Tailored oils produced from recombinant heterotrophic microorganisms.

(54) Title: SPECIFIC OILS PRODUCED FROM RECOMBINANT HETEROTROPHIC MICROORGANISMS.

(54) Title: TAILORED OILS PRODUCED FROM RECOMBINANT HETEROTROPHIC MICROORGANISMS.

(57) Summary

The present invention relates to a microalgae cell comprising an exogenous nucleic acid, wherein the exogenous nucleic acid (a) encodes a ketoacyl-ACP synthase enzyme or (b) disrupts or down-regulates the expression of an endogenous ketoacyl-ACP synthase , where the microalgae cell is a cell of the genus Prototheca. Methods and compositions are provided for the production of oil, fuels, oleochemicals, and other compounds in recombinant microorganisms, including oil-bearing microorganisms, and low-cost culture methods of such microorganisms. Microalgae cells containing exogenous genes encoding, for example, a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, a polysaccharide-degrading enzyme, a ketoacyl-ACP synthase enzyme, an acyl-ACP thioesterase fat, a acyl-CoA / fatty aldehyde reductase, a fatty aldehyde decarbonylase and / or an acyl carrier protein are useful in the manufacture of transportation fuels, such as renewable diesel, biodiesel and renewable jet fuel, as well as oleochemicals, such as functional fluids, surfactants, soaps and lubricants.

(57) Abstract

Methods and compositions for the production of oil, fuels, oleochemicals, and other compounds in recombinant microorganisms are provided, including oil-bearing microorganisms and methods of low cost cultivation of such microorganisms. Microalgal cells containing exogenous genes encoding, for example, a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, a polysaccharide-degrading enzyme, a keto acyl-ACP synthase enzyme, a fatty acyl-ACP thioesterase, a fatty acyl-CoA / aldehyde reducíase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and / or an acyl carrier protein are useful in manufacturing transportation fuels such as renewable diesel, biodiesel, and renewable jet fuel, as well as oleochemicals such as functional fluids, surfactants, soaps and lubricants.

Institute

Mexican Property

Industrial iIh

<img file="MX339639B_D0001.tif" />

PATENT TITLE NO. 339639

Headlines):

Home:

Denomination:

Classification:

Inventor (s):

SOLAZYME, INC,

225 Gateway Boulevard, South San Francisco, California, 94080, USA,

SPECIFIC OILS PRODUCED FROM RECOMBINANT HETEROTROPHIC MICROORGANISMS.

lnt.CI.8: C10L1 / 02: C10M101 / 04; C12N1 / 13: C12N15 / 09: C12N15 / 74: C12P7 / 64

SCOTT FRANKLIN: ARAVIND SOMANCHI; JANICE WEE: GEORGE RUDENKO; JEFFREY L. MOSELEY: WALT RAKITSKY

REQUEST

Numbers

MX / a / 2012/013777

International filing deadline: May 27, 2011

PRIORITY

Country:

US

US

US

US

Date:

May 2010, „August 18, 2010November 16, 2010 December 29, 2010

Number:

61/349,774

61/374,992

61/414,393

61/428,192

- * - s

- | W ψ A!

Validity: Twenty years · Expiration Date: May 27, 2031 • r '' '..................' ¹

The% jj> reference attempt is granted based on articles · V, 3 * fraction V, 6th useful fraction, and SS of the Industrial Property Law.

D # in accordance with article 23 of the Industrial Property Law, cost from the date of preseeation of the sotidhjjSJnternai dapchos. - 'the patent has a validity of twenty non-expendable years, it will be subject-toLpsoóWla' 'Sarita to keep people il. : Yes' -ST

Qaten subscribes this Bulo does so based on the dtapueato by the aniorios O tracciones III and 7 ° bis 2 of the Industrial Property Law (Diario flfici al déte Federación (DOF) 06/27/1991, réfcrtwWtalf 02 / Ó8 / 1994, 10/25/1996, 12/26/1997, 07/05/1999, ^ WWSOS ^ · - ^⁷²⁰⁰⁶Attraction V

07/01/2002, 07/15/2004, 07/28/2004 and 09/07/2007); Articles 1, 3, 4, 5, section V, subsection a), 16 sections I and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property (DOF) 12/27/1999, amended on 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1, 3 and 5, subsection a) of the Agreement that delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Heads of Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

Issue Date: June 2, 2016

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33763*,

SPECIFIC OILS PRODUCED 1Γ START LIR - RECOMBINANT HETEROTROPHIC MICROORGANISMS

CROSS REFERENCE TO RELATED REQUESTS

This application claims the benefit under 35 USC

119 (e) of the United States provisional patent application

United no. 61 / 349,774, filed May 28, 2010, U.S. Provisional Patent Application No.

61 / 374,992, filed August 18, 2010, U.S. Provisional Patent Application No. 61 / 414,393, filed on November 16, 2010, and U.S. Provisional Patent Application No. 61 / 428,192, filed on December 2010. Each of these applications is incorporated herein by reference in its entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application includes a sequence listing shown on pages 1-195, attached hereto.

FIELD OF THE INVENTION v

The present invention relates to the production of oils, fuels, and oleochemicals prepared from microorganisms. In particular, the description relates

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IA FRCWEOAR 'WllSTRIAL

<img file="MX339639B_D0004.tif" />

of tools, including cranberries. aldehydes.

with oleaginous microalgae, the same methods for the production of lipid compounds, fatty acid esters, alcohol acids, and alkanes, as well as methods and reagents to genetically modify them with the aim of improving production efficiency and modifying the type and composition of the oils they produce.

BACKGROUND OF THE INVENTION

Fossil fuel is a general term for buried geological fuel deposits of organic materials, formed from rotting plants and animals that have been converted to crude oil, coal, natural gas, or heavy oils from exposure to heat and pressure in the bark. of the earth for billions of years.

Fossil fuels are a finite, non-renewable resource.

The increase in energy demand by the global economy has also brought increased pressure on the cost of hydrocarbons. Aside from energy, many industries, including plastics and chemical manufacturers, rely heavily on the availability of hydrocarbons as a raw material for their manufacturing processes. Cost-effective alternatives to current sources of

<img file="MX339639B_D0005.tif" />

Supply could help mitigate the upward pressure on energy and raw material costs.

PCT publication no. 2008/151149 describes methods and materials for cultivating microalgae for oil production and particularly exemplifies the production of diesel fuel from oil produced by Chlorella protothecoides microalgae. There remains a need for improved methods of producing oil in microalgae, particularly methods that produce oils with shorter chain lengths and a higher degree of saturation and without pigments, with increased performance and efficiency. The present invention satisfies this need.

SUMMARY OF THE INVENTION

The present invention provides oil-bearing microbial cells, preferably microalgae cells, that have different lipid profiles, and includes recombinant cells that express exogenous genes encoding proteins such as acyl fatty-ACP thioesterases. The present invention further provides methods for preparing lipids and oil-based products, including fuels such as biodiesel, renewable diesel, and reactor fuel, from such cells.

<img file="MX339639B_D0006.tif" />

IMPIOUS"

In a first aspect, the present invention provides oil-bearing microbial cells, preferably microalgae cells, that have a lipid profile that is at least

1% or at least 5%, preferably at least 3%, C8: 0. In some cases, the lipid profile is at least 10% or at least 15%, preferably at least 12%, C8: 0. In some embodiments, the cell is a recombinant cell. In some cases, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards C8 chain length acyl fatty substrates. In some embodiments, exogenous genes encode an acyl-ACP thioesterase Cuphea palustris. In some cases, the cell is a cell

Prototheca. In some cases, the cell is from a genus or species of microalgae selected from the microalgae identified in Table 1.

In a second aspect, the present invention provides oleaginous microbial cells, preferably microalgae cells, that have a lipid profile that is at least 4% C10: 0. In some cases, the lipid profile is at least 20%, at least 25%, or at least 30%, preferably at least 24%, C10: 0. In some cases, the ratio of C10: 0 to

C12: 0 is at least 6: 1. In some embodiments, the cell is a recombinant cell. In some cases, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards CIO chain length acyl fatty-ACP substrates. In some embodiments, exogenous genes encode an acyl-ACP thioesterase protein from a species selected from the group consisting of Cuphea hookeriana and Ulmus americana. In some cases, the cell is a Prototheca cell. In some embodiments, the cell is from a genus or species selected from the microalgae identified in Table 1.

In a third aspect, the present invention provides oil-bearing microbial cells, preferably microalgae cells, that have a lipid profile that is at least

10% or at least 15%, preferably at least 13%, C12: 0. In some cases, the lipid profile is at least 30%, at least

35% or at least 4 0%, preferably at least 34%, C12-.0. In some cases, the ratio of C12 to C14 is at least 5: 1. In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards C12 chain length acyl fatty-ACP substrates. In some cases, the

ΙΜΡΙ

MEXICAN INSTITUI

<img file="MX339639B_D0007.tif" />

Df. IA INDUSTRIAL PROPERTY

<img file="MX339639B_D0008.tif" />

encode acyl-ACP thioesterase proteins from

Umbellularia californica and Cinnamomum camphora that have hydrolysis activity towards C12 chain length acyl fatty substrates. In some embodiments, the cell is a Prototheca cell.

In a fourth aspect, the present invention provides oleaginous microbial cells, preferably microalgae cells, that have a lipid profile that is at least

5% or at least 15%, preferably at least 10%, C14: 0. In some cases, the lipid profile is at least 40%, at least

45%, or at least 50%, preferably at least 43%, C14: 0. In some cases, the ratio of C14: 0 to C12: 0 is at least 7: 1.

In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards C14 chain length acyl fatty-ACP substrates. In some embodiments, the acyl-ACP thioesterase protein is from a species selected from the group consisting of Cinnamomum camphora and

Ulmus americana. In some cases, the cell is a cell

Prototheca. In some embodiments, the cell is of a

<img file="MX339639B_D0009.tif" />

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IkJíTiTi itc utrir »κιλ genus or species selected from the mirmaigag idpnt-ί f-icadas in Table 1.

In a fifth aspect, the present invention provides oil-bearing microbial cells, preferably microalgae cells, that have a lipid profile that is at least

10% or at least 20%, preferably at least 15%, C16: 0. In some cases, the lipid profile is at least 30%, at least

35% or at least 40%, preferably at least 37%, C16: 0. In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards C16 chain length acyl fatty-ACP substrates. In some embodiments, the recombinant cell comprises at least two exogenous genes encoding the acyl-ACP thioesterase protein of

Umbellularia cali fornica and Cinnamomum camphora that have hydrolysis activity towards C16 chain length acyl fatty substrates. In some cases, the cell is a Prototheca cell.

In a sixth aspect, the present invention provides oil-bearing microbial cells, preferably microalgae cells, that have a lipid profile that is at least

55% or at least 65%, preferably at least 60%, acids

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MEXICAN INSTITUTE Dt IA PROPERTY

INDUSTRIAL

<img file="MX339639B_D0010.tif" />

saturated fatty. In some cases the cells have a lipid profile that is at least 80%, at least 85%, or at least 90%, preferably at least 86%, saturated fatty acids. In some cases, the cell is a recombinant cell. In some embodiments, the recombinant cell comprises an exogenous gene encoding an acyl-ACP thioesterase protein that has hydrolysis activity towards C10C16 chain length acyl fatty-ACP substrates. In some embodiments, the cell comprises an exogenous gene that encodes a ketoacyl synthase protein. In some cases, the cell is a Prototheca cell.

In a seventh aspect, the present invention provides oil-bearing microbial cells, preferably microalgae cells, comprising a mutated endogenous desaturase gene, wherein the mutation inactivates the gene or desaturase. In some cases, the cell has a lipid profile that is at least 4 0% or at least 50%, preferably at least 45%, saturated fatty acids. In some cases, the cell has a lipid profile that is at least 15%, at least 20%, or at least 25%, preferably at least 19%,

C18: 0. In some embodiments, the cell comprises a mutated endogenous desaturase gene that results in at least a 2-fold increase in C18: 0 fatty acid, compared to a

<img file="MX339639B_D0011.tif" />

wild cell. In some cases, the microalgae cell has a lipid profile that is not greater than 1% or not greater than 5%, preferably not greater than 2%, C18: 2. In some embodiments, the microalgae cell has a lipid profile that is not greater than 5% or not greater than 10%, preferably not greater than 7%, 18: 1.

In some embodiments of the recombinant cells discussed in the present disclosure, the cell comprises a mutated endogenous desaturase gene, wherein the mutation inactivates the gene or desaturase.

In an eighth aspect, the present invention provides a method of preparing lipids. In one embodiment, the method comprises (a) culturing a cell as discussed above until the cell is at least 15% or at least

25%, preferably at least 20%, lipid by dry weight, and (b) separating the lipid from the water soluble biomass components.

In a ninth aspect, the present invention provides another method for preparing lipids. In one embodiment, the method comprises (a) cultivating an oil-bearing microbe, preferably a microalgae cell, containing exogenous genes encoding two distinct acyl-ACP thioesterases, wherein the lipid profile of the cell is distinct from (i)

<img file="MX339639B_D0012.tif" />

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MEXICAN INSTITUTE Dt LA PtOPlEOAO iNOUSTlIAl.

the cell profile without the exogenous genes and (ii) the cell profile with only one of the exogenous genes, and (b) separating the lipid from the water soluble biomass components. In some cases, at least one of the exogenous genes encodes an acyl fatty ACP thioesterase selected from the group consisting of the thioesterases identified in Table 4.

In a tenth aspect, the present invention provides a method of preparing an oil-based product. In one embodiment, the method comprises (a) culturing a cell as discussed above until the cell is at least 5% or at least 15%, preferably at least 10%, lipid by dry weight, (b) separating the lipid from the water soluble biomass components, and (c) subjecting the lipid to at least one chemical reaction selected from the group consisting of:

saponification; metathesis; acid hydrolysis; alkaline hydrolysis; enzymatic hydrolysis; catalytic hydrolysis;

hydrolysis with hot compressed water; a catalytic hydrolysis reaction where the lipid is separated into glycerin and fatty acids; an amination reaction to produce fatty nitrogen compounds; an ozonolysis reaction to produce mono and dibasic acids; a triglyceride separation reaction selected from

MEXICAN INSTITUTE

OF THE FRDFIEDAP

INDI ISTRIAI.

group consisting of enzymatic separation and pressure separation; a condensation reaction that follows a reaction

<td>hydrolysis;</td><td>a reaction</td><td>of</td><td>hydroprocessing;</td><td>a</td>

<td>reaction of</td><td colspan="2">hydroprocessing</td><td>and a reaction</td><td>of</td>

<td>deoxygenation</td><td>or a reaction</td><td>of</td><td>condensation before</td><td>of or</td>

simultaneous to the hydroprocessing reaction; a gas elimination reaction; a deoxygenation reaction selected from the group consisting of a hydrogenolysis reaction, hydrogenation, a consecutive hydrogenation hydrogenation reaction, a consecutive hydrogenolysis and hydrogenation reaction, and a combined hydrogenation hydrogenation reaction; a condensation reaction following a deoxygenation reaction; an esterification reaction; an interesterification reaction; a transesterification reaction; a hydroxylation reaction; and a condensation reaction following a hydroxylation reaction, whereby an oil-based product is produced.

In some cases, the oil-based product is selected from soap or a combustible product. In some embodiments, the oil-based product is a fuel product selected from the group consisting of biodiesel, renewable diesel, and jet fuel. In some

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<img file="MX339639B_D0013.tif" />

INSTITUTO MEXICANO Dt LA EROflEDAD cases, the fuel product is biodiesel with one or more of the following attributes: (i) 0.01-0.5 mcg / g, 0.025-0.3 mcg / g, preferably 0.05-0.244 mcg / g, total carotenoids; (ii) less than 0.01 mcg / g, less than 0.005 mcg / g, preferably less than 0.003 mcg / g, lycopene; (iii) less than 0.01 mcg / g, less than 0.005 mcg / g, preferably less than 0.003 mcg / g, beta carotene; (iv) 0.01-0.5 mcg / g, 0.0250.3 mcg / g, preferably 0.045-0.268 mcg / g, chlorophyll A; (v) 1-500 mcg / g, 35-175 mcg / g, preferably 38.3-164 mcg / g, gamma tocopherol; (vi) less than 1%, less than 0.5%, preferably less than 0.25%, brassicasterol, campesterol, stignasterol, or beta-sitosterol; (vii) 100-500 mcg / g, 225350 mcg / g, preferably 249.6-325.3 mcg / g, total tocotrienols; (viii) 0.001-0.1 mcg / g, 0.0025-0.05 mcg / g, preferably 0.003-0.039 mcg / g, lutein; or (ix) 10-500 mcg / g, 50-300 mcg / g, preferably 60.8-261.7 mcg / g, tocopherols. In some cases, the fuel product is renewable diesel that has a T10-T90 of at least 20 ° C, 40 ° C, or 60 ° C. In some cases, the fuel product is reactor fuel that meets HRJ-5 and / or ASTM specification D1655.

In an eleventh aspect, the present invention provides a triglyceride oil comprising (a) a lipid profile

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<img file="MX339639B_D0014.tif" />

INSTITUTO MEXICANO DE LA mOHBDAO of at least 3% C8: 0, at least 4% C10: 0, at least 13% C12: 0, at least 10% C14: 0, and / or at least 60% saturated fatty acids, and (b) one or more of the following attributes: (i)

0.01-0.5 mcg / g, 0.025-0.3 mcg / g, preferably 0.05-0.244 5 mcg / g, total carotenoids; (ii) less than 0.01 mcg / g, less than 0.005 mcg / g, preferably less than 0.003 mcg / g, lycopene; (iii) less than 0.01 mcg / g, less than 0.005 mcg / g, preferably less than 0.003 mcg / g, beta carotene; (iv)

0.01-0.5 mcg / g, 0.025-0.3 mcg / g, preferably 0.045-0.268 10 mcg / g, chlorophyll A; (v) 1-300 mcg / g, 35-175 mcg / g, preferably 38.3-164 mcg / g, gamma tocopherol; (vi) less than 1%, less than 0.5%, preferably less than 0.25%, brassicasterol, campesterol, stignasterol, or beta-statosterol; (vii) 100-500 mcg / g, 225-350 mcg / g, preferably 249.6-325.3 mcg / g, total tocotrienols;

(viii) 0.001-0.1 mcg / g, 0.0025-0.05 mcg / g, preferably

0.003-0.039 mcg / g, lutein; or (ix) 10-500 mcg / g, 50-300 mcg / g, preferably 60.8-261.7 mcg / g, tocopherols.

In a twelfth aspect, the present invention provides an isolated microalgae oil having a C8: C10 fatty acid ratio of at least 5: 1. In a related aspect, the present invention provides an oil isolated from microalgae with at least 50% to 75%,

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL preferably at least 60%, of saturated fatty acids. In another related aspect, the present invention provides an isolated microalgae oil having a C16: 14 fatty acid ratio of about 2: 1. In yet another related aspect, the present invention provides an isolated microalgae oil having a C12-.C14 fatty acid ratio of at least 5: 1. In some embodiments, the microalgae contains at least one exogenous gene. In some cases, the microalgae is of the genus Prototheca.

In a thirteenth aspect, the present invention provides a triglyceride oil comprising (a) a lipid profile of less than 5% or less than 2%, preferably less than 1%, &lt;C12; between l% -10%, preferably 2% -7%, C14: 0; between 20% -35%, preferably

23% -30%, C16: 0; between 5% -20%, preferably 7% -15%, C18: 0;

between 35-60%, preferably 40-55%, C18: 1; and between l -20%, preferably 2-15%, C18: 2 fatty acids; and (b) one or more of the following attributes: (i) 0.01-0.5 mcg / g, 0.025-0.3 mcg / g, preferably 0.05-0.244 mcg / g, total carotenoids; (ii) less than 0.01 mcg / g, less than 0.005 mcg / g, preferably less than 0.003 mcg / g, lycopene; (iii) less than 0.01 mcg / g, less than 0.005 mcg / g, preferably less than 0.003 mcg / g, beta carotene; (iv) 0.01-0.5 mcg / g, 0.02515

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MEXICAN INSTITUTE OF f, A PROPERTY INDI 15TR1AI.

<img file="MX339639B_D0015.tif" />

0.3 mcg / g, preferably 0.045-0.268 mcg / g, chlorophyll A; (v) 1-300 mcg / g, 35-175 mcg / g, preferably 38.3-164 mcg / g, gamma tocopherol; (vi) less than 1%, less than 0.5%, preferably less than 0.25%, brassicasterol, campesterol, 5 stignasterol, or beta-sitosterol; (vii) 100-500 mcg / g, 225350 mcg / g, preferably 249.6-325.3 mcg / g, total tocotrienols; (viii) 0.001-0.1 mcg / g, 0.0025-0.05 mcg / g, preferably 0.003-0.039 mcg / g, lutein; or (ix) 10-500 mcg / g, 50-300, preferably 60.8-261.7 mcg / g, tocopherols. 10 In some cases, triglyceride oil is isolated from a microbe that comprises one or more exogenous genes. In some embodiments, the one or more exogenous genes encode an acyl fatty-ACP thioesterase. In some cases, acyl fatty acp thioesterase has hydrolysis activity towards C14 chain length acyl fatty acp substrates. In some embodiments, the microbe further comprises a mutated endogenous desaturase gene, where the mutation inactivates the gene or desaturase.

In a fourteenth aspect, the present invention provides a method of producing a triglyceride oil comprising a lipid profile of less than 5%, or less than 2%, preferably less than 1%, &lt;C12; between l% -10%, preferably 2% -7%, C14: 0; between 20% -35%, preferably

<img file="MX339639B_D0016.tif" />

iMPir

23% -30%, C16: 0; between 5% -20%, preferably 7% -15%, C18: 0;

between 35% -60%, preferably 40-55%, C18: 1; and between l -20%, preferably 2-15%, C18: 2 fatty acids, where the triglyceride oil is isolated from a microbe comprising one or more exogenous genes. In some cases, the triglyceride oil comprises a lipid profile of 1-10%, preferably 3-5%, C14: 0; 20% -30%, preferably 25-27%,

C16: 0; 5% -20%, preferably 10-15%, C18: 0; and 35% -50%, preferably 40-45%, C18: 1. In some embodiments, the one or more exogenous genes encode an acyl fatty-ACP thioesterase. In some cases, acyl fatty acp thioesterase has hydrolysis activity towards C14 chain length acyl fatty acp substrates. In some cases, the microbe further comprises a mutated endogenous desaturase gene, where the mutation inactivates the gene or desaturase. In some cases, the one or more exogenous genes are a sucrose invertase. In some embodiments, the mutated endogenous desaturase gene is a stearoyl-acyl-bearing desaturase protein (SAD) (eg, seq.

199-200). In some embodiments, the mutated endogenous desaturase gene is a fatty acid desaturase (FAD).

In a fifteenth aspect, the present invention provides an oil microbial cell, preferably

ΙΜΡΙ

MfcXlCAN · OS INSTITUTE THE INOI ISTRIAL PROPERTY

<img file="MX339639B_D0017.tif" />

a microalgae cell, comprising a triglyceride oil, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of at least about 1% C8: 0, at least about 1%

C10: 0, at least about 1% C12: 0, at least about 2% C14: 0, at least about 30% C16: 0, at least about 5% C18: 0, at least about 60% C18: l, less than about 7% C18: 2, and at least about 35% saturated fatty acids. In some cases, the oil microbial cell comprises an exogenous gene, and optionally, an endogenous oil microbial cell desaturase is inactivated or mutated to have less enzyme activity.

In some cases, the fatty acid profile of triglyceride oil is similar to the fatty acid profile of a naturally occurring oil. In some cases, oil of natural origin is selected from the group consisting of cocoa butter, coconut oil, palm oil, palm kernel oil, shea butter, bait, and beef butter.

In some cases, the fatty acid profile of triglyceride oil comprises a profile selected from the group consisting of, the total combined amounts of C8: 0 and

CIO: 0 is at least about 10%, the total amount

<img file="MX339639B_D0018.tif" />

Combined IMPI of C10: 0, C12: 0, and C14: 0 is at least about 50%, the total combined amount of C16: 0,

C18: 0 and C18: 1 is at least about 60%, the combined total amount of C18: 0, C18: l and C18.-2 is at least about 60%, the combined total amount of C14: 0,

C16: 0, C18: 0, and C18: 1 is at least about 60%, and the combined total amount of C18: 1 and C18: 2 is less than about 30%. In some cases, the fatty acid profile of triglyceride oil comprises a ratio of fatty acids selected from the group consisting of ratio

C8: 0 to C10: 0 of at least about 5 to 1, CIO: 0 to C12: 0 ratio of at least about 6 to 1, ratio

C12: 0 to C14: 0 of at least about 5 to 1, ratio

C14: 0 to C12: 0 of at least about 7: 1, and ratio

C14: 0 to C16: 0 of at least about 1 to 2.

In some cases, the endogenous desaturase is selected from the group consisting of stearoyl ACP desaturase and delta 12 fatty acid desaturase. In some cases, the exogenous gene is selected from the group consisting of a gene encoding an acyl-ACP thioesterase. In some cases, the exogenous gene encodes an acyl-ACP thioesterase selected from the group consisting of those identified in Table 4. In some cases, the microbial cell

<img file="MX339639B_D0019.tif" />

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INSTITUTO MEXICANO DE LA RROHEDAU INDUSTRIAL oleaginosa also includes a gene that encodes a sucrose invertase.

In various embodiments, the oil microbial cell is a cell of a microalgae genus or species selected from Achnanthes orientalis, Agmenellum,

Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphora delicatissima capitata, Amphora sp.,

Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandií, Borodinella sp., Botryococcus braunii,

Botryococcus sudeticus, Cartería, Chaetoceros gracilis,

Chaetoceros muelleri, Chaetoceros muelleri subsalsum,

Chaetoceros sp. , Chlorella anitrata, Chlorella Antarctica,

Chlorella aureoviridis, Chlorella candida, Chlorella capsúlate, 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 kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var.

lutescens

Chlorella miniata

Chlorella minutissima

<img file="MX339639B_D0020.tif" />

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INSTITUTO MEXICANO DE IA PROPIEDAD INCIIISTPIAI.

Chlorella mutabilis, Chlorella nocturnal, Chlorella parva,

Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoid.es (which includes any strain of UTEX 1806, 411,

264, 256, 255, 250, 249, 31, 29, 25, and CCAP strains 211/17 and

211 / 8d), Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularás var. minimal, Chlorella regularas var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. elllpsoidea,

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.

autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f.

tertia, 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., Cryptomonas sp., Cyclotella cryptica,

Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp.,

Dunaliella bardawil, Dunaliella bíoculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella

ΙΜΡΙ

INSTITI IT »MEXICAN» E LA ΑΑΟΓΙΑΟΑΓ

INH.imiAl

<img file="MX339639B_D0021.tif" />

saline, Dunaliella terrícola, Dunaliella tertiolecta,

Dunaliella viridis, Dunaliella tertiolecta, 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 (UTEX LB 2614), Monoraphidium minutum, Monoraphidium sp. ,

Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicule pseudotenelloides, Navicule pelliculosa, Navicula saprophila,

Navicula sp., 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 sp., Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp. , Phagus, Phormidium, Platymonas sp. ,

Pleurochrysis portfolio, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii

IMPI

MEXICAN INSTRUMENT OR THE INDUSTRIAL COMPANY

<img file="MX339639B_D0022.tif" />

Pyramimonas

Scenedesmus sp. , Pyrobotrys, Sarcinoid chrysophyte, armatus, Spirogyra, Spirulina platensis,

Stichococcus sp. Synechococcus sp. , Tetrahedra, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and

Viridiella fridericiana.

In some cases, the oil microbial cell is a cell of the genus Prototheca. In some cases, the oil microbial cell is a cell of the genus

Prototheca moriformis.

In some cases, the oil microbial cell is

<td>an oil yeast cell.</td><td>In</td><td>Some</td><td>cases,</td><td>the</td>

<td>oil microbial cell is</td><td>a</td><td>cell</td><td colspan="2">bacterial</td>

<td>oleaginous.</td><td></td><td></td><td></td><td></td>

<td>In some cases, the oil</td><td>of</td><td>origin</td><td>natural</td><td>is</td>

<td>cocoa butter and the exogenous gene</td><td colspan="2">understands</td><td>a gene</td><td>of</td>

<td>Carthamus tinctorus thioesterase.</td><td>In</td><td>Some</td><td>cases,</td><td>the</td>

<td>oil of natural origin is oil</td><td>of</td><td colspan="2">coconut nut.</td><td>In</td>

In some cases, the naturally occurring oil is palm oil and the exogenous gene comprises an Elaeis guiniensis thioesterase gene, a Cuphea hookeriana thioesterase gene, a combination of a Cuphea hookeriana KAS IV gene and a gene

Cuphea wright ii FATB2, or a construct designed to interrupt an endogenous KAS II gene. In some cases, the

<img file="MX339639B_D0023.tif" />

Natural naturally occurring oil is palm kernel oil and the exogenous gene comprises a combination of a FATB2 gene from

Cuphea wrightii and a construct designed to interrupt an endogenous SAD2B gene. In some cases, the oil of natural origin is shea butter. In some cases, the oil of natural origin is beef bait. In some cases, the oil of natural origin is butter and the exogenous gene comprises a combination of the thiosterase U gene. californica and a construct designed to interrupt an endogenous SAD2B gene, a combination of a Garcinia mangostana thiosterase gene and a construct designed to interrupt a gene

Endogenous SAD2B, a Brassica napus thiosterase gene, or a Cuphea hookeriana thiosterase gene.

In a sixteenth aspect, the present invention provides an oleaginous microbial triglyceride oil composition, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of at least about 1% C8: 0, at least about 1%

C10: 0, at least about 1% C12: 0, at least about 2% C14: 0, at least about 30% C16: 0, at least about 5% C18: 0, at least about

60% C18: 1, less than about 7% C18: 2, and at least about 35% saturated fatty acids.

In several

<img file="MX339639B_D0024.tif" />

^Z4 IMPI

MEXICAN INSTITUTE ΠΕ THE PROPERTY, INDUSTRIAL modalities, the composition of triglyceride oil by cultivating a population of recombinant oil microbial oil cells or oil microbial cells in a culture medium, where the oil microbial cells as described above, particularly those previously described in relation with the fifteenth aspect of the invention.

In some cases, the composition of oleaginous microbial triglyceride oil further comprises an attribute selected from the group consisting of: (i) less than 0.3 mcg / g total carotenoids; (ii) less than 0.005 mcg / g lycopene; (iii) less than 0.005 mcg / g beta carotene; (iv) less than 0.3 mcg / g chlorophyll A; (v) less than 175 mcg / g gamma tocopherol, - (vi) less than 0.25% brassicasterol, campesterol, stignasterol, or beta-sitosterol; (vii) less than 350 mcg / g total tocotrienols; (viii) less than 0.05 mcg / g lutein; or (ix) less than 275 mcg / g tocopherols.

In a seventeenth aspect, the present invention provides a method of producing an oleaginous microbial triglyceride oil composition having a fatty acid profile selected from the group consisting of at least about 1% C8: 0, at least about 1% C10: 0 , at least about 1% C12: 0, at least about

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<img file="MX339639B_D0025.tif" />

2% C14: 0, at least about 30% C16: 0, at least about 5% C18: 0, at least about 60% C18: l, less than about 7% C18: 2, and at least about 35% fatty acids saturated, wherein the method comprises the steps of: (a) culturing a population of oil microbial oil cells in a culture medium up to at least 10% of the dry cell weight of the oil microbial oil cells is triglyceride oil; and (b) isolating the triglyceride oil composition from the oil microbial cells. In various embodiments, the triglyceride oil composition is produced by cultivating a population of recombinant microbial oil cells or recombinant oil microbial cells as described above, particularly those described above in connection with the fifteenth aspect of the invention.

In an eighteenth aspect, the present invention provides a method for preparing an oil-based product, wherein the method comprises the steps of: (a) subjecting the composition of oleaginous microbial triglyceride oil, as described above in connection with the sixteenth aspect of the invention, to at least one chemical reaction selected from the group consisting of:

<img file="MX339639B_D0026.tif" />

IMPI

INSTITU I · MEXICANO OE LA AROAUdad INnilSTRIAI saponification; metathesis; acid hydrolysis; alkaline hydrolysis; enzymatic hydrolysis; catalytic hydrolysis, hydrolysis with hot compressed water; a catalytic hydrolysis reaction where the lipid is separated into glycerin and fatty acids; an amination reaction to produce fatty nitrogen compounds; an ozonolysis reaction to produce mono and dibasic acids; a triglyceride separation reaction selected from the group consisting of enzymatic separation and pressure separation; a condensation reaction that follows a hydrolysis reaction; a hydroprocessing reaction; a hydroprocessing reaction and a deoxygenation reaction or a condensation reaction prior to or simultaneous with the hydroprocessing reaction; a gas elimination reaction; a deoxygenation reaction selected from the group consisting of a hydrogenolysis reaction, hydrogenation, a consecutive hydrogenation-hydrogenation reaction, a consecutive hydrogenolysis-hydrogenation reaction, and a combined hydrogenation-hydrogenation reaction; a condensation reaction following a deoxygenation reaction; an esterification reaction; an interesterification reaction; a transesterification reaction; a hydroxylation reaction;

IMPI Mexican Institute '

DE LA MtOREDAD fV / * indust »i * i. Xg'ff * and a condensation reaction following a hydroxylation reaction; and (b) isolating the reaction product from other components.

In some cases, the oil-based product is selected from the group consisting of a soap, a fuel, a dielectric fluid, a hydraulic fluid, a plasticizer, a lubricant, a heat transfer fluid, and a metalworking fluid. . In some cases, the oil-based product is a combustible product selected from the group consisting of: (a) biodiesel; (b) renewable diesel; and (c) reactor fuel.

In some cases, the fuel product is biodiesel with one or more of the following attributes: (i) less than 0.3 mcg / g total carotenoids; (ii) less than 0.005 mcg / g lycopene; (iii) less than 0.005 mcg / g beta carotene; (iv) less than 0.3 mcg / g chlorophyll A; (v) less than 175 mcg / g gamma tocopherol; (vi) less than 0.25% brassicasterol, campesterol, stignasterol, or beta-sitosterol; (vii) less than 350 mcg / g total tocotrienols; (viii) less than 0.05 mcg / g lutein; or (ix) less than 275 mcg / g tocopherols.

In some cases, the fuel product is renewable diesel that has a T10-T90 of at least 20 ° C, 40 ° C, or

60 ° C.

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MSXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX339639B_D0027.tif" />

In some cases, the fuel product is reactor fuel that meets HRJ-5 and / or ASTM specification D1655.

These and other aspects and embodiments of the invention are described in the accompanying figure, the brief description of which follows immediately, the detailed description of the invention below, and is exemplified in the examples below. Any or all of the features discussed above and throughout the application may be combined in various embodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a chromatogram of renewable diesel produced from oil. triglyceride of

Prototheca.

DETAILED DESCRIPTION OF THE INVENTION

The present invention arises from the discovery that Prototheca and certain related microorganisms have unexpectedly advantageous properties for the economical and high-volume production of oils, fuels, and other hydrocarbon or lipid compositions, as well as the discovery of methods and reagents for genetically alter these microorganisms to improve these properties. The oils produced by these

IMPI

MEXICAN INSTITUTE

OE ΙΑ ΓΚΙΚΙΠΑΓ »» 3 — INDUSTRIAL> ^, - ro Microorganisms can be used in fuel for transportation, in the oleochemical and / or food and cosmetic industries, among other applications. Transesterification of lipids produces esters of long chain fatty acids useful as biodiesel. Other enzymatic and chemical processes can be adjusted to produce fatty acids, aldehydes, alcohols, alkanes, and alkenes. In some applications, renewable diesel, jet fuel, or other hydrocarbon compounds are produced. The present invention also provides methods for cultivating microalgae with high productivity and high lipid yield, and / or for the most cost-effective production of the compositions described herein.

This detailed description of the invention is divided into sections for the convenience of the reader. Section I provides the definitions of the terms used herein. Section II provides a description of the culture conditions useful in the methods of the invention.

Section III provides a description of genetic engineering materials and methods. Section IV provides a description of the genetic modification of microorganisms (eg Prototheca) to allow the use of sucrose. Section V provides a

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INOI ISTRIAL

<img file="MX339639B_D0028.tif" />

description of the genetic modification of microorganisms (eg Prototheca) to modify lipid biosynthesis. Section VI describes methods for producing fuels and chemicals. Section VII describes the examples and embodiments of the invention. The detailed description of the invention is followed by examples illustrating various aspects and embodiments of the invention.

I. DEFINITIONS [0001] Unless defined otherwise, all technical and scientific terms used in the present description have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide a skilled person 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 Genetics, 5th Ed., R.

Rieger et al. (Eds.), Springer Verlag (1991); and Hale &amp;

Marham, The Harper Collins Dictionary of Biology (1991). As used throughout this document, the following terms have the meanings attributed to them, unless otherwise specified.

<img file="MX339639B_D0029.tif" />

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

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 a transgenic microalgae is active on the microalgae.

Acyl Carrier Protein or ACP is a protein that binds to a growing acyl chain during fatty acid synthesis as a thiol ester to the distal thiol portion of 4¹-phosphopantothein and comprises a component of the fatty acid synthase complex.

[0002] An acyl-CoA or acyl-CoA molecule, is a molecule comprising an acyl moiety covalently attached to coenzyme A through a thiol ester bond in the distal thiol of the 4 moiety¹-phosphopantothein from coenzyme A.

Area Percent refers to the area of ​​peaks observed using the FAME GC / FID detection methods, in which each fatty acid in the sample is converted to a fatty acid methyl ester (FAME) prior to detection. For example, a separate peak is observed for a fatty acid of

14 unsaturated carbon atoms (C14: 0) compared to any other fatty acid such as C14: 1. The peak area for each FAME class is directly proportional to its percent composition in the mix and is calculated based on the

<img file="MX339639B_D0030.tif" />

IMPI sum of all the peaks present in the sample (i.e.

[area under specific peak / total area of ​​all measured peaks] X 100). When referring to the lipid profiles of the oils and cells of the invention, at least 4% of

C8-C14 means that at least 4% of the total fatty acids in the cell or in the composition of the extracted glycerolipids, has a chain length that includes 8, 10, 12 or 14 carbon atoms.

Axenic is a culture of an organism free of contamination by other living organisms.

Biodiesel is a biologically produced fatty acid alkyl ester suitable for use as fuel in a diesel engine.

Biomass is the material produced by the growth and / or propagation of cells. Biomass can contain cells and / or intracellular content, as well as extracellular material, includes, but is not limited to, compounds secreted by a cell.

Bioreactor, is a total or partial enclosure where cells are grown, optionally in suspension.

Catalyst is an agent, such as a molecule or a macromolecular complex, capable of facilitating or promoting a chemical reaction of a reactant to its product without

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<img file="MX339639B_D0031.tif" />

become a part of the product. A catalyst increases the speed of a reaction, after which the catalyst can act on another reactant to form the product. A catalyst generally lowers the total activation energy required for the reaction, so that it occurs more quickly or at a lower temperature. Thus, a balance of the reaction can be achieved more quickly. Examples of catalysts include enzymes, which are biological catalysts; heat, which is a non-biological catalyst; and metals used in the refining processes of fossil oils.

Cellulosic material, is the product of the digestion of cellulose, including glucose and xylose, and optionally additional compounds such as disaccharides, oligosaccharides, lignin, furfurals and other compounds. Non-limiting examples of sources of cellulosic material include sugarcane bagasse, beet sugar pulp, corn stubble, wood chips, sawdust, and rod grass.

Co-culture, and variants of it such as co-cultivation and co-fermentation, refer to the presence of two or more cell types in the same bioreactor. The two or more cell types can be both microorganisms, such as microalgae, or can be a cultured microalgae cell

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<img file="MX339639B_D0032.tif" />

with a different cell type. Culture conditions may be those that promote the growth and / or spread 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 the cell growth for the rest.

Cofactor is any molecule, other than the substrate, necessary for an enzyme to perform its enzymatic activity.

Complementary DNA, or cDNA, defines a copy of

DNA of the mRNA, normally obtained by reverse transcription of the messenger RNA (mRNA) or by amplification (for example, via polymerase chain reaction (PCR)).

Cultured, and variants of this as cultured and fermented, refer to the intentional promotion of growth (increase in cell size, cell content, and / or cell activity) and / or spread (increase in the number of cells via mitosis) of one or several cells by using selected and / or controlled culture conditions. The combination of both growth and propagation can be called proliferation. Examples of selected and / or controlled conditions include the use of a defined medium (with characteristics ³⁵ IMPíSfc '

MEXICAN INSTITUTE • E LA RRORIEOAÍJ

IN9USTRIAL * »«. "*, Ti known as pH, ionic strength, and carbon source), and specific conditions of temperature, oxygen tension, carbon dioxide levels, and growth in a bioreactor. Cultivating does not refer to the growth or spread of microorganisms in nature or elsewhere without human intervention; for example, it is not a crop the natural growth of an organism that finally becomes a fossil to produce oil.

Cytolysis is the lysis of cells in a hypotonic environment. Cytolysis is caused by excessive osmosis, or by the movement of water, into a cell (hyperhydration). The cell cannot resist the osmotic pressure of the water inside it, and it explodes.

Delipid food and delipidated microbial biomass, is the microbial biomass after the oil (including lipids) is extracted or isolated from it, either through the use of mechanical extraction (that is, exerted by a press) or solvent extraction, or both of them. The delipidated food has a reduced amount of oils / lipids compared to the microbial biomass before the extraction / isolation of the oils / lipids, but contains some residual amount of oils / lipids.

<img file="MX339639B_D0033.tif" />

Expression vector or expression construct o

IMPI

INSTITUTO MEXICAN · »f THE NAME», 'NBUSTRIAL plasmid or recombinant DNA construct, refers to a nucleic acid that has been generated by human intervention, including recombinant or direct chemical synthesis methods, with a series of specific nucleic acids that allow the transcription and / or translation of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or a nucleic acid fragment. Typically, the expression vector includes a nucleic acid that is transcribed operably linked to a promoter.

