Methods and compositions for producing squalene using yeast

(56) References cited:

WO-A1 -2009/143490 WO-A2-2008/130372 WO-A2-2010/023551 • Dow AgroSciences LLC (formerly Mycogen Corporation): Using Yeast Fermentation to Produce Cost-Effective and Biodegradable Lubricants,, 27 May 2004 (2004-05-27), XP002657524, Retrieved from the Internet: URL:http://statusreports.atp.nist.gov/repo rts/95-01-0148PDF.pdf [retrieved on 2011-08-24] • LADYGINA N ET AL: A review on microbial synthesis of hydrocarbons, PROCESS BIOCHEMISTRY, ELSEVIER, NL, vol. 41, no. 5, 1 May 2006 (2006-05-01), pages 1001-1014, XP002545855, ISSN: 1359-5113, DÓI: D0l:10.1016/J.PROCBI0.2005.12.007 • BEOPOULOS ATHANASIOS ET AL: Control of lipid accumulation in the yeast Yarrowia lipolytica, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 74, no. 24,1 December 2008 (2008-12-01), pages 7779-7789, XP009112521, ISSN: 1098-5336, DÓI: DOI:10.1128/AEM.01412-08 • ATHANASIOS BEOPOULOS ET AL: Yarrowia lipolytica as a model for bio-oil production, PROGRESS IN LIPID RESEARCH, vol. 48, no. 6, 1 November 2009 (2009-11-01), pages 375-387, XP55005437, ISSN: 0163-7827, DÓI: 10.1016/j.plipres.2009.08.005

Note: Within nine months ofthe publication ofthe mention ofthe grant ofthe European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall nőt be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).

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EP 2 504 421 Β1 • PASRIJA RITU ET AL: Squalene epoxidase encoded by ERG1 affects morphogenesis and drug susceptibilities of Candida albicans., THE

JOURNAL OF ANTIMICROBIAL

CHEMOTHERAPY JUN 2005 LNKDPUBMED:15845783, vol. 55, no. 6, June 2005 (2005-06), pages 905-913, XP002661204, ISSN: 0305-7453 • BONANNO J B ET AL: Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis, PROCEEDÍNGS OF THE NATIONAL ACADEMYOF SCIENCES, NATIONAL ACADEMY OF SCIENCES, WASHINGTON, DC; US, vol. 98, no. 23, 1 January 2001 (2001-01-01), pages 12896-12901, XP002298128, ISSN: 0027-8424, DÓI: DOI:10.1073/PNAS.181466998 • SABIROVA JÚLIA S ET AL: The ’LipoYeasts’ project: using the oleaginous yeast Yarrowia lipolytica in combination with specific bacterial genes for the bioconversion of lipids, fats and oils intő high-value products., MICROBIAL BIOTECHNOLOGY JAN 2011 LNKDPUBMED:21255371, vol. 4, no. 1, January 2011 (2011-01), pages 47-54, XP002657526, ISSN: 1751-7915 • KARST F ETAL: ERGOSTEROL BIOSYNTHESIS IN SACCHAROMYCES CEREVISIAE MUTANTS DEFICIENT IN THE EARLY STEPS OF THE PATHWAY, MOLECULAR AND GENERAL GENETICS, SPRINGER VERLAG, BERLIN, DE, vol. 154, no. 3,1 January 1977 (1977-01-01), pages 269-277, XP009041810, ISSN: 0026-8925, DÓI: 10.1007/BF00571282

EP 2 504 421 Β1

Description

FIELD OF THE INVENTION [0001] Disclosed are methods and compositions for producing isoprenoids such as squalene using yeast.

BACKGROUND OF THE INVENTION [0002] The following description ofthe background ofthe invention is provided simply as an aid in understanding the invention and is nőt admitted to describe or constitute prior art to the invention.

[0003] Isoprenoids, such as squalene, are commercially important types of lipids. They have excellent lubricity, oxidative stability, low pour points, low freezing points, high flash points, and facile biodegradability. Squalene is currently produced by extractíon from olive oil or cold water shark liver oil at a high unit cost. Because of the high unit cost, economically feasible uses for squalene and squalane (the fully hydrogenated derivative of squalene) are in small markét applications such as watch lubricants, pharmaceuticals/nutraceuticals, cosmetics, perfumes and as Chemical intermediates for high-value products.

