGACCTTAAATCCTGGAAGCTCGAATCCGCGTTCGCAAACGAGGGCTTTCTCGGC TACCAATACGAATGCCCAGGCCTGATAGAGGTCCCAACAGAGCAAGATCCCAGC
AAGTCCTACTGGGTGATG'nTATrTCCATTAATCCAGGAGCACCGGCAGGAGGTT CrrTTAATCAGTACTTCGTCGGAAGCTTTAACGGAACTCATTTCGAGGCATnGAT aaccaatcaagagtagttgattttggaaaggactactatgccctgcagactttct tcaatactgacccgacctatgggagcgctcttggcattgcgtactág
GGAGTATrCCGCATTCGTTCCTACAAACCCTTGGAGGTCCTCCATGTCGCTCGTG AGGAAATTCTCTCTCAACACTGAGTACCAGGCCAACCCGGAAACCGAACTCATA AACCTGAAAGCCGAACCGATCCTGAACATTAGCAACGCTGGCCCCTGGAGCCGG TTTGCAACCAACACCACGTTGACGAAAGCCAACAGCTACAACGTCGATCTTTCGA ATAGCACCGGTACACTTGAATTTGAACTGGTGTATGCCGTCAATACCACCCAAAC
GATCTCGAAGTCGGTGTTCGCGGACCTTCCCCTCTGGTITAAAGGCCTGGAAGAC
CCCGAGGAGTACCTCAGAATGGGTTTCGAGGTTTCTGCGTCCTCCTTCTTCCTTGA TCGCGGGAACAGCAAAGTAAAATTTGTTAAGGAGAACCCATATTTTACCAACAG GATGAGCGTTAACAACCAACCATrCAAGAGCGAAAACGACCTGTCGTACTACAA
AGTGTATGGTTTGCTTGATCAAAATATCCTGGAACTCTACTTCAACGATGGTGAT GTCGTGTCCACCAACACATACTTCATGACAACCGGGAACGCACTGGGCTCCGTGA
ACATGACGACGGGTGTGGATAACCTGTrCTACATCGACAAATTCCAGGTGAGGG
AAGTCAAGTGA
SEQ ID N0:9
TCCGGTOTGTTGTAAGTCCA
SEQID NOGO
TTGTCGGAATGTCATATCAA m vrui i
SEQ ID NO: 13
TCCGÜTG TGTTGTA AGTCCA
SEQ ID NO: 14
MTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLF WGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAI WTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMT AAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVM FISINPGaT'AGGSFNQYFVGSFNGTHFF AFGNQSRVVnFGKDYYAFQTFFNTDPTYGS ALGIA WASNWEYSAFVPTNPWRSS.MSLVRKFSLNTEYQANPETELINLKAEPILNISN AGPWSRFATNTrLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLW'FK GLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSY YKVYGLLDQNÍLELYFNDGDWSTNTYFMTTGNALGSVNMI'TGVDNLFYIDKFQVR
EVK
SEQ1DNO:15
MLLQAFFLLLAGFAAKISAS
SEQID NO:16
MANKSLLLLLLLGSLASG
SEQID NO:17
MARLPLAALG
SEQ ID NO: 18
MANKLLLLLLLLLLPLAASG
SEQID NO:19
MII.QAFLinJ.AGFAAKlSAS.MTNETSDRPLVHFTPNKGWlNDPNG.LWYDEKI)AKWHLYF
QYNPNDTVWGTPLEWGHATSDDLTNWEDQP1AIAPKRNDSGAFSGSMVVDYNNTSGFFND TIDPRQRCVAIWTYNTTESEEQYISYSLDGGYTFTBYQKNPVLAANSTQFRDPKVFWYEPSQ KW4TAAKSQDYKIEIYSSDDLKSWKLESAFANEGFWYQYECPGUEVPTEQDPSKSYWV
MFISINT'GAPAGGSFNQYFVGSFNGTHFEAFDNQSRVA/DFGK.DYYALQTFFNTDPTYGSALG IAWASWEYSAFVFrNPWRSSMSLVRKFSLNTEYQANPETELlNLKAEPILNISNAGPWSRF ATNTTTLTKANSYNVDLSNSTGTLEFELVYAWITQI'ISICSVFADLSLWFKGLEDPEEYLRMG
SEQ ID NO:20
CTGACCCGACCCTATGGGAGCGCTCTTGGC
SEQ ID NO:21
CTTGACTTCCCTCACCTGGAATTTGTCG
SEQ ID NO:22
TGGCATCTGTACTTTCAATACAACCCGAACGA.TACTGTCTGGGGGACGCCATTGT TTTGGGGCCACGCCACGTCCGACGACCTGACCAATTGGGAGGACCAACCAATAG CrATCGCTCCGAAGAGGAACGACTCCGGAGCATTCrCGGGT<sup>-</sup>rCCA.TGGTGGTTGA CTACAACAATACTTCCGGCTTTTTCAACGATACCATTGACCCGAGACAAACGCTGC GTGGCCATATGGACTTACAACACACCGGAGTC.CGAGGAGCAGTACATCTCGTAT
AGCCTGGACGGTGGA'lACACnrrACAGAGTATCAGAAGAACCCTGTGCTTGCTG