Exogenous gene, is a nucleic acid that codes for the expression of an RNA and / or protein that has been introduced (transformed) in a cell. A transformed cell can be defined as a recombinant cell, into which an additional exogenous gene (s) can be introduced. The exogenous gene can be from a different species (heterologous), or from the same species (homologous) with respect to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the cell genome or is under different control, with respect to the endogenous copy of the gene. An exogenous gene can be present in more than one copy in the cell. An exogenous gene can³⁷ IMPIAS

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Dt LA FRONEDAI 'industrial be maintained in a cell as an insert in the genome or as an episomal molecule.

Exogenously provided, refers to a molecule provided to the culture medium of a cell culture.

Pressed with an ejector, it is a mechanical method to extract oil from raw materials such as soybeans and rapeseed. An ejector press is a screw machine which presses the material through a caged, barrel-shaped cavity 10. The raw material enters on one side of the press and the cake leaves on the other side, while the oil filters between the bars of the cage and is collected.

The machine uses friction and continuous pressure from the screw units to move and compress the raw material. The oil comes out through small openings that do not allow solids to pass through. Since the raw material is pressed, friction typically causes heating.

Acyl Fatty-ACP Thioesterase, is an enzyme that catalyzes the cleavage of a fatty acid from an acyl carrier protein (ACP) during lipid synthesis.

Acyl Fat-CoA / Aldehyde Reductase, is an enzyme that catalyzes the reduction of an acyl-CoA molecule to a primary alcohol.

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JN> TTT »fTO MtXICAMO re la wepiti'At INPUSTWIAL

<img file="MX339639B_D0034.tif" />

Acyl fatty-CoA reductase, is an enzyme that catalyzes the reduction of an acyl-CoA molecule to an aldehyde.

Fatty aldehyde decarbonylase, is an enzyme that catalyzes the conversion of a fatty aldehyde to an alkane.

Fatty aldehyde reductase, is an enzyme that catalyzes the reduction of an aldehyde to a primary alcohol.

Fixed carbon source is a carbon-containing molecule or molecules, typically an organic molecule that is present at room temperature and pressure in solid or liquid form, which can be used by a microorganism grown there.

Homogenate is biomass that has been physically separated.

Hydrocarbon is (a) a molecule that contains only hydrogen and carbon atoms, where the carbon atoms are covalently linked to form a linear, branched, cyclic, or partially cyclic backbone to which the hydrogen atoms are attached. The molecular structure of hydrocarbon compounds varies from the simplest, in the form of methane (CH₄), which is a constituent of natural gas, a very strong and complex, such as some molecules such as asphaltenes found in crude oil, petroleum, and bitumen. Hydrocarbons can be found in gaseous form,

ΙΜΡϊ @>

INSTITUTO MEXICANC DE IA FROMEDAP

IW'STRIAL S «» »liquid, or solid, or in any combination of these forms, and may have one or more double or triple bonds between the carbon atoms adjacent to the main chain. Therefore, the term includes linear, branched, cyclic, or partially cyclic linear alkanes, alkenes, lipids, and paraffins. Examples include propane, butane, pentane, hexane, octane, and squalene.

Hydrogen: carbon ratio is 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 CH₄ (4:1).

Hydrophobic fraction, is the portion, or fraction of a material that is more soluble in a hydrophobic phase compared to an aqueous phase. A hydrophobic fragment is substantially insoluble in water and is normally nonpolar.

Increased lipid yield, refers to the increase in the productivity of a microbial culture, for example, increasing the dry weight of cells per liter of culture, increasing the percentage of cells that make up lipids, or increasing the total amount of lipids per liter of crop volume per unit time.

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The inducible promoter is a promoter that mediates the transcription of operably linked genes in response to a particular stimulus. Examples of such promoters may be promoter sequences that are induced under conditions of changing pH or nitrogen levels.

In operative junction, it is the functional junction between two nucleic acid sequences, such as a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operative binding to an exogenous gene if it can mediate transcription of the gene.

In situ means in place or in its original position.

Limiting concentration of a nutrient is the concentration of a compound in a crop that limits the spread of an organism in the crop. A non-limiting concentration of a nutrient is a concentration that aids maximum propagation during a given period of the crop.

Thus, the number of cells produced during a given period of culture is lower 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

<img file="MX339639B_D0035.tif" />

<img file="MX339639B_D0036.tif" />

₄₁ IMPI ______ MEXICAN INSTITUTE

OF INDUSTRIAL RORTHY culture, when the nutrient is prpspnts in a concentration greater than that necessary for maximum propagation.

Lipase is a water soluble enzyme that catalyzes the hydrolysis of esters bound to lipid substrates insoluble in water. Lipases catalyze the hydrolysis of lipids in glycerols and fatty acids.

Lipid modifying enzyme, refers to an enzyme that alters the covalent structure of a lipid. Examples of lipid modifying enzymes include a lipase, an fatty acyl-ACP thioesterase, an fatty acyl CoA / aldehyde reductase, an fatty acyl-CoA reductase, a fatty aldehyde reductase, a desaturase, including a stearoyl acyl carrier desaturase protein. (SAD) and a fatty acyl destaurase (FAD), and a fatty aldehyde decarbonylase.

Lipid pathway enzyme, is any enzyme that plays a role in lipid metabolism, that is, in lipid synthesis, modification, or degradation, and any protein that chemically modifies lipids, as well as proteins carriers.

[0003] Lipids are a class of molecules that are soluble in non-polar solvents (such as ether and

<img file="MX339639B_D0037.tif" />

ΙΜΡΙ - chloroform) and are relatively or completely insoluble in water. Lipid molecules have these properties because they are made up of long hydrocarbon tails that are hydrophobic in nature. Examples of lipids 5 include fatty acids (saturated and unsaturated); glycerides or glycerolipids (such as monoglycerides, diglycerides, triglycerides or neutral fats, and phosphoglycerides or glycerophospholipids); non-glycerides (sphingolipids, sterol lipids including cholesterol and steroid hormones, prenol lipids including terpenoids, fatty alcohols, waxes, and polyketides); and complex lipid derivatives (sugar-bound lipids, or glycolipids, and protein-bound lipids). Fats are a subgroup of lipids called triacylglycerides.

Lysate is a solution that contains the content of lysed cells.

Lysis is the rupture of the plasma membrane and optionally of the cell wall of a biological organism sufficient to release at least some intracellular content, often by mechanical, viral or osmotic mechanisms that compromise its integrity.

Lysar is the disturbance of the cell membrane and optionally the cell wall of a biological organism or

<img file="MX339639B_D0038.tif" />

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Dt THE PROPERTY »» 'STRIaL cell sufficient to release at least some intracellular content.

Microalgae is a eukaryotic microbial organism that contains a chloroplast or plastid, and optionally is capable of photosynthesis, or a prokaryotic microbial organism that is capable of photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed source of carbon into energy, as do heterotrophs, which can live only at the cost of a fixed source of carbon. Microalgae include single-celled organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes, such as, for example, Volvox, which is a simple photosynthetic multicellular microbe from two different cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys.

Microalgae also include obligate heterotrophic microorganisms that have lost the ability to photosynthesize, such as certain dinoflagellate algae species and Prototheca genus species.

<img file="MX339639B_D0039.tif" />

Microorganism and microbe, are organisms

<img file="MX339639B_D0040.tif" />

unicellular microscopy.

Naturally co-expressed, referring to two proteins or genes means that the proteins or their genes co-express naturally in a tissue or organism from which they are derived, for example, because the genes encoding the two proteins are low control of a common regulatory sequence or because they are expressed in response to the same stimulus.

Osmotic shock is the breakdown of cells in a solution after a sudden reduction in osmotic pressure. Osmotic shock is sometimes induced to release the cellular components of these cells into a solution.

Polysaccharide degradation enzyme, is any enzyme capable of catalyzing the hydrolysis or saccharification of any polysaccharide. For example, cellulases catalyze the hydrolysis of cellulose.

Polysaccharides, or glycans, are carbohydrates made from monosaccharides linked by glycosidic bonds. Cellulose is a polysaccharide that is part of certain cell walls of plants. Cellulose can be depolymerized by enzymes to produce monosaccharides such as

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<img file="MX339639B_D0041.tif" />

glucose and xylose, as well as larger disaccharides and oligosaccharides.

Promoter is a nucleic acid control sequence that directs the transcription of a nucleic acid.

As used in the present description, a promoter includes the necessary nucleic acid sequences near the transcription start site, such as, in the case of a type II polymerase promoter, a TATA element. A promoter optionally also includes the distal enhancer or repressor elements, which can be located up to several thousand base pairs from the transcription start site.

Recombinant is a cell, nucleic acid protein or vector, which was modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, for example, recombinant cells express genes that are not found in native (non-recombinant) cells or express native genes differently than how these genes are expressed by a non-recombinant cell. A recombinant nucleic acid is a nucleic acid originally formed in vitro, generally by nucleic acid manipulation, for example using polymerases and endonucleases, or otherwise in a ⁴⁶ IMPI »*

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DE LA FROREDAD 'C * »w3¿LJÍ

INDUSTRIAL form that is not normally found in nature. Recombinant nucleic acids can be. produce, for example, to place two or more nucleic acids in operative linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by the ligation of molecules of

DNAs that do not normally bind in nature are considered recombinant for the purposes of the present invention. Once a recombinant nucleic acid is obtained and introduced into a host cell or organism, it can be replicated using the in vivo cellular machinery of the host cell; however, these nucleic acids, once produced by recombinant methods, although subsequently replicated intracellularly, are still considered recombinant for the purposes of the present invention. Similarly, a recombinant protein is a protein produced using recombination techniques, that is, through the expression of a recombinant nucleic acid.

Renewable diesel is a mixture of alkanes (like

C10: 0, C12: 0, C14: 0, C16: 0 and C18: 0) produced by the hydrogenation and deoxygenation of lipids.

Saccharification is a process to convert biomass, usually cellulosic or lignocellulosic biomass, into sugars

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MBÍICANO INSTITUTE DF LA ΤΜΟΤΙΪΟΛΟ inbiistwal monomers, such as glucose and xylose. Saccharified or depolymerized cellulosic material or biomass refers to the cellulosic material or biomass that is converted to monomeric sugars through saccharification.

The similar term, when used in the context of a comparison with a naturally occurring oil, without further qualification, means that the oil being compared to the naturally occurring oil contains approximately +/- 15%, or +/- 10 % of the two upper triglycerides of the oil of natural origin. For example, shea butter (B. Parkii oil) contains 41,256.8% C18: 0 and 34.0-46.9% C18: 1 as the two most common triglyceride components (see Table 5). A similar oil that is within +/- 10% can contain approximately

37% to approximately 62% C18: 0 and from 31% to approximately

52% C18: 1 as the two most common triglyceride components.

When used in this context, the similar term includes +/- 9%, +/- 8%, +/- 7%, +/- 6%, +/- 5%, +/- 4%, +/- 3%, + / 2%, or +/- 1%, and may also represent a comparison with the three or four higher triglycerides of the oil of natural origin, or two outside the three higher triglycerides, or three outside the four triglycerides superior.

<img file="MX339639B_D0043.tif" />

biological, such as a cell, by using the energy of sound waves.

Furfural species, it is 2-furancarboxaldehyde or a derivative that retains the same basic structural characteristics.

Stubble, are the dried stems and leaves of a crop that remain after the grain is harvested.

Sucrose utilization gene is a gene that, when expressed, aids the ability of a cell to use 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 hexokinases such as glucokinases and fructokinases.

II. CULTURE

The present invention generally relates to the cultivation of microorganisms (eg, microalgae, oil yeast, fungi, and bacteria), particularly recombinant microalgal strains, including Prototheca strains, for lipid production. For the convenience of the reader, this section is subdivided into subsections. The

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MEXICAN INSTITUTE OF PROPERTY

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Subsection 1 describes the Prototheca species and strains and how to identify new Prototheca species and strains and related microalgae by comparison of genomic DNA as well as other microorganisms. Subsection 2 describes useful bioreactors for cultivation. Subsection 3 describes the culture media. Subsection 4 describes oil production according to illustrative cultivation methods of the invention. These descriptions are also more generally applicable to other microorganisms.

one. Species and strains of Prototheca and other microorganisms

Prototheca is a remarkable microorganism for use in lipid production, because it can produce high levels of lipids, particularly lipids suitable for fuel production. Lipids produced by

Prototheca have shorter hydrocarbon chains and a higher degree of saturation than those produced by other microalgae. Furthermore, Prototheca's lipids are generally free of pigments (low to undetectable levels of chlorophyll and certain carotenoids) and in any case contain much less pigments than lipids of other microalgae. Furthermore, the recombinant Prototheca cells provided by the invention can be used to produce

<img file="MX339639B_D0045.tif" />

ΙΜΡΙ lipids with higher performance and efficiency, and at a reduced cost, in relation to the production of lipids from other microorganisms. Illustrative strains of

Prototheca for use in the methods of the invention include Prototheca moriformis, Prototheca krugani,

Prototheca stagnora or Prototheca zopfii. Furthermore, these microalgae grow heterotrophically and can be genetically modified such as Prototheca wickerhamii,

Prototheca stagnora (including UTEX 327), Prototheca portoricensis, Prototheca moriformis (including strains of

UTEX 1441, 1435), and Prototheca zopfii. Species of the genus Prototheca are obligate heterotrophs.

The Prototheca species for use in the invention can be identified by the amplification of certain target regions of the genome. For example, the identification of a specific species or variety of Prototheca can be accomplished by amplification and sequencing of nuclear DNA and / or chloroplasts 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 for phylogenetic analysis, such as amplification and sequencing of the

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INDUSTRIAL ^M rJT ribosomal internal transcribed spacer (ITS1 and ITS2 rDNA), _

23S rRNA, 18S rRNA, and other conserved regions of the genome, can be used by those skilled in the art to identify species not only of Prototheca but also other lipid and hydrocarbon-producing organisms with similar lipid profiles and production capacity. For examples of algae identification and classification methods, see also, for example, Genetics, August 2005;

170 (4): 1601-10 and RNA, April 2005; ll (4): 361-4.

In this way, comparison of genomic DNA can be used to identify suitable species of microalgae that are used in the present invention. Conserved genomic DNA regions, such as but not limited to DNA encoding 23S rRNA, can be amplified from microalgae species and compared to consensus sequences to select microalgae species that are taxonomically related to the preferred microalgae used in the present invention. Examples of comparing DNA sequences for species of the genus Prototheca are shown below. Comparison of genomic DNA can also be helpful in identifying microalgae species that have been misidentified in a collection of strains. Often a collection of strains will identify species

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industrial ^ 5f of microalgae based on their tenoLipltas — ymorfológicas characteristics. The use of these characteristics can lead to the incorrect categorization of the species or genus of a microalgae. Using genomic DNA comparison may be a better method of categorizing microalgae species based on their phylogenetic relationship.

Microalgae for use in the present invention typically have genomic DNA sequences encoding 23S rRNA with at least 99%, at least 95%, at least 90%, or at least 85% nucleotide identity with at least one of the sequences listed in sec. with no. Ident .:

11-19.

For sequence comparison to determine percent nucleotide or amino acid identity, typically a sequence acts as a reference sequence, against which the sequences being compared are compared.

When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, if necessary posterior coordinates are designated, and the program parameters of the sequence algorithm are designated. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequence (s) relative to the

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reference sequence, based on parameters - de - jM = © g £ ama. designated.

Optimal sequence alignment for comparison can be done, for example, by the Smith &amp; Waterman local homology algorithm, Adv. Appl. Math. 2: 482 (1981), by the Needleman &amp; Wunsch homology alignment algorithm, J. Mol. Biol. 48: 443 (1970), by searching for the Pearson &amp; Lipman similarity method,

Proc. Nati. Acad Sci., USA 85: 2444 (1988), for computerized implementations of these algorithms (GAP,

BESTFIT, FASTA, and TFASTA of the Wisconsin Software Package

Genetics, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et al., Supra).

Another example of an algorithm that is suitable for determining sequence identity and sequence similarity is the BLAST algorithm, which is described in

Altschul et al., J. Mol. Biol. 215: 403-410 (1990). The software for running BLAST analyzes is publicly available 20 through the National Center for Information.

Biotechnology (on the website www.ncbi.nlm.nih.gov). This algorithm involves first identifying high-scoring sequence pairs (HSP) by

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INDUSTRIAL Xte g identification of short words of length W in — X problem sequence, which combine or satisfy some threshold score greater than parameter T, when they are aligned with a word of the same length in a database sequence. Parameter T is referred to as the neighbor word score threshold (Altschul et al., Supra.).

The hits of neighboring words behave like seeds to start searches and find longer HSPs that contain them. The hits of neighboring words are then extended in both directions throughout each sequence until the cumulative alignment score is increased. For nucleotide sequences, cumulative alignment scores are calculated using parameters M (reward score for a pair of residues that combine; always> 0) and N (punishment score for residues that do not combine; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extensions of the hits of the neighboring words in each direction are stopped when: the cumulative alignment score decreases by an amount X from its maximum value reached;

cumulative score goes to zero or below zero due to accumulation of one or more alignments of

<img file="MX339639B_D0049.tif" />

negative scoring residuals; or the end of 'any of the sequences is reached. To identify whether the nucleic acid or the polypeptide is within the scope of the invention, the parameters defined in the BLAST programs are adapted. The BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strings. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. The TBLATN program (which using protein sequences to sequence nucleotides) uses by default a word length (W) of 3, a hope (E) of 10, and a BLOSUM score matrix 62. (see Henikoff &amp;

Henikoff, Proc. Nati Acad Sci., USA 89: 10915 (1989)).

In addition to calculating the percentage identity of the sequence, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin &amp; Altschul, Proc. Nati. Acad Sci., USA

90: 5873-5787 (1993)). A measure of the similarity provided by the BLAST algorithm is the probability of the smallest sum (P (N)), which provides an indication of the probability with which two nucleotide sequences or

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amino acids randomly match. For example, a nucleic acid is considered similar to a reference sequence if the probability of the smallest sum in a comparison between the test nucleic acid and a reference nucleic acid is less than about 0.1, more preferably if it is less than about 0.01. and more preferably if it is less than approximately 0.001.

Other considerations affecting the selection of microorganisms for use in the invention, in addition to the production of lipids or hydrocarbons suitable for the production of oils, fuels and oleochemicals, are: (1) high lipid content as a percentage of cell weight ; (2) ease of growth; (3) ease of genetic modification; and (4) ease of biomass processing. In particular modalities, the wild or genetically engineered microorganism produces cells that contain at least 40%, at least 45%, at least

50%, at least 55%, at least 60%, at least 65%, or at least 70% or more of lipids. Preferred organisms grow heterotrophically (in sugar in the absence of light).

Examples of algae that can be used to practice the present invention include, but are not limited to, the following algae listed in Table 1.

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Table 1. Examples of algae.

Achnanthes orientalis, Agmenellum, Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora delicatissima capitata,

Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Cartería, Chaetoceros gracilis, Chaetoceros muelleri subsalsum, Chaetoceros muelleri subsalumum, Chaetoceros muelleri subsalsum, Ch.

Antarctic, Chlorella aureoviridis, Chlorella candida,

Chlorella capsule, 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 kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var.

lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilís, Chlorella nocturnal, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides (including any of the UTEX 1806, 411, 264, 256,

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255, 250, 249, 31, 29, 25, and CCAP strains 211/17 and 211 / 8d),

Chlorella protothecoides var. acidicola, Chlorella you will regulate,

Chlorella will regulate var. minimum, Chlorella will regulate var.

umbricata, Chlorella relsiglii, 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. autotrophica,

Chlorella vulgaris var. viridis, Chlorella vulgaris var.

vulgaris, Chlorella vulgaris var. vulgaris f. tertia,

Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides,

Chlorella vulgaris, Chl orococcum infusionum, Chlorococcum sp.,

Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Cryptomonas sp., Cyclotella cryptica, cyclotella meneghiniana, cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate,

Dunaliella maritime, Dunaliella minuta, Dunaliella parva,

Dunaliella peircei, Dunaliella primolecta, Dunaliella salina,

Dunaliella terrícola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis,

<img file="MX339639B_D0053.tif" />

Eremosphaera sp., Ellipsoidon sp., Euglena, Franceía sp.,

Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp.,

Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana,

Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614), Monoraphidium minutum, Monoraphidium sp. ,

Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp. ,

Navicula acceptata, Navicula biskanterae, Navicule pseudotenelloides, Navicule pelliculosa, Navicula saprophila,

Navicula sp., 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 sp.,

Oscillatoria subbrevis, Pascheria acidophila, Pavlova sp.,

Phagus, Phormidium, Platymonas sp., Pleurochrysis portfolio,

Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis,

Prototheca moriformis, Prototheca zopfii, Pyramimonas sp.,

Pyrobotrys, Sarcinoid chrysophyte, Scenedesmus armatus,

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Spirogyra, Spirulina platensis, Stichococcus sp.,

Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana

Examples of oil yeast that can be used to practice the present invention include, but are not limited to, the following oil yeasts listed in Table 26.

Table 26. Examples of oil yeast.

Cryptococcus curvatus, · Cryptococcus terricolus, Candida sp.,

Lipomyces starkeyi, Lipomyces Upo fer, Endomycopsis vernalis,

Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica

Examples of other mushrooms that can be used to practice the present invention include, but are not limited to, the following mushrooms listed in Table 27.

Table 27. Examples of fungi.

Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus,

Aspergillus terreus, Pennicillium iilacinum, Hensenulo,

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Chaetomium, Cladosporium, Malbranchea, Rhizopu'3 ', ~ Y' ^ and Lhiuiw

In some embodiments of the present invention, the microorganism is a bacterium. Examples of exogenous gene expression in bacteria, such as E. coli, are well known; see for example Molecular Cloning: A Laboratory

Manual, Sambrook and others (3rd edition, 2001, Coid Spring

Harbor Press).

2. Bioreactor

Microorganisms are cultivated for two purposes, that of performing genetic manipulations and for the production of hydrocarbons (for example, lipids, fatty acids, aldehydes, alcohols and alkanes). The first type of culture is carried out on a small scale and initially, at least, under conditions in which the initial microorganism can grow. Cultivation for hydrocarbon production purposes is usually performed on a large scale (for example, 10,000 1, 40,000 1, 100,000 1 or greater bioreactors) in a bioreactor. Microalgae, including Prototheca species, are typically grown in the methods of the invention in a liquid medium within a bioreactor.

Typically, the bioreactor does not allow light to enter.

<img file="MX339639B_D0056.tif" />

The bioreactor or fermenter is used to filter oil microbial cells, preferably microalgae cells, through various phases of their physiological cycle.

Bioreactors offer many advantages for use in heterotrophic growth and propagation methods. To produce biomass for use in food, the microalgae are preferably fermented in large amounts of liquid, such as suspension cultures as shown in one example. Bioreactors such as steel fermenters can accommodate very large volumes of culture (bioreactors of 40,000 liters and more capacity are used in various embodiments of the invention). Bioreactors typically also allow control of culture conditions such as temperature, pH, oxygen tension, and carbon dioxide levels. For example, bioreactors are typically configured, for example using ports attached to pipes, to allow gaseous components such as oxygen or nitrogen to be bubbled through the culture liquid. Other parameters of the culture, such as the pH of the culture medium, the identity and concentration of the trace elements, and other constituents of the medium, can be easily manipulated using the bioreactor.

<img file="MX339639B_D0057.tif" />

The bioreactors can be configured so that the culture medium flows through the bioreactor throughout the period of time in which the microalgae reproduce and increase their number. In some embodiments, for example, the medium can be infused into the bioreactor after inoculation, but before the cells reach the desired density. In other cases, the bioreactor is filled with the culture medium at the start of a culture, and no further culture medium is infused after the culture is inoculated. In other words, the microalgae biomass is cultivated in an aqueous medium for a period of time in which the microalgae reproduce and increase their number; however, during this time period the amounts of the aqueous culture medium do not flow through the bioreactor.

Thus, in some embodiments, the aqueous culture medium does not flow through the bioreactor after inoculation.

Bioreactors equipped with devices such as stirring blades and impellers, balancing mechanisms, stirring bars, means for infusing pressurized gas, can be used to mix microalgae cultures. Mixing can be continuous or intermittent. For example, in some embodiments, a turbulent flow regime of gas inlet and media inlet is not maintained for

<img file="MX339639B_D0058.tif" />

reproduction of the microalgae until the desired increase in the number of said microalgae has been reached.

The bioreactor ports can be used to introduce, or extract gases, solids, semisolids and liquids from the reactor chamber containing the microalgae. Although many bioreactors have more than one port (for example, one for medium entry and one for sampling), it is not necessary for only one substance to enter or exit the port. For example, a port can be used to flow the culture medium into the reactor and subsequently for sampling, gas inlet, gas outlet, or other purposes. Preferably, a sampling port can be used repeatedly without compromising the axenic nature of the culture. A sampling port can be configured with a valve or other device that allows the sample circulation to be stopped or started or to provide a continuous sampling means. Typically, bioreactors have at least one port that allows the inoculation of a culture and said port can be used, in addition, for other purposes such as the entry of medium or gas.

The ports of the bioreactors allow to manipulate the gaseous content of the microalgae cultures. To illustrate, part of the volume of a bioreactor may be gas

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INSTm. »T '> MEXICANO OR ΙΆ RRORIEDAD instead of liquid, and the gas inlets of the bioreactor allow gases to be pumped into the bioreactor. Gases that can be pumped beneficially into a bioreactor include air, air / CO mixtures₂, noble gases, such as argon and other gases. Bioreactors are typically equipped to allow the user to control the rate of gas entry into the bioreactor. As noted above, increasing the gas flow within the bioreactor increases the culture mix.

[0004] The increase in gas flow also affects the turbidity of the crop. Turbulence can be achieved by placing a gas inlet port below the level of the aqueous culture medium so that the gas entering the bioreactor bubbles to the culture surface. One or more gas outlet ports allow gas to escape, thereby preventing pressure from gradually increasing in the bioreactor. Preferably, the gas outlet port leads to a valve in a single direction that prevents contaminating microorganisms from entering the bioreactor.

3. Means, medium

The microalgae culture medium is generally composed of a fixed source of nitrogen, a source

<img file="MX339639B_D0060.tif" />

fixes carbon, trace elements, upciuiially a buffer solution to maintain pH, and phosphate (typically provided as the phosphate salt). Other components can include salts such as sodium chloride, particularly for marine microalgae. Nitrogen sources include organic and inorganic nitrogen sources, including, for example, without limitation, molecular nitrogen, nitrate, nitrate salts, ammonia (pure or in salt form, such as (NH₄)₂SW₄ and NH₄OH), protein, soy flour, steep corn liqueur, and yeast extract. Examples of trace elements include zinc, boron, cobalt, copper, manganese, and molybdenum in, for example, the respective forms of ZnCl.₂, H₃BO₃, CoCl₂-6H₂0, CuCl₂-2H₂OR,

MnCl₂-4H₂O and (NH₄) ₆Mo₇0₂₄ 4H₂OR.

According to the methods of the present invention useful microorganisms are found in various places and environments throughout the world. As a consequence of its isolation from other species and its resulting evolutionary divergence, the particular growth medium for optimal growth and generation of lipid and / or hydrocarbon constituents may be difficult to predict. In some cases, certain strains of microorganisms may be unable to grow in a particular growth medium due to

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presence of some inhibitory component e- ° ^Ί = absence of an essential nutritional requirement required by a particular strain of the microorganism.

Solid and liquid growth media 5 are generally available from a wide variety of sources, and instructions for preparing a particular medium that is suitable for a wide range of varieties of microorganisms can be found, for example, on-site. web http://www.utex.org/, a site maintained by the University of Texas at Austin, 1 University Station A6700, Austin, Texas, 78712-0183, for its algae culture collection (UTEX). For example, various freshwater and saltwater media include those described in PCT publication no. 2008/151149, incorporated herein by reference.

In a particular example, Proteose medium is suitable for axenic cultures, and an 11 volume of medium (pH ~ 6.8) can be prepared by adding 1g of proteose peptone to 1 liter of Bristol Medium. The Bristol medium comprises 2.94 mM of

NaN0₃0.17 mM CaCl₂-2H₂Or, 0.3 mM MgSO₄-7H₂Or, 0.43 mM,

1.29 mM KH₂PO₄, and 1.43 mM NaCl in an aqueous solution.

For 1.5% agar medium, 15 g of agar can be added to 1 of the solution. The solution is covered and subjected to

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INSTITUTO MUC1CAN4 DE LA PRONIftAD industrial autoclave, and then store at refrigerated temperature before use. Another example is the insulation medium of

Prototheca (PIM), comprising 10g / l Potassium Hydrogen Phthalate (KHP), 0.9g / l Sodium Hydroxide, 0.1g / l Magnesium Sulfate, 0.2g / l Potassium Hydrogen Phosphate, 0.3g / l of ammonium chloride, 10g / l of glucose, 0.001g / l of thiamine hydrochloride, 20g / l of agar, 0.25g / l of 5-fluorocytosine, in a pH range of 5.0 to 5.2 (see

Pore, 1973, App. Microbiology, 26: 648-649). Other suitable means for use with the methods of the invention can be easily identified by consulting the URL identified above, or by consulting other organizations that maintain cultures of microorganisms, such as SAG, CCAP or CCALA. SAG refers to the Algae Culture Collection at the University of Gottingen (Gottingen, Germany), CCAP refers to the collection of algae and protozoa cultures managed by the Association

Scottish Marine Sciences (Scotland, UK), and CCALA refers to the collection of laboratory algae cultures at the Institute of Botany (Treboñ, Czech Republic).

Additionally, U.S. Patent No. 5,900,370 describes media formulations and conditions suitable for the heterotrophic fermentation of the Prototheca species.

<img file="MX339639B_D0063.tif" />

For oil production, it is lmpuiLariLu tt — i »selection of a fixed carbon source, since the cost of the fixed carbon source must be low enough for oil production to be economical. Thus, although suitable carbon sources include, for example, acetate, floridoside, fructose, galactose, glucuronic acid, glucose, glycerol, lactose, trickle, Nacetylglucosamine, rhamnose, sucrose, and / or xylose, the selection of raw materials containing Such compounds is an important aspect of the methods of the invention. Suitable raw materials useful in accordance with the methods of the invention include, for example, black liquor, corn starch, depolymerized cellulosic material, buttermilk, molasses, potato, sorghum, sucrose, beet sugar, sugar cane, rice, and wheat. Carbon sources can also be provided as a mixture of sucrose and depolymerized beet sugar pulp. One or more carbon sources can be supplied at a concentration of at least about 50 QM, at least about 100 QM, at least about 500 ÜM, 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. Carbon sources of

<img file="MX339639B_D0064.tif" />

Of particular interest for the purposes of the present invention include cellulose (in depolymerized form), glycerol, sucrose, and sorghum, each of which is discussed in more detail below.

In accordance with the present invention, the microorganisms can be grown using depolymerized cellulosic biomass as a raw material. Cellulosic biomass (eg, stubble like corn stubble) is cheap and readily available; however attempts to use this material as a raw material for yeast have failed. In particular, these raw materials have been found to have an inhibitory effect on yeast growth, and yeast cannot use the 5-carbon sugars produced from cellulosic materials (eg, xylose from hemicellulose). In contrast, microalgae can grow on processed cellulosic material.

Cellulosic materials generally include about 40-60% cellulose, about 20-40% hemicellulose; and 10-30% lignin.

Suitable cellulosic materials include residues of herbaceous and woody energy crops, as well as agricultural crops, i.e. parts of plants, mainly stems and leaves not removed from fields with

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MEXICAN INSTITUTE PE LA PtORIEPAP INDUSTRIAL

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the main food or fiber product. Examples include agricultural wastes such as sugarcane bagasse, rice husk, corn fiber (including stalks, leaves, husks and cobs), wheat straw, rice straw, beet sugar pulp, citrus pulp, citrus peel, forest waste such as wood and softwood thinning, hard and softwood, and waste from wood operations, wood waste such as factory waste (wood chips, sawdust) and pulp mill waste, urban waste, such as pieces of paper from urban solid waste, urban wood waste and green urban waste such as municipal grass clippings and wood construction waste. Additional cellulosic materials include dedicated cellulosic crops such as rod grass, hybrid poplar wood, miscanthus, reed fibers, and sorghum fibers. The 5-carbon sugars produced from these materials include xylose.

Cellulosic materials are treated to increase the efficiency with which the microbe can use the sugar (s) contained in the materials. The invention provides new methods for the treatment of materials

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<img file="MX339639B_D0066.tif" />

cellulosic after acid blast to make materials suitable for use in a heterotrophic culture of microbes (eg, microalgae and oil yeasts). As discussed above, lignocellulosic biomass is comprised of several fractions, including cellulose, a crystalline beta-bond polymer.

1,4-glycosidics (a 6-carbon sugar), hemicellulose, a more closely associated polymer composed predominantly of xylose (a 5-carbon sugar), and to a lesser extent, crafty, galactose, arabinose, lignin, a complex aromatic polymer consisting of synapyl alcohol and its derivatives, and pectins, which are linear chains of a linked 1,4-polygalacturonic acid. Due to the polymeric structure of cellulose and hemicellulose, the sugars (eg, glucose and monomeric xylose) in them are not found in such a way that they can be used (metabolized) efficiently by many microbes. For these microbes, further processing of the cellulosic biomass to generate the monomeric sugars that make up the polymers can go a long way in ensuring that the cellulosic materials are used efficiently as a raw material (carbon source).

<img file="MX339639B_D0067.tif" />

IMPI

MEXICAN INSTITUTE

OF THE INDUSTRIAL ERT IgllDAD

Cellulosic or cellulose biomass — SS — 3ΠΠΤ5Γ5 — a — ΠΓΓ process, called an explosion, in which the biomass is treated with dilute sulfuric acid (or other) at elevated pressure and temperature. This process conditions the biomass so that it can be efficiently subjected to the enzymatic hydrolysis of the cellulosic and hemicellulosic fractions in glucose and xylose monomers. The monomeric sugars that are obtained are called cellulose sugars. Cellulosic sugars can subsequently be used by microorganisms to produce various metabolites (eg lipids). The acid blasting step results in partial hydrolysis of the hemicellulose fraction to obtain moñosaccharides. These sugars are completely released from the biomass. by means of a subsequent treatment. In some modalities, this treatment consists of a hydrothermal treatment that includes washing the exploited material with hot water, which removes contaminants such as salts. This stage is not necessary for cellulosic ethanol fermentations because more dilute concentrations of sugars are used in these processes. In other modalities, post treatment is additional acid treatment. In still other modalities, post treatment is hydrolysis

<img file="MX339639B_D0068.tif" />

enzymatic of the exploited material. These treatments can also be used in any combination. The type of treatment can affect the type of sugar released (for example, five-carbon sugars versus six-carbon sugars) and the stage at which they are released in the process.

As a consequence, different sugar currents can be created, which can be five or six carbon atoms. These enriched streams of five or six carbon atoms can then be targeted to specific microorganisms with different carbon utilization capabilities.

The methods of the present invention typically involve fermentation at higher cell densities than is achieved in ethanolic fermentation. Due to the high densities of the cultures for the heterotrophic production of cellulosic oil, the fixed carbon source (for example, the sugar stream (s) of the cellulosic derivative) is preferably in a concentrated form. The glucose level of the depolymerized cellulosic material is preferably at least 300 g / liter, at least 400 g / liter, at least 500 g / liter or at least 600 g / liter before the culture step, which is optionally a fed batch culture, in which cells are fed with the

<img file="MX339639B_D0069.tif" />

material as they grow and accumulate lipids. In the production of cellulosic ethanol, cellulosic sugar streams are not used at or near these concentration ranges.

Thus, to generate and maintain the high cell densities during the production of lignocellulosic oil, the carbon raw material (s) must be supplied to the heterotrophic cultures in a highly concentrated form. However, any component in the feed stream that is not a substrate and that is not metabolized by the oil-bearing microorganism will accumulate in the bioreactor, leading to problems if the component is toxic or has inhibitory activity on the production of the desired end product. . Although lignin, and by-products derived from lignin, by-products derived from carbohydrates, such as furfurals and hydroxymethylfurfurals and the salts derived from the generation of cellulosic materials (during the explosion process and the subsequent neutralization process), and even unmetabolized sugars pentose / hexose, can present problems in ethanolic fermentations, These effects are significantly amplified in processes in which their concentration in the initial raw material is high. To achieve sugar concentrations in the range of 300g / l (or

ΙΜΡΪ

INSTITUTO MEXICANA, DE LA RRORIEDAF 'industrial

<img file="MX339639B_D0070.tif" />

above) For six-carbon sugars that can be used in large-scale productions of the lignocellulosic oils described in the present invention, the concentration of these toxic materials may be 20 times higher than the concentration typically present in ethanolic biomass fermentations. cellulosic.

The treatment with the explosion process of the cellulosic material uses significant amounts of sulfuric acid, heat and pressure, therefore releasing byproducts of specifically furfural and hydroxymethylfurfural carbohydrates. Furfurals and hydroxymethylfurfurals are produced during the hydrolysis of hemicellulose by dehydrating xylose in furfural and water. In some embodiments of the present invention, these by-products (eg, furfurals and hydroxymethylfurfurals) are removed from the saccharified lignocellulosic material prior to introduction into the bioreactor. In some embodiments of the present invention, the carbohydrate by-product removal process is performed by hydrothermal treatment of the exploited cellulosic materials. Furthermore, the present invention provides methods in which strains capable of tolerating compounds such as furfurals and

ΙΜΡΙ

<img file="MX339639B_D0071.tif" />

INWISTRIAL hydroxymethylfurfurals are used for the production of lignocellulosic oils. In another embodiment, the present invention also provides methods and microorganisms that are not only capable of tolerating furfurals in the fermentation medium, but are actually capable of metabolizing these by-products during the production of lignocellulosic oil.

The explosion process also generates significant levels of salts. For example, typical blast conditions can result in conductivities greater than 5 mS / cm when the exploited cellulosic biomass is resuspended in a 10: 1 water: solids (dry weight) ratio. In certain embodiments of the present invention, the diluted exploited biomass is subjected to enzymatic saccharification, and the obtained supernatant is concentrated up to 25 times for use in the bioreactor. The salt level (measured by conductivity) in the concentrated sugar stream (s) can be unacceptably high (up to 1.5 M Na⁺equivalents). Additional salts are generated with the neutralization of the exploited materials for the subsequent enzymatic saccharification process. The present invention provides methods for removing these salts so that the concentrated cellulosic sugar stream (s)

<img file="MX339639B_D0072.tif" />

resulting can be used in tieLeiútiuf us processes - to produce lignocellulosic oil. In some embodiments, the method of removing these salts is deionization with resins, such as, but not limited to, DOWEX Marathon

MR3. In certain embodiments, the resin deionization step occurs prior to sugar concentration or pH adjustment and hydrothermal treatment of the biomass prior to saccharification or any combination of the procedure; in other modalities this stage is carried out after one or more of these processes. In other embodiments, the blast process itself is changed to prevent generation of salts at unacceptable high levels.

For example, a suitable alternative for the explosion of cellulosic biomass with sulfuric acid (or another acid) is the manufacture of mechanical pulp to make the cellulosic biomass receptive to enzymatic hydrolysis (saccharification). In still other embodiments, native strains of microorganisms resistant to high levels of salt or genetically engineered strains with resistance to high levels of salt are used.

A preferred embodiment for the process of preparing exploited cellulosic biomass for use in the production of heterotrophic lignocellulosic oil using microbes

<img file="MX339639B_D0073.tif" />

ΙΜΡΙ oilseeds. A first stage involves adjusting- θ · ί · - · ρί · ί — of --- í-exploded cellulosic abiomass resuspended in the interval of

5.0-5.3 followed by washing the cellulosic biomass three times. This washing step can be performed using a variety of means including the use of desalting and ion exchange resins, reverse osmosis, hydrothermal treatment (as described above), or simply by repeated re-suspension in deionized water and centrifugation. This washing step results in a cellulosic current whose conductivity is between 100-300 pS / cm and the elimination of significant amounts of furfurals and hydroxymethylfurfurals. The decantation of this washing stage can be saved to concentrate the five-carbon sugars released from the hemicellulose fraction.

A second stage comprises the enzymatic saccharification of the washed cellulosic biomass. Accellerase (Genencor) is used in a preferred embodiment. A third stage involves the recovery of sugars by centrifugation or decantation and washing of the saccharified biomass. The resulting biomass (solids) is an energy-dense, lignin-rich component that can be used as fuel or disposed of as waste. The sugar stream recovered during the spin / decant and rinse process is collected. A fourth

<img file="MX339639B_D0074.tif" />

IMPI

MEXICAN INSTITUTE OF THE W «FIIDAD INOI ISTRIAL stage comprises microfiltration to remove solid contaminants and recover the permeate. A fifth stage comprises a concentration stage that can be performed using a vacuum evaporator. This step may optionally include the addition of antifoaming agents such as P'2000 (Sigma / Fluka), which is sometimes needed due to the protein content of the resulting sugar raw material.

In another embodiment of the methods of the invention, the carbon source is glycerol, including the acidified and unacidulated glycerol by-products obtained from the transesterification of biodiesel. In one embodiment, the carbon source includes glycerol and at least one other carbon source. In some cases, all glycerin and at least 15 other attached carbon source are supplied to the microorganism at the start of fermentation. In some cases, glycerin and the at least one other attached carbon source are supplied to the microorganism simultaneously at a predetermined ratio. In some cases, glycerol and at least one of the other fixed carbon sources are fed to the microbes at a predetermined rate during the course of fermentation.