[0004] There exist, however, significant potential markets for biodegradable lubricants, lubricant additives, and hydraulicfluids. Biodegradability of these products is particularly important for environmentally sensitive applications, such as agricultural applications, or where considerable lubricant or hydraulic fluids may be lost to the environment. The potential markets for biodegradable lubricants, lubricant additives, and hydraulic fluids are quite large, estimated to be on the order offive millión metric tons per annum.

[0005] Biodegradable lubricants, lubricant additives, and hydraulic fluids derived from vegetable and animal fats and oils are available, bút they have drawbacks. They typically solidify at relatively high temperatures (i.e., they solidify in cold weather) and have flash points that are too low for use in hot conditions, (i.e., they break down or combust under normál hot engine conditions).

[0006] Thus, a cost effective method of production of squalene is desired that would allow for large-scale manufacturing and widespread use of squalene and squalane in biodegradable lubricants, lubricant additives, and hydraulic fluids. [0007] Chang et al., (Appl. Microbiol. Biotechnoi., 2008, 78, 963-72) discloses the discovery of a wild type yeast, Pseudozyma sp. JCC207, that produces a large amount of squalene and several polyunsaturated fatty acids. Chang et al. describe isolating Pseudozyma sp. JCC207 from seawater collected near Guam, USA, and are unsure whether Pseudozyma sp. JCC207 is a new species or a variant of P. regulosa or P. aphidis. In the article, the efficiency of squalene production [oí Pseudozyma sp. JCC207] was investigated under different conditions.

[0008] Dow AgroSciences LLC, Using Yeast Fermentation to Product Cost-effective and Biodegradable Lubricants, http://statusreports.atp.nist.gov.reports/95-01-0148PDF.pdf, discloses that [t]he company proposed to use genetic engineering to altér the metabolic characteristics of an oleaginous (oily) yeast to increase the yeast’s ability to produce isoprenes through biosynthesis. Specifically, four enzymes were targeted: ACCase, hydroxymethylglutaryl CoA reductasc (HMGR), squalene synthetase, and squalene epoxidase.

[0009] U.S. Patent No. 5,460,949 discloses [a] method increasing the accumulation of squalene and specific sterols in yeast. In particular, it is disclosed that [s]qualene and sterol accumulation is increased by increasing the expression level of a gene encoding a polypeptide having the HMG-CoA reductase activity.

[0010] WO 2008/130372 A9 relates to methods for the biological production of certain sterol compounds, and to systems for producing oleaginous yeasts or fungi that are capable of producing certain sterol compounds.

SUMMARY OF THE INVENTION [0011] Subject matter of the present invention is a composition as defined in claim 1, and a method of producing squalene as defined in claim 2. The dependent claims relate to particular embodiments thereof.

[0012] One aspect ofthe present invention accordingly relates to a composition comprising a genetically converted yeast. The genetically converted yeast expresses one or more modified enzymes having one or more designed mutations, wherein the one or more designed mutations are at defined positions within said enzyme. The one or more modified enzymes comprise squalene epoxidase, and the squalene epoxidase has reduced activity and/or expression. The yeast produces increased quantities of squalene as compared to the native yeast, and it is a Yarrowia lipolytica strain selected from the group consisting of ATCC 90812, ATCC MYA-2613, or Yeastern polg.

[0013] Another aspect of the present invention relates to a method of producing squalene by a genetically converted yeast. The method comprises increasing or decreasing activity or expression of one or more enzymes in the isoprenoid biosynthesis pathway. The enzyme activity or expression is increased or decreased by one or more designed mutations, wherein the one or more designed mutations are at defined positions within said enzyme. The one or more modified enzymes comprise squalene epoxidase, and the squalene epoxidase has reduced activity and/or expression. The yeast

EP 2 504 421 Β1 produces increased quantities of squalene as compared to the native yeast, and it is a Yarrowia lipolytica strain selected from the group consisting of ATCC 90812, ATCC MYA-2613, or Yeastern polg.