C.AAATTCGACTCAGTTCCGAGATCCGAAGGTCTTTTGGTACGAGCCCTCGCAGAA /ΥΤΚΙ/Ύ A Tí*» λ ΠΥ'! Á E' Λ AA Λ rviV» A Á /Ui AG*TA Γ' * AAAAA .•ryj^ p<sub>Λ</sub>
TGACGACCTTAAATCCTGGAAGCTCGAATCCGCGTTCGCAAACGAGGGCTTTCTC GGCTACCAATACGAATGCCCAGGCCTGATAGAGGTCCCAACAGAGCAAGATCCC AGCAAGTCCTACTGGGTGATGTTTATITCCATTAATCCAGGAGCACCGGCAGGAG GTTCTTTTAATCAGTACTTCGTCGGAAGCTTTAACGGAACTCATTTCGAGGCATTT GATAACCAATCAAGAGTAGTTGATTTTGGAAAGGACTACTATGCCCTGCAGACTT TCTTCAATACTGACCCGACCTATOGGAGCGCTCTTGGCATTGCGTGGGCTTCTAA CTGGGAGTATTCCGCATTCGTTCCTACAAACCCTTGGAGGTCCTCCATGTCGCTC GTGAGGAAATTCTCTCTCAACACTGAGTACCAGGCCAACCCGGAAACCGAACTC ATAAACCTGAAAGCCGAACCGATCCTGAACATTAGCAACGCTGGCCCCTGGAGC
CGGTTTGCAACCAACACCACGTTGACGAAAGCCAACAGCTACAACGTCGATCTTT CGAATAGCACCGGTACACTTGAATTTGAACTGGTGTATGCCGTCAATACCACCCA
AACGATCTCGAAGTCGGTGTTCGCGGACCTTCCCCTCTGGTTTAAAGGCCTGGAA GACCCCGAGGAG'TACCTCAGAATGGGTTTCGAGGTTTCTGCGTCCTCCTTCTTCCT TGATCGCGGGAACAGCAAAGTAAAATTTGTTAAGGAGAACCCATATTTTACCAA
CAGGATGAGCGTTAACAACCAACCATTCAAGAGCGAAAACGACCTGTCGTACTA CAAAGTGTATGGTTTGCTTGATCAAAATATCCTGGAACTCTACTTCAACGATGGT
GATGTCGTGTCCACCAACACATACTTCATGACAACCGGGAACGCACTGGGCTCCG TGAACATGACGACGGGTGTGGATAACCTGTTCTACATCGACAAATTCCAGGTGA
GGGAAGTCAAGTGAgatctgtcgatcgacaagctcgagtttclccataataatgtgtgagÉagttcccagataagggaatta gggttcctatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgíaaaatacttctatcaataaaatttctaatt cclaaaaccaaaatccaglactaaaatgaccagatcc
Contents70
27 sheets
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70 members in 18 offices
Priority claims4
| Document | Office | Kind | Date |
|---|---|---|---|
| 94158107 | United States of America | P | |
| 95917407 | United States of America | P | |
| 96829107 | United States of America | P | |
| 2406908 | United States of America | P |
Members70
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| AU2008259834A1 | Australia | A1 | |
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| NZ595029A | New Zealand | A | |
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| US8476059B2 | United States of America | B2 | |
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| EP3546588A3 | European Patent Office (EPO) | A3 | |
| BRPI0811967B1 | Brazil | B1 |
Numbers
- Application
- 99800
Titles2
- English
- OIL PRODUCTION IN MICROORGANISMS
- Spanish
- PRODUCCIÓN DE ACEITE EN MICROORGANISMOS
Classification
- CPC, 33
- C10L1/026
- C12P5/00
- C12N1/32
- C12N9/0006
- C12N9/0008
- C12N9/1205
- C12N9/16
- C12N9/20
- C12N9/2408
- C12N9/88
- C12P7/6463
- C12P7/649
- Y02T50/678
- C12P7/6409
- C07H21/04
- C12N9/14
- C12N9/2431
- C12Y302/01026
- Y02E50/30
- C12P7/6458
- C11B1/00
- C12M1/00
- C10L1/06
- Y02E50/10
- C12P21/06
- C12P5/02
- C11C3/00
- Y02P20/52
- C10L2200/0484
- C10L2270/026
- C10L2270/04
- C12N1/12
- C12N15/79
- IPC, 4
- C12M1 00
- C12P5 00
- C12P7 6458
- C12P7 649