<img file="MX339639B_D0075.tif" />

Some microalgae undergo cell division more rapidly in the presence of glycerol than in the presence of glucose (see PCT Pub. No. 2008/151149). In these cases, two-stage growth processes in which cells are first fed with glycerol to rapidly increase cell density, and then fed with glucose to accumulate lipids, can improve the efficiency with which lipids are produced. The use of the glycerol by-product from the transesterification process provides significant economic benefits by putting it back into the production process. Other feeding methods such as glycerol and glucose mixtures are also provided. Feeding such mixtures also provides the same economic benefits. Furthermore, the invention provides methods for feeding the microalgae with alternative sugars, such as sucrose in various combinations with glycerol.

In another embodiment of the methods of the invention, the carbon source is invert sugar. Invert sugar is produced by the separation of sucrose into its monosaccharide, fructose and glucose components. Invert sugar production can be achieved through various methods that are known in the art. One such method is to heat

<img file="MX339639B_D0076.tif" />

an aqueous sucrose solution. Frequently catalysts are used to accelerate the conversion of sucrose to invert sugar. These catalysts can be biological, for example enzymes such as invertases and sucrases can be added to sucrose to accelerate the hydrolysis reaction to produce invert sugar. Acid is an example of a non-biological catalyst, when combined with heat it can accelerate the hydrolysis reaction. Once invert sugar is prepared, it is less prone to crystallization compared to sucrose, and thus provides advantages for storage and fed batch fermentation, which in the case of heterotrophic cultivation of microbes, including microalgae , there is a need for a concentrated carbon source. In one embodiment, the carbon source is invert sugar, preferably in a concentrated form, preferably at least 800g / liter, at least 900g / liter, at least 1,000g / liter or at least 1,100g / liter prior to the step of culture, which is optionally a fed batch culture. Invert sugar, preferably in a concentrated form, is fed to cells over time as cells grow and accumulate lipids.

<img file="MX339639B_D0077.tif" />

ιμριγ ^

MEXICAN INSTITUTE

In another embodiment of the invpnrjón methods, the carbon source is sucrose, including a complex sucrose-containing raw material, such as thick cane juice from sugarcane processing. Due to the higher 5 densities of the cultures for heterotrophic oil production, the fixed carbon source (eg sucrose, glucose, etc.) is preferably in a concentrated form, preferably at least 500 g / liter, at least 600 g / liter, at least 700 g / liter or at least 800 g / liter of the fixed carbon source before the cultivation stage, which is optionally a fed batch culture in which the material is fed to the cells over time as the cells grow and accumulate lipids.

In some cases, the carbon source is sucrose in the form of thick cane juice, preferably in a concentrated form, preferably at least 60% solids or approximately 770 g / liter of sugar, at least 70% solids or approximately 925 g / liter of sugar, or at least

80% solids or approximately 1125 g / liter of sugar before the cultivation stage, which is optionally a fed batch culture. The thick juice from the concentrated cane feeds the cells over time as the cells grow and accumulate lipids.

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M £ X1CAN &lt;i INSTITUTE

OF INDUSTRIAL PROPERTY

In one modality, the culture medium¹ UlC'lliytí, —also, 'at least one sucrose utilization enzyme. In some cases, the culture medium includes a sucrose invertase. In one embodiment, the sucrose invertase enzyme is a secretable sucrose invertase enzyme encoded by an exogenous sucrose invertase gene expressed by the population of microorganisms. Thus, in some cases, as described in more detail in Section IV, below, the microalgae was genetically modified to express a sucrose utilizing enzyme, such as a sucrose transporter, - a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.

Complex sucrose-containing raw materials include molasses waste from sugarcane processing; the use of these low-value sugar cane processing waste products provides significant cost savings from the production of hydrocarbons and other oils. Sorghum is another complex sucrose-containing raw material that is useful in the methods of the invention, including sorghum syrup and pure sorghum. Sorghum syrup is produced from the juice of sweet sorghum cane. Its sugar profile is mainly made up of glucose (dextrose), fructose and sucrose.

<img file="MX339639B_D0078.tif" />

<img file="MX339639B_D0079.tif" />

IMPI

MEXICAN INSTITUTE ». _,. . -. . OF RROFIÍDAD

Four. Industrial oil production

For the production of oil according to the method of the invention, it is preferred to cultivate the cells in the dark, as is the case, for example, when using very large fermenters (40,000 liters and of greater capacity) that do not allow the light reach the crop. Prototheca species are cultivated and propagated for oil production in a medium containing a fixed carbon source and in the absence of light; such growth is known as heterotrophic growth.

As an example, an inoculum of lipid-producing oil-bearing microbial cells, preferably microalgae cells are introduced into the medium; there is a lag period (lag phase) before the cells start to spread. After the latency period, the propagation speed increases at a constant rate and enters the log or exponential phase. The exponential phase, in turn, is followed by a slowdown in propagation due to decreased nutrients such as nitrogen, increased toxic substances, and quorum detection mechanisms. After this deceleration, the propagation stops and the cells enter a stationary phase or constant state, depending on the particular environment provided by the Mexican Institute.

ΠΕ THE OWN

INDUSTRIAL '• J **** · * · * .— to cells. To obtain h-io ™ ^³ τ-i &lt;-a in lipids, the crop typically harvests well after the end of the exponential phase, which can end early by allowing nitrogen or another key nutrient (other than carbon) to be depleted, forcing cells to convert excess carbon sources into lipids. The parameters of the culture conditions can be manipulated to optimize the total oil production, the combination of lipid species produced and / or the

1θ production of a specific oil.

As mentioned above, a bioreactor or fermenter is used to allow cells to undergo the various phases of their growth cycle. As an example, an inoculum of the lipid-producing cells can be introduced into a medium, followed by a latency period (lag phase) before the cells begin to grow.

After the latency period, the growth rate increases steadily and enters the log or exponential phase. The exponential phase, in turn, is followed by a slowdown in growth due to decreased nutrients and / or increased toxic substances. After this slowdown, growth stops, and cells enter a stationary phase or constant state,

<img file="MX339639B_D0080.tif" />

ΙΜΡΙ

MHKICAN INSTITUTE · depending on the particular environment provided to the cells. The production of lipids by the cells described in the present may occur during or after the exponential phase, including the stationary phase where nutrients are supplied, or are still available, to allow continued lipid production in the absence of cell division. .

Preferably, the microorganisms that grow using the conditions described herein and known in the art comprise at least about 20% by weight of lipids, preferably at least about 40% by weight, more preferably at least about 50% by weight. weight, and most preferably at least approximately

60% by weight. Process conditions can be adjusted to increase the performance of lipids suitable for a particular use and / or to reduce production costs. For example, in certain embodiments, a microalgae is grown in the presence of a limiting concentration of one or more nutrients, such as nitrogen, phosphorus, or sulfur, while providing excess fixed carbon energy such as glucose. Nitrogen limitation tends to increase the performance of microbial lipids, above the performance of microbial lipids in a culture in the

<img file="MX339639B_D0081.tif" />

ΙΜΡΙ

INSTITUTO MEXICANC UE LA FRORtDAP 'NnnSTRlAL that nitrogen is supplied in excess. In particular embodiments, the lipid yield increase is at least about: 10%, 50%, 100%, 200%, or 500%. The microbe can be grown in the presence of a limiting amount of a nutrient for a portion of the total culture period or for the entire period. In particular modalities, the nutrient concentration moves between a limiting concentration and a non-limiting concentration, at least twice during the total cultivation period. The lipid content of the cells can be increased by continuing the culture for longer periods of time, while providing excess carbon, limiting or not nitrogen.

[0005] In another embodiment, the yield in lipid production is increased by culturing a lipid-producing microorganism (eg, microalgae) in the presence of one or more cofactors of an enzyme of the lipid metabolic pathway (eg, an enzyme for the synthesis of fatty acids). In general, the concentration of the cofactor (s) is sufficient to increase the performance of microbial lipids (for example, fatty acids) above the performance of microbial lipids in the absence of the cofactor (s). In a modality

IMPI

MEXICAN INSTITUTE OF THE rtORBOAC INDUSTRIAL

<img file="MX339639B_D0082.tif" />

In particular, the cofactor (s) are provided to the culture by including a microbe in the culture (eg, microalgae) containing an exogenous gene encoding the cofactor (s). Alternatively, the cofactor (s) can be provided to a culture by including a microbe (eg, microalgae) containing an exogenous gene that encodes a protein that participates in cofactor synthesis. In certain embodiments, suitable cofactors include any vitamins required by a lipid pathway enzyme, such as, for example: biotin, pantothenate. Genes encoding cofactors suitable for use in the invention or involved in the synthesis of such cofactors are well known and can be introduced into microbes (eg, microalgae), using constructs and techniques as described above.

Specific examples of bioreactors, culture conditions, and heterotrophic growth and propagation methods described herein, can be suitably combined to improve the efficiency of microbial growth and the production of lipids and / or proteins.

Microalgae biomass with a high percentage of accumulated oils / lipids per dry weight is generated using

<img file="MX339639B_D0083.tif" />

different cultivation methods

DE IA RROglgOAP ινρι.ητμιαι.

that are known in the raa (see Pub. PCT no. 2008/151149). The microalgae biomass generated by the cultivation methods described herein and useful in accordance with the present invention comprises at least 10% microalgae oil per dry weight. In some embodiments, the microalgae biomass comprises at least 25%, at least 50%, at least 55%, or at least 60% microalgae oil per dry weight. In some embodiments, the microalgae biomass contains 10-90% microalgae oil, 25-75% microalgae oil, 40-75% microalgae oil, or 50-70% microalgae oil per dry weight.

The microalgae oil from the biomass described herein, or extracted from the biomass for use in the methods and compositions of the present invention, may comprise glycerolipids with one or more different esterified fatty acid side chains. Glycerolipids comprise one esterified glycerol molecule for one, two or three fatty acid molecules, which can be of different lengths and have different degrees of saturation. The length and saturation characteristics of fatty acid molecules (and microalgae oils) can be manipulated to modify the properties or

<img file="MX339639B_D0084.tif" />

ratios of fatty acid molecules in the microalgae ace of the present invention, via culture conditions or via lipid genetic engineering, as described in more detail below in Section IV. Therefore, specific mixtures of algae oil can be prepared from a single species of algae, by mixing biomass or algae oil from two or more species of microalgae, or by a mixture of algae oil of the invention. with oils from other sources such as soybean, rapeseed, cane, palm, palm kernel, coconut, corn, plant residues, Chinese tallow, olive, sunflower, cottonseed, chicken fat, beef tallow, porcine tallow, microalgae , macroalgae, microbes, cufea, flax, peanut, select white fat, lard, camelina sativa, mustard seeds, cashew nuts, oats, lupine, kenaf, marigold, hemp, coffee, flaxseed (flax), hazelnut, euphorbia, pumpkin seeds, coriander, camellia, sesame, safflower, rice, tung tree, cocoa, copra, opium poppy, castor beans, walnut, jojoba, macadamia, walnuts

Brazil, avocado, oil, or a distilled fraction of any of the above oils.

The composition of the oil, that is, the properties and proportions of the fatty acids that make up the

IMPI

ΤΓΠ5ΤΓΠ TO MEXICANO □ E LA FR «INDUSTRIAL FBDAD

<img file="MX339639B_D0085.tif" />

glycerolipids, can also be manipulated by combining the biomass or oil of at least two different species of microalgae. In some modalities, at least two of the different microalgae species have different glycerolipid profiles. The various microalgae species can be cultivated together or separately, as described herein, preferably under heterotrophic conditions, to generate the respective oils. Different species of microalgae may contain different percentages of the different fatty acids that make up the glycerolipids in the cell.

Generally, Prototheca strains have little or no C8-C14 chain length fatty acids. For example, Prototheca moriformis (UTEX 1435), Prototheca krugani (UTEX 329), Prototheca stagnora (UTEX 1442), and

Prototheca zopfii (UTEX 1438) do not contain (or contain undetectable amounts) of C8 fatty acids, between 0-0.01% CIO fatty acids, between 0.03-2.1% C12 fatty acids and between 1.0-1.7% C14 fatty acids.

In some cases, Prototheca strains containing a transgene encoding an acyl fatty ACP thioesterase that has activity towards the acyl fatty substrate ACP of C8 or C8-10 chain lengths have at least .- •. = S ' 2. 3

IMP I

MEXICAN INSTITUTE

DK LA MONffMD V INDUSTRIAL

1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 12%, or at least 15% or more, fatty acids of length chain C8. In other cases, Prototheca strains containing a transgene encoding an acyl fatty ACP thioesterase that has activity towards the acyl fatty acp substrate of CIO chain lengths are at least 1%, at least 5%, at least 10 %, at least 15%, at least 20%, at least 24%, or at least 25% or more, CIO chain length fatty acids. In other cases, strains of

Prototheca containing a transgene encoding an acyl fatty acp-thioesterase that has activity towards the C12 chain length acyl fatty acp substrate has at least

1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 34%, at least 35% or at least 40% or more, C12 chain length fatty acids. In other cases, Prototheca strains containing a transgene encoding an acyl fatty ACP thioesterase that has activity towards the C14 chain length acyl fatty ACP substrate have at least 1%, at least 2%, at least 3 %, at least 4%, at least 5%, at least 6%, at least 7%, at least 10%, at least 15%, at least 30%, at least 43%, or at least 45% or more, C14 chain length fatty acids.

<img file="MX339639B_D0086.tif" />

IMPI

INSTITUT # MEXICANO • P LA rtOMBDAD

INDUSTRIAL

In non-limiting examples, Prototheca strains containing a transgene encoding an acyl fatty acp thioesterase that has activity towards the C8 chain length acyl fatty acp substrate have between 1% -25%, or between 1% - 15%, preferably 1.8-12.29%, of C8 chain length fatty acids. In other non-limiting examples, Prototheca strains containing a transgene encoding an acyl fatty ACP thioesterase that has activity towards the CIO chain length fatty acyl CIO-ACP substrate are between 1% -50%, or between l % -25%, preferably 1.9123.97% CIO chain length fatty acids. In other non-limiting examples, Prototheca strains containing a transgene encoding an acyl fatty acp thioesterase that has activity towards the acyl fatty acp substrate of C12 chain length is between 5% -50%, or between 10% -40, preferably 13.55-34.01%, of C12 chain length fatty acids. In other non-limiting examples, strains of

Prototheca containing a transgene encoding an acyl fatty acp-thioesterase that has activity towards the C14 chain length acyl fatty acp substrate has between 1% 60%, or between 2% -45%, preferably 2.59-43.27%, of C14 chain length fatty acids. In other non-limiting examples, the Prototheca strains containing a transgene

<img file="MX339639B_D0087.tif" />

ΙΜΡΙ encoding a fatty acyl-ACP thioesterase that has broad specificity towards variable carbon chain length acyl fatty-ACP substrates has up to 30%, up to 35%, or preferably up to 39.45% of 5-length fatty acids chain C16. In some cases, strains of

Prototheca containing a transgene encoding an acyl fatty acp thioesterase that has activity towards the acyl fatty acp substrate of chain lengths between C8 and C14 are between 1% -75%, or between 2% -60%, preferably 2.6910 57.98%, of medium chain fatty acids (C8-C14). In some cases, Prototheca strains containing a transgene encoding an acyl fatty-ACP thioesterase that has activity towards the acyl fatty-ACP substrates of chain lengths between C12 and C14 are at least 3 0%, at least 40 %, or at least 49% of C12-C14 fatty acids. In some cases, keeping the transgenic Prototheca strains under constant and high selective pressure to conserve exogenous genes is advantageous in increasing desired fatty acids of a specific chain length. High levels of exogenous gene retention can also be achieved by inserting exogenous genes into the nuclear chromosomes of cells using homologous vector recombination and the methods described herein. The cells

IMPI

MEXICAN INSTITUTE. • E LA RRORIJT-AD INDUSTRIAL

<img file="MX339639B_D0088.tif" />

Recombinants containing exogenous genes integrated into nuclear chromosomes are an object of the invention.

The microalgae oil can also include other components produced by the microalgae, or incorporated into the microalgae oil of the culture medium. These other constituents may be present in a greater or lesser amount depending on the culture conditions used to cultivate the microalgae, the microalgae species, the extraction method used to recover the microalgae oil from the biomass and other factors that may affect the composition of microalgae oil. Non-limiting examples of these constituents include carotenoids, present at 0.01-0.5 mcg / g, 0.025-0.3 mcg / g, preferably 0.05 to 0.244 micrograms / gram, of oil; chlorophyll A present from 0.01-0.5 mcg / g, 0.025-0.3 mcg / g, preferably 0.045 to 0.268 micrograms / gram, of oil;

total chlorophyll of less than 0.1 mcg / g, less than 0.05 mcg / g, preferably less than 0.025 micrograms / gram, of oil; gamma tocopherol present from 1-300 mcg / g, 35-175 mcg / g, preferably 38.3-164 micrograms / gram, of oil;

total tocopherols present of 10-500 mcg / g, 50-300 mcg / g, preferably 60.8 to 261.7 microgram / gram, of oil;

less than 1%, less than 0.5%, preferably less than 0.25%

ΙΜΡΙ

MEXICAN INSTITUTE • E LA PROPItOAO INDUSTRIAL

<img file="MX339639B_D0089.tif" />

brassicasterol, campesterol, stigmasterol, or beta-sytosterol; total tocotrienols less than 400 mcg / g, preferably less than 300 micrograms / gram, of oil; or total tocotrienols present at 100-500 mcg / g, 225-350 mcg / g, preferably 249.6 to 325.3 micrograms / gram, of oil.

The other components may include, without limitation, phospholipids, tocopherols, tocotrienols, carotenoids (for example, alpha-carotene, beta-carotene, lycopene, etc.), xanthophylls (for example, lutein, zeaxanthin, alpha-10 cryptoxanthine and beta-cryptoxanthin), and various organic or inorganic compounds. In some cases, the oil extracted from Prototheca species ranges from 0.001-0.01 mcg / g,

0.0025-0.05 mcg / g, preferably 0.003 to 0.039 microgram lutein / gram, oil, less than 0.01 mcg / g, less than

0.005 mcg / g, preferably less than 0.003 micrograms of lycopene / gram, of oil; and less than 0.01 mcg / g, less than

0.005 mcg / g, preferably less than 0.003 microgram of beta carotene / gram, of oil.

In some embodiments, the present invention 20 provides an oil microbial cell comprising a triglyceride oil, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of:

at least about 1%, at least about 2%, at

IΜ ΡI $ 5 ^^

INSTITUT · MEXICANO »F LA RRORIEDAD

INDUSTRIAL minus about 5%, at least about 7%, at least about 10%, or at least about 15%,

C8: 0; at least about 1%, at least about

5%, at least about 15%, at least about

20%, at least about 25%, or at least about

30%, C10: 0; at least about 1%, at least

<td>approximately</td><td>5%, at least approximately</td><td> 10%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>15%, at least approximately</td><td> 20%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>25%, at least approximately</td><td> 30%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>35%, at least approximately</td><td> 40%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>45%, at least approximately</td><td> 50%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>55%, at least approximately</td><td> 60%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>65%, at least approximately</td><td> 70%,</td><td>to the</td><td>less</td>

<td>approximately</td><td colspan="2">75%, or at least about 80</td><td> %,</td><td>C12: 0;</td>

at least about 2%, at least about 5%, at

<td>less</td><td>approximately</td><td> 10%,</td><td>to the</td><td>less about</td><td>15%, at</td>

<td>less</td><td>approximately</td><td> 20%,</td><td>to the</td><td>less about</td><td>25% at</td>

<td>less</td><td>approximately</td><td> 30%,</td><td>to the</td><td>less about</td><td>35% at</td>

<td>less</td><td>approximately</td><td> 40%,</td><td>to the</td><td colspan="2">minus approximately 45%, or at</td>

<td>less</td><td>approximately</td><td> 50%,</td><td colspan="3">C14: 0; at least about</td>

<td> 30%,</td><td>to the</td><td>less</td><td>approximately</td><td> 35%,</td><td>to the</td><td>less</td><td>approximately</td>

<td> 40%,</td><td>to the</td><td>less</td><td>approximately</td><td> 45%,</td><td>to the</td><td>less</td><td>approximately</td>

<td> 50%,</td><td>to the</td><td>less</td><td>approximately</td><td> 55%,</td><td>to the</td><td>less</td><td>approximately</td>

ΙΜΡΪ

MEXICAN INSTITUTE OF LA PRORÍT »A¡ · 'INDUSTRIAL

<img file="MX339639B_D0090.tif" />

60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about

<td>90%, C16-.0;</td><td>to the</td><td>minus about 5%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 10%,</td><td>at least about 15%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 20%,</td><td>at least about 25%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 30%,</td><td>at least about 35%,</td><td>to the</td><td>less</td>

about 40%, at least about 45%, or at least about 50%, C18: 0; at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%,

<td>C18: l; less</td><td colspan="2">than</td><td>about 7%,</td><td colspan="2">less</td><td>than</td>

<td>approximately</td><td> 5%,</td><td colspan="2">less than about</td><td> 3%,</td><td colspan="2">less than</td>

<td>approximately</td><td> 1%,</td><td colspan="2">or about 0%, C18:</td><td>2; and</td><td>to the</td><td>less</td>

<td>approximately</td><td> 35%,</td><td>to the</td><td>less about</td><td> 40%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 45%,</td><td>to the</td><td>less about</td><td> 50%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 55%,</td><td>to the</td><td>less about</td><td> 60%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 65%,</td><td>to the</td><td>less about</td><td> 70%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 75%,</td><td>to the</td><td>less about</td><td> 80%,</td><td>to the</td><td>less</td>

<td colspan="3">about 85%, or saturated fatty acids.</td><td colspan="2">at least about</td><td> 90</td><td>%, of</td>

100

In some embodiments, the oil microbial cell

<img file="MX339639B_D0091.tif" />

It comprises triglyceride oil, which comprises the fatty acid profile selected from the group consisting of:

<td>total combined quantities</td><td>from C8: 0 and CIO: 0 from to</td><td>less</td>

<td>about 10% at least</td><td>approximately 20%,</td><td>less</td>

<td>about 3 0%, at least</td><td>approximately 40%, at</td><td>less</td>

<td>about 50% at least</td><td>approximately 60%, at</td><td>less</td>

<td>about 7 0%, at least</td><td>approximately 80%, at</td><td>less</td>

<td colspan="3">about 90%, or about 100%; quantities</td>

<td>combined totals of C10: 0,</td><td>C12: 0, and C14: 0 from to</td><td>less</td>

<td>about 50% at least</td><td>approximately 60%, at</td><td>less</td>

<td>about 70% at least</td><td>approximately 80%, at</td><td>less</td>

<td colspan="3">about 90%, or about 100%; quantities</td>

<td>combined totals of C16: 0,</td><td>C18: 0 and C18: 1 from to</td><td>less</td>

<td>about 60% at least</td><td>approximately 70%, at</td><td>less</td>

<td colspan="3">about 80%, at least about 90%, or</td>

approximately 100%; total combined amounts of C18: 0,

C18: 1 and C18: 2 of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; total combined amounts of C14: 0, C16: 0, C18: 0 and C18: 1 of at least about 60%, at least about 70s%, at least about 80%, at least about 90%, or

101

<img file="MX339639B_D0092.tif" />

approximately 100%; and total combined amounts of

C18: 1 and C18: 2 of less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or about 0%.

In some embodiments the oil microbial cell comprises triglyceride oil with a fatty acid profile comprising a ratio of fatty acids selected from the group consisting of: a C8: 0 ratio to

C10: 0 of at least about 5 to 1, at least 6 to 1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least 10 to

one; a C10: 0 to C12: 0 ratio of at least about 6 to

1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least to 1; a C12: 0 to C14: 0 ratio of at least about 5 to 1, at least 6 to 1, at least 7 to 1, at least 8 to 1, at least 9 to 1, or at least 10 to 1; A relationship

C14: 0 to C12: 0 of at least 7 to 1, at least 8 to 1, at least 9 to

1, or at least 10 to 1; and a C14: 0 to C16: 0 ratio of at least 1 to 2, at least 1 to 3, at least 1 to 4, at least 1 to 5, at least 1 to 6, at least 1 to 7, at least 1 at 8, at least 1 at

9, or at least 1 to 10.

In some embodiments, the present invention provides an oil microbial cell comprising

ΙΜ

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<img file="MX339639B_D0093.tif" />

INSTITUTO MEXICANI1 γ DE LA PROntDAD \ a triglyceride oil composition, wherein the fatty acid profile of the triglyceride oil is selected from the group consisting of: at least about 1%, at least about 2%, at least about 5%, at less than about 7%, at least about 10%, or at least about 15%, C8: 0; at least about 1%, at least about 5%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% C10: 0; at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80%, C12: 0; at least about 2%, at least about 5%, at least about 10%, at least about

15%, at least about 20%, at least about

25%, at least about 30%, at least about

35%, at least about 40%, at least about

45%, or at least about 50%, C14: 0; at least

103

<img file="MX339639B_D0094.tif" />

i ívi ri

MEXICAN INSTITUTE OE INOIISTRJAL PROPERTY

<td>approximately</td><td> 30%,</td><td>at least about 35%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 40%,</td><td>at least about 45%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 50%,</td><td>at least about 55%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 60%,</td><td>at least about 65%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 70%,</td><td>at least about 75%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 80%,</td><td>at least about 85%, or</td><td>to the</td><td>less</td>

<td>approximately</td><td> 90%,</td><td colspan="2">C16: 0; at least about 5</td><td>% al</td>

<td>less</td><td>approximately</td><td>10% at</td><td>less</td><td>approximately</td><td> 15%,</td><td>to the</td>

<td>less</td><td>approximately</td><td>20% at</td><td>less</td><td>approximately</td><td> 25%,</td><td>to the</td>

<td>less</td><td>approximately</td><td>30% at</td><td>less</td><td>approximately</td><td> 35%,</td><td>to the</td>

less than about 40%, at least about 45%, or at least about 50%, C18: 0; at least about

<td> 60%,</td><td>to the</td><td>less</td><td>approximately 65%, at</td><td>less</td><td>approximately</td>

<td> 70%,</td><td>to the</td><td>less</td><td>approximately 75%,</td><td>less</td><td>approximately</td>

<td> 80%,</td><td>to the</td><td>less</td><td>about 85%, or at</td><td>less</td><td>approximately</td>

90%, C18: l; less than about 7%, less than

<td>approximately</td><td colspan="2">5% less than</td><td>approximately</td><td> 3%,</td><td colspan="2">less than</td>

<td>approximately</td><td colspan="3">1%, or about 0%, C18:</td><td>;2; and</td><td>to the</td><td>less</td>

<td>approximately</td><td>35% at</td><td>less</td><td>approximately</td><td> 40%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>45% at</td><td>less</td><td>approximately</td><td> 50%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>55% at</td><td>less</td><td>approximately</td><td> 60%,</td><td>to the</td><td>less</td>

<td>approximately</td><td>65% at</td><td>less</td><td>approximately</td><td> 70%,</td><td>to the</td><td>less</td>

about 75%, at least about 80%, at least

104

<img file="MX339639B_D0095.tif" />

ΙΜΡΙ

MEXICAN INSTITUTE J ^ · PE LA ΕΜΟΡΙίΠΑΓ; Λ

Τ ΓΓ \ ν * ΓΙΓΜΛ »&lt;

INDD'STIUAL about 85%, or at least about 90%, of saturated fatty acids.

In some embodiments, the oleaginous microbial triglyceride oil composition comprises triglyceride oil comprising a fatty acid profile in which: the total combined amount of C10: 0, C12: 0 and C14: 0 is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C16: 0, C18: 0 and C18: l is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C18: 0,

C18: l and C18: 2 is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C14: 0, C16: 0, C18: 0 and C18: l is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% ; the total combined amount of C8: 0 and

C10: 0 is less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than

<img file="MX339639B_D0096.tif" />

105 about 25%, less than about 2 0%, more than about 15%, less than about 10%, less than about 5%, or about 0%.

In some embodiments, the oleaginous microbial triglyceride oil composition comprises triglyceride oil with a fatty acid profile comprising a ratio of fatty acids selected from the group consisting of: a C8: 0 to C10: 0 ratio of at least about 5 to

1, at least about 6 to 1, at least about 7 to, at least about 8 to 1, at least about 1, or at least about 10 to 1; a C10: 0 to C12: 0 ratio of at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to

one; a C12: 0 to C14: 0 ratio of at least about 5 to

1, at least about 6 to 1, at least about 7 to, at least about 8 to 1, at least about 1, or at least about 10 to 1; a C14: 0 to C12: 0 ratio of at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1; a C14: 0 to C16: 0 ratio of at least about 1 to 2, at least about 1 to

3, at least about 1 to 4, at least about 1

106

<img file="MX339639B_D0097.tif" />

to 5, at least about 1 to 6, at least, about 7, at least about 1 to 8, at least about 1 to 9, or at least about 1 to 10.

In some embodiments, the present invention provides a method of producing an oleaginoidal microbial triglyceride oil composition with a fatty acid profile selected from the group consisting of: at least about 1%, at least about 2%, at least about 5%, at least about 7%, at least about 10%, or at least about 15%, C8: 0; at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%, C10: 0; at least about

1%, at least about 5%, at least about

10%, at least about 15%, at least about

20%, at least about 25%, at least about

30%, at least about 35%, at least about

40%, at least about 45%, at least about

50%, at least about 55%, at least about

60%, at least about 65%, at least about

70%, at least about 75%, or at least about

80%, C12: 0; at least about 2%, at least

<img file="MX339639B_D0098.tif" />

107

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INSTITUTO MWüCANO Ot LA MOWSDAU INOUSTMIAL

<td>approximately</td><td> 5%,</td><td>to the</td><td>less</td><td>approximately</td><td> 10%.</td><td>to the</td><td>mPTins ..</td>

<td>approximately</td><td> 15%,</td><td>to the</td><td>less</td><td>approximately</td><td> 20%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 25%,</td><td>to the</td><td>less</td><td>approximately</td><td> 30%,</td><td>to the</td><td>less</td>

<td>approximately</td><td> 35%,</td><td>to the</td><td>less</td><td>approximately</td><td> 40%,</td><td>to the</td><td>less</td>

about 45%, or at least about 50%, C14: 0;

<td>to the</td><td>minus about 30%,</td><td>to the</td><td>less</td><td>approximately</td><td> 35%,</td>

<td>to the</td><td>minus about 40%,</td><td>to the</td><td>less</td><td>approximately</td><td> 45%,</td>

<td>to the</td><td>minus about 50%,</td><td>to the</td><td>less</td><td>approximately</td><td> 55%,</td>

<td>to the</td><td>minus about 60%,</td><td>to the</td><td>less</td><td>approximately</td><td> 65%,</td>

<td>to the</td><td>minus about 70%,</td><td>to the</td><td>less</td><td>approximately</td><td> 75%,</td>

<td>to the</td><td>minus about 80%,</td><td colspan="4">at least about 85%, or</td>

<td>to the</td><td>minus about 90%,</td><td>C16:</td><td>0; to the</td><td colspan="2">less about</td>

<td> 5%,</td><td>at least about</td><td> 10%</td><td>to</td><td colspan="2">less about</td>

<td> 15%,</td><td>to the</td><td>minus about 20%,</td><td>to the</td><td>less</td><td>approximately</td>

<td> 25%,</td><td>to the</td><td>minus about 30%,</td><td>to the</td><td>less</td><td>approximately</td>

<td> 35%,</td><td>to the</td><td>minus about 40%,</td><td>to the</td><td>less</td><td>approximately</td>

<td> 45%,</td><td>or</td><td>at least about</td><td colspan="3">50% C18: 0; at least</td>

about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, C18: 1; less than about 7%, less than about 5%, less than about 3%, less than about 1%, or about 0%, C18: 2; and

108

Mexicano Mexican Institute of Industrial _ Industrial Proneness * »

<img file="MX339639B_D0099.tif" />

at least about 35 'at least about 45' at least ^ .pTú.-imndnm ^ tA 4 0%.

at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least minus approximately 90%, of saturated fatty acids, where the method comprises the steps of: (a) culturing a population of oil microbial cells in a culture medium up to at least 10% of the dry cell weight of the oil microbial cells is triglyceride oil; and (b) isolating the triglyceride oil composition from the oil microbial cells.

In some embodiments, the method of producing oleaginous microbial triglyceride oil compositions produces triglyceride oils comprising a fatty acid profile in which: the total combined amount of C10: 0,

C12: 0 and C14: 0 is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C16: 0,

C18: 0 and C18: 1 is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the amount

109

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<img file="MX339639B_D0100.tif" />

combined total of C18: 0, C18: l and C18: 2 is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the total combined amount of C14: 0,

C16: 0, C18: 0 and C18: l is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%; the

<td>combined quantity</td><td>total</td><td>of</td><td>C8: 0 and CIO: 0</td><td>is</td><td>less</td><td>than</td>

<td>about 50%,</td><td>less</td><td>than</td><td>approximately</td><td> 45%,</td><td>less</td><td>than</td>

<td>about 40%,</td><td>less</td><td>than</td><td>approximately</td><td> 35%,</td><td>less</td><td>than</td>

<td>about 30%,</td><td>less</td><td>than</td><td>approximately</td><td> 25%,</td><td>less</td><td>than</td>

<td>about 20%,</td><td>less</td><td>than</td><td>approximately</td><td> 15%,</td><td>less</td><td>than</td>

<td>about 10%</td><td colspan="2">, less</td><td colspan="3">that about 5%,</td><td>or</td>

about 0%.

In some embodiments, the method of producing oleaginous microbial triglyceride oil compositions produces triglyceride oils with a fatty acid profile comprising a ratio of triglyceride oils selected from the group consisting of: a C8: 0 ratio to

C10: 0 of at least about 5 to 1, at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1; a C10: 0 to C12: 0 ratio

110

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<img file="MX339639B_D0101.tif" />

at least about 6 to 1, at least about 7 to, at least about 8 to 1, at least about 1, or at least about 10 to 1; a C12: 0 to C14: 0 ratio of at least about 5 to 1, at least about 6 to 1, at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1; a C14: 0 to C12: 0 ratio of at least about 7 to 1, at least about 8 to 1, at least about 9 to 1, or at least about 10 to 1; and a C14: 0 to C16: 0 ratio of at least about 1 to 2, at least about 1 to 3, at least about 1 to 4, at least about 1 to 5, at least about 1 to 6, at least about 1 to 7, at least about 1 to 8, at least about 9, or at least about 1 to 10.

III. GENETIC ENGINEERING METHODS AND MATERIALS

The present invention provides methods and materials for genetically modifying microorganisms, including Prototheca cells and recombinant host cells, useful in the methods of the present invention, including but not limited to, Prototheca moriformis host cells,

Recombinant Prototheca zopfii, Prototheca krugani, and Prototheca stagnora. The description of these methods and lll

IMPI

DELA WilFIBDAD MEXICAN INSTITUTE

INDUSTRIAL

<img file="MX339639B_D0102.tif" />

Materials is divided into subsections for the convenience of the reader. In subsection 1, the transformation methods are described. In subsection 2, genetic engineering methods are described through the use of homologous recombination. In subsection 3, expression vectors and components are described.

In certain embodiments of the present invention, it is preferred to genetically modify a microorganism to improve lipid production, modify the properties or proportions of components generated by the microorganism, or improve or re-provide growth characteristics in a variety of raw material materials.

Chlorella, particularly Chlorella protothecoides,

Chlorella minutissima, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., And Chlorella emersonii are the preferred microorganisms for use in the genetic modification methods described in the present disclosure, although other Chlorella species as well as other varieties of microorganisms may be used.

Promoters, cDNA, and 3'UTR, as well as other elements of the vectors, can be generated through cloning techniques using fragments isolated from native sources (see, for example, Molecular Cloning: A Laboratory

112

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PROPERTY i ^ .. ~ industpial Sjria

Manual, Sambrook and others (3rd edition, 2001, Coid Spring

Harbor Press; and US Patent 4,683,202).

Alternatively, the elements can be generated synthetically using known methods (see, for example,

Gene. 1995 Oct 16; 164 (1): 49-53).

one. Engineering Methods - Transformation [0006] Cells can be transformed by any suitable technique including, for example, biolistics, electroporation (see Maruyama et al., (2004) Biotechnology Techniques 8: 821-826), transformation by glass beads and transformation by whiskers of silicon carbide. Another method that can be used involves protoplast formation and the use of CaCl₂ and polyethylene glycol (PEG) to introduce recombinant DNA into microalgae cells (see Kim et al. (2002), Mar. Biotechnol. 4: 63-73, reporting the use of this method for the transformation of Chorella ellipsoidea). Microalgae co-transformation can be used to introduce two different vector molecules into a cell simultaneously (see eg Protist 2004

Dec; 155 (4): 381-93).

[0007] Biolistic methods (see, for example, Sanford,

Trends In Biotech. (1988) 6: 299 302, U.S. Patent

United no. 4,945,050; electroporation (Fromm et al., Proc.

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<img file="MX339639B_D0103.tif" />

Nati. Acad Sci., (USA) (1985) 82: 5824 5828); Using a laser beam, microinjection or any other method capable of introducing DNA into a microalgae can also be used for the transformation of a Prototheca cell.

Any convenient technique for introducing a transgene into a microorganism, such as Chorella, can be used in the present invention. Dawson et al. (1997) (supra) describe the use of microprojectile bombardment to introduce the nitrate reductase (NR) gene from Chlorella vulgaris into mutants of Chlorella sorokiniana

NR-deficient, resulting in stable transformants.

Briefly, 0.4 micron tungsten beads were plasmid coated; 3 X 10⁷C. sorokiniana cells were spread in the third center of a non-selective agar plate and bombarded with the PDS-1000 / He Biolistic Partióle system

Delivery® (Bio-Rad).

A preferred method of introducing a transgene into a microorganism, such as Chlorella, is the method described by Kim et al. (2002), Mar. Biotechnol. 4: 63-73. Kim reports the transformation of Chorella elllpsoidea protoplasts using

CaCl₂ and polyethylene glycol (PEG). In particular, protoplasts were prepared by culturing C. ellipsoidea cells at a density of 1-2 X 10⁸/ Ml. The cells are

114

MEXICAN INSTITUTE '/ L · DE LA FROPIEDAC

INDUSTRIAL recovered and washed by centrifugation for 5 minutes at 1600 g and resuspended in 5 ml of phosphate buffer (Ph 6.0) containing 0.6 M sorbitol, 0.6 M mannitol, 4% (w / v) cellulose (Calbiochem), 2 % (weight / volume) of macerase (Calbiochem), and 50 units of pectinase (Sigma). The cell suspension was incubated at 25 ° C for 16 hours in the dark with gentle shaking. The resulting protoplasts were recovered by centrifugation at 400g for 5 minutes. The pellet was delicately resuspended in 5 ml of f / 2 medium containing 0.6M sorbitol and 0.6M mannitol and centrifuged at 400g for 5 minutes. This pellet was resuspended in 1 ml of a 0.6 M sorbitol / mannitol solution containing 50 mM CaCl₂. Then 5 mg of the transgene DNA was added, along with 25 ug calf thymus DNA (Sigma), to 10⁷-10⁸ protoplasts at 0.4 Mi. After 15 minutes at room temperature, 2 00 di of PNC (40% polyethylene glycol 4000, 0.8 M NaCl, 50 Mm CaCl) were added₂) and carefully mixed for 30 minutes at room temperature. Following this, 0.6 ml of the f / 2 medium supplemented with a 0.6 M sorbitol / mannitol solution, 1% yeast extract and 1% glucose was added, and the transformed cells were incubated at 25 ° C for 12 hours in the dark. for the regeneration of the cell wall.

A

115

<img file="MX339639B_D0104.tif" />

ΙΜΡΙ

INSTITUTO MEXICANL DE LA ntOHEDAD INDUSTRIAL similar method was used by Huang et al. (2007) (supra) to introduce a transgene encoding mercuric reductase in

Chlorella sp. DT.

Furthermore, electroporation was used to transform microorganisms, such as Chorella. As reported

Maruyama et al. (2004), Biotechnology Techniques 8: 821-826 (incorporated by reference in its entirety herein), this technique was used to introduce a transgene into Chlorella saccharophila c-211-la protoplasts prepared from cells in stationary phase. The transient expression of the introduced plasmid was observed under a resistance field of between 600 and 900 V / cm, and a pulse duration of around 400 ms, where a membrane permeability greater than 70-kDa FITCdextran was verified.

Examples of expression of transgenes in microorganisms, such as 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), pgs.

335-341; Appl Microbiol Biotechnol (2006) 72: 197-205; Marine Biotechnology 4, 63-73, 2002; Current Genetics 39: 5, 365-370 (2001); Plant Cell Reports 18: 9, 778-780, (1999); biology

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<img file="MX339639B_D0105.tif" />

Plantarium 42 (2): 209-216, (1999);

20, (2 005)). See also the examples in the present description.

Examples of expression of transgenes in oil yeast (eg, Yarrowia lipolytica) can be found in the literature (see, eg, Bordes et al., J Microbiol Methods, Jun 27 (2007)). Examples of expression of transgenes in 'fungi (for example, Mortierella alpine, Mucor circinelloid.es, 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): 215-23 (1992); Current Microbiology, 30 (2): 83-86 (1995); Sakuradani,. NISR Research Grant, Studies

Metabolic Manipulation 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. (3rd edition, 2001, Coid

Spring Harbor Press.

Vectors for the transformation of microorganisms in accordance with the present invention can be prepared by familiar techniques known to those experienced in ¹¹⁷ IMPI

INSTITUTO MEXICANA DE IA RRORIEDAD INDUSTRIAL the subject. The nucleotide sequence of the construct used for transformation of multiple Chlorella species corresponds to sec. with no. ID: 8. In one embodiment, an illustrative vector design for expression of a lipase gene in a microorganism such as a microalgae contains a gene encoding a lipase in operable linkage with an active promoter in microalgae.