[0014] According to a particular embodirnent, the genetically converted yeast is derived from an oleaginous yeast. [0015] According to a particular embodirnent, the one or more modified enzymes further comprise a modified enzyme selected from the group consisting of acetyl-CoA carboxylase (ACCase), HMG-CoA reductase, squalene synthase, ATP citrate lyase, ATP citrate synthase, mevalonate kinase, glycerol kinase and 5-aminolevulinate synthase.

[0016] According to a particular embodirnent, the activity or expression is reduced to about 90%; or about 80%; or about 70%; or about 60%; or about 50%; or about 40%; or about 30%; or about 20%; or about 10%; or about 5% ofthe activity or expression ofthe corresponding native yeast.

[0017] According to a particular embodirnent, the activity or expression is between 90-95%; or 80-90%; or 70-80%; 60-70%; or 50-60%; or 40-50%; or about 30-40%; or about 20-30%; about 10-20%; or about 5-10%; or about 2-5% of the activity or expression ofthe corresponding native yeast.

[0018] According to a particular embodirnent, the yeast is the Yeastern polg strain of Yarrowia lipolytica.

[0019] According to a particular embodirnent, an antifungal agent is present in the composition or is added to the yeast in the method. For example, an antifungal agent is present in the composition or is added to the yeast in the method at a concentration between 0.5 to 100 μg/ml, between 1 to 25 μg/ml, or between 10 to 15 μg/ml. According to a particular embodirnent, the antifungal agent may be an allylamine antifungal agent. For example, the antifungal agent may be an allylamine antifungal agent present at a concentration between 0.5 to 100 μg/ml, such as between 1 to 25 μg/ml, or between 10 to 15 μg/ml. According to a particular embodirnent, the antifungal agent may be terbinafine. For example, the terbinafine may be present at a concentration between 0.5 to 100 μg/ml, such as between 1 to 25 μg/ml, or between 10 to 15 μg/ml.

[0020] According to a particular embodirnent, the method comprises cultivating the yeast with an antifungal agent; wherein the yeast is the Yeastern polg strain of Yarrowia lipolytica and wherein the antifungal agent is terbinafine. For example, the terbinafine may be present at a concentration of about 12.5 μg/ml or greater.

[0021] As disclosed herein, increased amounts of an isoprenoid (for example, squalene) produced by a genetically converted or non-genetically converted yeast may be the result of mutating, modifying and/or altering the activity ofone or more enzymes within the isoprenoid biosynthesis pathway. For example acetyl-CoA carboxylase (or ACCase), HMG-CoA reductase, squalene epoxidase, squalene synthase, ATP citrate synthase, mevalonate kinase (e.g., Y. lipolytica mevalonate kinase (Genolevures YALIOB16038g)), glycerol kinase (e.g., Y. lipolytica glycerol kinase (Genolevures YALI0F00484g)) and/or 5-aminolevulinate synthase (e.g., encoded by Saccharomyces cerevisiae HEM1 gene) may be modified, mutated or have altered activity.

[0022] As disclosed herein, a genetically converted yeast expressing a modified enzyme may be produced by introducing a mutation in the enzyme through use of a gene repair oligonucleobase as described herein. Such methods may include introducing a gene repair oligonucleobase containing a specific mutation for a target gene of interest intő a yeast cell by any of a number of methods well-known in the art (e.g., eleetroporation, LiOAc, biolistics, spheroplasting, and/or Agrobacterium (see, for example, McClelland, C.M., Chang, Y.C., and Kwon-Chung, K.J. (2005) Fungal Genetics and Biology 42:904-913) and identifying a cell having the mutated enzyme.

[0023] As disclosed herein, a method of producing isoprenoids, preferably squalene, may include providing a genetically converted or non-genetically converted yeast as described herein and extracting squalene from the yeast. The method may include exposing yeast (either genetically converted or non-genetically converted) to an antifungal agent (for example, an allylamine antifungal agent such as terbinafine) and extracting squalene from the yeast. The method may include exposing a genetically converted yeast such as described herein to an antifungal agent (for example, an allylamine antifungal agent such as terbinafine) and extracting squalene from the yeast. The method may include exposing a non-genetically converted yeast such as described herein to an antifungal agent (for example, an allylamine antifungal agent such as terbinafine) and extracting squalene from the yeast.