Alternatively, if the vector does not contain a promoter in operative linkage to the gene of interest, the gene can be transformed in cells such that it becomes operably linked to an endogenous promoter at the point of vector integration. The promoter-free transformation method was shown to work on microalgae (see for example Plant Journal 14: 4, (1998), pp. 441-447). The vector may further contain a second gene that encodes a protein that, for example, imparts resistance to an antibiotic or herbicide, i.e., a selectable marker.

Optionally, one or both genes is / are followed by a 3 'untranslated sequence containing a polyadenylation signal. The expression cassettes encoding the two genes can be physically linked in one vector or in separate vectors. Co-transformation of microalgae can also be used, in which different vector molecules are used

<img file="MX339639B_D0106.tif" />

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ΙΜΡΙ simultaneously to transform cells (see, eg, 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 where cells lacking the resistance cassette would not grow.

2. Engineering Methods - Homologous Recombination

Homologous recombination is the ability of complementary DNA sequences to align and exchange regions of homology. Transgenic (donor) DNA containing sequences homologous to the target genomic (template) sequences is introduced into the body and then recombination within the genome takes place at the site of the corresponding homologous genomic sequence. The mechanistic steps in this process, in most cases, include: (1) pairing the homologous DNA segments, (2) the introduction of double-stranded breaks in the donor DNA molecule, (3) invasion of the standard DNA molecule by the free ends of the donor's DNA followed by DNA synthesis; and (4) resolution of repair events for double-strand breaks that result in the end products of recombination.

<img file="MX339639B_D0108.tif" />

The ability to carry out recombination

119

Homologous IMPI in a host organism has many practical implications, so it can be carried out at the molecular genetic level and is useful in generating an oleaginous microbe that can produce specific oils. By its very nature homologous recombination is a precise gene-targeting event, therefore most transgenic lines that are generated with the same targeting sequence will be essentially identical in terms of phenotype, thus requiring the evaluation of far fewer transformation events. Homologous recombination also targets gene insertion events on the host chromosome, resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci could impact gene expression, even from heterologous / UTR promoters, homologous recombination may be a method of searching for a locus in an unfamiliar genomic environment and evaluating the impact of these environments on gene expression.

A particularly useful application of genetic engineering using homologous recombination is to co-opt host-specific regulatory elements such as promoters / UTRs to drive expression of heterologous genes.

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<img file="MX339639B_D0109.tif" />

in a highly specific way. For example, ablation or gene inactivation of the desaturase gene / gene families could be expected to increase the total percentage of saturated fatty acids produced in the host cell by a heterologous gene encoding a selective marker. The

Example 11 describes the homologous recombination directed at the constructs and an example of such ablations or inactivations of the desaturase gene generated in Prototheca moriformis.

Because homologous recombination is a precise gene-directed event, it can be used to precisely modify any nucleotide (s) within a gene or sequence of interest, as long as enough flank regions are identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences that affect gene expression of

RNA and / or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activities such as substrate specificity, affinities, and Km, and thus affect the desired change in host cell metabolism. . Homologous recombination provides a powerful means of manipulating the host genome, resulting in targeted mutagenesis, gene conversion,

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<img file="MX339639B_D0110.tif" />

gene deletion, gene duplication, gene reversal, and the exchange of gene expression regulatory elements such as promoters, enhancers, and

3'UTR.

Homologous recombination can be accomplished through the use of targeting constructs containing pieces of endogenous sequences to target the gene or region of interest in the endogenous host cell genome. Such targeting sequences can be located either 5 'from the gene or region of interest, 3' from the gene / region of interest, or even flank the gene / region of interest. Such targeting constructs can be transformed within the host cell, either as a supercoiled DNA plasmid with an additional vector backbone, a PCR product without a vector backbone, or as a linear molecule. In some cases, it may be advantageous to first expose the homologous sequences within the transgenic DNA (donor DNA) with a restriction enzyme. This stage can increase the efficiency of recombination and decrease the occurrence of unwanted events. Other methods to increase recombination efficiency include the use of PCR to generate transformed transgenic DNA containing linear ends homologous to the target genomic sequences.

<img file="MX339639B_D0111.tif" />

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For non-limiting illustration purposes, donor DNA sequence regions that are useful for homologous recombination include the KE858 region of DNA in

Prototheca moriformis. KE858 is a 1.3 kb genomic fragment, spanning part of the coding region for a protein that shares homology with the transfer RNA (tRNA) family of proteins. Southern blotting demonstrated that the KE858 sequence was present in a single copy in the Prototheca moriformis genome (UTEX 1435). This region and the examples of use of this region for homologous targeted recombination are described in PCT application no. PCT / US2009 / 66142. Another donor DNA region that is useful in portions of the 6S region genomic sequence. The use of this sequence in homologous recombination in Prototheca morifomis is described below in the examples.

3. Vectors and vector components

Vectors for transforming microorganisms in accordance with the present invention can be prepared by familiar techniques known to those skilled in the art in view of the description herein. A vector typically contains one or more genes in which each gene encodes for the expression of a desired product (the

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<img file="MX339639B_D0112.tif" />

gene product) and operably binds to one or more control sequences that regulate gene expression or direct the gene product to a location in the recombinant cell. To assist the reader, this subsection is divided into subsections. Subsection A describes the control sequences that are typically found in vectors, as well as new control sequences that are provided in the present invention. Subsection B describes genes that are typically found in vectors, as well as new codon optimization methods and genes that are prepared by using the same provided by the invention.

A. Control sequences

Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location inside or outside the cell. Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence.

Another control sequence is a 3 'untranslated sequence that is located at the end of a coding sequence that encodes a polyadenylation signal. The sequences of

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<img file="MX339639B_D0113.tif" />

Controls that target gene products to a particular location include those that encode signal peptides, which target the protein to which they are attached to a particular location inside or outside the cell.

Therefore, an exemplary vector design for expression of an exogenous gene in a microalgae contains a sequence that encodes a product of the desired gene (for example, a selection marker, an enzyme that modifies the lipid pathway, or a sucrose utilizing enzyme) in an operative link with an active promoter in microalgae. Alternatively, if the vector does not contain a promoter in operative linkage to the coding sequence of interest, the coding sequence can be transformed within cells such that it becomes operably linked to an endogenous promoter at the point of vector integration. . The promoter-free transformation method was shown to work on microalgae (see for example Plant Journal 14: 4, (1998), pp. 441-447).

[0008] Many promoters are active on microalgae, including promoters that are endogenous to transforming algae, as well as promoters that are not endogenous to transforming algae (ie promoters of other algae, promoters of higher plants , and the promoters of

<img file="MX339639B_D0114.tif" />

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IMPI plant virus or algae virus). Illustrative exogenous or endogenous promoters that are active in microalgae (as well as genes for resistance to functional antibiotics in microalgae) are described in PCT publication no.

2008/151149 and references cited there).

The promoter that is used to express an exogenous gene can be the promoter that naturally binds to that gene or it can be a heterologous gene. Some promoters are active in more than one species of microalgae. Other promoters are species specific. Illustrative promoters include promoters such as Chlamydomonas reinhardtii β-tubulin, which is used in the examples below, and viral promoters, such as cauliflower mosaic virus (CMV) and Chlorella virus, which were shown to be active. in multiple microalgae species (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). Another promoter that is suitable to use for the expression of exogenous genes in Prototheca is the Chlorella sorokiniana glutamate dehydrogenase / 5'UTR promoter. Optionally, at least 10, 20, 30, 40 are used,

50, or 60 nucleotides or more of these sequences containing a promoter. Illustrative promoters useful for

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<img file="MX339639B_D0115.tif" />

Exogenous gene expression in Prototheca are listed in the sequence listing of this application, such as the Chlorella HUPl gene promoter (sec. with ID #: 1) and the Chlorella ellipsoidea nitrate reductase promoter (sec.

with no. Ident .: 2). Chlorella visrus promoters can also be used to express genes in Prototheca, such as sec. with numbers Ident .: 1-7 of the patent of

United States 6,395,965. Additional promoters active in Prototheca can be found, for example, in Biochem Biophys Res Commun. 1994 Oct 14; 204 (1): 187-94; Plant Mol Biol. 1994 Oct; 26 (1): 85-93, - Virology. 2004 Aug 15, -326 (1): 1509; and Virology. 2004 Jan 5, -318 (1): 214-23. Other useful promoters are described in detail in the Examples below.

A promoter can be generally characterized as constitutive or inducible. The constitutive promoters are generally active or function to guide expression at all times (or at certain points in the cell's life cycle) at the same level. Inducible promoters, on the other hand, are significantly activated (or made inactive) or up-regulated or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention. Inducible promoters useful in the invention include

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those that mediate transcription of an operatively linked gene in response to a stimulus, such as an exogenously supplied small molecule (eg, glucose, as in seq. with ID #: 1), temperature (hot or cold) , the lack of nitrogen in the culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, the transcription of an operably linked gene that is transcribed at a low level. The examples below describe additional inducible promoters that are useful in Prototheca cells.

The inclusion of the termination region control sequence is optional, and if employed, then the choice will be one of convenience, since the termination region is relatively interchangeable. The termination region can be native to the transcription initiation region (the promoter), can be native to the DNA sequence of interest, or can be obtained from another source. See for example

Chen and Orozco, Nucleic Acids Res. (1988) 16: 8411.

The present invention also provides control sequences and recombinant genes and vectors containing them which provides compartmentalized expression of a gene for

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IMPI ^INSTn?^T· «EXICaNO

INBUSTRmy interest. Organelles for orientation are chloroplasts, plastids, mitochondria, and the endoplasmic reticulum. Furthermore, the present invention provides control sequences and the recombinant genes and vectors that contain them which provides for the secretion of a protein outside the cell.

Proteins that are expressed in the nuclear genome of

Prototheca can be directed at plastids by signals directed at plastid. Sequences targeting endogenous plastids for Chlorella are known, such as genes in the Chlorella nuclear genome that encode proteins that target plastids; see, eg, accession numbers AY646197 and AF499684 at GenBank, and in one embodiment, such control sequences are used in the vectors of the present invention to direct expression of a protein to a Prototheca plastid.

The examples below describe the use of algae plastid targeting sequences to target heterologous proteins to the correct compartment in the host cell.

The cDNA libraries were made using Prototheca moriformis and Chlorella protothecodies cells and are described in PCT application no. PCT / US2009 / 066142.

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INWISTRIAL

In another embodiment of the present invention, the expression of a polypeptide in Prototheca is directed to the endoplasmic reticulum. The inclusion of an appropriate retention or classification signal in an expression vector ensures that the proteins are retained in the endoplasmic reticulum (ER) and do not go down into the Golgi. For example, the vector IMPACTVECT0R1.3, from Wageningen UR-Plant Research International, includes the well-known KDEL retention or classification signal. With this vector, RE retention has a practical advantage since it was reported to improve expression levels 5 times or more. The main reason for this appears to be that ER contains lower concentrations and / or different proteases responsible for posttranslational degradation of expressed proteins than those 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.

In another embodiment of the present invention, a polypeptide is targeted for secretion outside the cell in the culture medium. See Hawkins et al., Current

Microbiology Vol. 38 (1999), pgs. 335-341 for examples of active secretion signals in Chlorella that can

<img file="MX339639B_D0118.tif" />

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MEXICAN INSTITUTE DF. INDUSTRIAL PROPERTY used, in accordance with the methods of the invention, in

Prototheca.

[0009] Many promoters are active on microalgae, including promoters that are endogenous to transforming algae, as well as promoters that are not endogenous to transforming algae (ie promoters of other algae, promoters of higher plants , and promoters of plant viruses or algae viruses). Exogenous and / or endogenous promoters that are active in microalgae, and genes for resistance to functional antibiotics in microalgae are described, for example, by Curr Microbiol. Dec 1997; 35 (6): 356-62 (Chlorella vulgaris); Mar Biotechnol (NY).

2002 Jan; 4 (1): 63-73 (Chlorella ellipsoidea); Mol Gen Genet.

nineteen ninety six Oct 16, -252 (5): 572-9 (Phaeodactylum tricornutum); Plant

Mol Biol. 1996 Apr; 31 (1): 1-12 (Volvox carteri); Proc Nati

Acad Sci US A. 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 weissflog'ii); Plant Physiol. 2002 May; 129 (1): 712. (Porphyridium sp.); Proc Nati Acad Sci USA. 2003 Jan

21, -100 (2): 438-42. (Chlamydomonas reinhardtii); Proc Nati Acad

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Sci USA. 1990 Feb; 87 (3): 1228-32. (Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jun 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); Sea

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.

Bacteriol. (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. 2 002, -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 Gen 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:

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317-322 (Chlorella); for additional promoters see also Table 1 of US Patent 6,027,900).

The promoter that is used to express an exogenous gene can be the promoter that naturally binds to that gene or it can 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 reinhardtii and viral promoters such as cauliflower mosaic virus (CMV) and Chlorella virus which were shown to be active in multiple microalgae species (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, a

<td>nucleic acid</td><td>of that type that</td><td>understands</td><td>any</td><td>part</td><td>of</td>

<td colspan="2">the sec. with no. of ident. :</td><td>150, or the</td><td>promoter</td><td>RBCS2</td><td>of</td>

<td>Ch1amydomonas</td><td>reinhardtii (sec</td><td>with no.</td><td colspan="3">of ident. : 151).</td>

<td>Optionally,</td><td colspan="2">at least 10, 20 are used,</td><td> 30, 40,</td><td>50, or</td><td> 60</td>

<td>nucleotides or</td><td>more of these</td><td>sequences</td><td colspan="2">that contain</td><td>a</td>

promoter. Preferred endogenous promoters for species of the genus Chlorella are sec. with no. of ident.:1 and sec. with no. of ident.:2.

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[0010] Preferred promoters useful for

IMPI exogenous gene expression in Chlorella are listed in the sequence listing for this application, such as the Chlorella HUP1 gene promoter (sec. With ID #: 1) and the Chlorella ellipsoidea nitrate reductase promoter ( section with ID number: 2). Chlorella visus promoters can also be used to express genes in Chlorella, such as sec. with numbers Ident: 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 (1): 187-94; Plant Mol Biol. 1994 Oct; 26 (1): 85-93;

Virology. 2004 Aug 15; 326 (1): 150-9; and Virology. 2004 Jan

5;318 (1) :214-23 .

B. Optimization of genes and codon

Typically, a gene includes a promoter, the coding sequence, and termination control sequences. When assembled by recombinant DNA technology, a gene can be called an expression cassette and can be flanked by restriction sites for proper insertion into a vector that is used to introduce the recombinant gene into a host cell.

The expression cassette can be flanked by sequences of

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Genome DNA or other target nucleic acids to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette may remain unintegrated, in which case, the vector typically includes an origin of replication that is capable of providing replication of the

Heterologous vector DNA.

A common gene present in a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to differentiate itself from cells that do not express the protein. Such a gene and its corresponding gene product is called a selection marker. Any of a wide variety of selection markers can be employed in a transgenic construct useful for transforming Prototheca. Examples of suitable selectable markers include the G418 resistance gene, the nitrate reductase gene (see Dawson et al. (1997), Current Microbiology 35: 356-362), the hygromycin phosphotransferase gene (HPT; see Kim et al. (2002),

Mar. Biotechnol. 4: 63-73), the neomycin phosphotransferase gene, the ble gene, which confers resistance to phleomycin (Huang et al. (2007), Appl. Microbiol.

Biotechnol.

72: 197-205), aminoglycoside-3'-0135

IMPI

MEXICAN INSTITUTE • and INDUSTRIAL PROPERTY

<img file="MX339639B_D0123.tif" />

phosphotransferase (sec. with ID no .: 194), which confers resistance to kanamycin. Methods for determining the sensitivity of microalgae to antibiotics are well known. For example, Mol Gen Genet. 1996 Oct

16;252(5):572-9.

Other selectable markers that are not based on antibiotics can also be used in a transgene construct useful for transforming microalgae generally, including Prototheca species. Genes that confer the ability to use certain carbon sources that were previously unable to be used by the microalgae can also be used as a selectable marker. By way of illustration, Prototheca moriformis strains typically grow poorly, if at all, in sucrose. Using a construct that contains a sucrose invertase gene can confer the ability of positive transformants to grow on sucrose as a carbon substrate. Additional details on the use of sucrose as a selectable marker in conjunction with other selectable markers are discussed in Section IV below.

For the purposes of the present invention, the expression vector that is used to prepare a host cell

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<img file="MX339639B_D0124.tif" />

Recombinant of the invention will include at least two, and often three, genes, if one of the genes is a selection marker. For example, a genetically engineered Prototheca of the invention can be made by transformation with vectors of the invention that comprise, in addition to a selection marker, one or more exogenous genes, such as, for example, the sucrose invertase gene or the acylc ACP-thioesterase gene. One or both genes can be expressed through the use of an inducible promoter, which allows controlling the relative expression time of these genes to improve lipid performance and conversion to fatty acid esters. The expression of the two or more exogenous genes can be under the control of the same inducible promoter or under the control of different inducible (or constitutive) promoters. In the latter situation, the expression of a first exogenous gene can be induced for a first period of time (during which the expression of a second exogenous gene may or may not be induced) and the expression of a second exogenous gene can be induced by a second time period (during which the expression of the first exogenous gene may or may not be induced).

In other modalities, the two or more exogenous genes (in addition to any selection marker) are: an acyl

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fatty ACP thiosterase and an acyl fatty CoA / aldehyde reductase, the combined actions of which yield an alcohol product.

In addition other combinations of exogenous genes are provided;

including, without limitation, an acyl fatty acp thiosterase and an acyl fatty CoA reductase to generate aldehydes. In one embodiment, the vector provides the combination of an acyl fatty acp thiosterase, an acyl fatty CoA reductase and a fatty aldehyde decarbonylase to generate allenes. In each of these modalities, one or more of the exogenous genes can be expressed through the use of an inducible promoter.

Other illustrative vectors of the invention expressing two or more exogenous genes include those encoding both a sucrose transporter and a sucrose invertase enzyme and those encoding a selection marker and a secreted sucrose invertase. Recombinant Prototheca transformed with any type of vector produces lipids at a lower manufacturing cost due to the engineering-generated ability to use sugarcane (and sugars derived from cane sugar) as a carbon source. Insertion of the two exogenous genes described above can be combined with disruption of polysaccharide biosynthesis through targeted and / or random mutagenesis, which directs flow

<img file="MX339639B_D0126.tif" />

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IMPI increasing carbon to lipid production.

Individually and in combination, trophic conversion, modifications to alter lipid production, and treatment with exogenous enzymes alter the lipid composition produced by a microorganism. The alteration may be a change in the amount of lipids produced, the amount of one or more hydrocarbon species produced relative to other lipids, and / or the types of lipid species that are produced in the microorganism. For example, the microalgae can be modified to produce a higher amount and / or percentage of TAG.

For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mRNA with codons that are preferably used by the host cell to be transformed. Thus, proper expression of the transgenes may require that the use of the transgene codon coincide with the specific codon with bias in the organism in which the transgene is expressed. The exact mechanisms underlying this effect are many, but include proper balancing of available aminoacylated tRNA stores with proteins that are synthesized in the cell, coupled with more efficient translation of messenger RNA (mRNA) transgenics when satisfied. this ¹³⁹ IMPI

MEXICAN INSTITUTE Of LA fROflíDAt. ·

INDUSTRIAL need. When codon usage in the transgene is not optimized, the available tRNA stores are not sufficient to allow efficient translation of the heterologous mRNA resulting in stagnation and ribosomal termination and possible instability of the transgenic mRNA.

The present invention provides codon optimized nucleic acids useful for the successful expression of recombinant proteins in Prototheca. The use of codons in Protothecase species was analyzed by studying the sequences of

Isolated cDNA from Prototheca moriformis. This analysis represents the question about 24,000 codons and resulted in Table 2 below.

Table 2. Use of preferred codons in strains of

<img file="MX339639B_D0127.tif" />

Prototheca.

<td>To</td><td>GCG</td><td> 345 (0.36)</td><td>Asn</td><td>AAT</td><td> 8 (0.04)</td>

<td></td><td>GCA</td><td> 66 (0.07)</td><td></td><td>AAC</td><td> 201 (0.96)</td>

<td></td><td>GCT</td><td> 101 (0.11)</td><td></td><td></td><td></td>

<td></td><td>GCC</td><td> 442 (0.46)</td><td>Pro</td><td>CCG</td><td> 161 (0.29)</td>

<td></td><td></td><td></td><td>CCA</td><td> 49</td><td> (0.09)</td>

<td>Cys</td><td>TGT</td><td> 12 (0.10)</td><td></td><td>CCT</td><td> 71 (0.13)</td>

<td></td><td>TGC</td><td> 105 (0.90)</td><td></td><td>CCC</td><td> 267 (0.49)</td>

<img file="MX339639B_D0128.tif" />

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<td>Asp</td><td>GAT</td><td> 43</td><td> (0.12)</td><td>Gln</td><td>CAG</td><td> 226 (0.82)</td>

<td></td><td>GAC</td><td> 316</td><td> (0.88)</td><td></td><td>CAA</td><td> 48 (0.18)</td>

<td>Glu</td><td>GAG</td><td> 377</td><td> (0.96)</td><td>Arg</td><td>AGG</td><td> 33 (0.06)</td>

<td></td><td>GAA</td><td> 14</td><td> (0.04)</td><td></td><td>AGA</td><td> 14 (0.02)</td>

<td></td><td></td><td></td><td></td><td>CGG</td><td> 102</td><td> (0.18)</td>

<td>Phe</td><td>TTT</td><td> 89</td><td> (0.29)</td><td></td><td>CGA</td><td> 49 (0.08)</td>

<td></td><td>TTC</td><td> 216</td><td> (0.71)</td><td></td><td>CGT</td><td> 51 (0.09)</td>

<td></td><td></td><td></td><td></td><td>CGC</td><td> 331</td><td> (0.57)</td>

<td>Gly</td><td>GGG</td><td> 92</td><td> (0.12)</td><td></td><td></td><td></td>

<td></td><td>GGA</td><td> 56</td><td> (0.07)</td><td>To be</td><td>AGT</td><td> 16 (0.03)</td>

<td></td><td>GGT</td><td> 76</td><td> (0.10)</td><td></td><td>AGC</td><td> 123 (0.22)</td>

<td></td><td>GGC</td><td> 559</td><td> (0.71)</td><td></td><td>TCG</td><td> 152 (0.28)</td>

<td></td><td></td><td></td><td></td><td>TCA</td><td> 31</td><td> (0.06)</td>

<td>His</td><td>CAT</td><td> 42</td><td> (0.21)</td><td></td><td>TCT</td><td> 55 (0.10)</td>

<td></td><td>CAC</td><td> 154</td><td> (0.79)</td><td></td><td>CBT</td><td> 173 (0.31)</td>

<td>lie</td><td>ATA</td><td></td><td> 4 (0.01)</td><td>Thr</td><td>ACG</td><td> 184 (0.38)</td>

<td></td><td>ATT</td><td> 30</td><td> (0.08)</td><td></td><td>HERE</td><td> 24 (0.05)</td>

<td></td><td>ATC</td><td> 338</td><td> (0.91)</td><td></td><td>ACT</td><td> 21 (0.05)</td>

<td></td><td></td><td></td><td></td><td>ACC</td><td> 249</td><td> (0.52)</td>

<td>Lys</td><td>AAG</td><td> 284</td><td> (0.98)</td><td></td><td></td><td></td>

(0.02)

Val

GTG 308 (0.50)

AAA

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<td></td><td></td><td></td><td></td><td>GTA_</td><td></td><td>9 ín.ni)</td>

<td>Leu</td><td>TTG</td><td> 26</td><td> (0.04)</td><td></td><td>GTT</td><td> 35 (0.06)</td>

<td></td><td>TTA</td><td></td><td> 3 (0.00)</td><td></td><td>GTC</td><td> 262 (0.43)</td>

<td></td><td>CTG</td><td> 447</td><td> (0.61)</td><td></td><td></td><td></td>

<td></td><td>CTA</td><td></td><td></td><td> 20 (0.03)</td><td></td><td>Trp TGG</td>

<td></td><td> 107</td><td> (1.00)</td><td></td><td></td><td></td><td></td>

<td></td><td>CTT</td><td> 45</td><td> (0.06)</td><td></td><td></td><td></td>

<td></td><td>CTC</td><td> 190</td><td> (0.26)</td><td>Tyr</td><td>TAT</td><td> 10 (0.05)</td>

<td></td><td></td><td></td><td></td><td>TAC</td><td> 180</td><td> (0.95)</td>

<td>Met</td><td>ATG</td><td> 191</td><td> (1.00)</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td colspan="2">Termination</td><td>TGA / TAG / TAA</td>

In other embodiments, the gene in the recombinant vector was codon optimized with reference to a microalgal strain

<td>it's not</td><td>a</td><td>Prototheca strain</td><td>. For example,</td><td>the</td><td>methods</td><td>of</td>

<td>to register</td><td>the</td><td>genes for the</td><td>expression in</td><td colspan="2">microalgae</td><td>I know</td>

<td>describe</td><td>in</td><td>the patent of</td><td>United States</td><td> 7,</td><td> 135,290.</td><td>The</td>

<td colspan="3">additional information for</td><td>optimization</td><td>by</td><td colspan="2">codon is</td>

<td>available,</td><td colspan="2">for example in</td><td colspan="2">the database</td><td>of use</td><td>of</td>

2θ GenBank codons.

Other non-limiting examples of codon usage in

Chlorella pyrenoidosa, Dunaliella salina, and Chlorella

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[0011] Table 28. Use of codons in Chlorella pyrenoidosa

<td>Phe</td><td>UUU</td><td> 39</td><td> (0.82)</td><td>To be</td><td>UCU 50 (1.04)</td>

<td></td><td>UUC</td><td colspan="2"> 56 (1.18)</td><td>UCC</td><td> 60 (1.25)</td>

<td>Leu</td><td>UUA</td><td> 10</td><td> (0.20)</td><td>UCA</td><td> 6 (0.96)</td>

<td></td><td>UUG</td><td> 46</td><td> (0.91)</td><td>UCG</td><td> 43 (0.89)</td>

<td>Tyr</td><td>UAU</td><td> 15</td><td> (0.59)</td><td>Cys</td><td>UGU 46 (0.77)</td>

<td></td><td>UAC</td><td> 36</td><td> (1.41)</td><td>UGC</td><td> 73 (1.23)</td>

<td>ter</td><td>UAA</td><td> 9</td><td> (0.00)</td><td>ter UGA</td><td> 43 (0.00)</td>

<td>ter</td><td>UAG</td><td> 15</td><td> (0.00)</td><td>Trp</td><td>UGG 69 (1.00)</td>

<td>Leu</td><td>CUU</td><td> 49</td><td> (0.97)</td><td>Pro</td><td>CCU 80 (0.98)</td>

<td></td><td>CUC</td><td> 73</td><td> (1.45)</td><td>CCC</td><td> 88 (1.08)</td>

<td></td><td>CUA</td><td> 22</td><td> (0.44)</td><td>CCA</td><td> 93 (1.14)</td>

<td></td><td>CUG</td><td colspan="2"> 103 (2.04)</td><td></td><td>GCC 65 (0.80)</td>

<td>His</td><td>CAU</td><td> 50</td><td> (0.88)</td><td>Arg</td><td>CGU 39 (0.76)</td>

<td></td><td>CAC</td><td> 3</td><td> (1.12)</td><td>CGC</td><td> 63 (1.23)</td>

<td>Gln</td><td>CAA</td><td> 59</td><td> (0.84)</td><td>CGA</td><td> 46 (0.90)</td>

<td></td><td>CAG</td><td> 2</td><td> (1-16)</td><td>CGG</td><td> 47 (0.92)</td>

<td>lie</td><td>AUU</td><td> 24</td><td> (0.69)</td><td>Thr</td><td>ACU 32 (0.67)</td>

<td></td><td>AUC</td><td> 61</td><td> (1.76)</td><td>ACC</td><td> 76 (1.60)</td>

<td></td><td>AUA</td><td> 19</td><td> (0.55)</td><td>HERE</td><td> 41 (0.86)</td>

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<td>Met</td><td>AUG</td><td> 42</td><td> (1.00)</td><td>ACG</td><td> 41</td><td> (0.86)</td>

<td>Asn</td><td>AAU</td><td> 26</td><td> (0.75)</td><td>To be</td><td>AGU</td><td> 23 (0</td>

<td></td><td>AAC</td><td> 3</td><td> (1.25)</td><td>AGC</td><td> 67</td><td> (1.39)</td>

<td>Lys</td><td>AAA</td><td> 32</td><td> (0.54)</td><td>Arg</td><td>AGA</td><td> 51 (1</td>

<td></td><td>AAG</td><td> 86</td><td> (1.46)</td><td>AGG</td><td> 61</td><td> (1.19)</td>

<td>Val</td><td>GUU</td><td> 36</td><td> (0.75)</td><td>To</td><td>GCU</td><td> 57 (0</td>

<td></td><td>GUC</td><td> 54</td><td> (1.13)</td><td>GCC</td><td> 97</td><td> (1.34)</td>

<td></td><td>GUA</td><td> 30</td><td> (0.63)</td><td>GCA</td><td> 89</td><td> (1.23)</td>

<td></td><td>GUG</td><td> 71</td><td> (1.49)</td><td>GCG</td><td> 47</td><td> (0.65)</td>

<td>Asp</td><td>GAU</td><td> 60</td><td> (0.95)</td><td>Gly</td><td>GGU</td><td> 35 (0</td>

<td></td><td>GAC</td><td> 66</td><td> (1.05)</td><td>GGC</td><td> 78</td><td> (1.33)</td>

<td>Glu</td><td>GAA</td><td> 41</td><td> (0.68)</td><td>GGA</td><td> 54</td><td> (0.92)</td>

<td></td><td>GAG</td><td> 80</td><td> (1.32)</td><td>GGG</td><td> 67</td><td> (1.15)</td>

48)

00)

79)

60)

Table 29. Use of preferred codons in Dunaliella salina.

<td></td><td>TTC</td><td>(Phe)</td><td>TAC</td><td>(Tyr)</td><td>TGC</td><td>(Cys)</td>

<td></td><td></td><td>TAA</td><td colspan="2">(termination)</td><td></td><td></td>

<td></td><td>TGG</td><td>(Trp)</td><td>CCC</td><td>(Pro)</td><td>CAC</td><td>(His)</td>

<td> 20</td><td></td><td>CGC</td><td>(Arg)</td><td></td><td></td><td></td>

<td></td><td>CTG</td><td>(Leu)</td><td>CAG</td><td>(Gln)</td><td>ATC</td><td>(He)</td>

ACC (Thr)

<img file="MX339639B_D0130.tif" />

ATG (Met)

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<td></td><td>AAC</td><td>(Asn)</td><td>AGC</td><td>(To be)</td>

<td></td><td></td><td>AAG</td><td>(Lys)</td><td></td>

<td></td><td>GCC</td><td>(To)</td><td>GAC</td><td>(Asp)</td>

<td></td><td></td><td>GTG</td><td>(Val)</td><td></td>

<td> 5</td><td>GAG</td><td>(Glu)</td><td></td><td></td>

GGC (Gly)

Table 30.

protothecoids.

Use of preferred codons in Chlorella

<td>TTC</td><td>(Phe)</td><td>TAC</td><td>(Tyr)</td>

<td colspan="3">termination)</td><td></td>

<td>TGG</td><td>(Trp)</td><td>CCC</td><td>(Pro)</td>

<td></td><td>CGC</td><td>(Arg)</td><td></td>

<td>CTG</td><td>(Leu)</td><td>CAG</td><td>(Gln)</td>

<td></td><td>ACC</td><td>(Thr)</td><td></td>

<td>GAC</td><td>(Asp)</td><td>CBT</td><td>(To be)</td>

<td>GCC</td><td>(To)</td><td>AAC</td><td>(Asn)</td>

<td>GAG</td><td>(Glu)</td><td></td><td></td>

<td>TGC</td><td>(Cys)</td><td>TGA</td><td> (</td>

<td></td><td>CAC</td><td>(His)</td><td></td>

<td></td><td>ATC</td><td>(He)</td><td></td>

<td>ATG</td><td>(Met)</td><td>AAG</td><td>(Lys)</td>

<td>GGC</td><td>(Gly)</td><td>GTG</td><td>(Val)</td>

C. Inducible expression

The present invention further provides the use of an inducible promoter to express a gene of interest. In particular, the use of an inducible promoter to express a

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[0012] Inducible promoters useful in the invention include those that mediate transcription of an operatively linked gene in response to a stimulus, such as an exogenously supplied small molecule (eg, glucose, as in seq. ident .: 1), temperature (hot or cold), light, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, the transcription of an operably linked gene that is transcribed at a low level. In the latter case, the level of lipase transcription preferably does not significantly interfere with the growth of the microorganism in which it is expressed.

Expression of transgenes in Chlorella can be induced inducibly through promoters such as the promoter that drives the hexose transporter gene

<img file="MX339639B_D0131.tif" />

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IMPí enChlorella (sec. With ID number: 1). This promoter is strongly activated by the presence of glucose in the culture media.

D. Expression of two or more exogenous genes

Furthermore, a genetically modified microorganism, such as a microalgae, could comprise and express two or more exogenous genes, such as, for example, a lipase and a lytic gene, for example, one that encodes a polysaccharide degradation enzyme. One or both genes can be expressed through the use of an inducible promoter, which allows controlling the relative expression time of these genes to improve lipid performance and conversion to fatty acid esters. The expression of the two or more exogenous genes can 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 exogenous gene can be induced for a first period of time (during which the expression of a second exogenous gene may or may not be induced) and the expression of a second exogenous gene can be induced by a second time period (during which the expression of the first exogenous gene may or may not be induced). Vectors and methods for modifying the microbes they produce are provided herein.

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Additionally provided are genetically modified microbial strains (eg, microalgae, oil yeast, bacteria, or fungi) that express two or more exogenous genes, such as, for example, an acyl fatty ACP thioesterase and an acyl fatty CoA / aldehyde reductase, the combined action of which produces an alcoholic product. In addition, other exogenous gene combinations are provided, including, without limitation, an acyl fatty acp thiosterase and an acyl fatty CoA reductase to generate aldehydes. Furthermore, this application provides the combination of an acyl fatty acp thioesterase, an acyl fatty CoA reductase and a fatty aldehyde decarbonylase to generate alkanes. One or more of the exogenous genes can be expressed through the use of an inducible promoter.

Examples of other suitable modifications for use in the present invention include genetically modified strains of microalgae to express two or more exogenous genes, one encoding a transporter of a fixed carbon source (such as sucrose) and a second encoding an enzyme. sucrose invertase. Fermentable organisms

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INDUSTRIAL> l ^ * W »g ​​produce hydrocarbons at a lower manufacturing cost than those obtained by previously known biological hydrocarbon production methods. Insertion of the two exogenous genes described above can be combined with disruption of polysaccharide biosynthesis through targeted and / or random mutagenesis, which directs the increasing flow of carbon to hydrocarbon production. Individually and in combination, trophic conversion, modifications to alter hydrocarbon production, and treatment with exogenous enzymes alter the composition of hydrocarbons produced by a microorganism. 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, the microalgae can be modified to produce a higher amount and / or percentage of TAG.

E. Compartmentalized expression

The present invention further provides for the compartmentalized expression of a gene of interest. In particular, in particular modalities, it may be advantageous to direct the

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lipase expression to one or more cellular compartments where it is sequestered from most of the cellular lipids until the start of the transesterification reaction. The preferred organelles for targeting are chloroplasts, mitochondria, and the endoplasmic reticulum.

(1) Expression in chloroplasts

In one embodiment of the present invention, expression of a polypeptide in a microorganism is directed to chloroplasts. Methods for directing the expression of a heterologous gene to the chloroplast are known and can be used in the present invention. Methods for targeting foreign gene products 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 United States patent.

no. 4,940,835 for the use of transit peptides for nuclear translocation of gene products in the chloroplast. Methods for directing protein transport to the chloroplast were further reviewed in Kenauf

TIBTECH (1987) 5:40 47. Chlorella endogenous chloroplast targeting sequences are known, such as the genes in the Chlorella nuclear genome that encodes

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proteins that target the chloroplast; see, for example, the GenBank accession numbers AY646197 and AF499684.

Wageningen UR- Plant Research International sells an IMPACTVECTOR1.4 vector, which uses the secretion signal of the small protein subunit of Chrysanthemum morifolium to deliver a heterologous protein into the environment of the chloroplast stroma (cytoplasmic), moving through a double membrane system. The protein fuses with the first 11 amino acids of the mature rubisco protein to allow adequate processing of the signal peptide (Wong et al., Plant Molecular Biology 20: 81-93 (1992)). The signal peptide contains a natural intron of the gene

RbcS.

In another approach, the chloroplast genome is genetically engineered to express the heterologous protein. Stable transformation of Chlamydomonas reinhardtii chloroplasts (a green algae) has been described using bombardment of recipient cells with high-speed tungsten microprojectiles coated with foreign DNA. See, for example, Boynton et al., Science (1988) 240: 1534 1538; Blowers and others Plant Cell (1989)

1: 123 132 and Debuchy et al., EMBO J. (1989) 8: 2803 2809. The transformation technique, using microprojectiles from

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INDUSTRIAL * «», 'g tungsten, is described in Klein et al., Nature (London) (1987) 7:70 73. Other methods of transforming chloroplast for plants and microalgae are known. See for example United States 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 United States Patent No.

6,320,101 (published November 20, 2001 by Kaplan et al .; incorporated herein by reference), all 10 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 cells through particle bombardment with the aim of introducing at least one heterologous nucleic acid molecule into the chloroplasts. Heterologous nucleic acid is selected so that it is integrated into the chloroplast genome through homologous recombination that is easily carried out by the enzymes of chloroplasts. To this end, heterologous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid sequence that is derived from the chloroplast genome. Furthermore, heterologous nucleic acid typically includes a selection marker. Details

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<img file="MX339639B_D0135.tif" />

Additional related to this technique are found in United States Patent Nos. 4,945,050 and 5,693,507 which are incorporated herein by reference. A polypeptide can be produced in this way by the chloroplast protein expression system.

United States Patent No. 7,135,620 (published November 14, 2006 by Daniell et al., Incorporated herein by reference) describes chloroplast expression vectors and related methods. Expression cassettes are DNA constructs that include a suitable coding sequence and control sequences to provide correct 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 to be provided 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 an inverted repeat 3 'region of a chloroplast gene that can stabilize the RNA of the introduced genes, thereby improving the expression of foreign genes. The

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INDUSTRIAL cassette can optionally include an antibiotic resistance gene.

Typically, the expression cassette is flanked by convenient restriction sites for insertion into a suitable genome. The expression cassette can be flanked by DNA sequences from the 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 typically includes an origin of replication of the chloroplast, which is capable of providing replication of the 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 could include promoters obtainable from chloroplast genes, such as the spinach or pea psbA gene, or the rbcL and atpB promoter region from maize and rRNA promoters. Examples of 20 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:

¹⁵⁴ IMPI mexicanc institute OF industuial PAOftíDAD

4985 5002, - Zurawaki et al., Nucleic Acids Res. (1981) 9: 3251

3270; and Zurawski et al., Proc. Nat'1 Acad Sci. USA (1982)

79: 7699 7703. Other promoters can be identified and the relative strength of the promoters thereby identified identified, by placing a promoter of interest 5 'to a marker gene minus the promoter and observing its efficacy in relation to the transcription obtained from from, for example, the psbA gene promoter, a relatively strong chloroplast promoter. The efficiency of heterologous gene expression can be further enhanced 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 chloroplast of

Chlorella can be used for the expression of exogenous genes in Chlorella chloroplast such as those found in GenBank accession number NC_001865 (Chlorella vulgaris complete chloroplast genome).

Where it is desired to provide the inducible expression of the heterologous gene, an inducible promoter and / or an untranslated 5 'region containing sequences that are provided for regulation by can be included in the expression cassette.

<img file="MX339639B_D0137.tif" />

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level of transcription and / or translation (at the 3'TT end For example, the 5 'untranslated region may be from a gene where expression is light dimmable. Similarly, the 3' inverted repeat regions can be use to stabilize RNA in heterologous genes Inducible genes can be identified by improved expression in response to a particular stimulus of interest and low 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 non-expression occurs in little or no light. Light-regulated promoters from green microalgae are known (see, eg, Mol Genet Genomics. 2005 Dec, -274 (6): 62536).

The termination region employed will be primarily one of convenience, since the termination region appears to be relatively interchangeable between chloroplasts and bacteria. The termination region can be native to the transcription initiation region, can be native to the DNA sequence of interest, or can be obtained from another source. See for example Chen and

Orozco, Nucleic Acids Res. (1988) 16: 8411.

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Expression cassettes can be transformed into a plant cell of interest by any of a number of methods. These methods include, for example, biolistic methods (see, for example, Sanford, Trends In Biotech.

(1988) 6: 299 302, US Patent No. 4,945,050;

electroporation (Fromm et al., Proc. Nati. Acad Sci., (United States) (1985) 82: 5824 5828); use of a laser beam, microinjection or any other method capable of introducing

DNA in a chloroplast.

Additional descriptions of chloroplast expression vectors suitable for use in microorganisms such as microalgae are found in

United States no. 7,081,567 (published July 25,

2006, de Xue et al.); 6,680,426 (published January 20,

2004, by Daniell et al.) And 5,693,507 (published December 2, 1997, by Daniell et al.).

Proteins expressed in the Chlorella nuclear genome can be directed to the chloroplast using signals directed to the chloroplast. Chlorella-endogenous chloroplast targeting sequences are known, such as genes in the Chlorella nuclear genome that encode chloroplast-targeting proteins; see, for example, the GenBank accession numbers AY646197 and AF499684. The

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<img file="MX339639B_D0139.tif" />

Proteins can also be expressed in the chloroplast of

Chlorella by insertion of genes directly into the chloroplast genome. Transformation of the chloroplast typically occurs through homologous recombination, and can be performed if the sequences of the chloroplast genome are known from the creation of targeted vectors (see, for example, the entire genome sequence of a Chlorella chloroplast; number access to GenBank

NC_001865). See the previous sections in this for details of the transformation of the chloroplast.

(2) Expression in the mitochondria

In another embodiment of the present invention, expression of a polypeptide in a microorganism is directed to the mitochondria. Methods for the selection of foreign gene products in mitochondria have been described (Boutry et al., Nature (London) (1987) 328: 340 342) including in green microalgae (see, eg, Mol Gen Genet. 1993

Jan; 236 (2-3): 235-44).

For example, an expression vector that encodes the appropriate secretion signal can direct a heterologous protein to the mitochondria. The vector IMPACTVECTOR1.5, from

Wageningen UR- Plant Research International, uses the yeast CoxIV secretion signal, which was shown to release

158 proteins in the mitochondrial matrix.

IMPI

<img file="MX339639B_D0140.tif" />

Protein fuses

<img file="MX339639B_D0141.tif" />

with the first 4 amino acids of the yeast CoxIV protein to allow adequate processing of the signal peptide (Kohler et al., Plant J 11: 613-621 (1997)). Other mitochondrial targeted 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 nuclear genome of

Chlorella can be targeted to the mitochondria using targeted mitochondrial signals. See the previous sections in the present description for the details of selection and transformation of the mitochondrial protein.

(3) Expression in the endoplasmic reticulum

In another embodiment of the present invention, expression of a polypeptide in a microorganism is directed to the endoplasmic reticulum. The inclusion of an appropriate retention or classification signal in an expression vector ensures that the proteins are retained in the endoplasmic reticulum (ER) and do not go down into the Golgi. For example, the vector IMPACTVECTOR 1.3, from Wageningen URPlant Research International, includes the well-known KDEL retention or classification signal. With this vector, the

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Retention in the ER has a practical advantage since it was reported to improve expression levels 5 times or more. The main reason for this appears to be that ER contains lower concentrations and / or different proteases responsible for post-translational degradation of proteins that are expressed than those 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.

Although the methods and materials of the invention allow the introduction of any exogenous gene into a microorganism, for example Prototheca, genes related to the use of sucrose and the modification of the lipid pathway are of particular interest, as discussed in the following sections.

IV. SELECT MARKERS

one. USE OF IA SACAROSA

In one embodiment, the recombinant Prototheca cell of the invention further contains one or more exogenous sucrose utilization genes. In various embodiments, the one or more genes encode one or more proteins selected from the group consisting of a fructokinase, a glucokinase, a hexokinase, a sucrose invertase, a transporter of

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IMPI sucrose. For example, expression of a sucrose transporter and a sucrose invertase allows Prototheca to transport sucrose into the cell from the culture medium and hydrolyzes sucrose to produce glucose and fructose. Optionally, a fructokinase may also be expressed in cases where endogenous hexokinase activity is insufficient for maximum fructose phosphorylation. Examples of suitable sucrose transporters are the Genbank accession numbers.

CAD91334, CAB92307, and CAA53390. Examples of suitable fructokinases are Genbank accession numbers P26984, P26420 and CAA43322.

In one embodiment, the present invention provides a Prototheca host cell that secretes a sucrose invertase. Secretion of a sucrose invertase avoids the need for the expression of a transporter that can transport sucrose into the cell. This is because the secreted invertase catalyzes the conversion of a sucrose molecule to a glucose molecule and a fructose molecule, both of which can be transported and used by the microbes provided by the invention. For example, the expression of a sucrose invertase (such as sec. With ident number: 3) with a secretion signal (such

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I

161 as sec. with no. Ident: 4 (yeast), sec. with no.

Ident: 5 (from upper floors), sec. with no. from ident:

(consensus signal of eukaryotic secretion), and sec. with no. Ident: 7 (combination of sequence signals from higher plants and eukaryotic consensus) generates invertase activity outside the cell. Expression of that protein, as allowed by the genetic engineering methodology described herein, allows cells that are already capable of using extracellular glucose as an energy source to use sucrose as an extracellular energy source.

Prototheca species expressing an invertase in sucrose-containing media are a preferred species of microalgae for oil production. The expression and extracellular directionality of this fully active protein allows the resulting host cells to grow in sucrose, whereas their untransformed counterparts cannot. Therefore, the present invention provides recombinant Prototheca cells with codon optimized for the invertase gene, includes but is not limited to the yeast invertase gene, integrated into its genome such that the invertase gene is expressed as assessed by invertase activity and hydrolysis of sucrose. The present invention also provides the genes for the

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INSTITUTO MEXICANO DF IA PMIHBUAD INDUSTRIAL invertasa useful as selection markers in cells

Recombinant prototheca, as these cells are capable of growing in sucrose, whereas their untransformed counterparts cannot, and methods for the selection of recombinant host cells through the use of an invertase as a powerful selection marker for molecular genetics algae.

Successful expression of sucrose invertase in

Prototheca also illustrates another aspect of the present invention as it demonstrates that heterologous (recombinant) proteins can be expressed in the algae cell and successfully transit outside the cell and in the culture medium in a fully active and functional form. Therefore, the present invention provides methods and reagents for expressing a wide and diverse range of heterologous proteins in microalgae and secreting them out of the host cell. Such proteins include, for example, industrial enzymes, such as, for example, lipases, proteases, cellulases, pectinases, amylases (eg, sec. No.

Ident .: 190-191), esterases, oxidoreductases, transferases, lactases, isomerases and invertases, as well as therapeutic proteins such as, for example, growth factors, cytokines, full-size antibodies that

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they comprise two light and two heavy chains, Fab, scFv (single stranded variable fragment), camel-like antibodies, antibody fragments, antibody fragment fusions, antibody-receptor fusions, insulin, interferons, and insulin-like growth factors.

The successful expression of a sucrose invertase in

Prototheca also illustrates another aspect of the present invention, as it provides methods and reagents for the use of fungal transit peptides in algae to direct protein secretion in Prototheca; and the methods and reagents for determining whether a peptide can function, and its ability to function, as a transit peptide in Prototheca Prototheca cells. The methods and reagents of the invention can be used as a tool and platform to identify other peptides to successfully transport proteins to the outside of a cell, and that yeast invertase has great utility in these methods. As demonstrated in this example, removal of the yeast endogenous invertase transit peptide and its replacement by other transit peptides, either endogenous to host algae or from other sources (eukaryotes, prokaryotes, and viruses), can identify whether any peptide of interest

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can function as a transit peptide to guide

m.sTRiM.

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proteins on their way out of the cell.

Examples of suitable sucrose invertases include those identified by Genbank accession numbers.

CAB95010, NP_012104 and CAA06839. Non-limiting examples of suitable invertases are listed below in Table 3. Amino acid sequences for each listed invertase were included in the sequence listing below. In some cases, the exogenous sucrose utilization gene suitable for use in the methods and vectors of the invention encode a sucrose invertase having at least 40, 50, 60,

75, or 90% or greater amino acid identity with a sucrose invertase selected from Table 3.

Table 3. Sucrose invertases.

<td>DESCRIPTION</td><td>Organism</td><td>Access to</td><td>Sec. With no. of</td>

<td>Reverse</td><td>Chicorium intybus</td><td>Y11124</td><td>Sec. With no. of</td>

<td></td><td></td><td></td><td>ident .: 20</td>

<td>Reverse</td><td>Schizosaccharomyces</td><td>AB011433</td><td>Sec. With no. of</td>

<td></td><td>pombe</td><td></td><td>ident.:21</td>

<td>beta-</td><td>Anomalous pichia</td><td>X80640</td><td>Sec. With no. of</td>

<td>fructofuranosidase</td><td></td><td></td><td>ident.:22</td>

<td>(invertase)</td><td></td><td></td><td></td>

<td>Reverse</td><td>Debaryomyces</td><td>X17604</td><td>Sec. With no. of</td>

<td></td><td>occidentalis</td><td></td><td>ident .: 23</td>

<td>Reverse</td><td>Oryza sativa</td><td>AF019113</td><td>Sec. With no. of</td>

<img file="MX339639B_D0152.tif" />

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<td></td><td></td><td></td><td colspan="2">ident .: 24</td>

<td>Reverse</td><td>Allium strain</td><td>AJ006067</td><td>Sec. With no.</td><td>of</td>

<td></td><td></td><td></td><td>ident .: 25</td><td></td>

<td>Reverse</td><td>Beta vulgaris</td><td>AJ278531</td><td>Sec. With no.</td><td>of</td>

<td></td><td>subsp. Vulgaris</td><td></td><td>ident .: 26</td><td></td>

<td>beta-</td><td>Bifidobacterium</td><td>AAT28190</td><td>Sec. With no.</td><td>of</td>

<td>fructofuranosidase</td><td>brief UCC2003</td><td></td><td>ident .: 27</td><td></td>

<td>(invertase)</td><td></td><td></td><td></td><td></td>

<td>Reverse</td><td>Saccharomyces</td><td>NP_012104</td><td>sec. with no.</td><td>of</td>

<td></td><td></td><td></td><td>sec. with no.</td><td>of</td>

<td></td><td>cerevisiae</td><td></td><td>ident.:28</td><td></td>

<td></td><td></td><td></td><td>(amino acid)</td><td></td>

<td>Invest A</td><td>Zymomonas mobilis</td><td>AAO38865</td><td>Sec. With no.</td><td>of</td>

<td></td><td></td><td></td><td>ident .: 29</td><td></td>

<td>Reverse</td><td>Arabadopsis</td><td>NP_566464</td><td>Sec. With no.</td><td>of</td>

<td></td><td>thaliana</td><td></td><td>ident .: 188</td><td></td>

The secretion of an invertase into the culture medium by

Prototheca enables cells to grow also in residual molasses from sugarcane processing as they do in pure reagent-grade glucose; The use of this low-value waste product from sugar cane processing can provide significant cost savings in the production of lipids and other oils. Therefore, the present invention provides a culture of microorganisms containing a population of Prototheca microorganisms, and a culture medium comprising (i) sucrose

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IAL »'STRIAL and (ii) an enzyme sucrose invertase. In various forms, sucrose in cultivation comes from sorghum, sugar beets, sugar cane, molasses, or depolymerized cellulosic material (which may optionally contain lignin).

In another aspect, the methods and reagents of the invention significantly increase the number and types of raw materials that recombinant Prototheca can use. While the microbes exemplified herein are altered in such a way that they can use sucrose, the methods and reagents of the invention can be applied so that the raw materials, such as cellulosics, are usable by a modified host microbe of the invention with the ability to secrete cellulases, pectinases, isomerases, or the like, such that the breakdown products of enzyme reactions are no longer simply tolerated, rather, they are used as a carbon source by the host. An example of this is described below and in the examples of microbes manipulated to express a secretable α-galactosidase, which confers the ability to hydrolyze agalactosyl bonds in oligosaccharides such as those contained in raffinose and stachyose which are two oligosaccharides that are necunetranized. in agricultural waste streams.

. Alpha-galactosidase expression

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Although the expression of a sucrose invertase, as described above, confers the ability on Prototheca cells to use sucrose more efficiently as a carbon source (through the enzymatic hydrolysis of the a-bond between the fructose and glucose molecules in the disaccharide sucrose), Expression of other enzymes that hydrolyze other types of α-bonds in oligosaccharides may confer the ability for Prototheca cells to use other sources of carbon. The expression of these enzymes (and the resulting ability to use carbon sources that Prototheca and other microalgae cells would not ordinarily be capable of) can be used as a selective marker for these transgenic Prototheca cells by allowing the selection of positive clones that are able to grow on these carbon sources.

In one embodiment, the recombinant Prototheca cell of the invention further contains one or more exogenous genes encoding polysaccharide degrading enzymes. In various embodiments, the one or more genes that encode polysaccharide degrading enzymes is a gene that encodes a secreted agalactosidase. Expression of an exogenously secreted α-galactosidase in a Prototheca cell confers the ability of such a transformed strain to grow on sugars

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Μ LA ΜΟΜΒΟΑΓ)

INDUSTRIAL (carbon sources) containing D-galactosyl linkages, such as o-linkages between galactose monosaccharide units and glucose. Prototheca strains expressing an exogenous, secreted agalactosidase will be able to use disaccharides such as melibiosa (disaccharide composed of aD-galactose-glucose).

Sugars such as raffinose (a trisaccharide made up of α-linked galactose-glucose-fructose) and stachyose (a tetrasaccharide made up of two linked 10-D-galactose units, followed by α-linked glucose and fructose) occur in significant proportions in streams from agricultural waste such as beet pulp (raffinose) and soybean meal (stachyose). Such agricultural residues represent a significant unused carbon source for oil conversion by microbes (including Prototheca) capable of using them.

[0013] Prototheca are unable to use oligosaccharides such as raffinose and stachyose in any significant amount at all. In the case of raffinose and stachyose, although transgenic strains expressing a sucrose invertase (as described above) have the ability to hydrolyze the α-link between fructose and glucose in an α-galactosyl derivative of sucrose, but the

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The rest of the oligosaccharide remains unused, since sucrose invertase does not cleave the remaining α-bonds in such sugars and the resulting disaccharides are not usable.

In another embodiment, the recombinant Prototheca cell of the invention comprises an exogenous gene encoding a sucrose invertase and an exogenous gene encoding an agalactosidase. Thus, strains expressing a sucrose invertase and an α-galactosidase will be able to fully hydrolyze oligosaccharides such as raffinose and stachyose, allowing consumption of the monomer components. Additionally, genes encoding agalactosidase can be used as a selectable marker for transformation. Clones containing the exogenous agalactosidase gene will have the ability to grow on melibiosa. Examples of α-galactosidase genes suitable for use in Prototheca strains include the MEL1 gene from

Saccharomyces carlbergehsis, the AglC gene from Aspergilus niger.

Remarkably, not all α-galactosidase genes are functional in Prototheca species, even if the genes are optimized according to the use of preferred codons in Prototheca strains. The examples below demonstrate the ability of transgenic Prototheca cells to grow in melibiosa when transformed with an optimized MEL1 gene.

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by codon from S. carlbergensis and the AglC gene from A. niger, but not a gene that encodes α-galactosidase from the top plant, Cyamopsis tetragonobola (guar seed).

3. Thiamine auxotrophic supplementation

Prototheca strains that include Prototheca moriformis are known to be thiamine auxotrophic (see, for example, Ciferri, 0. (1956) Nature, v.178, pp. 1475-1476), meaning that these strains require thiamine in the nutrient medium for growth. Thiamine auxotrophy may be the result of mutations or loss of enzyme expression in the thiamine biosynthetic pathway. Complemented transgenic strains expressing the enzyme (s) missing from the thiamine biosynthetic pathway can then grow without adding thiamine, thus reducing the cost of the nutrient medium as well as making the resulting microalgae biomass more desirable from the point of animal nutrition. Supplementation with an enzyme of the thiamine biosynthetic pathway can also be used as a selectable marker since the transgenic gene confers the ability to grow on plates / media that do not contain thiamine.

In one embodiment, the recombinant Prototheca cell of the invention further contains one or more exogenous genes that

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encode the enzyme of the thiamine biosynthetic pathway. In another embodiment, the recombinant Prototheca cell of the invention comprises an exogenous gene encoding hydroxymethylpyrimidine phosphate synthases (eg, sec.

with no. Ident .: 192) from algae, plants or cyanobacterial sources. In still other embodiments, hydroxymethylpyrimidine phosphate synthase is encoded by a THIC gene. In still other modalities, the THIC gene is THIC C169 from Coccomyxa, THIC from Arabidopsis thaliana, THIC PCC 6803 from Synechocystis sp, or THIC from Salmonella enterica subsp. Enteric serovar Typhimurium str. (section with ID number: 193). The examples below detail the modification of Prototheca moriformis UTEX 1435 with the restored thiamine prototype.

Other selectable markers

Any of a wide variety of selectable markers can be used in the construct of a transgene useful for the transformation of microorganisms, such as 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

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OF THE INDUSTRY NAME !.

confers resistance to phleomycin. Methods for determining the sensitivity of microalgae to antibiotics are well known. For example. Mol Gen

Genet. 1996 Oct 16, -252 (5): 572-9.

More specifically, Dawson et al. (1997), Current

Microbiology 35: 356-362 (incorporated by reference in its entirety herein), describe the use of the Chlorella nitrate reductase (NR) gene as a selectable marker for NR-deficient Chlorella soroklniana mutants. Kim et al. (2002), Mar. Biotechnol.4: 6373 (incorporated by reference herein in its entirety), describe 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

V. LIPIDIC ENGINEERING

In addition to altering the capacity of microorganisms (for example, microalgae.

oil yeast, fungi, or bacteria), such as Prototheca to use raw materials as sucrose-containing raw materials, the present invention further provides

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ΤΤ! .ΠΌ MUCAMO M LA rttOMfPAD

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Recombinant microorganisms (eg Prototheca) that were modified to alter the properties and / or proportions of lipids produced. The pathway can also be, or alternatively, modified to alter the properties and / or proportions of various lipid molecules that are produced through enzymatic processing of lipids and intermediates in the fatty acid pathways. In various embodiments, the recombinant microorganisms (eg Prototheca cells) of the invention have, compared to their untransformed counterparts, an optimized lipid yield per unit volume and / or per unit time, chain length of carbon (for example, for renewable diesel production or for chemical industry applications requiring lipid raw materials), reduced number of double or triple bonds, optionally zero, and an increase in the hydrogen: carbon ratio of a particular lipid species or a different lipid population. Additionally, desirable hydrocarbon-producing microorganisms can be manipulated to produce such components in higher amounts, or with greater specificity.

In the case of microalgae, some wild cells already have good growth characteristics but do not produce

<img file="MX339639B_D0160.tif" />

174 the desired types or amounts of lipids ^ Tros- example ^ include, but are not limited to, Pyrobotrys, Phormidium,

Agmenellum, Cartería, Lepocinclis, Pyrobotrys, Nitzschia,

Lepocinclis, Anabaena, Euglena, Spirogyra, Chlorococcum,

Tetraedron, Oscillatoria, Phagus, and Chlorogonium, which have the desired growth characteristics in municipal drainage or wastewater. Such cells, as well as Chlorella, Prototheca, and other microbe species, can be engineered to have improved lipid production characteristics. Desired characteristics include optimizing lipid yield per unit volume and / or per unit time, carbon chain length (for example, for biodiesel production or for industrial applications requiring hydrocarbon feedstocks), reducing the number of double or triple bonds, optionally to zero remove or remove rings and cyclic structures and increase the hydrogen ratio: carbon of a particular lipid species or a different lipid population. Furthermore, the microalgae that produce suitable hydrocarbons can be further modified to have even more desirable hydrocarbon yields. Examples of such microalgae include species of the genus Chlorella and the genus

Prototheca.

IMPI

MEXICAN INSTITUTE

MEXICAN INSTITUTE

OF THE Ε «0ίΙΕΟΑΓ,

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In particular modalities, one or more enzymes that control branch points in the metabolismTsmÓ "" of '"± a · ~ fatty acid synthesis were regulated up or down to improve lipid production. Up-regulation can be achieved, for example, by transforming cells with expression constructs in which a gene encoding the enzyme of interest is expressed, for example, through the use of a strong promoter and / or enhancer elements that increase transcription. Such constructs may include a selection marker such that transformants can be screened, which can result in an amplification of the construct and an increase in the level of expression 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 the conversion of pyruvate to acetyl-CoA (examples, some from microalgae, include accession numbers in Genbank 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 stage in the synthesis of fatty acids. In

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INSTITUTO MEXICANO consequently, this enzyme can be regulated áaeeaSáí &amp; nSí to increase the production of acids — fatty— (eg, some of microalgae, include access numbers of Genbank

BAA94752; AAA75528; AAA81471; YP_537052; YP_536879; NP_045833 and BAA57908). Fatty acid production can also be increased by upregulation of acyl carrier protein (ACP), which transports growing acyl chains during fatty acid synthesis (eg, some from microalgae, include accession numbers in Genbank AOTOF8; P51280; NP_849041; YP_874433). Glycerol-3-phosphate acyl transferase catalyzes the rate limiting step of fatty acid synthesis. Upregulation of this enzyme can increase fatty acid production (some microalgae, for example, include accession numbers in Genbank AAA74319;

AAA33122; AAA37647; P44857 and ABO94442).

Up-regulation or down-regulation of genes can be applied to global regulators that control gene expression of fatty acid biosynthetic pathways. Consequently, one or more global regulators of fatty acid synthesis are positively or negatively regulated, as appropriate, to inhibit or increase, respectively, the expression of a plurality of genes

<img file="MX339639B_D0162.tif" />

177

ΙΜΡΙ synthetic fatty acids and, in úH-íma-'nnt-mnia increase lipid production. Examples include sterol regulatory element binding proteins (SREBPs), such as SREBP-la and SREBP-lc (for examples see Genbank accession numbers NP_035610 and Q9WTN3).

The present invention further provides recombinant microorganisms (eg, Prototheca cells) that were modified to contain one or more exogenous genes encoding lipid-modifying enzymes such as, for example, fatty acyl-ACP thioesterases (eg.

C. callophylla (sec. With ID no .: 145 and sec. With no.

Ident .: 146; see also Table 4), fatty acyl CoA / aldehyde reductase (see Table 6), acyl fatty CoA reductase (see Table 7), fatty aldehyde decarbonylase (see Table 8), fatty aldehyde reductase, desaturase (such as desaturase of stearoyl-ACP (for example, an R.

communis SAD, sec. with no. Ident .: 147 and sec. with no. Ident .: 148) optimized for codon and fatty acyl desaturases and squalene synthases (see accession number to GenBank

AF205791). In some embodiments, genes encoding acyl fatty ACP thioesterase and a naturally co-expressing acyl carrier protein are transformed within a Prototheca cell, optionally with one or more genes that

178

<img file="MX339639B_D0163.tif" />

encode other enzymes for lipid modification. In other embodiments, ACP and acyl fatty acp thioesterase may have an affinity for each other that gives an advantage when the two are used together in the microbes and methods of the present invention, regardless of whether they co-express naturally. or not in a particular tissue or organism. Therefore, the present invention contemplates both pairs of these naturally co-expressing enzymes and those that share an affinity to interact with each other to facilitate cleavage of a carbon chain of specific length from ACP.

In still other modalities, an exogenous gene encoding a desaturase is transformed within the microorganism (eg, a Prototheca cell) in conjunction with one or more genes encoding other lipid modifying enzymes to provide modifications with respect to saturation of lipids. In other embodiments, an endogenous desaturase gene is overexpressed (eg, by introducing additional copies of the gene) into the microorganism (eg, a Prototheca cell). Stearoyl ACP desaturase (see, for example, GenBank accession numbers AAF15308; ABM45911; and AAY86086), for example, catalyzes the conversion of stearoyl acp to oleoyl ACP. Regulation

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INDUSTRIAL

<img file="MX339639B_D0164.tif" />

Upregulation of this gene can increase the proportion of monounsaturated fatty acids produced by a cell, whereas downregulation can reduce the proportion of monounsaturated fatty acids. For illustrative purposes, stearoyl5 ACP desaturases (SAD) are responsible for the synthesis of C18 fatty acids: the starting of C18: 0 precursors. Another family of desaturases are acyl fatty desaturases (FAD), which include delta 12 fatty acid desaturases (Δ12 FAD).

These desaturases further provide modifications with respect to lipid saturation. For illustrative purposes, delta 12 fatty acid desaturases are responsible for the synthesis of C18: 2 fatty acids from C18: 1 precursors. Likewise, the expression of one or more glycerolipid desaturases can be controlled to alter the ratio of unsaturated to saturated fatty acids such as ω-6 desaturase fatty acid, ω-6-desaturase fatty acids, or ω-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 modifications at specific locations within a substrate of specified carbon length, or substrates having a length of carbons within a specified range. In another modality, if the

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<img file="MX339639B_D0165.tif" />

desired fatty acid profile is. an increase in monounsaturates (such as C16: 1 and / or C18: l) the over-expression of a SAD or the expression of a heterologous SAD can be coupled with silencing or inactivation (eg, through mutation, iRNA, knockout of an endogenous desaturase gene, etc.) of an acyl fatty desaturase (FAD).

In other embodiments, the microorganisms (eg, Prototheca cells) were modified to have a mutated endogenous desaturase gene, where the mutation inactivates the gene or enzyme desaturase. In some cases, the mutated endogenous desaturase gene is a fatty acid desaturase (FAD). In other cases, the mutated endogenous desaturase gene is a stearoyl acyl desaturase (SAD) transporter protein. Example 11 below describes the directed ablation or knockout of stearoyl-ACP desaturases and delta 12 fatty acid desaturases.

In some cases, it may be advantageous to pair one or more genetic engineering techniques to achieve a transgenic cell that produces the desired lipid profile. In one embodiment, a microorganism (eg, a Prototheca cell) comprises an endogenous desaturase mutated gene and one or more exogenous genes. In non-limiting examples, a Prototheca cell with a mutated desaturase gene

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Endogenous IMNICTRIAL _ may further express an exogenous acyl fatty acp-thioesterase gene and / or a sucrose invertase gene.

Example 11 below describes a transgenic cell of

Prototheca that contains a directed or knockout ablation of an endogenous SAD and further expresses a C14-preferred thioesterase and a sucrose invertase from Cinnamomum camphora. In this case, the transgenic Prototheca cell produces a lipid profile that closely approximates the lipid profile found in the sebum. Tallow is typically derived from molten beef or sheep fat, is solid at room temperature, and is used in a variety of applications in the food, cosmetic, and chemical industries. The fatty acid profile of sebum is: 4% C14: 0; 26%

C16: 0; 3% C16: l; 14% C18: 0; 41% C18: l; 3% C18: 2; and 1% C18: 3.

As shown in Example 11 below, clones of transgenic Prototheca cells with targeted or knockout ablation of endogenous SAD and expressing a thioesterase that prefers C. camphora C14 have a lipid profile of: less than 1% C12 and fatty acids of shorter carbon chain length; 2.74% to 6.13% C14: 0; 23.07% to 25.69%

C16: 0; 7.02% to 11.08% C18: 0; 42.03% to 51.21% C18: l; and 9.37% a

13.45% C18: 2 expressed as a percentage of area). In some cases, transgenic Prototheca cells have profiles

<img file="MX339639B_D0167.tif" />

182

Lipid IMPI of: 3-5

C14: 0; 25-27

C16: 0; 10-15

C18: 0; and 4045% C18: 1.

Thus, in particular embodiments, the microbes of the present invention are genetically engineered to express one or more exogenous genes selected from an acyl acp thioesterase, an acyl-CoA / aldehyde reductase, an acyl fatty-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, or a naturally expressed acyl transporter protein. Suitable expression methods are described above in relation to expression of a lipase gene, including, but not limited to, inducible expression and compartmentalized expression. An acyl fatty ACP thioesterase cleaves a fatty acid from an acyl carrier protein (ACP) during lipid synthesis. Through additional enzymatic processing, the cleaved fatty acid combines with a coenzyme to produce an acyl-CoA molecule. This acylCoA is the substrate for the enzymatic activity of acyl fatty-CoA reductase to yield an aldehyde, as well as for an acyl fatty CoA / aldehyde reductase to produce an alcohol. The aldehyde that is produced by the action of the acyl fatty CoA reductase identified above is the substrate for additional enzyme activity by either an aldehyde

183

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fatty reductase to produce an alcohol, or a fatty aldehyde decarbonylase to produce an alkane or alkene.

In some embodiments, the fatty acids, glycerolipids, or corresponding primary alcohols, aldehydes, allenes, or alkenes, which are generated by the methods described herein, contain 8, 10, 12, or 14 carbon atoms. The preferred fatty acids for the production of diesel, biodiesel, renewable diesel, or jet fuel, or the corresponding primary alcohols, aldehydes, allenes and alkenes, for industrial applications contain from 8 to 14 carbon atoms. In certain embodiments, the above fatty acids, as well as the other corresponding hydrocarbon molecules, are saturated (without any carbon-carbon double or triple bond), monounsaturated (single double bond); unsaturated poly (two or more double bonds); it is linear (non-cyclical) or branched.

For fuel production, higher saturation is preferred.

The enzymes described directly above have preferential specificity for the hydrolysis of a substrate containing a specific number of carbon atoms. For example, an acyl fatty acp thioesterase may have a preference for cleavage of a fatty acid that has

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INDUSTRIAL carbon atoms of acp. In some embodiments, length-specific ACP and thioesterase may have an affinity for each other that makes them especially useful in combination (eg, exogenous acp and thioesterase genes can naturally co-express in a given tissue or organism from which they are derived). Thus, in various embodiments, the recombinant Prototheca cells of the invention may contain an exogenous gene that encodes a protein with specificity to catalyze enzymatic activity (eg, cleavage of a fatty acid from an ACP, reduction of a acyl-CoA to an aldehyde or an alcohol, or the conversion of an aldehyde to an alkane) with respect to the number of carbon atoms contained in the substrate. Enzymatic specificity may, in various embodiments, be for a substrate having 8 to carbon atoms, preferably 8 to 18 carbon atoms, and more preferably 8 to 14 carbon atoms. A preferred specificity is for a substrate that has less, ie 12, better than more, ie 18 carbon atoms.

Other suitable acyl-ACP fatty thioesterases for use with the microbes and methods of the invention include, but are not limited to, those listed in Table 4.

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Table 4. Acyl Fatty-ACP Thioesterases and GenBank Access Numbers.

fatty acyl-ACP thioesterase from Umbellularia cali fornica (GenBank # AAC49001) (ID no.: 203) fatty acyl-ACP thioesterase from Cinnamomum camphora (GenBank # Q39473) fatty acyl-ACP thioesterase from Umbellularia californica (GenBank # 041635) Myristica fragrans fatty acyl-ACP thioesterase (GenBank # AAB71729) (sec. With ID #: 224) Myristica fragrans fatty acyl-ACP thioesterase (GenBank # AAB71730) (sec. No. ID: 222) Fatty acyl-ACP thioesterase from Elaeis guineensis (GenBank # ABD83939) (Sec. ID: 204) Fatty acyl-ACP thioesterase from Elaeis-guineensis (GenBank # AAD42220) Fatty acyl-ACP thioesterase Populus tomentosa (GenBank # ABC47311) (Sec. ID #: 207) Acyl Fatty-ACP Thioesterase from Arabidopsis thaliana (GenBank # NP_172327) (Sec. ID #: 208) Acyl Fatty-ACP Thioesterase from Arabidopsis thaliana (GenBank # CAA85387), (sec. with ID no .: 2 09)

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Arabidopsis fatty acyl-ACP thioesterase (GenBank # CAA85388) (sec. with ID #: 210) Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank # Q9SQI3) (sec. with ID #: 211) acyl fatty-ACP thioesterase from Cuphea lanceolata (GenBank # CAA54060) (sec. with ID #: 212) acyl fatty-ACP thioesterase from Cuphea hookeriana (GenBank # AAC72882) (sec. with ID #: 202) acyl fatty-ACP thioesterase from Cuphea calophylla subsp.

mesostemon (GenBank # ABB71581) (ID no.: 213) Fatty acyl-ACP thioesterase from Cuphea lanceolata (GenBank # CAC19933) Fatty acyl-ACP thioesterase from Elaeis guineensis (GenBank # AAL15645) (sec no. ID: 206) Fatty acyl-ACP thioesterase from Cuphea hookeriana (GenBank # Q39513) Fatty acyl-ACP thioesterase from Gossypium hirsutum (GenBank # AAD01982) (section with ID no .: 214) Fatty acyl-ACP thioesterase from Vitis vinifera (GenBank # CAN81819) (sec. With no. ID: 215) Garlicia mangostana acyl fatty acp-thioesterase (GenBank # AAB51525)

<img file="MX339639B_D0171.tif" />

187 fatty acyl-ACP thioesterase from Brassica júncea (GenBank # ABI18986) (sec. with ID #: 216) acyl fatty-ACP thioesterase from Madhuca longifolia (GenBank # AAX51637) (sec. with ID #: 217) fatty acyl-ACP thioesterase from Brassica napus (GenBank # ABH11710) fatty acyl-ACP thioesterase from Oryza sativa (indica cultivar group) (GenBank # EAY86877) (sec. with ID no .: 218) fatty acyl-ACP thioesterase from Oryza sativa (cultivar group japanica) (GenBank # NP_001068400) (sec. with no. Ident .:

219) Fatty acyl-ACP thioesterase from Oryza sativa (indica cultivar) (GenBank # EAY99617) (ID no .: 220) Fatty acyl-ACP thioesterase from Cuphea hookeriana (GenBank # AAC49269) Fatty acyl-ACP thioesterase of Ulmus Americana (GenBank # AAB71731) Fatty acyl-ACP thioesterase from Cuphea lanceolata (GenBank # CAB60830) (sec. with ID no .: 221) Fatty acyl-ACP thioesterase from Cuphea palustris (GenBank # AAC49180) Fatty acyl-ACP Germanic Iris thioesterase (GenBank

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DE LA FROFIÍDAO # AAG4 3858) ____ „_____ fatty acyl-ACP thioesterase from Germanic Iris (GenBank # AAG43858.1) fatty acyl-ACP thioesterase from Cuphea palustris (GenBank # AAC49179) fatty acyl-ACP thioesterase from Myristica

AAB71729) fatty acyl-ACP thioesterase from Myristica fragrans (GenBank #

AAB717291.1) Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank # U39834) (ID no .: 197) Umbilluaria cali fornica fatty acyl-ACP thioesterase (GenBank # M94159) (Sec. No. ident .: 285) Fatty acyl-ACP thioesterase from Cinnamomum camphora (GenBank # U31813) (sec. with ID no .: 223) Fatty acyl-ACP thioesterase from Cuphea wrightii (GenBank # U56103) (sec. ident .: 183) Ricinus communis fatty acyl-ACP thioesterase (GenBank # ABS30422) (sec. no. ID: 198)

The examples below describe the directionality and successful expression of heterologous acyl fatty acp-thioesterases.

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of Cuphea hookeriana, Umbellularia cali fornica, Cinnamomun camphora, Cuphea palustris, Cuphea lanceolata, Iris germanica, Myristica fragrans and Ulmus americana in Prototheca species. Additionally, alterations in the fatty acid profile were confirmed in the expression of the host cells of these heterologous acyl fatty ACP thioesterases. These results were unexpected given the lack of sequence identity between algae and higher plant thioesterases in general, and between Prototheca moriformis acyl fatty ACP thioesterases and the heterologous acyl fatty ACP thioesterases listed above. As shown in the Examples, expression of these heterologous thioesterases in Prototheca generates a transgenic microalgae that is capable of producing oil / lipids with truly unique fatty acid profiles that are not currently available from commercial seed crops, even at through the mixing of various seed cultivation oils. Table 5 shows the fatty acid profiles of common commercial seed oils. All commercial seed oil data below was compiled from the United States Pharmacopeia Food and Chemical Codes, 7^ma Ed. 2010-2011. The data of the

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INDUSTRIAL tallow are from the National Research Council: Fat content and composition of animal products (1976).

Table 5. Lipid profiles of commercial seed oils (in percentages).

<td></td><td>C8: 0</td><td>cio : 0</td><td>Cl2: 0</td><td>C14: 0</td><td>C16: 0</td><td>C18: 0</td><td>C18 :one</td><td>C18: O- diOH</td><td>C18: l -OH</td><td>C18: 2</td><td>C18: 3 a</td>

<td>R. communis</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0.9-</td><td> 1.0-</td><td> 3.7</td><td> 0.4-</td><td> 83.6-</td><td> 0</td><td> 0.2-0.6</td>

<td>(Oil of</td><td></td><td></td><td></td><td></td><td> 1.6</td><td> 1.8</td><td> -</td><td> 1.3</td><td> 89.0</td><td></td><td></td>

<td>castor bean)</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 6.7</td><td></td><td></td><td></td><td></td>

<td>C. nucífera</td><td> 5.0</td><td> 4.0</td><td> 44-52</td><td> 15-21</td><td> 8.0-</td><td> 1.0-</td><td> 5.0</td><td> 0</td><td> 0</td><td> 0-2.5</td><td> 0</td>

<td>(Oil of</td><td> -</td><td> -</td><td></td><td></td><td> 11.0</td><td> 4.0</td><td> -</td><td></td><td></td><td></td><td></td>

<td>walnut</td><td> 9.0</td><td> 8.0</td><td></td><td></td><td></td><td></td><td> 8.0</td><td></td><td></td><td></td><td></td>

<td>coconut)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Z. mays</td><td> 0</td><td> 0</td><td> 0</td><td> &lt; 1.0</td><td> 8.0-</td><td> 0.5-</td><td> 19-</td><td> 0</td><td> 0</td><td> 38-65</td><td> &lt; 2.0</td>

<td>(Oil of</td><td></td><td></td><td></td><td></td><td> 19.0</td><td> 4.0</td><td> 50</td><td></td><td></td><td></td><td></td>

<td>corn)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>G.</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td> 0.5-</td><td> 17-29</td><td> 1.0-</td><td> 13-</td><td> 0</td><td> 0</td><td> 40-63</td><td> 0.1-2.1</td>

<td>barbadense</td><td></td><td></td><td></td><td> 2.0</td><td></td><td> 4.0</td><td> 44</td><td></td><td></td><td></td><td></td>

<td>(Oil of</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>seed of</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>cotton)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>B. rapa, B</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td> &lt; 0.2</td><td> &lt; 6.0</td><td> &lt; 2.5</td><td> &gt;50</td><td> 0</td><td> 0</td><td> &lt; 40</td><td> &lt; 14</td>

<td>napus, B.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>júncea</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>(Canda)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>0. European</td><td> 0</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td>β .5-</td><td> 0.5-</td><td> 56-</td><td> 0</td><td> 0</td><td> 3.5-</td><td> &lt; 1.2</td>

<td>(Olive)</td><td></td><td></td><td></td><td></td><td> 20.0</td><td> 5.0</td><td> 85</td><td></td><td></td><td> 20.0</td><td></td>

<td>A. hypoqaea</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td> &lt; 0.2</td><td> 7.0-</td><td> 1.3-</td><td> 35-</td><td> 0</td><td> 0</td><td> 13.0-</td><td> &lt; 0.6</td>

<td>(Peanut)</td><td></td><td></td><td></td><td></td><td> 16.0</td><td> 6.5</td><td> 72</td><td></td><td></td><td> 43</td><td></td>

<td>AND.</td><td> 3.0</td><td> 2.5</td><td> 40-52</td><td> 14.0-</td><td> 7.0-</td><td> 1.0-</td><td> 11.</td><td> 0</td><td> 0</td><td> 0.5-</td><td> 0</td>

<td>guineensis</td><td> -</td><td> -</td><td></td><td> 18.0</td><td> 10.0</td><td> 3.0</td><td> 0-</td><td></td><td></td><td> 4.0</td><td></td>

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<td>(Almond</td><td> 5.0</td><td> 6.0</td><td></td><td></td><td></td><td></td><td> 19.</td><td></td><td></td><td></td><td></td>

<td>Palm)</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0</td><td></td><td></td><td></td><td></td>

<td>AND.</td><td> 0</td><td> 0</td><td> 0</td><td> 0.5-</td><td> 32.0-</td><td> 2.0-</td><td> 34-</td><td> 0</td><td> 0</td><td> 7.2-</td><td> 0</td>

<td>guineensis</td><td></td><td></td><td></td><td> 5.9</td><td> 47.0</td><td> 8.0</td><td> 44</td><td></td><td></td><td> 12.0</td><td></td>

<td>(Palm)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>C.</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td> &lt; 0.1</td><td> 2.0-</td><td> 1.0-</td><td> 7.0</td><td> 0</td><td> 0</td><td> 72-81</td><td> &lt; 1.5</td>

<td>tinctorus</td><td></td><td></td><td></td><td></td><td> 10.0</td><td> 10.0</td><td> -</td><td></td><td></td><td></td><td></td>

<td>(Safflower)</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 16.</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0</td><td></td><td></td><td></td><td></td>

<td>H. annus</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td> &lt; 0.5</td><td> 3.0-</td><td> 1.0-</td><td> 14-</td><td> 0</td><td> 0</td><td> 20-75</td><td> &lt; 0.5</td>

<td>(Sunflower)</td><td></td><td></td><td></td><td></td><td> 10.0</td><td> 10.0</td><td> 65</td><td></td><td></td><td></td><td></td>

<td>G. max</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td> &lt; 0.5</td><td> 7.0-</td><td> 2.0-</td><td> 19-</td><td> 0</td><td> 0</td><td> 48-65</td><td> 5.0-10.0</td>

<td>(Bean from</td><td></td><td></td><td></td><td></td><td> 12.0</td><td> 5.5</td><td> 30</td><td></td><td></td><td></td><td></td>

<td>soy)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>L.</td><td> 0</td><td> 0</td><td> &lt; 0.1</td><td> &lt; 0.5</td><td> 2.0-</td><td> 2.0-</td><td> 8.0</td><td> 0</td><td> 0</td><td> 40-80</td><td> &lt; 5.0</td>

<td>usitatissim</td><td></td><td></td><td></td><td></td><td> 9.0</td><td> 5.0</td><td> -60</td><td></td><td></td><td></td><td></td>

<td>um</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>(Solin-</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>linseed)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>B. parkii</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 3.8-</td><td> 41.2-</td><td> 34 .</td><td> 0</td><td> 0</td><td> 3.7-</td><td> 0</td>

<td>(Seed of</td><td></td><td></td><td></td><td></td><td> 4.1</td><td> 56.8</td><td> 0-</td><td></td><td></td><td> 6.5</td><td></td>

<td>shea)</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 46.</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 9</td><td></td><td></td><td></td><td></td>

<td>Butter</td><td></td><td> 0-1</td><td> 0-1</td><td> 0-4</td><td> 22-30</td><td> 24-37</td><td> 29-</td><td></td><td></td><td> 0-3</td><td></td>

<td>ca cao</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 38</td><td></td><td></td><td></td><td></td>

<td>Tallow</td><td></td><td></td><td></td><td> 3-4</td><td> 23-28</td><td> 14-23</td><td> 36-</td><td></td><td></td><td> 1-4</td><td> &lt; 1</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 43</td><td></td><td></td><td></td><td></td>

<td>Lardo</td><td></td><td></td><td></td><td> 1-2</td><td> 22-26</td><td> 13-18</td><td> 39-</td><td></td><td></td><td> 8-15</td><td> 0.5-1.5</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td>

As an example, none of these common seed oils contain high amounts of C8 or CIO fatty acids,

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INSTITUTO MEXICANO 3 mlafkohedad r / j industrial ΝμΓ * · «¹* where coconut oil and palm kernel oil are the major sources, but both in a 1: 1 ratio (C8: C10 fatty acids). As shown in the examples, the

Prototheca transformed with Cuphea palustris thioesterase of preference C: 8 was able to achieve not only a C8 fatty acid level above 12%, but also, the ratio of C8: C10 fatty acids was approximately 5: 1.