[0024] In the methods and compositions disclosed herein that include an antifungal agent (for example, an allylamine antifungal agent such as terbinafine), the antifungal agent (for example terbinafine or other antifungal agent) may be added or present in a concentration at or above about 1 μg/ml; or about 5 μg/ml; or about 10 μg/ml; or about 11 μg/ml; or about 12 μg/ml; or about 12.5 μg/ml; or about 13 μg/ml; or about 15 μg/ml; or about 16 μg/ml; or about 20 μg/ml; or about 25 μg/ml; or about 30 μg/ml; or about 40 μg/ml; or about 50 μg/ml or greater. In the methods and compositions disclosed herein that include an antifungal agent (for example, an allylamine antifungal agent such as terbinafine), the antifungal agent (for example terbinafine or other antifungal agent) may be added or present in a concentration between about 0.5 to 100 μg/ml; or 0.5 to 50 μg/ml; or 1 to 50 μg/ml; or 5 to 50 μg/ml; or 8 to 50 μg/ml; or 10 to 50 μg/ml; or 12 to 50 μg/ml; or 15 to 50 μg/ml; or 15 to 50 μg/ml; or25 to 50 μg/ml; or 1 to 25 μg/ml; or5 to 25 μg/ml; or 10 to 25 μg/ml; or 10 to 20 μg/ml; or 10 to 15 μg/ml.

[0025] Disclosed herein is a genetically converted yeast that produces isoprenoids. In certain examples, the genetically converted yeast produces squalene.

[0026] Further disclosed herein is a genetically converted yeast, wherein the yeast is genetically converted such that

EP 2 504 421 Β1 it produces increased levels of squalene as compared to the corresponding native yeast. In certain examples, the genetically converted yeast expresses one or more modified enzymes having one or more mutations. In certain examples the expression level of one or more enzymes in the genetically converted yeast is increased or decreased relatíve to the corresponding native yeast. In related examples, the genetically converted yeast expresses one or more modified enzymes having one or more mutations and the expression level ofone or more enzymes in the genetically converted yeast is increased or decreased relatíve to the corresponding native yeast. In certain preferred examples a genetically converted yeast as disclosed herein is genetically converted by introducing a mutation intő an enzyme using a gene repair oligobase. In somé examples a genetically converted yeast as disclosed herein is genetically converted by introducing one or more mutations ator around the translation start site ofa gene encoding an enzyme to increase or decrease expression of the enzyme, for example, as described in US Patent Application Nos. 10/411,969 and 11/625,586. In certain examples, the enzyme modified in a genetically converted yeast as disclosed herein includes one or more enzymes selected from the group consisting of acetyl-CoA carboxylase (or ACCase), HMG-CoA reductase, squalene epoxidase, squalene synthase, ATP citrate lyase, ATP citrate synthase, mevalonate kinase (e.g., Y. lipolytica mevalonate kinase (Genolevures YALI0B16038g)), glycerol kinase (e.g., Y. lipolytica glycerol kinase (Genolevures YALI0F00484g) and 5-aminolevulinate synthase.

[0027] A nucleobase comprises a base, which is a purine, pyrimidine, or a derivative or analóg thereof. Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2’-deoxyriboside. Nucleosides can be linked by one of several linkage moieties, which may or may nőt contain phosphorus. Nucleosides that are linked by unsubstituted phosphodiester linkages are termed nucleotides. Nucleobases as used herein include peptide nucleobases, the subunits of peptide nucleic aeids, and morpholine nucleobases as well as nucleosides and nucleotides.

[0028] An oligonucleobase is a polymer of nucleobases, which polymer can hybridize by Watson-Crick base pairing to a DNA having the complementary sequence. An oligonucleobase chain has a single 5’ and 3’ terminus, which are the ultimate nucleobases ofthe polymer. A particular oligonucleobase chain can contain nucleobases ofall types. An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that are complementary and hybridized by Watson-Crick base pairing. Nucleobases are either deoxyribo-typeorribo-type. Ribo-type nucleobases are pentosefuranosyl containing nucleobases wherein the 2’ carbon is a methylene substituted with a hydroxyl, alkyloxy or halogén. Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases that do nőt contain a pentosefuranosyl moiety.