Changes in fatty acid levels are useful in producing oils that contain a fatty acid profile adapted for a variety of commercial applications.

Furthermore, changes in the relationships between different lengths of fatty acid chains is something that is not commercially available in oils that did not go through even more expensive chemical processes (such as esterification, distillation, fractionation, and reesterification). As another example, palm oil is the oil with the highest C16: 0 fatty acid content (32-47%), but palm oil has very few C14: 0 fatty acids. Prototheca containing thioesterase U. americana reaches approximately 33-38% C16: 0 fatty acids and approximately 10-16% C14: 0 fatty acids (approximately a 2: 1 ratio C16: 0 to C14: 0). This fatty acid profile is unattainable through the mixing of

<img file="MX339639B_D0174.tif" />

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MEXICAN INSTITUTE OF I. » INDUSTRIAL FROFIEDAD commercially available oils because ios oils^-&lt;35 · Seeds that are high in 16: 0 fatty acids usually do not contain many 14: 0 fatty acids.

The examples below further describe, for the first time, the successful directionality and expression of at least two acyl fatty-ACP thioesterases in one clone. Alterations in fatty acid profiles were confirmed in these clones and depending on which two thioesterases were co-expressed in one clone, the fatty acid profiles were impacted differently. As an example, from Table 5 above, both coconut oils and palm kernel oil have C12: C14 ratios of approximately 3: 1.

As described in the examples below, a Prototheca transformant containing two heterologous thioesterase genes was able to produce levels of fatty acids

C12: C14 at a ratio of approximately 5: 1. The type of C12: C14 fatty acid ratio was, until now, unattainable at commercial levels (i.e., through mixing of seed oils).

Another novel aspect of oils produced by transgenic microalgae is the degree of saturation of fatty acids. Palm oil is currently the largest source of saturated oil, with total saturates at

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unsaturated from 52% to 48%. As shown in the examples below, Prototheca with heterologous thioesterases from U.

americana γ C. camphora reached total saturation levels above 60% in the oil they produced.

In addition, it is shown in the examples below that Prototheca with heterologous thioesterases from U. americana reached total saturation levels above 86% in the oil it produced.

Acyl fatty CoA / aldehyde reductases suitable for use with the microbes and methods of the invention include, but are not limited to, those listed in Table 6.

Table 6. Acyl Fat-CoA / Aldehyde Reductases listed by GenBank Accession Numbers.

AAC45217, YP_047869, BAB85476, YP_001086217, YP_580344,

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_01115500, YP_001141848, NP_336047, NP_216059, YP_882705, YP_88607, YP_88607, YP_88608

AAR88762, ABK28586, NP_197634, CAD30694, NP_001063962,

BAD46254, NP_001030809, EAZ10132, EAZ43639, EAZ07989,

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NP_001062488, CAB88537, NP_001052541, CAH66597, CAE02214,

CAH66590, CAB88538, EAZ39844, AAZ06658, CAA68190, CAA52019, and BAC84377

Acyl fatty CoA reductases suitable for use with the microbes and methods of the invention include, but are not limited to, those listed in Table 7.

Table 7. Acyl Fat-CoA Reductases Listed by GenBank Access Numbers.

NP_187805, ABO14927, NP_001049083, CAN83375, NP_191229,

EAZ42242, EAZ06453, CAD30696, BAD31814, NP_190040, AAD38039,

CAD30692, CAN81280, NP_197642, NP_190041, AAL15288, and

NP 190042

Fatty aldehyde decarbonylases suitable for use with the microbes and methods of the invention include, but are not limited to, those listed in Table 8.

Table 8. Fatty aldehyde decarbonylases listed by GenBank accession numbers.

NP_850932, ABN07985, CAN60676, AAC23640, CAA65199, AAC24373,

CAE03390, ABD28319, NP_181306, EAZ31322, CAN63491, EAY94825,

EAY86731, CAL55686, XP_001420263, EAZ23849, NP_200588,

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NP_001063227, CAN83072, AAR90847, and AAR97643

Combinations of naturally co-expressed acyl fatty ACP thioesterases and acyl carrier proteins are suitable for use with the microbes and methods of the invention.

Additional examples of lipid or hydrocarbon modifying enzymes include amino acid sequences contained in, referenced in, or encoded by nucleic acid sequences contained or referenced in, any of the following United States 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

GenBank with access numbers: AAO18435; ZP_00513891;

Q38710; AAK60613; AAK60610; AAK60611; NP_113747; CAB75874;

AAK60612; AAF20201; BAA11024; AF205791; and CAA03710.

Other enzymes in the lipid biosynthetic pathway are further suitable for use with microbes and methods of the invention. For example, keto acyl-ACP synthase (Kas) enzymes work in conjunction with some of the enzymes listed above in the lipid biosynthetic pathway. There are different classes of Kas enzymes: Kas I participates in

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successive stages of condensation between la9 — eadenaja „, de_ acil

Increasingly increasing ACP and malonyl-ACP. Kas II typically participates in the final condensation stage and carries from C16: 0-ACP to C18: 0-ACP and incorporates malonyl-ACP. As such, in higher plants and some species / strains of microalgae that predominantly synthesize fatty acids

C16-C18: 0 (and its unsaturated derivatives), Kas II enzymes interact with FatA gene products (acyl-ACP thioesterases).

Acyl-ACP thioesterases are terminators of fatty acid biosynthesis in higher plants (and some species of microalgae), - and in most of these plant species, this is carried out by members of the FatA gene family , whose role is to finish the elongation in step C16: 0 to C18: 0. In species that synthesize shorter fatty acid chains (such as Cuphea,

Elaeis, Myristica, or Umbellularia), a different group of acyl-ACP thioesterases encoded by FatB genes carry out this termination step (see, for example, the codon-optimized coding region of Cocos nucifera FatB3-B, seq. Ident .: 189). The interaction between enzymes

Kas II and acyl-ACP thioesterases is important for the correct elongation of the fatty acid chain. Like a

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INSTITUTO MEXICANO Dt LA MOHEDAL INDUSTRIAL consequently, in higher plant species (and microalgae species) that evolved FatB genes capable of shorter chain lipid biosynthesis, there is a corresponding coevolution of an additional class of genes

Kas, called Kas IV genes. The KasIV genes are responsible for the chain length elongation of a specific size range of fatty acids, 4-14 carbons in length.

Other enzymes suitable for use with the microbes and the methods of the invention include those that have at least 70% amino acid identity with one of the proteins, listed in Tables 4, 6-8, and that exhibit the Corresponding desired enzymatic activity (eg cleavage of a fatty acid from an acyl carrier protein, reduction of an acyl-CoA to an aldehyde or alcohol, or conversion of an aldehyde to an alkane). In additional embodiments, the enzyme activity is present in a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about a

99% identity with one of the sequences described above, all of which are incorporated

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by reference herein as fully provided.

By selecting the desired combination of exogenous genes to be expressed, the product generated by the microbe can be adapted, which can be extracted from the aqueous biomass. For example, the microbe may contain: (i) an exogenous gene encoding an acyl fatty-ACP thioesterase; and, optionally, (ii) a co-expressed acyl carrier protein or other acyl carrier protein that has an affinity for acyl fatty-ACP thioesterase (or vice versa), and optionally (iii) an exogenous gene encoding an acyl fatty-CoA / aldehyde reductase or an acyl fatty -CoA reductase and, optionally, (iv) an exogenous gene encoding a fatty aldehyde reductase or a fatty aldehyde decarbonylase. The microbe, under the culture conditions described herein, synthesizes a fatty acid bound to an ACP and the acyl fatty-ACP thioesterase catalyzes the cleavage of the fatty acid from the ACP to produce, by further enzymatic processing, a molecule of acyl-CoA. When present, acyl fatty CoA / aldehyde reductase catalyzes the reduction of acyl CoA to an alcohol. Similarly, acyl fatty CoA reductase, when present, catalyzes the reduction of acyl CoA to an aldehyde. In those modalities in which a

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IMPI '^Tf5Éw '_P'ííí' '^SPOUT • Exogenous nwistrial gene encoding an acyl fatty-PnA reductase is present and is expressed to yield 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 conversion of the aldehyde to an alkane or alkene, when present.

In another embodiment, the microbe may contain: (i) an exogenous gene encoding an acyl fatty-ACP thioesterase; (ii) optionally, a naturally co-expressed acyl transporter protein or an acyl transporter protein that has an affinity for acyl fatty acid-ACP thioesterase; (iii) a mutated endogenous desaturase gene, wherein the mutation renders the desaturase gene or protein desaturase inactive, such as a desaturase knockout;

(iv) overexpression of an endogenous stearoyl acyl desaturase transporter protein or expression of a heterologous SAD; and (v) any combination of the above.

Genes encoding such enzymes, such as acyl fatty ACP thioesterases, can be obtained from cells that are already known to exhibit significant lipid production, such as Chlorella protothecoides. Genes already known have a role in lipid production, by

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For example, a gene encoding an enzyme that saturates double bonds can be transformed individually in recipient cells. However, in the practice of the invention it is not necessary to make a priori assumptions as to which genes are necessary. Methods for identifying genes that can alter (enhance) lipid production in microalgae are described in PCT publication no. 2008/151149.

Therefore, the present invention provides a microorganism (eg, a Prototheca cell) that was genetically engineered to express a lipid pathway enzyme at an altered level compared to a wild cell of the same species. In some cases, the cell produces more lipids compared to the wild type cell when both cells are cultured under the same conditions. In some cases, the cell is genetically modified and / or selected to express a lipid pathway enzyme at a higher level than that of wild-type cells. 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 is genetically modified and / or

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selects 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 cell is genetically engineered and / or selected to express a global regulator of fatty acid synthesis at an altered level compared to the wild-type cell, thereby expressing levels of a plurality of genes Synthetic fatty acids are altered compared to 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-ACPdesaturase and a glycerolipid desaturase. In some cases, the cell is genetically modified and / or selected to express a lower level of an enzyme in a lipid pathway, or to not express a specific enzyme in a lipid pathway at all (i.e., where a lipid pathway enzyme is knockout, or is replaced with an exogenous gene).

Some microalgae produce significant amounts of non-lipid metabolites, such as, for example,

<img file="MX339639B_D0184.tif" />

polysaccharides. Because polysaccharide biosynthesis can use a significant proportion of the total metabolic energy available to cells, mutagenesis of lipid-producing cells followed by detection of reduced or eliminated polysaccharide production generates new strains that are capable of producing high lipid yields.

In other embodiments, the present invention is directed to an oil-producing microbe containing one or more exogenous genes, where the exogenous genes encode the protein (s) selected from the group consisting of an acyl fatty ACP thioesterase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, an fatty acyl-CoA / aldehyde reductase, a fatty aldehyde decarbonylase, a desaturase, and an acyl carrier protein. In another embodiment, an endogenous desaturase gene is overexpressed in a mic containing one or more of the above exogenous genes. In one embodiment, the exogenous gene is in operative link with a promoter, which is inducible or repressible in response to a stimulus. In some cases, the stimulus is selected from the group consisting of a small molecule that is supplied exogenously, heat, cold, and limited nitrogen in the culture medium. In some cases, the exogenous gene is

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expressed in a cellular compartment. In some embodiments, the cell compartment is selected from the group consisting of a chloroplast, a plastid, and a mitochondria. In some modalities the microbe is Prototheca moriformis, Prototheca krugani, Prototheca stagnora or Prototheca zopfii.

In one embodiment, the exogenous gene encodes a fatty acyl-ACP thioesterase. In some cases, the thioesterase that is encoded by the exogenous gene catalyzes the cleavage of an 8 to 18 carbon fatty acid from an acyl carrier protein (ACP). In some cases, thioesterase that is encoded by the exogenous gene catalyzes the cleavage of a 10 to 14 carbon fatty acid from an ACP. In one embodiment, the thioesterase that is encoded by the exogenous gene catalyzes the cleavage of a 12-carbon fatty acid from a

ACP.

In one embodiment, the exogenous gene encodes an acyl fatty CoA / aldehyde reductase. In some cases, reductase encoded by the exogenous gene catalyzes the reduction of an 8 to 18 carbon acyl fatty CoA to the corresponding primary alcohol. In some cases, reductase encoded by the exogenous gene catalyzes the reduction of a 10-14 carbon acyl fatty CoA to the corresponding primary alcohol. In one embodiment, reductase encoded by the exogenous gene

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It catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol.

The present invention also provides a cell for

Recombinant prototheca containing two exogenous genes, wherein a first exogenous gene encodes an acyl-ACP thioesterase and a second exogenous gene encodes a protein that is selected from the group consisting of an acyl-CoA reductase, an acyl fatty-CoA / aldehyde reductase , and an acyl carrier protein. In some cases, the two exogenous genes are each in an operable link with a promoter, which is induced in response to a stimulus. In some cases, each promoter is inducible in response to an identical stimulus, such as limited or no nitrogen in the culture medium. The limitation or total absence of nitrogen in the culture medium stimulates oil production in some microorganisms, such as the Prototheca species, and can be used as a trigger to induce oil production at high levels. When used in combination with the genetic engineering methods described herein, lipid as a percentage of the dry weight of cells can be brought to high levels such as at least 30%, at least 40%, at least 50%, at least 60%, at least 70% and at least 75%; the methods described herein provide cells with

<img file="MX339639B_D0187.tif" />

206 those lipid levels where the lipid is at least 1% -

<td colspan="4">5%, preferably at least 4%,</td><td>C8-C14,</td><td>to the</td><td>minus 0.</td><td>.25% -l%,</td>

<td>preferably</td><td>to the</td><td>less</td><td> 0.3%</td><td>, C8,</td><td>to the</td><td>less</td><td>l% -5%,</td>

<td>preferably</td><td>to the</td><td>less</td><td> 2%,</td><td>CIO,</td><td>to the</td><td>less</td><td>l% -5%,</td>

<td>preferably</td><td>to the</td><td>less</td><td> 2%,</td><td>C12, and</td><td>to the</td><td>less</td><td>l% -5%,</td>

<td>preferably</td><td colspan="2">at least 2%,</td><td>C14.</td><td colspan="2">In some</td><td colspan="2">modalities the</td>

cells are above 10%, above 15%, above 20%, or above 25% lipid per dry cell weight and contains lipid that is at least 5%, at least

10% or at least 15% C8-C14, at least 10%, at least 15%, at least 20%, at least 25% or at least 30% C8-C14, at least 20%, at least 25%, at minus 30%, at least 35% or at least 40%, C8C14, 5% -40%, preferably 10-30%, C8-C14 and 10% -40%, preferably 20-30%, C8-C14.

The novel oils described herein are distinct from other naturally occurring oils high in medium chain fatty acids, such as palm oil, palm kernel oil, and coconut oil. For example, levels of contaminants such as carotenoids are much higher in palm oil and palm kernel oil than in the oils of the invention. Palm and palm kernel oils, in particular, contain alpha and beta carotenes and lycopene in

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much higher amounts than there are in the oils of the invention. In addition, more than 20 different carotenoids are found in palm kernel oil, while the Examples demonstrate that the oils of the invention contain very few carotenoid species and very low levels. Additionally, the levels of vitamin E compounds such as tocotrienols are much higher in palm, palm kernel and coconut oils than in the oils of the invention.

In one embodiment, the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 18-carbon fatty acid from an ACP. In some embodiments, the exogenous second gene encodes an acyl fatty CoA / aldehyde reductase that catalyzes the reduction of an 8 to 18 carbon acyl fatty CoA to a corresponding primary alcohol. In some cases, thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 10-14 carbon fatty acid from an ACP, and reductase encoded by the second exogenous gene catalyzes the reduction of an acyl-fatty CoA from 10-14 carbon to the corresponding primary alcohol, where thioesterase and reductase act on the same carbon chain length. In one embodiment, thioesterase encoded by the first exogenous gene catalyzes the cleavage of

<img file="MX339639B_D0189.tif" />

208

ΙΜΡΙ a 12-carbon fatty acid from an ACP, and reductase encoded by the second exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol. In some embodiments, the exogenous second gene encodes an acyl fatty CoA reductase5 that catalyzes the reduction of an 8 to 18 carbon acyl fatty CoA to a corresponding aldehyde. In some embodiments, the second exogenous gene encodes an acyl transporter protein that is naturally co-expressed with acyl fatty-ACP thioesterase.

In some embodiments, the second exogenous gene encodes an acyl-CoA reductase, and the microbe also contains a third exogenous gene encoding a fatty aldehyde decarbonylase. In some cases, thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18 carbon fatty acid from an ACP, reductase encoded by the exogenous second gene catalyzes the reduction of an acyl fatty acid-CoA from 8 to 18 carbons to a corresponding fatty aldehyde, and the decarbonylase encoded by the third exogenous gene catalyzes the conversion of an 8 to 8-carbon fatty aldehyde to a corresponding alkane, where thioesterase, reductase, and decarbonylase act on the same carbon chain length.

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209

In some embodiments, the second exogenous gene encodes

IMPÍ. ' an acyl carrier protein, and the microbe further contains an exogenous third gene encoding a protein selected from the group consisting of an acyl-CoA reductase and an acyl fatty-CoA / aldehyde reductase. In some cases, the exogenous third gene encodes an acyl fatty CoA reductase, and the microbe also contains an additional fourth exogenous gene encoding a fatty aldehyde decarbonylase.

The present invention also provides methods for producing an alcohol comprising culturing a population of recombinant microorganisms (eg, cells of

Prototheca) in a culture medium, where the cells contain (i) a first exogenous gene that encodes an acyl fatty-ACP thioesterase, and (ii) a second exogenous gene that encodes an acyl fatty-CoA / aldehyde reductase, and cells synthesize a fatty acid linked to an acyl carrier protein (ACP), acyl fatty-ACP thioesterase catalyzes the cleavage of fatty acid from ACP to produce, through subsequent transformation, an acyl fatty-CoA, and fatty acyl-CoA / aldehyde reductase catalyzes the reduction of acyl-CoA to an alcohol.

The present invention further provides methods for producing a lipid molecule in a microorganism (for

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example, a Prototheca cell). In one embodiment, the method comprises culturing a population of Prototheca cells in a culture medium, wherein the cells contain (i) a first exogenous gene encoding an acyl-ACP thioesterase, and (ii) a second exogenous gene encoding an acyl. fatty-CoA reductase, and where microbes synthesize a fatty acid linked to an acyl carrier protein (ACP), acylACP thioesterase catalyzes the cleavage of fatty acid from the

ACP to produce, through subsequent transformation, fatty acyl-CoA reductase, and fatty acyl-CoA reductase catalyzes the reduction of acyl-CoA to an aldehyde.

The present invention also provides methods for producing a fatty acid molecule having a specified carbon chain length in a microorganism (eg, a Prototheca cell). In one embodiment, the method comprises culturing a lipid-producing population of Prototheca cells in a culture medium, where the microbes contain an exogenous gene encoding an acyl fatty ACP thioesterase, which has a specific or preferential activity for a certain length of carbon chain, such as 8, 10, 12, or 14 carbon atoms, and where the microbes synthesize a fatty acid bound to an acyl carrier protein (ACP) and thioesterase catalyzes the

IM

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MEXICAN PROPERTY INSTITUTE fatty acid cleavage from ACP when the fatty acid was synthesized to the specific length of the carbon chain.

In the various modalities described above, the microorganism (eg, a Prototheca cell) can 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 stearoyl ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase. In other cases, the, the microorganism (eg Prototheca cell) contains a lipid modifying enzyme selected from the group consisting of an acyl fatty ACP thioesterase, an acyl fatty CoA / aldehyde reductase, an acyl fatty CoA reductase , a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and / or an acyl carrier protein.

A number of illustrative cassettes or transformation constructs that are used to express a variety of lipid pathway enzymes and lipid modifying enzymes that are discussed in the present disclosure are presented in the Examples. Other useful constructs, without limitation, are listed in Table 37, below.

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Table 37. Illustrative transformation constructs, codon-optimized coding regions, and enzymes.

<td>Transformation construct / Region</td><td>sec.</td>

<td>coding / enzyme</td><td>with no. of ident.</td>

<td>C10: 0 specific C thioesterase construct. hookerian</td><td> 243</td>

<td>coding region for C. hookeriana C10: 0 specific thioesterase (codon optimized)</td><td> 244</td>

<td>C. hookeriana KAS IV enzyme construct</td><td> 245</td>

<td>coding region for C. hookeriana the enzyme KAS IV (codon optimized)</td><td> 246</td>

<td>C. hookeriana KAS IV enzyme</td><td> 247</td>

<td>C. hookeriana C10: 0 specific thioesterase plus C. hookeriana KAS IV enzyme construct</td><td> 248</td>

<td>coding region for C. lanceolata CIO: 0 specific thioesterase with acid desaturase fatty UTEX 1435 Ü12</td><td> 249</td>

<td>U-specific C12: 0 thioesterase construct.</td><td> 250</td>

<img file="MX339639B_D0194.tif" />

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<td>ra 1 7 fnrn i ca</td><td></td>

<td>coding region for U. californios C12: 0</td><td> 251</td>

<td>specific thioesterase (codon optimized)</td><td></td>

<td>G. mangostana C16: 0 thioesterase construct</td><td> 252</td>

<td>coding region for G. mangostana C16: 0</td><td> 253</td>

<td>thioesterase (codon optimized)</td><td></td>

<td>B. napus C18: 0 thioesterase construct</td><td> 254</td>

<td>coding region for B. napus C18: 0 thioesterase</td><td> 255</td>

<td>(optimized by codon)</td><td></td>

<td>Stearoyl-ACP desaturase construct of 0.</td><td> 256</td>

<td>europaea</td><td></td>

<td>coding region for 0. europaea estearoil-ACP</td><td> 257</td>

<td>desaturase (codon optimized)</td><td></td>

<td>C. hookeriana thioesterase construct C16: 0</td><td> 258</td>

<td>coding region for C. hookeriana C16: 0</td><td> 259</td>

<td>thioesterase (codon optimized)</td><td></td>

<td>E. guineensis C16: 0 thioesterase construct</td><td> 260</td>

<td>coding region for E. guineensis C16: 0</td><td> 261</td>

<td>thioesterase (codon optimized)</td><td></td>

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<img file="MX339639B_D0195.tif" />

<td>C. tinctorius ACP-thioesterase in the construct of the Ü12 fatty acid desaturase locus</td><td> 262</td>

<td>coding region for C. tinctorius ACP- thioesterase (codon optimized)</td><td> 263</td>

<td>broad specificity thioesterase construct C14: 0-C18: 0 of M. fragrans</td><td> 264</td>

<td>coding region for M. fragrans C14: 0-C18: 0 broad specificity thioesterase (optimized by codon)</td><td> 265</td>

<td>coding region for specific thioesterase C: 14: 0 from M. fragrans</td><td> 266</td>

<td>Specific thioesterase C14: 0 of M. fragrans with the transit peptideDl2 FAD</td><td> 267</td>

<td>Ricinus communis ACP-thioesterase construct</td><td> 268</td>

<td>coding region for Ricinus ACP-thioesterase communis (codon optimized)</td><td> 269</td>

<td>C. camphora thioesterase construct C14: 0</td><td> 270</td>

<td>coding region for thioesterase C14: 0 C. camphora (codon optimized)</td><td> 271</td>

<td>C14: 0 specific C thioesterase construct.</td><td> 272</td>

<img file="MX339639B_D0196.tif" />

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<td rowspan="2"></td><td></td>

<td></td>

<td>C14: 0 specific C thioesterase construct. camphora</td><td> 273</td>

<td>specific thioesterase C10: 0-C16: 0 of U. Americana at a SAD locus</td><td> 274</td>

<td>coding region for specific thioesterase CIO: 0-C16: 0 of U. Americana (codon optimized)</td><td> 275</td>

<td>C. wrightii KASA1 + C. wrightii FatB2 thioesterase + suc2 construct</td><td> 276</td>

<td>coding region for C. wrightii KASA1 (optimized by codon)</td><td> 277</td>

<td>coding region for FatB2 thioesterase from C. wrightii (codon optimized)</td><td> 278</td>

SAW. FUELS AND CHEMICAL PRODUCTION PRODUCTION

For the production of fuel according to the methods of the invention, the lipids produced by the cells of the invention are collected, or are collected in another way, by any convenient means. Lipids can be isolated by extraction of whole cells. First the cells are broken, and then the intracellular and associated lipids

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ΙΜΡΙ

INSTITUTO MEXICANO DE IA RRORPPAD INDUSTRIAL cell membrane / wall as well as extracellular hydrocarbons can be separated from the cell mass, such as by using centrifugation as described above. Intracellular lipids produced in microorganisms, in some modalities, are removed after lysing the cell of the microorganism. Once extracted, the lipids are further refined to produce oils, fuels, or oleochemicals.

After the completion of the culture, the 10 microorganisms can be separated from the fermentation broth. Optionally, the separation is carried out by centrifugation to generate a concentrated paste. Centrifugation does not remove significant amounts of intracellular water from the microorganisms and is not a drying step. The biomass can optionally be washed with a washing solution (eg DI water) to get rid of the fermentation broth and cell debris.

Optionally, the washed microbial biomass can also be dried (oven dried, lyophilized, etc.) prior to cell disruption. Furthermore, cells can be lysed without separation of part or all of the fermentation broth, when the fermentation is complete. For example, cells may be in a ratio of less than 1: 1, v: v of

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217

ΙΜΡΙ cells with respect to the pvtrarplnlar liquid they are lysed.

The lipid-containing microorganisms can be lysed to produce a lysate. As detailed herein, the step of lysing a microorganism (also known as cell lysis) can be accomplished by any convenient means, including heat-induced lysis, addition of a base, addition of an acid, use of 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 done to release intracellular molecules that have been produced by the microorganism. Each of these methods for lysis of a microorganism can be used as a single method or in simultaneous or sequential combination. The magnitude of cell disruption can be observed by microscopic analysis.

Using one or more of the methods described herein, more than 70% cell disruption is typically observed.

Preferably the cell breakdown is more than 80%, more preferably it is more than 90% and more preferably a

100%.

<img file="MX339639B_D0199.tif" />

In particular modalities, the microorganism is lysed

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IMPI after growth, for example to increase cell lipid and / or hydrocarbon exposure to extraction or further processing. The timing of lipase expression (eg, via an inducible promoter) or celqlar lysis can be adjusted to optimize lipid and / or hydrocarbon performance. A series of lysis techniques are described below.

These techniques can be used individually or in combination.

In an embodiment of the present invention, the lysis step of a microorganism comprises heating a cell suspension containing the microorganism. In this embodiment, the fermentation broth containing the microorganisms (or a suspension of microorganisms isolated from the fermentation broth) is heated until the microorganisms, i.e. the cell walls and membranes of the microorganisms are degraded or ruptured . Typically, the 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 more efficient cell lysis. The

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Cell lysing by heat treatment can be performed by boiling the microorganism. Alternatively, heat treatment (without boiling) can be done in an autoclave. The thermally treated lysate can be cooled for further treatment. Cell disruption can also be accomplished by steam treatment, that is, by adding steam under pressure. Vapor treatment of microalgae for cell disruption is described, for example, in US Patent No. 6,750,048. In some embodiments, steam treatment can be accomplished by spraying steam into the fermenter and maintaining the broth at a desired temperature for less than about 90 minutes, preferably less than about 60 minutes, and more preferably less than about 30 minutes. .

In another embodiment of the present invention, the lysis step of 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 protein compounds of the used microorganisms. Bases that are useful for solubilizing proteins are known in the art of chemistry. The exemplary bases that are useful in the methods

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of the present invention include, but - *^ίη i-im-it-arge _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 No. 6,750,048.

In another embodiment of the present invention, the lysis step of a microorganism comprises adding an acid to a cell suspension containing the microorganism. Acid lysis can be carried out using an acid at a concentration of 10 to 500 mN or preferably 40 to 160 nM. Acid lysis is preferably carried out above room temperature (for example, 40-160 °, and preferably at a temperature of 50-130 °. For moderate temperatures (eg, room temperature at 100 ° C and particularly room temperature at 65 °), acid treatment can be usefully combined with sonication or other cell disruption methods.

In another embodiment of the present invention, the lysis step of a microorganism comprises the lysis of the microorganism by using an enzyme. Preferred enzymes for lysis of a microorganism are proteases and polysaccharide degrading enzymes such

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such as hemicellulase (for example, hemicellulase from

Aspergillus niger; Sigma Aldrich, St. Louis, MO; # H2125), pectinase (eg, Rhizopus sp. Pectinase; Sigma

Aldrich, St. Louis, MO; # P2401), Mannaway 4.0 L (Novozymes), cellulase (for example, Trichoderma viride cellulose; Sigma

Aldrich, St. Louis, MO; # C9422), and driselase (for example, driselase from Basidiomycetes sp.; Sigma Aldrich, St. Louis,

MO; # D9515.

In other embodiments of the invention, lysis is carried out using an enzyme, such as, for example, a cellulase such as a polysaccharide degradation enzyme, optionally derived from Chlorella or a virus of

Chlorella, or a protease, such as the protease of

Streptomyces griseus, chymotrypsin, proteinase K, the proteases listed in Degradation of Polylactide by

Commercial Proteases, Oda Y. et al., Journal of Polymers and the Environment, Volume 8, Number 1, January 2000, pp. 2932 (4), Alcalase 2.4 FG (Novozymes), and Flavourzyme 100 L (Novozymes). Any combination of a protease and a polysaccharide degrading enzyme can also be used, including any combination of the above proteases and polysaccharide degrading enzymes.

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In another embodiment, lysis can be performed using an ejector press. In this process, the biomass is forced through a high-pressure screw-type device, using the cells and causing the intracellular lipids to be released and separate from the protein and fiber (and the other components) in the cell.

In another embodiment of the present invention, the lysis step of a microorganism is performed by ultrasound, that is, sonication. In this way, the cells can also be used with high frequency sound. Sound can be produced electronically and is transported through a metal tip to an appropriately concentrated cell suspension. This sonication (or ultrasound) alters cellular integrity based on the formation of cavities in the cell suspension.

In another embodiment of the present invention, the lysis step of a microorganism is carried out by mechanical lysis. Cells can be mechanically lysed and optionally homogenized to facilitate collection of hydrocarbons (eg, lipids). For example, a pressure disruptor can be used to pump a mixture containing cells through a restricted orifice valve. High pressure is applied (up to 1500

<img file="MX339639B_D0204.tif" />

223 bar), followed by instantaneous expansion through the outlet nozzle. Cell rupture is carried out by three different mechanisms: impact on the valve, high shear of the liquid in the orifice, and the sudden drop in pressure after discharge, causing an explosion of the cell. The method releases the intracellular molecules.

Alternatively a ball mill can be used. In a ball mill, cells are shaken in suspension with small abrasive particles, such as granules. The cells rupture due to shear forces, grinding between the granules, and collisions with the granules. The granules break down the cells to release the cellular contents. Cells can also be broken by shear forces, such as with the use of mixing (such as with a high-speed blender or Waring as examples), French press, or even centrifugation in the case of weak cell walls, to break the cells.

In another embodiment of the present invention, the lysis step of a microorganism is performed by applying an osmotic shock.

In another embodiment of the present invention, the step of lysis of a microorganism comprises infection of the microorganism with a lytic virus. A wide variety of

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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 level of skill in the art. For example, the virus paramecium bursaria chlorella (PBCV-l) is the prototype of a group (family

Phycodnaviridae, genus Chlorovirus) of large, icosahedral, plaque-forming, double-stranded DNA viruses that replicate in and lyse certain green, single-celled, eukaryotic chlorella-like algae. Consequently, any sensitive microalgae can be lysed by infecting the culture with a suitable chlorella virus. Methods of infecting Chlorella species with a chlorella virus are known. See, for example, Adv. Virus Res.

2006; 66: 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. 2006 Mar; 80 (5): 2437-44; and

Annu. Rev. Microbiol. 1999; 53: 447-94.

In another embodiment of the present invention, the lysis step of a microorganism comprises autolysis. In this embodiment, a microorganism of the invention is genetically engineered to produce a lytic protein that lyses the microorganism. This lytic gene can be expressed with

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225 IMPI

INSTITUTO MEXICANO M LA PROPIEDAD INDUSTRIAL an inducible promoter, whereby cells can first be grown to a desirable density in a terminator, followed by induction of the promoter to express the lytic gene to lyse the cells. In one embodiment, the lytic gene encodes a polysaccharide degradant. In certain other embodiments, the lytic gene is a gene from a lytic virus. For example, a lytic gene from a Chlorella virus can be expressed in an algae cell; see virology 260, 308-315 (1999); FEMS Microbiology Letters 180 (1999) 45-53; Virology 263, 376-387 (1999); and Virology 230,

361-368 (1997). Expression of the lytic genes is preferably performed using an inducible promoter, such as an active promoter in microalgae that is induced by a stimulus, such as the presence of a small molecule, light, heat, and other stimuli.

There are several methods to separate the lipids from the cellular uses produced by the previous methods. For example, lipids and lipid derivatives, such as fatty aldehydes, fatty alcohols, and hydrocarbons, such as alenes, can be extracted with a hydrophobic solvent such as hexane (see Frenz et al.,

1989, Enzyme Microb. Technol., 11: 717). Lipid and lipid derivatives can also be extracted using₂₂₆ IMPI »Mexican Institute ot the MuHetiAP industrial liquefaction (see, for example, Sawavama and others, 1999,

Biomass and Bioenergy 17: 33-39 and Inoe et al., 1993, Biomass

Bioenergy 6 (4): 269-274); liquefaction of oil (see, for example, Minowa et al., 1995, Fuel 74 (12): 1735-1738); and extraction of C0₂ supercritical (see, for example, Mendes et al., 2003, Inorganánica Chimica Acta 356: 328-334). Miao and Wu describe a protocol for the recovery of lipids from microalgae from a culture of Chlorella prototheocoides, in which the cells were collected by centrifugation, washed with distilled water and dried by lyophilization. The resulting cell powder was pulverized in a mortar and then extracted with n-hexane (Miao and Wu, Biosource Technology (2006) 97: 841-846).

In this way, the lipids, lipid derivatives 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. Typically, 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

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lipids and / or hydrocarbon components form a solution with the organic solvent. In some cases, the solution may be further refined to recover the desired lipids or specific hydrocarbon components. Hexane extraction methods are well known in the art.

Lipids and lipid derivatives, such as fatty aldehydes, fatty alcohols, and hydrocarbons, such as alkanes produced by cells, as described herein, can be modified through the use of one or more enzymes, including a lipase, as described above. When hydrocarbons are in the extracellular environment of cells, one or more enzymes can be added to that environment under conditions in which the enzyme modifies the hydrocarbon or completes its synthesis from a hydrocarbon precursor. Alternatively, the hydrocarbons can be partially or completely isolated from the cellular material prior to the addition of one or more catalysts such as enzymes. These catalysts aggregate exogenously, and their activity occurs outside the cell or in vitro.

In this way, lipids and hydrocarbons produced by cells in vivo or modified

<img file="MX339639B_D0208.tif" />

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INSTITUTO MEXICANO Ot LA RRORIÍOA INDUSTRIAL enzymatically in vitro, as described herein, can optionally be processed by conventional means. Processing may include cracking (molecular decomposition) to reduce size, and therefore increase the hydrogen: carbon ratio of hydrocarbon molecules. Catalytic and thermal cracking methods are routinely used in the processing of hydrocarbons and triglyceride oils. Catalytic methods involve the use of a catalyst, such as a solid acid catalyst. The catalyst may be silica-alumina or a zeolite, which result in the heterolytic or asymmetric cleavage of a carbon-carbon bond to result in a carbocation and a hydride anion. These reactive intermediates then undergo either a rearrangement or transfer of the hydride with another hydrocarbon. The reactions, therefore, can regenerate the intermediaries to give rise to a self-multiplying chain mechanism. Hydrocarbons can also be processed to reduce, optionally to zero, the number of carbon-carbon double or triple bonds thereof. Hydrocarbons can also be processed to remove or remove a ring or ring structure in them. Hydrocarbons can also be processed to increase the

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INSTITUTO MEXICANO DI LA WOWIDAf »hydrogen: carbon ratio. This can^{, N}° include the addition of hydrogen (hydrogenation) and / or ¿1 cracking of hydrocarbons in other smaller hydrocarbons.

Thermal methods involve the use of elevated temperature and pressure to reduce the size of the oil. An elevated temperature of approximately

800 ° C and a pressure of approximately 700kPa. These conditions generate light, a term sometimes used to refer to hydrogen-rich hydrocarbon molecules (as opposed to photon flux), while also generating, by condensation, heavier hydrocarbon molecules which are relatively devoid of hydrogen. The methodology provides homolytic or symmetric cleavage, and produces alkenes that can be optionally enzymatically saturated, as described above.

Catalytic and thermal methods are standard in hydrocarbon processing and oil refining plants. In this way, the hydrocarbons that are produced by the cells, as described herein, can be collected and processed or refined by conventional means. See Hillen et al., (Biotechnology and

Bioengineering, Vol. XXIV: 193-205 (1982)) for a report on hydrocracking of hydrocarbons produced by

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OE THE PROPERTY jggl

INDUSTRIAL BT-jl · »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 in biodiesel, a transesterification process is used as described later in this

Section.

The hydrocarbons produced through the methods of the present invention are useful in a variety of industrial applications. For example, the production of linear alkylbenzene sulfonate (LAS), an anionic surfactant used in almost all types of detergents and cleaning products, uses hydrocarbons that generally comprise a chain of 10-14 carbon atoms. See, for example, US Patent Nos .: 6,946,430; 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; 5,833,999; 6,468,955; and 6,407,044.

Increasing interest is directed to the use of components of biologically derived hydrocarbons in fuels, such as biodiesel, renewable diesel and

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reactor fuel, since renewable biological raw materials can replace the raw materials derived from fossil fuels that are available, and their use is therefore desirable. There is an urgent need for methods for the production of hydrocarbon components from biological materials. The present invention satisfies this need by providing methods for the production of biodiesel, renewable diesel, and reactor fuel using the lipids generated by the methods described herein as a biological material for producing biodiesel, renewable diesel, and reactor fuel.

[0014] Traditional diesel fuels are petroleum distillates rich in paraffin hydrocarbons.

They have boiling ranges as wide as

370 ° to 780 ° F, both of which are suitable for combustion in a compression starter, such as a diesel-powered vehicle. The American Society of Testing and Materials (ASTM) sets the grade of diesel according to the boiling range, along with the allowable ranges for other fuel properties, such as cetane number, cloud point, flash point, viscosity, aniline point, sulfur content, water content,

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ash content, corrosion of the copper strip, and carbon residues. Technically, any distilled material from biomass-derived or other hydrocarbons that meets the appropriate ASTM specification can be defined as diesel fuel (ASTM D975), reactor fuel (ASTM D1655), or as biodiesel or if it is a Fatty Acid Methyl Ester (ASTM D6751).

After extraction, the lipids and / or hydrocarbon components recovered from the microbial biomass described herein can be subjected to chemical treatment to make a fuel for use in diesel vehicles and jet engines.

[0015] Biodiesel is a liquid that varies in color between gold and dark brown - depending on the raw material used in production. It is practically immiscible in water, has a high boiling point and low vapor pressure. Biodiesel refers to a processed fuel equivalent to diesel for use in vehicles with a diesel engine. Biodiesel is biodegradable and non-toxic. An added benefit of biodiesel over conventional diesel fuels is less engine wear. Typically, biodiesel comprises C14-C18 alkyl esters. Various processes convert biomass or lipid

<img file="MX339639B_D0212.tif" />

233 produced and isolated as described herein, in diesel fuels. A preferred method of producing biodiesel is transesterification of a lipid as described herein. A preferred alkyl ester for use as biodiesel is a methyl ester or ethyl ester.

[0016] Biodiesel produced by a method described herein can be used alone or mixed with conventional diesel fuel at any concentration in most modern diesel engine vehicles. When mixed with conventional diesel fuel (petroleum diesel), biodiesel can be present from about 0.1% to about 99.9%. Much of the world uses a system known as the B factor to establish the amount of biodiesel in any fuel mixture. For example, fuel that contains 20% biodiesel is labeled B20. Pure biodiesel is referred to as

B100.

[0017] Biodiesel can also be used as heating fuel in domestic and commercial boilers. Existing oil boilers may contain rubber parts, and may require conversion to run on biodiesel. The conversion process is relatively simple, which involves the exchange of

<img file="MX339639B_D0213.tif" />

2. 3. 4 rubber parts by synthetic parts due to my biodiesel is a strong solvent. Due to its strong solvent power, burning biodiesel will increase the efficiency of the boilers. Biodiesel can be used as an additive in diesel formulations to increase the lubricity of pure Ultra-Low Sulfur Diesel (ULSD) fuel, which is advantageous as it has virtually no sulfur content. Biodiesel is a better solvent than petrodiesel and can be used to separate deposits of debris in the fuel lines of vehicles that have previously run on petrodiesel.

Biodiesel can be produced by transesterification of triglycerides contained in oil-rich biomass. Therefore, in another aspect of the present invention there is provided a method of producing biodiesel. In a preferred embodiment, the method for producing biodiesel comprises the steps of (a) cultivating a lipid-containing microorganism using the methods described herein, (b) lysing a lipid-containing microorganism to produce a lysate, (c) isolating the lipids from the lysed microorganism and (d) transesterify the lipid composition, whereby biodiesel is produced. Methods for the growth of a microorganism, lysing a

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INDUSTRIAL microorganism to produce a lysate, treat the Used in a medium comprising an organic solvent to form a heterogeneous mixture and separate the treated lysate into a lipid composition, have been described above and can also be used in the biodiesel production method.

The lipid profile of biodiesel is usually very similar to the lipid profile of oil as a raw material. Other oils provided by the methods and compositions of the invention can be transesterified to produce biodiesel with lipid profiles that include (a) at least 1-5%, preferably at least 4%, C8-C14; (b) at least 0.25% -l%, preferably at least 0.3%, C8; (c) at least 1-5%, preferably at least 2%, CIO; (d) at least 1-5%, preferably at least 2%, C12; and (3) at least 20% 40%, preferably at least 30%, C8-C14.

The lipid compositions can be transesterified to produce esters of long chain fatty acids, useful as biodiesel. Preferred transesterification reactions are described below and include base catalyzed transesterification and transesterification using recombinant lipases. In a base catalyzed transesterification process, triglycerides are reacted with an alcohol, such as

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methanol or ethanol, in the presence of an alkaline catalyst, typically potassium hydroxide. This reaction forms methyl or ethyl esters and glycerin (glycerol) as a by-product.

[0018] Animal and vegetable oils are typically made from triglycerides that are esters of free fatty acids with the trihydric alcohol, glycerol. In transesterification, glycerol in a triacylglyceride (TAG) is replaced by a short-chain alcohol such as methanol or ethanol. A typical reaction scheme is as follows:

—0 OCR₁ base cat —Q _0CRn -w R, COOEt + FGCOOEt + R₃COOEt + C₃H₅(OH)₃ * 3 EtOH ',.,. .

_q 0PR₃ Ethyl esters of Ghcenna fatty acids

TriaJicérido

In this reaction, the alcohol is deprotoned with a base to make it a stronger nucleophile. Commonly, ethanol or methanol are used in large excess (up to 50 times).

Typically, this reaction will either take place too slowly or will not occur at all. Heat, as well as an acid or a base, can be used to help the reaction to proceed more quickly. The acid or base is not consumed by the transesterification reaction,

<img file="MX339639B_D0216.tif" />

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INSTITUTO MÜXICaNÍ DE LA PROPIEDAD industrial so they are not reactive but catalysts. Almost all biodiesel has been produced using the base catalysis technique, since it only requires low temperatures and pressures and produces over 98% conversion efficiency (as long as the starting oil is low in humidity and free of fatty acids).

Transesterification is also carried out, as mentioned above, 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 ° C, and a molar ratio of TAG to alcohol less than 1: 1, preferably approximately 3: 1. Lipases suitable for use in transesterification include, but are not limited to, those listed in Table 9. Other examples of

Useful steps for transesterification are found in, for example, US Patent Nos. 4,798,793; 4,940,845 5,156,963; 5,342,768; 5,776,741 and W089 / 01032. Such lipases include, but are not limited to, lipases produced by microorganisms from Rhizopus, Aspergillus, Candida, Mucor,

Pseudomonas, Rhizomucor, Candida, and Humicola and lipas from the pancreas.

<img file="MX339639B_D0217.tif" />

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Table 9. Lipases suitable for use in transesterification.

Lipase from Aspergillus niger ABG73614, Lipase B from Antarctic (novozym-435) CAA83122, Lipase from Candida 5 cylindraceaAAR24090, Lipase from Candida lipolytica (Lipase L; Amano Pharmaceutical Co., Ltd.), Lipase from Candida rugosa (eg. -OF; Meito Sangyo Co., Ltd.), Mucor laiehei lipase (Lipozyme IM 20), Pseudomonas fluorescens AAA25882 lipase, lipase from Rhizopus japónicas (Lilipase 10 A-10FG) Q7M4U7_1, lipase from Rhizomucor miehei B34959, lipasa de Rhizomucor F) AAF32408, Serratia marcescens lipase (SM Enzyme) ABI13521, Thermomyces lanuginosa lipase CAB58509, Lipase P (Nagase ChemteX Corporation), and Lipase QLM (Meito Sangyo Co., Ltd., Nagoya, Japan)

A challenge in using a lipase for the production of fatty acid esters suitable for biodiesel is that the price of lipase is much higher than the price of sodium hydroxide (NaOH) used by strong base 20 processes. This challenge was addressed using an immobilized lipase, which can be recycled. However, the activity of the immobilized lipase must be maintained after recycling for a minimum number of cycles to allow a process based on

239 IMPI Ol

MEXICAN INSTITUTE

D € THE PROPERTY Í%> jS

INOUSTMIAL ^ 5J3E lipase that competes with the strong base process in terms of cost of production. United States Patent No.

6,398,707 (issued June 4, 2002 to Wu et al.) Describes methods of increasing the activity of immobilized lipases and the regeneration of immobilized lipases with reduced activity. Some suitable methods include immersing an immobilized lipase in an alcohol having a number of carbon atoms not less than for a period of time, preferably 0.5-48 hours, and most preferably 0.5-1.5 hours. Some suitable methods also include washing a deactivated immobilized lipase with an alcohol having a carbon atom number of not less than 3, and then immersing the deactivated immobilized lipase in a vegetable oil for

0.5-48 hours.

In particular embodiments, a recombinant lipase is expressed in the same microorganisms that produce the lipids on which the lipase acts. Suitable recombinant lipases include those listed in Table 9 above and / or having GenBank accession numbers listed in Table 9 above, or a polypeptide having at least 70% amino acid identity with one of the lipases listed above

<img file="MX339639B_D0218.tif" />

240 in Table 9 and that exhibits activity

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MRXICAND INSTITUTE of INDUSTRIAL property

<img file="MX339639B_D0219.tif" />

In further embodiments, the enzyme activity is present in a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at minus about a

99% identity with one of the sequences described above, all of which are incorporated by reference herein as fully set forth. The DNA encoding the lipase and the selectable marker is preferably codon optimized cDNA. Methods of recoding genes for expression in microalgae are described in United States Patent No. 7,135,290.

The international common standard for biodiesel is EN

14214. ASTM D6751 is the most common biodiesel standard referenced in the United States and Canada. Germany uses DIN EN 14214 and the United Kingdom requires compliance with BS EN 14214. Basic industrial tests to determine whether products conform to these standards typically include gas chromatography, HPLC, and others. Biodiesel that meets quality standards is not very toxic, with a toxicity index (DL₅₀) greater than 50 ml / kg.

<img file="MX339639B_D0220.tif" />

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OF THE PROPERTY <% _ industrial >>

Although biodiesel that meets ASTM standards has to be non-toxic, there may be contaminants that tend to crystallize and / or precipitate out of the solution as sediment. Sediment or precipitates can cause problems such as decreased fuel flow, clogged fuel lines, clogged filters, etc. There are processes well known in the art that specifically deal with the removal of these pollutants and sediments from biodiesel, in order to produce a higher quality product. Sediment or precipitates can cause problems such as decreased fuel flow, clogged fuel lines, clogged filters, etc. There are processes well known in the art that specifically deal with the removal of these pollutants and sediments from biodiesel, in order to produce a higher quality product. Examples of these processes include, but are not limited to, oil pretreatment to remove contaminants such as phospholipids and free fatty acids (eg, degumming, caustic refining, and silica adsorbent filtration) and cold filtration. This process cools the biodiesel and filters any of the sediments or

<img file="MX339639B_D0221.tif" />

242 precipitates that may form when

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use at a lower temperature. This process cools the biodiesel and filters out any sediments or precipitates that may form when the fuel is used at a lower temperature. This process is well known in the art and is described in US Patent Application Publication No. 2007-0175091. Suitable methods may include cooling the biodiesel to a temperature below about 38 ° C, so that impurities and contaminants precipitate out as particles in the biodiesel liquid. Diatomaceous earth or other filter material can then be added to the cooled biodiesel to form a paste, which can then be filtered through a pressure sheet or other filter to remove particulates. Filtered biodiesel can then be passed through a polishing filter to remove remaining sediment and diatomaceous earth, in order to produce the final biodiesel product.

Example 13 describes the production of biodiesel using Prototheca moriformis triglyceride oil. Filterability by cold immersion by ASTM D6751 method

The biodiesel produced in Example 13 was 120

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INSTITUI »; AEXlCANL PE LA PMOWEDAD industrial seconds for a volume of 3 00ml. Eata ^ prneba in the filtration of 300 ml of B100, chilled at 40 ° F for hours, allowed to warm to room temperature, and vacuum filtered with 0.7 micron glass fiber with stainless steel support. The oils of the invention can be transesterified to generate biodiesel with a cold immersion time of less than 120 seconds, less than 100, and less than 90 seconds.

Subsequent processes can also be used if biodiesel is used particularly at cold temperatures. These processes include winterization and fractionation. There are several approaches to winterize biodiesel. There are several approaches to winterize biodiesel. One method is to mix biodiesel with petroleum diesel. Another approach is to remove saturated methyl esters indiscriminately, by mixing them in additives, allowing the saturated fats to crystallize and then filtering the crystals. Another approach is to remove saturated methyl esters indiscriminately, by mixing them in additives, allowing the saturated fats to crystallize and then filtering the crystals. Fractionation selectively separates methyl esters into components or

<img file="MX339639B_D0224.tif" />

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IMPI individual fractions, allowing the removal or inclusion of specific methyl esters.

Another valuable fuel provided by the methods of the present invention is renewable diesel, which comprises alloys, such as C10: 0, C12: 0, C14: 0, C16: 0 and

C18: 0, and therefore is distinguished from biodiesel. High quality renewable diesel is obtained in accordance with the ASTM standard

D975. The lipids produced by the methods of the present invention can serve as raw material to produce renewable diesel. The lipids produced by the methods of the present invention can serve as raw material to produce renewable diesel. 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 one embodiment, the method for the production of renewable diesel comprises (a) cultivating a lipid-containing microorganism using the methods described herein, (b) lysing the microorganism to produce a lysate, (c) isolating the lipid from the lysed microorganism, and (d)

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deoxygenate and hydrotreat the lipid to produce an alkane, thus producing renewable diesel. Lipids suitable for the manufacture of renewable diesel can be obtained through the extraction of microbial biomass using an organic solvent such as hexane, or through other methods, such as those described in the

United States no. 5,928,696. Some suitable methods may include mechanical pressing and centrifugation.

In some methods, the microbial lipid is first cracked in conjunction with hydrotreatment to reduce the length of the carbon chain 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 evaporate and distill the desired components into the diesel fuel in order to comply with an ASTM D975 standard, while the heavier components than desired are left to comply with the norm

D975. Hydrotreating, hydrocracking, deoxygenation, and isomerization methods of chemically modified oils, including triglyceride oils, are well known in the art. See for example the European patent applications EP1741768 (Al); EP1741767 (Al); EP1682466

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EP1795576 (Al); and the

6,630,066; 6,596,155;

(To the); EP1640437 (Al); EP1681337 (Al);

United States patents 7,238,277;

6,977,322; 7,041,866; 6,217,746; 5,885,440; 6,881,873.

In one embodiment of the renewable diesel production method, lipid treatment to produce an alkane is carried out by hydrotreating the lipid composition. In hydrothermal processing, biomass is typically reacted in water at elevated temperature and pressure to form oils and solid waste. Conversion temperatures are typically 300 ° to 660 ° F, with pressure sufficient to hold water primarily as a liquid, 100 to 170 atmospheres as standard. Reaction times are on the order of 15 to 30 minutes. After the reaction is complete, the organics are separated from the water. In this way a distillate suitable for diesel is produced.

In some methods of preparing renewable diesel, the first stage of triglyceride treatment is hydroprocessing to saturate the 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

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247

IMPI ~ tuwtniTn hydrogenation. The isomerization is then optionally carried out, also in the presence of hydrogen and a catalyst. The naphtha components are preferably removed by distillation. As examples, see United States Patents 5,475,160 (triglyceride hydrogenation), 5,091,116 (deoxygenation, hydrogenation, and gas extraction), 6,391,815 (hydrogenation), and 5,888,947 (isomerization).

An appropriate method for the hydrogenation of triglycerides includes preparing an aqueous solution of copper, zinc, magnesium and lanthanum salts and another alkali metal solution or preferably, ammonium carbonate. The two solutions can be heated to a temperature of about 20 ° C to about 85 ° C and are measured together in a beaker in ratios such that the pH in the beaker is kept between 5.5 and 7.5 with the aim of forming a catalyst. Additional water can be used initially in the precipitation vessel or added at the same time as the salt solution and the precipitation solution. The resulting precipitate can then be thoroughly washed; dry, calcine at 300 ° C and activate on hydrogen at temperatures in the range of 100 ° C to 400 ° C. One or

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more triglycerides can then be contacted and reacted with hydrogen in the presence of the catalyst described above in a reactor. The reactor may be a drip bed reactor, a fixed bed gas-solid reactor, a packed bubble column reactor, a continuous agitation tank reactor, a slurry phase reactor, or any other suitable type of reactor. known in the art. The process can be carried out in batches or continuously. Reaction temperatures are typically in the range of about 170 ° C to about 250 ° C, while reaction pressures are typically in the range of about 300 psig to about 2000 psig. Furthermore, the molar ratio of hydrogen to triglycerides in the process of the present invention is typically in the range of from about 20: 1 to about 700: 1. The process is typically carried out at a weight hourly space velocity (WHSV) in the range of about 0.1 h '¹ at about 5 h '¹. One skilled in the art will recognize that the time period required for the reaction will vary as a function of the temperature used, the molar ratio of hydrogen to triglycerides, and the partial pressure of hydrogen. Products produced by such

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Hydrogenation processes include fatty alcohols, glycerol, traces of paraffins, and unreacted triglycerides. These products are normally separated by conventional means such as, for example, distillation, extraction, filtration, crystallization, and the like.

[0019] Oil refineries use hydroprocessing to remove impurities by treating the sources with hydrogen. Hydroprocessing conversion temperatures are typically 300 ° to 700 ° F. Pressures are typically 40 to 100 atmospheres. Reaction times are typically in the order of 10 to minutes. Solid catalysts are used to increase certain reaction rates, improve the selectivity of certain products, and optimize hydrogen consumption.

Suitable methods for deoxygenation of an oil include heating an oil to a temperature in the range of about 350 ° F to about 550 ° F and keeping the hot oil continuously in contact with nitrogen, under at least one pressure that is at the range from about atmospheric pressure to above, for at least about 5 minutes.

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Suitable methods for isomerization include the use of alkaline isomerization and other oil isomerization known in the art.

Hydrotreating and hydroprocessing ultimately lead to a reduction in the molecular weight of the triglyceride source. The triglyceride molecule is reduced to four hydrocarbon molecules under hydroprocessing conditions: one propane molecule and three heavier hydrocarbon molecules, typically in the range of C8 to C18.

Thus, in one embodiment, the product of one or more chemical reaction (s) carried out on the lipid compositions of the invention, is a mixture of allenes comprising renewable diesel according to the standard ASTM D975. Hydrocarbon production by micro-organisms is reviewed by Metzger et al., Appl Microbiol

Biotechnol (2005) 66: 486-496 and A Look Back in the US

Department of Energy's Aquatic Species Program: Biodiesel from Algae, NREL / TP-580-24190, John Sheehan, Terri Dunahay,

John Benemann and Paul Roessler (1998).

The distillation properties of a diesel fuel are described in terms of T10-T90 (temperature at 10% and 90%, respectively, distilled volume). Diesel

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Renewable was produced from triglyceride oil from

Prototheca moriformis and is described in Example 13. The

T10-T90 of the material produced in Example 13 was 57.9 ° C.

Hydrogen treatment methods, isomerization, and other covalent modification of oils described herein, as well as distillation and fractionation methods (such as cold filtration) described herein, may be employed. to generate renewable diesel compositions with other T10T90 ranges, such as, 20, 25, 30, 35, 40, 45, 50, 60 and 65 ° C by using triglyceride oils that are produced according to the methods described herein.

The TIO of the material produced in Example 13 was

242.1 ° C. Hydrogen treatment methods, isomerization, and other covalent modification of oils described herein, as well as distillation and fractionation methods (such as cold filtration) described herein, can be employed to generate renewable diesel compositions with other TIO values, such as TIO between 180 and 295, between 190 and 270, between 210 and 250, between 225 and 245, and at least 290.

The T90 of the material produced in Example 13 was

300 ° C. Hydrogen treatment methods, the

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INSITO ΙΤΟ MEXICANO • Ε la MoneoAij ι · ϊ5γ ha *

ΙΝ »Ι ISTRIAL ^ te ^M rPr isomerization, and other covalent modification of the oils described herein, as well as the distillation and fractionation methods (such as cold filtration) described herein, can be employed to generate renewable diesel compositions with other T90 values, such as T90 between 280 and 380, between 290 and 360, between 300 and 350, between 310 and 340, and at least 290.

The FBP of the material produced in Example 13 was

300 ° C. Hydrogen treatment methods, isomerization, and other covalent modification of oils described herein, as well as distillation and fractionation methods (such as cold filtration) described herein, can be employed to generate renewable diesel compositions with other values

<td>15 FBP, such as</td><td>FBP</td><td>between 290 and 400,</td><td>between 300</td><td colspan="2">and 385, between</td>

<td>310 and 370, between</td><td> 315</td><td>and 360, and at least</td><td> 300.</td><td></td><td></td>

<td colspan="2">Other oils</td><td>p roporc i onado s</td><td>for the</td><td>methods</td><td>and</td>

<td>compositions</td><td>of</td><td>the invention</td><td>they can</td><td>undergo</td><td>to</td>

combinations of hydrotreating, isomerization, and other covalent modification including oils with lipid profiles including (a) at least 1-5%, preferably at least 4%, C8-C14; (b) at least 0.25% -l%, preferably at least 0.3%, C8; (c) at least l% -5%, preferably at least

<img file="MX339639B_D0232.tif" />

253

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2%, CIO; (d) at least l% -5%, preferably at least 2%,

C12; and (3) at least 20% -40%, preferably at least 30% C8C14.

[0020] A traditional ultra low sulfur diesel can be produced from any form of biomass by a two-step process. First, the biomass is converted into a synthetic gas, a gaseous mixture rich in hydrogen and carbon monoxide. The synthetic gas is then catalytically converted to liquids. Typically, the production of liquids is accomplished using the synthesis of

Fischer-Tropsch (FT). This technology is applied to coal, natural gas, and heavy oils. Therefore, in another preferred embodiment of the method for the production of renewable diesel, the treatment of the lipid composition to produce an alkane is carried out by indirect liquefaction of the lipid composition.

The present invention also provides methods for producing reactor fuel. The reactor fuel is light to straw colored. The most common fuel is an unleaded / paraffin oil-based fuel classified as Airplane Al, which is produced to a set of internationally standardized specifications. Jet fuel is a mixture of a large number of

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different hydrocarbons, possibly as many as a thousand or more.

The range of its size (molecular weight or carbon numbers) is limited by the requirements for the product, for example, the freezing point or the smoke point. Kerosone type jet fuel (including

Jet A and Jet Al) have a carbon number distribution between approximately 8 and 16 carbon numbers. Naphtha or wide-cut type jet fuel (including

Jet B) typically has a carbon number distribution of between about 5 and 15 carbons.

Both planes (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 freeze inhibitor (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 sparks. The

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 fuels

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<img file="MX339639B_D0235.tif" />

EGME.

In one embodiment of the invention, a reactor fuel is produced by mixing algae fuels with existing fuels. The lipids produced by the methods of the present invention can serve as a raw material to produce jet fuel. Therefore, in another aspect of the present invention, a method of producing reactor fuel is provided. 10 Two methods are provided herein for producing aircraft fuel from the lipids produced by the methods of the present invention: fluid catalytic cracking (FCC), and hydrodeoxygenation (HDO).

[0021] Fluid catalytic cracking (FCC) is a method that is used to produce olefins, especially propylene, from heavy fractions of crude oil. The lipids produced by the method of the invention can be converted to olefins. The process involves the flow of lipids produced through an FCC zone and a stream of the olefin compound product is collected, which is useful as reactor fuel. The lipids produced are contacted with a cracking catalyst under cracking conditions to provide a product stream that

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<img file="MX339639B_D0236.tif" />

It includes defined and hydrocarbons, useful as reactor fuel.

In one embodiment, the method for producing reactor fuel comprises (a) culturing a lipid-containing microorganism using the methods described herein, (b) lysing the lipid-containing microorganism to produce a lysate, (c) isolating the lipid from the lysate, and (d) treating the lipid composition, thereby producing the reactor fuel. In one embodiment of the method for producing a reactor fuel, the lipid composition may flow through a catalytic cracking zone of the fluid, which, in one embodiment, may comprise contacting the lipid composition with a cracking catalyst. under cracking conditions to provide a product stream comprising olefins C₂-C₅.

In certain embodiments of this method, it may be desirable to remove contaminants that may be present in the lipid composition. Therefore, before flowing the lipid composition through a fluid catalytic cracking zone, the lipid composition is pretreated. Pretreatment may include contacting the lipid composition with a resin of

257

<img file="MX339639B_D0237.tif" />

IMPI ion exchange. Resin exchanged⁰ ionic ^ 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 flows, either up or down. Other pretreatments may include 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 solution of the acid, usually at room temperature and atmospheric pressure.

The lipid composition, optionally pretreated, is flowed to an FCC zone, where the hydrocarbon components are cracked to olefins. Catalytic cracking is carried out by contacting the lipid composition in a reaction zone with a catalyst composed of a finely divided particulate material. The reaction is catalytic cracking, unlike hydrocracking, and is carried out in the absence of added hydrogen or hydrogen consumption. As the cracking reaction proceeds, substantial amounts of coke are deposited on the catalyst. The catalyst is regenerated at high temperatures by burning the catalyst coke in a regeneration zone. Coke-containing catalyst,

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<img file="MX339639B_D0238.tif" />

which is referred to herein as coked catalyst, is continuously transported from the reaction zone to the regeneration zone to regenerate and be replaced by an essentially coke-free regenerated catalyst from the regeneration zone. The fluidization of the catalyst particles by means of various gas streams allows the transport of the catalyst between the reaction zone and the regeneration zone. The methods for cracking the hydrocarbons, such as those for the lipid composition described herein, in a fluidized stream of the catalyst, transport of the catalyst between the reaction and regeneration zones, and combustion of coke in the regenerator, are well known to those skilled in the art of FCC processes. Exemplary FCC Applications and Useful Catalysts to Crack the Lipid Composition to Produce Olefins C₂-C₅ are described in United States Patent Nos. 6,538,169 and 7,288,685, which are incorporated in their entirety by reference.

Appropriate FCC catalysts generally comprise at least two components that may or may not be in the same matrix. In some embodiments, both components can be distributed throughout the entire

<img file="MX339639B_D0239.tif" />

259 reaction. The first component generally includes any of the known catalysts used in the fluidized catalytic cracking material, such as an amorphous active clay type catalyst and / or a high activity crystalline molecular sieve. Molecular sieve catalysts may be preferred over amorphous catalysts, due to their greatly improved selectivity for desired products. In some preferred embodiments, zeolites can be used as a molecular sieve in FCC processes. Preferably, the component of the first catalyst comprises a large pore zeolite, such as a Y-type zeolite, an active alumina material, a binder material, comprising either silica or alumina, and an inert filler such as kaolin.

In one embodiment, the cracking of the lipid composition of the present invention is performed in the elevator section, or alternatively the elevator section, of the FCC zone. The lipid composition is introduced into the elevator via a nozzle, resulting in rapid evaporation of the lipid composition. Before contacting the catalyst, the lipid composition will normally have a temperature of about 149 ° C to about 316 ° C (300 ° F to

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<img file="MX339639B_D0240.tif" />

600 ° F). The catalyst is flowed from a mixing container to the elevator, where it comes in contact with the lipid composition for a time of 2 seconds or less.

The mixed catalyst and the vapors of the reacted lipid composition are discharged from the top of the riser through an outlet connection, and separated into a stream of steam from the cracked product as defined and a collection of catalyst particles covered with amounts substantial coke which is generally referred to as coked catalyst. In an effort to minimize the contact time of the lipid composition and the catalyst, which can promote further conversion of the desired products to other unwanted products, any spacer arrangement, such as a spiral arm arrangement, can be use to remove the coked catalyst from the product stream quickly. The separator, for example the spiral arm separator, is located in an upper portion of a chamber with an extraction zone located in the lower portion of the chamber. The catalyst separated by the spiral arm arrangement drips into the extraction zone. The vapor stream of the cracked product comprising the cracked hydrocarbons including light and

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<img file="MX339639B_D0241.tif" />

Some catalysts leave the chamber through a duct that is in communication with the cyclones. Cyclones remove catalyst debris 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 separation vessel and then to the extraction area. The extraction zone removes the hydrocarbons adsorbed from the surface of the catalyst by the countercurrent contact with the steam.

[0022] The low partial pressure of hydrocarbons operates in favor of the production of light olefins. Consequently, the riser pressure is set to approximately 172 to 241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of approximately 35 to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressure of approximately 69 to 138 kPa (10 to 20 psia). twenty This relatively low partial pressure for hydrocarbons is achieved through the use of steam as a diluent insofar as the diluent is 10-55wt-% by weight of the lipid composition, and preferably of

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<img file="MX339639B_D0242.tif" />

approximately 15wt-% by weight of the lipid composition.

Other diluents, such as dry gas, can be used to achieve equivalent hydrocarbon partial pressures.

[0023] The temperature of the cracking current at the elevator outlet will be approximately 510 ° C to 621 ° C (950 ° F to 1150 ° F). However, riser outlet temperatures above 566 ° C (1050 ° F) produce more dry and sharper gas. While elevator outlet temperatures below 566 ° C (1050 ° F) produce less ethylene and propylene. Accordingly, it is preferred to carry out the FCC process at a preferred temperature of from about 566 ° C to about 630 ° C, preferred pressure from about 138 kPa to about 240 kPa (20 to 35 psia). Another condition for the process is the ratio of the catalyst to the lipid composition, which 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 reactor fuel, the lipid composition is introduced into the elevator section of a FCC reactor. The temperature in the elevator section will be very hot and in the range from about 700 ° C (1292 ° F) to rPt ^ a ^ r to about 760 ° C (1400 ° F) with a ratio^lft^5T¿^lto^ttaTt '

<img file="MX339639B_D0243.tif" />

Mexican Institute

ΠΕ THE PROPERTY L ·. τ _ _J HMftURTliAli__ composition of approximately 100 to approximately 150. 'and do you anticipate that the introduction of the lipid composition into the elevator section will produce considerable amounts of propylene and ethylene.

In another embodiment of the method for producing a reactor fuel with the lipid composition or lipids produced as described herein, the structure of the lipid composition or the lipids is broken by a process known as hydrodeoxygenation (HDO).

HDO means the 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 all the sulfur and nitrogen compounds are removed. The removal of sulfur is called hydrodesulfurization (HDS). The pre-treatment and the purity of the raw materials (lipid or lipid composition) contribute to the useful life of the catalyst.

Generally in the HDO / HDS stage, hydrogen is mixed with the source stock (lipid or lipid composition) and then the mixture is passed through a bed of catalyst, as a co-current flow, either as a single phase or a two-phase source stock. After the

<img file="MX339639B_D0244.tif" />

264 HDO / MDS stage, the product fraction is 'separated and a separate isomerization reactor'. An isomerization reactor for biological starting material is described in the literature (Fl 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenation of a hydrocarbon source, for example, the lipid composition or lipids herein, can also be performed by passing the lipid composition or lipids as a stream of co-current with hydrogen gas to through a first hydrogenation zone, and subsequently the hydrocarbon effluent is further hydrogenated in a second hydrogenation zone, passing hydrogen gas to the second hydrogenation zone as a counter current flow with respect to the hydrocarbon effluent.

Illustrative Applications of HDO and Useful Catalysts to Crack the Lipid Composition to Produce Definas C₂-C_s are described in the patent of the

United States no. 7,232,935, which is incorporated by reference in its entirety.

Typically, in the hydrodeoxygenation step, the structure of the biological component, such as the lipid composition or lipids thereof, breaks down, the

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INDVSTRlAl

<img file="MX339639B_D0245.tif" />

Oxygen, nitrogen, phosphorous and sulfur compounds, and light hydrocarbons as gas are removed, and olefinic bonds are hydrogenated. In the second stage of the process, that is, in the so-called isomerization stage, isomerization is carried out for branching the hydrocarbon chain and the performance of the paraffin is improved at low temperatures.

In the first stage, i.e. in the HDO stage of the cracking process, the hydrogen gas and the lipid composition or lipids herein to be hydrogenated are passed to an HDO catalyst bed system, either as co-current or counter-current flows, said catalyst bed system comprising one or more catalyst beds (es), preferably 1-3 catalyst beds. The HDO stage typically works in a co-current manner. In the case of an HDO catalyst bed system, which comprises two or more catalyst beds, one or more of the beds can operate using the countercurrent flow principle. In the HDO stage, the pressure varies between 20 and 150 bar, preferably between 50 and 100 bar, and the temperature varies between 200 and 500 ° C, preferably in the range of 300-400 ° C. In the stage of

HDO, hydrogenation catalysts can be used

<img file="MX339639B_D0246.tif" />

VTT and / or VIB of the Pd, Pt, Ni,

266 known to contain metals from the periodic system. Preferably hydrogenation are catalysts sopi

NiMo or a CoMo catalyst, being silica. NiMo / Al catalysts are typically used₂0₃ and CoMo / Al₂0₃.

Prior to the HDO step, the lipid composition or lipids herein, optionally, can optionally be treated by prehydrogenation under milder conditions, thus avoiding side reactions of the double bonds. Said prehydrogenation is carried out in the presence of a prehydrogenation catalyst at temperatures of 50,400 ° C and at hydrogen pressures of 1-200 bar, preferably at a temperature between 150 and 250 ° C and at a hydrogen pressure between 10 and 100 bar. The catalyst may contain Group VIII and / or VIB metals from the periodic system. Preferably, the prehydrogenation catalyst is a Pd, Pt, Ni, NiMo supported catalyst or a CoMo catalyst, the support is alumina and / or silica.

[0024] A gas stream from the stage of

HDO containing hydrogen is cooled and then compounds of carbon monoxide, carbon dioxide, nitrogen, phosphorous and sulfur, gaseous light hydrocarbons and others

<img file="MX339639B_D0247.tif" />

267

IMPI ^I* ICANO OF THE PROPERTY industrial impurities, are removed from it. After compression, the purified hydrogen or recycled hydrogen is returned back to the first catalyst bed and / or between the catalyst beds to compensate for the removed gas stream. The water is removed from the condensed liquid. Liquid is passed to the first catalyst bed or between the catalyst beds.

[0025] After the HDO step, the product undergoes an isomerization step. It is important to the process that the 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, in which the reaction product from the HDO stage can be purified by separation with steam or a suitable gas, such as light hydrocarbons, nitrogen or hydrogen. The optional separation step is carried out as a countercurrent in a unit located upstream of the isomerization catalyst, in which the gas and liquid are in contact with each other, or before the existing isomerization reactor in one unit. independent separation using the countercurrent principle.

<img file="MX339639B_D0248.tif" />

the hydrogenated lipids from the mixture of n-oarafin. I know

After the step of and the lipid composition or present, and, optionally, they pass to a reactive isomerization unit comprising one or more beds of the catalyst (s). The catalyst beds of the isomerization stage may operate as a co-current or counter current.

[0026] It is important to the process that the countercurrent flow principle is applied in the isomerization step. In the isomerization step, this is done by carrying out any optional separation steps or the isomerization reaction step or both in countercurrent form. In the isomerization stage, the pressure varies in the range of 20-150 bar, preferably in the range of 20-100 bar, 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 the molecular sieve and / or a Group VII metal and / or a vehicle. Preferably, the isomerization catalyst contains SAPO-11 or SAPO41 or ZSM22 or ZSM-23 or ferrierite and Pt, Pd or Ni and Al₂OR₃ and SiO₂. Typical isomerization catalysts are, for example,

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<img file="MX339639B_D0249.tif" />

Pt / SAPO-ll / Al₂OR₃, Pt / ZSM-22 / Al₂OR₃, Pt / ZSM-23 / Al₂OR₃ and Pt / SAPOll / SiO₂. The isomerization step and HDO step can be carried out in the same pressure vessel or in separate pressure vessels. The optional prehydrogenation can be carried out in a separate pressure vessel or in the same pressure vessel as the HDO and isomerization steps.

Thus, in one embodiment, the product of one or more chemical reactions is a mixture of alkanes that comprises

HRJ-5. In another embodiment, the product of one or more chemical reactions is an alkane mixture comprising reactor fuel according to ASTM D1655. In some embodiments, the composition that meets ASTM 1655 aircraft fuel specifications has a sulfur content that is less than 10 ppm. In other embodiments, the composition that meets ASTM 1655 aircraft fuel specifications has a TIO value of

<td colspan="2">distillation curve</td><td>less</td><td>of</td><td>205 ° C.</td><td>. In another modality,</td><td>the</td>

<td>composition</td><td>That</td><td>adjusts</td><td>to</td><td>the</td><td>Specifications</td><td>of the</td>

<td>fuel</td><td colspan="3">ASTM 1655 aircraft</td><td>has</td><td colspan="2">a boiling point</td>

<td>final (FBP)</td><td>less</td><td colspan="2">than 300 ° C</td><td>In</td><td>another modality,</td><td>the</td>

<td>composition</td><td>That</td><td>adjusts</td><td>to</td><td>the</td><td>Specifications</td><td>of the</td>

ASTM 1655 aircraft fuel has a point of

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<img file="MX339639B_D0250.tif" />

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INSTITUTO MEXICANO DE IA PROPIEDAD indi ISTRIAL inflammation of at least 38 ° C. In another embodiment, the composition that conforms to ASTM 1655 aircraft fuel specifications has a density between

775K / M³ and 840K / M³. In yet another embodiment, the composition that conforms to ASTM 1655 reactor fuel specifications has a freezing point that is below -4 7 ° C. In another embodiment, the composition that conforms to fuel specifications reactor

ASTM 1655 has a net heat of combustion that is at least

42.8 MJ / K. In another embodiment, the composition that conforms to ASTM 1655 reactor fuel specifications has a hydrogen content that is at least 13.4 mass%. In another embodiment, the composition that conforms to ASTM 1655 reactor fuel specifications has thermal stability, as assessed by quantitative gravimetric JFTOT at 260 ° C, which is below

3mm Hg. In another embodiment, the composition that meets ASTM 1655 aircraft fuel specifications has an existing gum that is below 7 mg / dl.

Therefore, the present invention describes a variety of methods in which the chemical modification of lipids of microalgae is performed to yield useful products in a variety of applications

271

<img file="MX339639B_D0251.tif" />

industrial and other. Examples of the processes for modifying oils produced by the methods described herein include, but are not limited to, hydrolysis of the oil, hydroprocessing of the oil, and esterification of the oil. Other chemical modification of the microalgae lipid includes, but is not limited to, epoxidation, oxidation, hydrolysis, sulfation, sulfonation, ethoxylation, propoxylation, amidation, and saponification. Modification of the microalgae oil produces basic oleochemicals that can be further modified into selected derived oleochemicals for a desired function. In a manner similar to that described above in connection with the fuel production process, these chemical modifications can also be made in the oils generated by the microbial cultures described herein. Examples of basic oleochemicals include, but are not limited to, soaps, fatty acids, fatty esters, fatty alcohols, fatty nitrogen compounds, fatty acid methyl esters, and glycerin. Examples of derived oleochemicals include, but are not limited to, fatty nitriles, esters, acid dimers, quats, surfactants, fatty alkanolamides, fatty alcohol sulfates, resins, emulsifiers, fatty alcohols, olefins,

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drilling muds, polyols, polyurethanes, polyacrylates, rubber, candles, cosmetics, metal soaps, soaps, alpha-sulfonated methyl esters, fatty alcohol sulphates, fatty alcohol ethoxylates, fatty alcohol ether sulfates, imidazolines, surfactants, detergents, esters , quats, ozonolysis products, fatty amines, fatty alkanolamides, ethoxysulfates, monoglycerides, diglycerides, triglycerides (including medium chain triglycerides), lubricants, hydraulic fluids, greases, dielectric fluids, mold release agents, metal working fluids, heat transfer fluids, other functional fluids, industrial chemicals (eg cleaners, textile processing aids, plasticizers, stabilizers, additives), surface coatings, paints and lacquers, electrical wiring insulation, and higher alkanes.

The hydrolysis of the fatty acid constituents from the glycerolipids produced by the methods of the invention produces free fatty acids that can be derivatized to produce other useful chemicals. Hydrolysis occurs in the presence of water and a catalyst that can be an acid or a base. The released fatty acids can be derivatized to produce a variety of products, as reported in the following:

<img file="MX339639B_D0253.tif" />

273 ΙΜΡΙ

MfcXICANO INSTITUTE

OE LA RRORICBa ·

INDUSTRIAL United States Patent Nos. 5,304,664 (Highly Sulfated Fatty Acids); 7,262,158 (Cleaning Compositions);

7,115,173 (Fabric softening compositions); 6,342,208 (Emulsions for skin treatment); 7,264,886 (Compositions that repel water); 6,924,333 (Paint additives); 6,596,768 (Lipid-enriched ruminant feed raw material); and 6,380,410 (Surfactants for detergents and cleaners).

With respect to hydrolysis, in one embodiment of the invention, a triglyceride oil is first optionally hydrolyzed in a liquid medium such as water or sodium hydroxide, with the aim of obtaining glycerin and soaps.

There are several suitable triglyceride hydrolysis methods, including but not limited to, saponification, acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis

<td>(called</td><td>here</td><td colspan="4">as fractionation), and hydrolysis using</td>

<td colspan="2">compressed water</td><td>with heat.</td><td>An expert in</td><td>the</td><td>matter</td>

<td>will recognize</td><td>than</td><td>an oil of</td><td>triglycerides</td><td>not</td><td>needs to</td>

<td>hydrolyze</td><td>with</td><td>the objective</td><td>to produce</td><td>a</td><td>product</td>

<td>oleochemical;</td><td colspan="2">rather the</td><td>oil can</td><td colspan="2">become</td>

directly to the desired oleochemical by another known process. For example, triglyceride oil

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<img file="MX339639B_D0254.tif" />

It can be converted directly to a fatty acid methyl ester by esterification.

In some embodiments, the catalytic hydrolysis of the oil produced by the methods described herein, by fractionation of the oil into glycerin and fatty acids. As mentioned above, fatty acids can then be processed through various other modifications to obtain oleochemical derivatives. For example, in one embodiment, the fatty acids can undergo an amination reaction to produce fatty nitrogen compounds. In another embodiment, fatty acids can undergo ozonolysis to produce mono and dibasic acids.

In other embodiments, hydrolysis can occur through the separation of oils produced in the present disclosure to create oleochemicals. In some preferred embodiments of the invention, a triglyceride oil can be separated before other processes are carried out. One skilled in the art will recognize that there are many suitable methods for triglyceride fractionation, including but not limited to enzyme fractionation and pressure fractionation.

Generally, the enzymatic fractionation methods of oil use enzymes, lipases, which act as

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Industrial i

<img file="MX339639B_D0255.tif" />

Biocatalysts in a mixture of water and ana-itc Rl enzymatic fractionation then fractionates the oil or fat, respectively, into glycerol and free fatty acids.

Glycerol can then migrate to the water phase, while the organic phase is enriched with free fatty acids.

Enzymatic fractionation reactions generally take place in the delimitation between the organic phase and the aqueous phase, where the enzyme is present only in the delimitation of the phases. The triglycerides that gather in the delimitation of the phases then contribute to or participate in the fractionation reaction. As the reaction proceeds, the density or concentration of fatty acids, still chemically bound as glycerides, compared to free fatty acids, decreases in phase delineation, so that the reaction is slower. In certain modalities, enzymatic fractionation can occur at room temperature. A person skilled in the art will know the appropriate conditions for the fractionation of the oil into the desired fatty acids.

As an example, the reaction rate can be accelerated by increasing the surface of

<img file="MX339639B_D0256.tif" />

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Say LA ΤΤΟΡ | «Ί_Αρ

INDUSTMIAL delimitation of the interface. Once the reaction is complete, the fatty acids are then separated from the organic phase released from the enzyme, and the residue, which still contains chemically bound fatty acids such as glycerides, is either fed back or recycled and mixed with the oil or fat to undergo fractionation. In this way, the recycled glycerides undergo an additional enzymatic fractionation process. In some embodiments, free fatty acids are extracted from an oil or partially fractionated fat in such a manner. Thus, if the chemically bound fatty acids (triglycerides) are

<td>they return or</td><td>I know</td><td>feed back</td><td>in</td><td>the</td><td>process</td><td>of</td>

<td>division,</td><td>the</td><td>consumption of the</td><td>enzyme</td><td>I know</td><td colspan="2">can reduce</td>

<td>drastically.</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>15 degree</td><td>of</td><td>division</td><td colspan="3">is determined as</td><td>the</td>

ratio of the measured acidity value divided by the theoretically possible acidity value, which can be calculated for a given oil or fat.

Preferably, the acidity value is measured by titration, according to common standard methods.

Alternatively, the density of the aqueous phase of glycerol can be taken as a measure of the degree of fractionation.

In one modality, the fractionation process as

<img file="MX339639B_D0257.tif" />

277

IMPI describes herein also arior-uaHn for the fractionation of the mono-, di- and triglycerides found in the so-called residual soap from the alkaline refining processes of the oils produced. In this way, the residual soap can be converted quantitatively, without prior saponification of neutral oils, into fatty acids. To this end, the fatty acids that are chemically bound in the soaps are released, preferably before fractionation, through an acid addition. In certain embodiments, a buffer solution is used in addition to water and enzyme for the fractionation process.