[0029] An oligonucleobase strand generically includes both oligonucleobase chains and segments or regions of oligonucleobase chains. An oligonucleobase strand has a 3’ end and a 5’ end. When an oligonucleobase strand is coextensive with a chain, the 3’ and 5’ ends of the strand are alsó 3’ and 5’ termini of the chain.

[0030] The term gene repair oligonucleobase is used herein to denote oligonucleobases, including mixed duplex oligonucleotides, non-nucleotide containing molecules, single stranded oligodeoxynucleotides and other gene repair molecules as described in detail below.

[0031] In somé examples, a genetically converted yeast or non-genetically converted yeast as disclosed herein is derived from an oleaginous yeast. In certain preferred examples, a genetically converted yeast or non-genetically converted yeast as disclosed herein is derived from a yeast selected from the group consisting of Cryptococcus curvatus, Yarrowia lipolytica, Rhodotorula glutinus, and Rhodosporidium toruloides. In somé preferred examples, the genetically converted yeast or non-genetically converted yeast is derived from a yeast selected from the group consisting of Cryptococcus curvatus, Yarrowia lipolytica, and Rhodotorula glutinus. In related examples, the genetically converted yeast or non-genetically converted yeast is derived from a yeast selected from the group consisting of Cryptococcus curvatus, and Rhodotorula glutinus. In certain preferred examples, the genetically converted yeast or non-genetically converted yeast is nőt derived from Yarrowia lipolytica. The genetically converted yeast used in the methods and compositions according to the present invention is a Yarrowia lipolytica strain selected from the group consisting of

ATCC 90812, ATCC MYA-2613, and Yeastern polg.

[0032] In certain preferred examples, an enzyme that is modified in a genetically converted yeast as disclosed herein is acetyl-CoA carboxylase (or ACCase). In somé preferred examples, acetyl-CoA carboxylase in a genetically converted yeast is modified such that its activity and/or expression is decreased relatíve to the corresponding native yeast; or such that the activity and/or expression is eliminated. In other examples, the acetyl-CoA carboxylase may be modified so that its substrate selectivity is altered. In somé preferred examples, the genetically converted yeast is modified such that the activity and/or expression of acetyl-CoA carboxylase is reduced relatíve to the corresponding native yeast bút the activity is nőt eliminated. In somé preferred examples, the genetically converted yeast is modified such that the activity and/or expression of acetyl-CoA carboxylase in the genetically converted yeast is reduced to about 90%; or about 80%; or about 70%; or about 60%; or about 50%; or about 40%; or about 30%; or about 20%; or about 10%; or about 5% ofthe activity and/or expression of the corresponding native yeast. In related examples, the genetically converted yeast is modified such that the activity and/or expression of acetyl-CoA carboxylase in the genetically converted yeast is between about 90-95%; or about 80-90%; or about 70-80%; or about 60-70%; or about 50-60%; or about 40-50%; or about

EP 2 504 421 Β1

30-40%; or about 20-30%; or about 10-20%; or about 5-10%; or about 2-5% of the activity and/or expression of the corresponding native yeast.

[0033] In certain preferred examples, an enzyme that is modified in a genetically converted yeast as disclosed herein is HMG-CoA reductase. In somé preferred examples, HMG-CoA reductase in a genetically converted yeast is modified such that its activity and/or expression is increased relatíve to the corresponding native yeast. In other examples, the HMG-CoA reductase may be modified so that it substrate selectivity is altered. In certain preferred examples, the genetically converted yeast is modified such that the activity and/or expression of HMG-CoA reductase in the genetically converted yeast is increased to at least 1.2-fold; or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or 10-fold; or 15-fold; or 20-fold; or 50-fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than the activity and/or expression ofthe corresponding native yeast.