In one embodiment, the oils produced according to the methods of the invention can also be subjected to

15 'saponification as a method of hydrolysis. Vegetable and animal oils are typically made from triaglycerols (TAG), which are esters of fatty acids with the trihydric alcohol, glycerol. In an alkaline hydrolysis reaction, glycerol in a TAG is removed, leaving three carboxylic acid anions, which can associate with alkali metal cations such as sodium or potassium, to produce fatty acid salts. In this scheme, the constituents of carboxylic acids dissociate from the

IMPI

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278 glycerol fraction and are replaced ^ with biH-m-yi ir groups »The amount of base (eg KOH) used in the reaction is determined by the desired degree of saponification. If the goal is, for example, to produce a soap product comprising some of the oils originally present in the TAG composition, an amount of base sufficient to convert all TAGs into fatty acid salts is introduced into the reaction mixture. This reaction is normally carried out in an aqueous solution and occurs slowly, but can be accelerated by adding heat. Precipitation of the fatty acid salts can be facilitated by the addition of salts, such as the water soluble alkali metal halides (eg, NaCl or KC1), to the reaction mixture.

Preferably, the base is an alkali metal hydroxide, such as NaOH or KOH. Alternatively, other bases, such as alkanolamines, including for example triethanolamine and aminomethylpropanol, can be used in the reaction scheme. In some cases, these alternatives may be preferable to produce a clear soapy product. In one embodiment, the lipid composition subject to saponification is a mimetic tallow (i.e., a lipid composition similar to that of a tallow) that is produced as described in

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it, or a mixture of a mimetic tallow with another triglyceride oil.

In some methods, the first stage of chemical modification may be hydroprocessing to saturate the double bonds, followed by deoxygenation at elevated temperature in the presence of hydrogen and a catalyst. In other methods, hydrogenation and deoxygenation can occur in the same reaction. In still other methods, deoxygenation occurs before hydrogenation. The isomerization can then be carried out optionally, also in the presence of hydrogen and a catalyst. Finally, gases and naphtha components can be removed if desired. For example, see United States Patent Nos. 55,475,160 (triglyceride hydrogenation); 5,091,116 (deoxygenation, hydrogenation, and gas removal);

6,391,815 (hydrogenation); and 5,888,947 (isomerization).

In some embodiments of the invention, triglyceride oils are partially or fully deoxygenated. Deoxygenation reactions form desired products, including but not limited to, fatty acids, fatty alcohols, polyols, ketones, and aldehydes. In general, without being limited by any particular theory, deoxygenation reactions involve a combination of several pathways of

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different reactions, including but not limited to:

hydrogenolysis, hydrogenation, consecutive hydrogenation-hydrogenolysis, consecutive hydrogenolysis-hydrogenation, and combined hydrogenation-hydrogenolysis reactions, resulting in at least partial removal of oxygen from fatty acids or fatty acid esters to produce reaction products , such as fatty alcohols, which can be easily converted to the desired chemicals by post-processing. For example, in one embodiment, a fatty alcohol can be converted to olefins by the FCC reaction or to higher alkanes through a condensation reaction.

One such chemical modification is hydrogenation, which is the addition of hydrogen to the double bonds in the constituent fatty acids of glycerolipids or free fatty acids. The hydrogenation process allows the transformation of liquid oils into semi-solid or solid fats, which may be more suitable for specific applications.

The hydrogenation of oils that are produced by the methods described in the present description can be carried out in conjunction with one or more of the methods and / or materials that are provided in the present description,

281 χ ιτι jt i

INSTITUTO MiXICAN. - &lt;

oe LA eHOFUOA »industrial as reported in the following: US Patent Nos. 7,288,278 (Food or drug additives);

5,346,724 (Lubrication Products); 5,475,160 (fatty alcohols); 5,091,116 (Edible oils); 6,808,737 (Structural fats for margarine and spreads); 5,298,637 (Fat substitutes with reduced calories); 6,391,815 (Hydrogenation Catalyst and Sulfur Adsorbent); 5,233,099 and 5,233,100 (fatty alcohols); 4,584,139 (Hydrogenation catalysts); 6,057,375 (Foam Suppressing Agents); and 7,118,773 (Edible Spread Paste Emulsions).

A person skilled in the art will recognize that various processes can be used to hydrogenate carbohydrates. A convenient method includes contacting the carbohydrates with hydrogen or hydrogen mixed with a suitable gas and a catalyst, under conditions sufficient in a hydrogenation reactor to form a hydrogenated product. The hydrogenation catalyst can generally include Cu, Re, Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or any combination thereof, either alone or with promoters, such as W, Mo, Au, Ag, Cr, Zn, Mn, Sn, Β, P, Bi, and alloys or any combination thereof. Other effective hydrogenation catalyst materials

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They include both supported nickel or rhenium modified ruthenium. In one embodiment, the hydrogenation catalyst also includes any of the supports, depending on the desired functionality of the catalyst. Hydrogenation catalysts can be prepared by methods known to those skilled in the art.

In some embodiments, the hydrogenation catalyst includes a supported Group VIII metal catalyst and a metal sponge material (eg, a nickel sponge catalyst). Raney nickel provides an example of an activated nickel sponge catalyst suitable for use in this invention. In another embodiment, the hydrogenation reaction in the invention is carried out using a catalyst comprising a nickel-rhenium catalyst or a modified nickel-tungsten catalyst. An example of a suitable catalyst for the hydrogenation reaction of the invention is a carbon supported nickel-rhenium catalyst.

In one embodiment, a suitable Raney nickel catalyst can be prepared by treating an alloy of approximately equal amounts by weight of nickel and aluminum with an aqueous alkaline solution, for example, containing approximately 25% by weight of

<img file="MX339639B_D0262.tif" />

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INpUSTtlAL sodium hydroxide. Aluminum is selectively dissolved by the aqueous alkaline solution, resulting in a sponge-like material comprising mostly nickel with small amounts of aluminum. The initial alloy includes promoter metals (namely molybdenum and chromium) in amounts such that about 1 to 2% by weight is held in the catalyst in the form of a nickel sponge.

In another embodiment, the hydrogenation catalyst is prepared using a ruthenium (III) nitrosyl nitrate / ruthenium (III) chloride solution in water to impregnate a suitable support material. The solution is then dried to form a solid that has a water content of less than about 1% by weight. The solid can then be reduced to atmospheric pressure in a stream of hydrogen at 300 ° C (without calcining) or 400 ° C (calcined) in a rotary ball furnace for 4 hours. After cooling and giving rise to the inert catalyst with nitrogen, the

5% by volume of the oxygen in the nitrogen is passed over the catalyst for 2 hours.

In certain embodiments, the disclosed catalyst includes a catalyst support. The catalyst support stabilizes and supports the catalyst. The type of catalyst support used depends on the chosen catalyst and the

284 reaction conditions. Suitable supports for the invention include, but are not limited to, carbon, silica, silica-alumina, zirconia, titania, ceria, vanadium, nitride, boron nitride, heteropoly acids, hydroxyapatite, zinc oxide, chromium, zeolite, carbon nanotubes , carbon fullerene and any combination of these.

The catalysts used in this invention can be prepared by conventional methods known to those skilled in the art. Suitable methods may include, but are not limited to, incipient wetting, evaporative impregnation, chemical vapor deposition, coating washing, sputtering techniques, and the like.

The conditions for carrying out the hydrogenation reaction may vary depending on the type of raw material and the desired products. One skilled in the art, with the benefit of this disclosure, will recognize the appropriate reaction conditions. In general, the hydrogenation reaction is carried out at temperatures from 80 ° C to 250 ° C, and preferably from 90 ° C to 200 ° C, and more preferably from 100 ° C to 150 ° C. In some embodiments, the hydrogenation reaction is carried out at pressures of 500 kPa to 14,000 kPa.

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The hydrogen used in the hydrogenolysis reaction of the present invention can include external hydrogen, recycled hydrogen, hydrogen generated in situ, and any combination thereof. As used herein, the term "external hydrogen" refers to hydrogen that does not originate from the biomass reaction itself, but is added to the system from another source.

In some embodiments of the invention, it is desirable to convert the starting carbohydrates into a smaller molecule that will be easier to convert to the desired higher hydrocarbons. A convenient method for this conversion is through a hydrogenolysis reaction. Several processes are known to perform the hydrogenolysis of carbohydrates. An appropriate method includes contacting a carbohydrate with hydrogen or hydrogen mixed with a suitable gas and a hydrogenolysis catalyst in a hydrogenolysis reactor, under conditions sufficient to form a reaction product comprising smaller molecules or polyols. As used herein, the term "smaller molecules" or "polyols" includes any molecule that has a smaller molecular weight, which may include fewer carbon or oxygen atoms than carbohydrates.

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OF INDUSTRIAL PROPERTY IA starting carbon. In one embodiment, the reaction products are smaller molecules that include polyols and alcohols. A person with common knowledge in the field would be able to select the appropriate method to carry out the hydrogenolysis reaction.

In some embodiments, a 5- or 6-carbon sugar or sugar alcohol can be converted to propylene glycol, ethylene glycol, and glycerol with a hydrogenolysis catalyst. The hydrogenolysis catalyst can

<td>include</td><td>Cr,</td><td>Mo,</td><td>W, Re, Mn,</td><td>Cu, Cd, Fe, Co, Ni, Pt,</td><td>P.S,</td><td>Rh,</td>

<td>Ru, Go,</td><td>You</td><td>, AND</td><td>alloys</td><td>or any combination</td><td>of</td><td>the</td>

<td>themselves,</td><td>already</td><td>be</td><td colspan="2">alone or with promoters such as</td><td>Au,</td><td>Ag,</td>

<td>Cr, Zn,</td><td>Mn,</td><td>Sn,</td><td>Bi, B, 0 and</td><td colspan="3">alloys or any combination</td>

thereof. The hydrogenolysis catalyst may also include a transition metal carbonaceous pyropolymer catalyst (eg, chromium, molybdenum, tungsten, rhenium, manganese, copper, cadmium) or group VIII metals (eg, iron, cobalt, nickel , platinum, palladium, rhodium, ruthenium, iridium and osmium). In certain embodiments, the hydrogenolysis catalyst can include any of the above metals, combined with an alkaline earth metal oxide or adhered to a catalytically active support. In certain modalities, the

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catalyst in the hydrogenolysis reaction

<img file="MX339639B_D0266.tif" />

catalyst support as described above for the hydrogenation reaction.

The conditions for carrying out the hydrogenolysis reaction may vary depending on the type of raw material and the desired products. One skilled in the art, with benefit of this disclosure, will recognize the appropriate conditions to use to carry out the reaction. In general, the hydrogenolysis reaction is carried out at temperatures from 110 ° C to 300 ° C, and preferably from 170 ° C to

220 ° C, and more preferably from 200 ° C to 225 ° C. In some embodiments, the hydrogenolysis reaction is performed under basic conditions, preferably at a pH of 8 to 13, and even more preferably at a pH of 10 to 12. In some embodiments, the condensation reaction is carried out at pressures. in a range between 60 KPa and 16500 KPa, and preferably in a range between 1700 KPa and 14000 KPa, and even more preferably between 4800 KPa and 11000 KPa.

The hydrogen used in the hydrogenolysis reaction of the present invention can include external hydrogen, recycled hydrogen, hydrogen generated in situ, and any combination thereof.

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In some embodiments, the aforementioned reaction products can be converted to higher hydrocarbons through a condensation reaction in a condensation reactor. In such embodiments, condensation of the reaction products occurs in the presence of a catalyst capable of forming higher hydrocarbons. Although not intended to be limited by theory, it is believed that the production of higher hydrocarbons occurs through a stepwise addition reaction, which includes the formation of carbon-carbon bonds or carbon-oxygen bonds. The resulting reaction products include any number of compounds containing these residues, as described in more detail below.

In certain embodiments, suitable condensation catalysts include an acid catalyst, a basic catalyst, or an acid / basic catalyst. As used herein, the term "acid / basic catalyst" refers to a catalyst that has an acidic functionality and a base. In some embodiments, the condensation catalyst may include, but is not limited to, zeolites, carbides, nitrides, zirconia, alumina, silica, aluminosilicates, phosphates, oxides of titanium, oxides of

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zinc, vanadium oxides, lanthanum oxides, yttrium oxides, scandium oxides, magnesium oxides, cerium oxides, baric oxides, calcium oxides, hydroxides, heteropoly acids, inorganic acids, modified acid resins, modified basic resins, and any Combination of these In some embodiments, the condensation catalyst may further include a modifier. Suitable modifiers include La, Y, Se, Ρ, B, Bi, Li, Na,

K, Rb, Cs, Mg, Ca, Sr, Ba, and any combination of these.

In some embodiments, the condensation catalyst may further include a metal. Suitable metals include

Cu, Ag, Au, Pt, Ni, Fe, Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Go,

Re, Mn, Cr, Mo, W, Sn, Os, alloys, and any combination of these.

In certain embodiments, the catalyst described in the condensation reaction may include a catalyst support as described above for the hydrogenation reaction. In certain embodiments, the condensation catalyst is self-sufficient. As used herein, the term "self-sufficient" means that the catalyst does not need any other material to support it. In other embodiments, the condensation catalyst is used in conjunction with a separate support

<img file="MX339639B_D0269.tif" />

290

IMPI appropriate to suspend the rat.alizadnr. In one embodiment, the support for the condensation catalyst is silica.

The conditions under which the condensation reaction occurs can vary depending on the type of raw material and the desired products. One skilled in the art, with benefit of this disclosure, will recognize the appropriate conditions to use to carry out the reaction. In some embodiments, the condensation reaction is carried out at a temperature at which the thermodynamics of the proposed reaction is favorable. The temperature for the condensation reaction can vary depending on the specific starting polyol or alcohol. In some embodiments, the temperature of the condensation reaction is in a range from 80 ° C to 500 ° C, and preferably from

125 ° C to 450 ° C, and more preferably from 125 ° C to 250 ° C. In some embodiments, the condensation reaction is carried out at pressures in a range from 0 kPa to 9000 kPa, and preferably in a range from 0 kPa to 7000 KPa, and even more preferably from 0 kPa to 5000 KPa.

The higher alkanes formed by the invention include, but are not limited to, straight or branched chain alkanes having 4 to 30 carbon atoms, straight or branched chain alkenes having 4 to 30 carbon atoms.

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carbon, cycloalkanes having 5 to 30 carbon atoms, cycloalkenes having 5 to 30 carbon atoms, aryls, fused aryl, alcohols, and ketones. Suitable allenes include, but are not limited to, butane, pentane, pentene, 2-methylbutane, hexane, hexene, 2-methylpentane, 3methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, heptene, octane, octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane, 2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, niene, decane, decene, undecane, undecene, dodecane, dodecene, tridecane, tridecene, tetradecane , tetradecene, pentadecane, pentadecene, nonildecano, nonildecene, eicosano, eicoseno, uneicosan, uneicosen, doeicosan, doeicosen, trieicosan, trieicosen, tetraeicosan, tetraeicosen, and isomers thereof. Some of these products may be suitable for use as fuels.

In some embodiments, the cycloalkanes and cycloalkenes are unsubstituted. In other embodiments, the cycloalkanes and cycloalkenes are mono-substituted. In still other embodiments, the cycloalkanes and cycloalkenes are multi-substituted. In embodiments comprising substituted cycloalkanes and cycloalkenes, the substituted group includes, but is not limited to, a straight-chain or branched alkyl having 1 to 12 carbon atoms, an alkylene²⁹² IMPI ιη «γπτοτο m * icanc DE LA MOHEDA! · Straight or branched chain having 1 to 12 carbon atoms, a phenyl, and any combination of these. Cycloalkanes and cycloalkenes include, but are not limited to, cyclopentane, cyclopentene, cyclohexane, cyclohexene, methyl cyclopentane, methyl cyclopentene, ethyl cyclopentane, ethyl cyclopentene, ethyl cyclohexane, ethyl cyclohexene, isomers, and any combination of these.

In some embodiments, the formed arils are unsubstituted. In another embodiment, the arils formed are mono-substituted. In embodiments comprising substituted aryl, the substituted group includes, without limitation, a straight or branched chain alkyl having to 12 carbon atoms, a straight or branched chain alkylene having 1 to 12 carbon atoms, a phenyl, and any combination of these. Aryls suitable for the invention include, but are not limited to, benzene, toluene, xylene, ethyl benzene, for xylene, meta xylene, and any combination of these.

The alcohols produced in the invention have from 4 to carbon atoms. In some embodiments, alcohols are cyclic. In other embodiments, the alcohols are branched. In another embodiment, the alcohols are straight chain. Suitable alcohols for the invention include,

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but not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptildecanol, octildecanol, eildeanol, uneicosanol, doeicosanol, trieicos, these.

The ketones produced in the invention have from 4 to 30 carbon atoms. In one embodiment, ketones are cyclic. In another embodiment, ketones are branched.

In another embodiment, ketones are straight chain. Ketones suitable for the invention include, but are not limited to, butanone, pentanone, hexanone, heptanone, octanone, nonanone, decanone, undecanone, dodecanone, tridecanone, tetradecanone, pentadecanone, hexadecanone, heptildecanone, octildecanone, noniIdeeanone, eicosanone, , trieicosanone, tetraeicosanone, and their isomer.

Another such chemical modification is interesterification. Naturally produced glycerolipids do not have a uniform distribution of fatty acid constituents. In the context of oils, interesterification refers to the exchange of acyl radicals between two esters of different

<img file="MX339639B_D0272.tif" />

294

IMPI glycerolipids. The interesterification process provides a mechanism by which the fatty acid constituents of a glycerolipid mixture can be rearranged to modify the distribution pattern. Interesterification is a known chemical process, and generally involves heating (to about

200 ° C) of a mixture of oils for a period of time (eg, 30 minutes) in the presence of a catalyst, such as an alkali metal or alkylated alkali metal (eg, sodium methoxide). This process can be used to randomly select the distribution pattern of the fatty acid constituents of an oil mixture, or it can be directed to produce a desired distribution pattern. This chemical lipid modification method can be carried out on the materials provided herein, such as microbial biomass, with a lipid dry cell weight percentage of at least 20%.

Targeted interesterification, in which a specific distribution pattern of fatty acids is sought, can be performed by maintaining the oil mixture at a temperature below the melting point of some TAGs that could be obtained. This results in the selective crystallization of these TAGs, which eliminates them

295

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efficiently from the reaction mixture where they crystallize. The process can continue until most of the fatty acids in the oil have precipitated, for example. A targeted interesterification process can be used, for example, to produce a product with a lower caloric content through the replacement of long chain fatty acids with their shorter chain counterparts. Targeted interesterification can also be used to produce a product with a mixture of fats that can provide the desired melting characteristics and structural features sought in food additives or products (eg, margarine) without resorting to hydrogenation, which It can produce unwanted trans isomers.

The interesterification of oils produced by the methods described in the present disclosure can be carried out in conjunction with one or more of the methods and materials, or to produce products, as reported in the following: US Patent Nos. 6,080,853 (Nondigestible Fat Substitutes); 4,288,378 (Peanut Butter Stabilizer); 5,391,383 (Edible atomized oil); 6,022,577 (Edible fats for food products); 5,434,278 (Edible fats

296

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MEXICAN INSTITUTE UB LA PROPIEDAD INDUSTRIAL

<img file="MX339639B_D0274.tif" />

for food products); 5,268,192 (Low Calorie Nut Products); 5,258,197 (low calorie edible compositions); 4,335,156 (Edible fat products);

7,288,278 (Food or drug additives) 7,115,760 (Fractionation process); 6,808,737 (structural fats); 5,888,947 (Engine lubricants); 5,686,131 (Edible Oil Blends); and 4,603,188 (Curable urethane compositions).

In an embodiment in accordance with the invention, the transesterification of the oil, as described above, is followed by the reaction of the transesterified product with polyol, as disclosed in US Patent No. 6,465,642, to produce the fatty acid polyester polyol. This process of esterification and separation can comprise the following steps d:

reacting a lower alkyl ester with the polyol in the presence of soap; remove soap residue from product mix; wash with water and dry the product mix to remove impurities; bleach the product mix for refining; separating at least a portion of the unreacted lower alkyl ester from the fatty acid polyester polyol in the product mixture; and recycling the unreacted lower alkyl ester separately.

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Transesterification can also be performed on the microbial biomass with short chain fatty acid esters, as reported in the US patent.

6,278,006. In general, transesterification can be carried out by adding a short chain fatty acid ester to an oil in the presence of a suitable catalyst and heating the mixture. In some embodiments, the oil comprises from about 5% to about 90% of the reaction mixture by weight. In some embodiments, the short chain fatty acid esters can make up about 10% to 50% of the reaction mixture by weight. Non-limiting examples of catalysts include basic catalysts, sodium methoxide, acidic catalysts, including inorganic acids such as sulfuric acid and acidified clays, organic acids such as methane sulfonic acid, benzenesulfonic acid and toluenesulfonic acid, and acidic resins, such as Amberlyst 15 .

Metals like sodium and magnesium, and metal hydrides are also useful catalysts.

Another such chemical modification is hydroxylation, which involves the addition of water to a double bond which results in saturation and incorporation of a hydroxyl radical. The hydroxylation process provides

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OE IA BROBUDAn.

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a mechanism for the conversion of one or more tuteners of fatty acids from a glycerolipid to a hydroxyl fatty acid. Hydroxylation can be carried out, for example, by the method reported in US Patent No. 5,576,027. Hydroxylated fatty acids, including castor oil and its derivatives, are useful as components in various industrial applications, including food additives, pigments, wetting agents, defoaming agents, waterproofing additives, plasticizing agents, cosmetic emulsifiers and / or deodorant agents, as well as in electronics, pharmaceuticals, adhesive ink paints, and lubricants. An example of how a glyceride can be hydroxylated is as follows: the fat can be heated, preferably to approximately

30-50 ° C combined with heptane and held at temperature for thirty minutes or more; then acetic acid can be added to the mixture followed by an aqueous solution of sulfuric acid followed by an aqueous solution of hydrogen peroxide which is added in small increments to the mixture for one hour; after aqueous hydrogen peroxide, the temperature can increase to at least about 60 ° C and stir for at least six hours;

299 after stirring the mixture is allowed to stand and a lower aqueous layer formed by the reaction can be removed while the upper layer of heptane formed by the reaction can be washed with hot water at a temperature of about 60 ° C, the heptane layer washed it can be neutralized with an aqueous solution of potassium hydroxide at a pH of about 5 to 7 and then it is removed by vacuum distillation; The reaction product can be vacuum dried at 100 ° C and the dry product is steam deodorized under vacuum conditions and filtered at approximately 50 ° C.

60 ° C using diatomaceous earth.

Hydroxylation of microbial oils produced by the methods described in the present description can be carried out in conjunction with one or more of the methods and / or materials, or to produce products, as reported in the following: US patents United Nos.

6,590,113 (Oil-based coatings and inks);

4,049,724 (Hydroxylation processes); 6,113,971 (olive oil butter); 4,992,189 (Lubricants and lubricant additives); 5,576,027 (Hydroxylated milk); and 6,869,597 (cosmetics).

Hydroxylated glycerolipids can be converted to stolides. Stolides consist of a glycerolipid in the

300

IΜ Ρ ί

MÜX1CANO INSTITUTE

FROM THE RROTFDAI V «^ SSLJ®

INDUSTRIAL as a hydroxylated fatty acid constituent has been esterified to another fatty acid molecule. The conversion of hydroxylated glycerolipids to stolides can be carried out by heating a mixture of glycerolipids and fatty acids, and contacting the mixture with a mineral acid, as described by Isbell et al., JAOCS 71 (2): 169 -174 (1994). Stolides are useful in a variety of applications, including without limitation those reported in the following: US Patent Nos. 7,196,124 (Elastomeric materials and floor covering); 5,458,795 (thickened oils for high temperature applications); 5,451,332 (Fluids for industrial applications); 5,427,704 (Fuel additives); and 5,380,894 (Lubricants, greases, plasticizers, and printing inks).

Another such chemical modification is olefin metathesis. In olefin metathesis, a catalyst cuts the alkylidene carbons into an alkene (olefin) and forms new alkenes by pairing each of them with different alkylidene carbons. The olefin metathesis reaction provides a mechanism for processes such as truncating alkyl chains of unsaturated fatty acids to alkenes by ethenolysis, crosslinking of fatty acids to

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through alkene bonds by auto-metathesis, and incorporation of new groups in fatty acids by inter-metathesis with derivatized alkenes.

In conjunction with other reactions, such as transesterification and hydrogenation, olefin metathesis can transform unsaturated glycerolipids into various end products. These products include glycerolipid oligomers for waxes, - short-chain glycerolipids for lubricants; bifunctional alkyl homo and hetero chains for chemicals and polymers; short chain esters for biofuel; and short-chain hydrocarbons for reactor fuel. Olefin metathesis can be carried out on derivatives of triacylglycerols and fatty acids, for example, using catalysts and methods that were reported in US Patent No. 7,119,216, US Patent Publication No. 2010/0160506, and United States Patent Publication No. 2010/0145086.

Bio-oil olefin metathesis generally comprises adding a Ru catalyst solution at a load of about 10 to 250 ppm under inert conditions to unsaturated fatty acid esters in the presence (intermetathesis) or absence (auto-metathesis) of other alkenes.

Reactions are typically allowed to proceed for hours.

<img file="MX339639B_D0278.tif" />

302 days and ultimately yield a distribution of alkene products. An example of how olefin metathesis can be carried out in a fatty acid derivative is as follows: A solution of the first generation Grubbs catalyst (dichloro [2 (1-methylethox-aO) phenyl] methyleneα-C] (tricycloheil -phosphamine) in toluene at a catalyst charge of 222 ppm can be added to a container containing degassed and dry methyl oleate. The container can then be pressurized with approximately 60 psig of ethylene gas and maintained at or below approximately 30 ° C for 3 hours, so that approximately 50% yield of methyl 9-decenoate can be produced.

The olefinic metathesis of product oils by the methods described herein can be performed in conjunction with one or more of the methods and / or materials, or to produce products, as reported in the following documents: sol. Patent Patent PCT / US07 / 081427 (α-olefin fatty acids) and sol. of United States Patent Nos. 12 / 281,938 (petroleum creams), 12 / 281,931 (paintball gun capsules), 12 / 653,742 (plasticizers and lubricants), 12 / 422,096 (bifunctional organic compounds), and 11 / 795,052 (candle wax).

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Other chemical reactions that can be performed on microbial oils include reacting triglycerides with a cyclopropane agent to increase fluidity and / or oxidative stability, as disclosed in US Patents 6,051,539 (manufacture of waxes from triacylglycerols) , 6,770,104 (epoxidation of triacylglycerols), as disclosed in The effect of fatty acid composition on the acrylation kinetics of epoxidized triacylglycerols, Journal of the American Oil

Chemists' Society, 79: 1, 59-63, (2001) and in Free Radical

Biology and Medicine, 37: 1, 104-114 (2004).

The generation of oil-bearing microbial biomass for fuels and chemicals as described above, results in the production of delipidated biomass flour. Delipidized flour is a by-product of algae oil preparation and is useful for animal feed for farm animals, eg ruminants, poultry, pigs and for aquaculture. The resulting flour, although the oil content is low, still contains high-quality protein, carbohydrates, fiber, ash, oil residues and other nutrients suitable for animal feed. Because cells are predominantly lysed by the process of separating the

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oils, delipid flour is easily digestible by these animals. The delipidized flour can optionally be combined with other ingredients, such as grains, in an animal feed. Because delipid flour has a powder consistency, it can be compressed into granules using an extruder or extender or other type of machine, which are commercially available.

The invention, which is described in detail above, is exemplified in the following examples, which are offered to illustrate but not to limit the claimed invention.

VII. EXAMPLES

EXAMPLE 1: Methods for growing Prototheca

The Prototheca strains were cultivated to achieve a high percentage of oil per dry weight of cells. Cryopreserved cells were thawed at room temperature and 500 µΐ of cells were added to 4.5 ml of medium (4.2 g / 1 K₂HPO₄, 3.1 g / 1 NaH₂PO₄0.24 g / 1 MgSO₄ · 7H₂Or, 0.25 g / 1 citric acid monohydrate, 0.025 g / 1 of

CaCl₂ 2H₂Or, 2 g / 1 of yeast extract) plus 2% glucose and grown for 7 days at 28 ° C with shaking (200 rpm) in a 6-well plate. The dry weight of the cells is

<img file="MX339639B_D0281.tif" />

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IMPIAS determined by centrifuging 1 ml of culture at 14,000 rpm for 5 min in a pre-weighed Eppendorf tube. The culture supernatant was discarded and the resulting cell pellet was washed with 1 ml of deionized water. The culture was centrifuged again, the supernatant was discarded, and the cell pellet was placed at -80 ° C until frozen. The samples were then lyophilized for 24 hours and the dry weight of the cells was calculated. For the determination of total lipids in the cultures, 3 ml of culture were withdrawn and subjected to analysis using a system of

Ankom (Ankom Inc., Macedonian, NY), according to the manufacturer's protocol. The samples were subjected to solvent extraction with an Amkom XT10 extractor according to the manufacturer's protocol. Total lipids were determined as the difference in mass between the acid hydrolyzed dry samples and the solvent extracted dry samples. Measurements of the oil percentage of the dry weight of the cells are shown in Table 10.

Table 10. Percentage of oil per dry weight of the cell

<td>Species</td><td>Strain</td><td>% Oil</td>

<td>Prototheca stagnora</td><td>UTEX 327</td><td> 13.14</td>

<td>Prototheca moriformis</td><td>UTEX 1441</td><td> 18.02</td>

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<img file="MX339639B_D0282.tif" />

<td>Prototheca moriformis</td><td>UTEX 1435</td><td> 27.17</td>

Microalgae samples from multiple strains of the genus Prototheca were genotyped. Genomic DNA was isolated from the algal biomass as follows. Liquid culture cells (approximately 200 mg) were centrifuged 5 min to resuspended then centrifuged 5 min to

<td> 14,000</td><td>x g. The</td><td>cells</td><td>I know</td>

<td>Water</td><td>distilled</td><td>sterile,</td><td>I know</td>

<td> 14,000</td><td>xg and se</td><td>scrapped</td><td>the</td>

supernatant. A single glass bead of ~ 2mm diameter was added to the biomass and the tubes were placed at -80 ° C for at least 15 minutes. Samples were removed and 150 μΐ of milling buffer (Sarkosil 1%, 0.25 M sucrose, 50 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH, added.

8.0, RNase A 0.5 pg / μΐ). The pellets were resuspended with brief agitation, followed by the addition of 40 µΐ of

5M NaCl. The samples were shaken briefly, followed by the addition of 66 µΐ of 5% CTAB (cetyl trimethylammonium bromide) and a final brief shake. The samples were then incubated at 65 ° C for 10 minutes after which they were centrifuged at 14,000 xg for 10 minutes. The supernatant was transferred to a new tube and extracted once with 300 μΐ of phenol: chloroform: isoamyl alcohol at 12: 12: 1, followed by

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INDUSTRIAL centrifugation for 5 minutes at 14,000 x g. The resulting aqueous phase was transferred to a fresh tube containing 0.7 vol of isopropanol (~ 190 pl), mixed by inversion, and incubated at room temperature for 30 minutes or overnight at 4 ° C. DNA was recovered through centrifugation at 14,000 xg for 10 minutes. The resulting precipitate was washed twice with 70% ethanol, followed by a final wash with 100% ethanol. The pellets were air dried for 20-30 minutes at room temperature, followed by resuspension in 50 µl of 10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μΐ of the total algae DNA, prepared as described above, was diluted 1:50 in 10 mM Tris, pH

8.0. PCR reactions, the final volume of 20 µΐ, were set as follows. Ten µΐ of iProof HF 2x Master Mix (BIO-RAD) was added to 0.4 µΐ of primer

SZ02613 (5'-TGTTGAAGAATGAGCCGGCGAC-3 '' (sec. With ID #: 9) at a standard concentration of 10mM). This primer sequence runs from position 567-588 at no.

Gen Bank access code L43357 and is highly conserved in the genomes of higher plants and algae plastids. This was followed by the addition of 0.4 μΐ of primer SZ02615 (5'CAGTGAGCTATTACGCACTC-3 '(sec. With ID #: 10) to the

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JσΟ MEXICAN INDUSTRIAL EROMEDAD standard concentration of 10 mM). This primer sequence is complementary to position 1112-1093 at no. Gen Bank access code L43357 and is highly conserved in the genomes of higher plants and algae plastids. Next, 5 µΐ of the diluted total DNA and 3.2 µΐ dH were added₂O. The PCR reactions were carried out as follows:

98 ° C, 45``, 98 ° C, 8 '', 53 ° C, 12``, 72 ° C, 20 '' for 35 cycles followed by 72 ° C for 1 min and held at 25 ° C. For purification of the PCR products, 20 µΐ of 10 mM Tris, pH 8.0 was added to each reaction, followed by extraction with 40 µΐ of phenol: chloroform: isoamyl alcohol

12:12: 1, shaking and centrifugation at 14,000 xg for minutes. The PCR reactions were applied to S400 columns (GE Healthcare) and centrifuged for 2 minutes at

3,000 x g. Subsequently purified PCR products were cloned into TOPO in the PCR8 / GW / TOPO and positive clones were selected on LB / Spec plates. The purified plasmid DNA was sequenced in both directions using forward and reverse M13 primers. In total, twelve strains of

Prototheca were selected to have their 23S rRNA DNA sequenced and the sequences are listed in the sequence listing. A summary of strains and sequence listing numbers are included below. The sequences are

<img file="MX339639B_D0285.tif" />

309 analyzed for general divergence to go-go ^{one to} UTEX 1435 sequence (section with ID number: 15).

Two pairs emerged (UTEX 329 / UTEX 1533 and UTEX 329 / UTEX 1440) as the maximum divergence pairs. In both cases, pairwise alignment resulted in 75.0% pairwise sequence identity. The percentage of sequence identity with UTEX 1435 was also included below:

Species Strain sec. with no. of ident.

nt identity

Prototheca kruegani with no. ident .: 11

UTEX 329

75.2

Prototheca wickerhamii UTEX 1440 sec. with no. ident .: 12

Prototheca stagnora with no. ident .: 13

Prototheca moriformis with no. ident .: 14

UTEX 1442

UTEX 288

75.7

75.4

Prototheca moriformis UTEX 1439; 1441; 100 with no. ident .: 15 sec

1435; 1437

Prototheca wikerhamii UTEX 1533

99.8 sec. with no. ident .: 16

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<img file="MX339639B_D0286.tif" />

<td>Prototheca</td><td>moriformis</td><td>UTEX</td><td> 1434</td><td> 75.9</td><td>sec.</td><td>with</td>

<td>no. ident</td><td> . : 17</td><td></td><td></td><td></td><td></td><td></td>

<td>Prototheca</td><td>zopfii</td><td>UTEX</td><td> 1438</td><td> 75.7</td><td>sec.</td><td>with</td>

<td>no. ident</td><td> . : 18</td><td></td><td></td><td></td><td></td><td></td>

<td>Prototheca</td><td>moriformis</td><td>UTEX</td><td> 1436</td><td> 88.9</td><td>sec.</td><td>with</td>

no. ident .: 19 [0027] Lipid samples from a subset of the strains listed above were analyzed for lipid profile using HPLC. The results are shown below in Table 11.

Table 11. Diversity of lipid chains in Prototheca species

<td>Strain</td><td>C14: 0</td><td>C16: 0</td><td>C16: l</td><td>C18: 0</td><td>C18: l</td><td>C18: 2</td><td>C18: 3</td><td>C20: 0</td><td>C20: l</td>

<td>UTEX 327</td><td> 0</td><td> 12.01</td><td> 0</td><td> 0</td><td> 50.33</td><td> 17.14</td><td> 0</td><td> 0</td><td> 0</td>

<td>UTEX 1441</td><td> 1.41</td><td> 29.44</td><td> 0.70</td><td> 3.05</td><td> 57.72</td><td> 12.37</td><td> 0.97</td><td> 0.33</td><td> 0</td>

<td>UTEX 1435</td><td> 1.09</td><td> 25.77</td><td> 0</td><td> 2.75</td><td> 54.01</td><td> 11.90</td><td> 2.44</td><td> 0</td><td> 0</td>

Oil extracted from Prototheca moriformis UTEX 1435 (via solvent extraction or using an expeller press was analyzed for carotenoids, chlorophyll,

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FROM LA rROFUDAI INDUSTRIAL tocopherols, other sterols and tocotrienols. The results are summarized below in Table 12.

Table 12. Analysis of carotenoid, chlorophyll, tocopherol / sterols and tocotrienol in the oil extracted from

Prototheca moriformis (UTEX 1435).

<td></td><td>Oil</td><td>Oil</td>

<td></td><td>pressing</td><td>extracted from</td>

<td></td><td>(mcg / ml)</td><td>solvent</td>

<td></td><td></td><td>(mcg / ml)</td>

<td>cis-Lutein</td><td> 0.041</td><td> 0.042</td>

<td>trans-Lutein</td><td> 0.140</td><td> 0.112</td>

<td>trans-</td><td> 0.045</td><td> 0.039</td>

<td>Zeaxanthin</td><td></td><td></td>

<td>cis-Zeaxanthin</td><td> 0.007</td><td> 0.013</td>

<td>t-alpha-</td><td> 0.007</td><td> 0.010</td>

<td>Crytoxanthin</td><td></td><td></td>

<td>t-beta-</td><td> 0.009</td><td> 0.010</td>

<td>Crytoxanthin</td><td></td><td></td>

<td>t-alpha-carotene</td><td> 0.003</td><td> 0.001</td>

<td>c-alpha-Carotene</td><td>none</td><td>none</td>

<td></td><td>detected</td><td>detected</td>

<td>t-beta-Carotene</td><td> 0.010</td><td> 0.009</td>

<td>9-cis-beta-</td><td> 0.004</td><td> 0.002</td>

<td>Carotene</td><td></td><td></td>

<td>Lycopene</td><td>none</td><td>none</td>

312

<td></td><td>detected</td><td>detected</td>

<td>Total of Carotenoids</td><td> 0.267</td><td> 0.238</td>

<td>Chlorophyll</td><td>&lt;0.01 mg / kg</td><td>&lt;0.01 mg / kg</td>

<td colspan="3">Tocopherols and Sterols</td>

<td></td><td>Oil</td><td>Oil</td>

<td></td><td>pressing</td><td>extracted from</td>

<td></td><td>(mg / lOOg)</td><td>solvent (mg / lOOg)</td>

<td>gamma Tocopherol</td><td> 0.49</td><td> 0.49</td>

<td>Campesterol</td><td> 6.09</td><td> 6.05</td>

<td>Stigmasterol</td><td> 47.6</td><td> 47.8</td>

<td>Beta-sitosterol</td><td> 11.6</td><td> 11.5</td>

<td>Others sterless</td><td> 445</td><td> 446</td>

<td colspan="2">Tocotrienols</td><td></td>

<td></td><td>Oil</td><td>Oil</td>

<td></td><td>pressing</td><td>taken from</td>

<td></td><td>(mg / g)</td><td>solvent (mg / g)</td>

<td>alpha Tocotrienol</td><td> 0.26</td><td> 0.26</td>

<td>beta Tocotrienol</td><td> &lt; 0.01</td><td> &lt; 0.01</td>

<td>gamma Tocotrienol</td><td> 0.10</td><td> 0.10</td>

MEXICAN INSTITUTE • E LA PROFIEOAD industrial

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IMPÍS-MEXICAN INSTITUTE BE IA BRORIEDAO INDUSTRIAL

<td>such Tocotrienol</td><td> &lt; 0.01</td><td> &lt; 0.01</td>

<td>Total of</td><td> 0.36</td><td> 0.36</td>

<td>Tocotrienols</td><td></td><td></td>

Oil extracted from Prototheca moriformis, from four separate batches, was refined and bleached using standard vegetable oil processing methods. In summary, the crude oil extracted from Prototheca moriformis was clarified in a horizontal decanter, where the solids were separated from the oil. The rinsed oil was then transferred to a tank with citric acid and water and allowed to settle for approximately 24 hours. After 24 hours, the mixture in the tank formed 2 separate layers. The bottom layer was composed of water and gums which were then decanted off before transferring the de-gummed oil into the bleach tank. The oil was then heated together with another dose of citric acid. The bleaching clay was then added to the bleaching tank and the mixture was further heated under vacuum in order to evaporate any water that was present. The mixture was then pumped through a leaf filter in order to remove the bleaching clay. The filtered oil was then passed through a

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INDUSTRIAL polishing filter final 5pm and then collected for storage until use. The refined and bleached oil (RB) was then analyzed for carotenoids, chlorophyll, sterols, tocotrienols, and tocopherols. The results of these analyzes are summarized in Table 13 below. Nd denotes none detected and the detection sensitivity is listed below:

Detection sensitivity

Carotenoids (mcg / g) nd = &lt;0.003 mcg / g Chlorophyll (mcg / g) nd = &lt;0.03 mcg / g

Sterol (%) nd = 0.25%

Tocopherols (mcg / g); na = 3 mcg / g

Table 13. Analysis of carotenoids, chlorophyll, sterols, tocotrienols and tocopherol from oil of Prototheca moriformis refined and bleached

<td></td><td>Lot Ά</td><td>Lot B</td><td>Lot C</td><td>Lot D</td>

<td colspan="5">Carotenoids (mcg / g)</td>

<td>Lutein</td><td> 0.025</td><td> 0.003</td><td>ND</td><td> 0.039</td>

<td>Zeaxanthin</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td>

<td>cis-</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td>

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<td>lutein / zeaxanthin</td><td></td><td></td><td></td><td></td>

<td>trans-alpha-</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td>

<td>cryptoxanthin</td><td></td><td></td><td></td><td></td>

<td>trans-beta-</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td>

<td>cryptoxanthin</td><td></td><td></td><td></td><td></td>

<td>trans-alpha-</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td>