[0034] An enzyme that is modified in the genetically converted yeast used in the methods and compositions according to the present invention is squalene epoxidase. In somé preferred examples, squalene epoxidase in a genetically converted yeast is modified such that its activity and/or expression is decreased relatíve to the corresponding native yeast; or such that the activity and/or expression is eliminated. In other examples, the squalene epoxidase may be modified so that its substrate selectivity is altered. In somé preferred examples, the genetically converted yeast is modified such that the activity and/or expression of squalene epoxidase is reduced relatíve to the corresponding native yeast bút the activity is nőt eliminated. In certain examples, the squalene epoxidase is modified to include one or more mutations or homologs ofone or more mutations associated with increased sensitivity to terbinafine. In certain examples, the yeast is nőt Saccharomyces cerevisiae and the squalene epoxidase is modified to include the homologs of one or more of the following mutations associated with increased sensitivity to terbinafine in the Saccharomyces cerevisiae ERG1 gene: G30S, L37P, and R269G (see, e.g., Turnowsky, 2005, 2007 and 2008). In certain examples, the yeast is Y. lipolytica and the squalene epoxidase is modified to include the homologs of one or more of the following mutations associated with increased sensitivity to terbinafine in the Saccharomyces cerevisiae ERG1 gene: G30S, L37P, and R269G (see, e.g., Turnowsky, 2005, 2007 and 2008). In somé examples, the yeast squalene epoxidase gene is modified as described herein by synthesis and replacement of the wild-type gene or by introduction of mutations by RTDS. In somé preferred examples, the genetically converted yeast is modified such that the activity and/or expression of squalene epoxidase in the genetically converted yeast is reduced to about 90%; or about 80%; or about 70%; or about 60%; or about 50%; or about 40%; or about 30%; or about 20%; or about 10%; or about 5% ofthe activity and/or expression ofthe corresponding native yeast. In related examples, the genetically converted yeast is modified such that the activity and/or expression of squalene epoxidase in the genetically converted yeast is between about 90-95%; orabout 80-90%; or about 70-80%; or about 60-70%; or about 50-60%; or about 40-50%; or about 30-40%; or about 20-30%; or about 10-20%; or about 5-10%; or about 2-5% of the activity and/or expression ofthe corresponding native yeast.

[0035] In certain preferred examples, an enzyme that is modified in a genetically converted yeast as disclosed herein is squalene synthase. In somé preferred examples, squalene synthase in a genetically converted yeast is modified such that its activity and/or expression is increased relatíve to the corresponding native yeast. In other examples, the squalene synthase may be modified so that it substrate selectivity is altered. In certain preferred examples, the genetically converted yeast is modified such that the activity and/or expression of squalene synthase in the genetically converted yeast is increased to at least 1.2-fold; or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or 10-fold; or 15-fold; or 20-fold; or 50fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than the activity and/or expression ofthe corresponding native yeast.

[0036] In certain preferred examples, an enzyme that is modified in a genetically converted yeast as disclosed herein is ATP citrate lyase. In somé examples, eitheror both subunits of ATP citrate lyase genes (fór example, Yarrowia lipolytica ATP citrate lyase; Genoleveres YALI0D24431g and YALI0E34793g) are modified as described herein. In certain examples, the activity of ATP citrate lyase in a modified yeast is increased by the insertion and/or heterologous expression ofan animal ATP lyase gene which comprises a singie subunit holoenzyme. In somé preferred examples, ATP citrate lyase in a genetically converted yeast is modified such that its activity and/or expression is increased relatíve to the corresponding native yeast. In certain preferred examples, the genetically converted yeast is modified such that the activity and/or expression of ATP citrate lyase in the genetically converted yeast is increased to at least 1.2-fold; or 1.5fold; or 2-fold; or 3-fold; or 4- fold; or 5- fold; or 10-fold; or 10-fold; or 20-fold; or 50-fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than the activity and/or expression ofthe corresponding native yeast.

[0037] In certain examples ofthe compositions and methods disclosed herein, the enzyme that is modified in a genetically converted yeast or non-genetically converted yeast is ATP citrate synthase. Preferably, its activity and/or expression is increased relatíve to the corresponding native yeast.

[0038] In certain preferred examples, an enzyme that is modified in a genetically converted yeast as disclosed herein is mevalonate kinase (e.g., Y .lipolytica mevalonate kinase (Genolevures YALI0B16038g)). In somé preferred examples, nevalonate kinase (e.g., Y. lipolytica mevalonate kinase (Genolevures YALI0B16038g)) in a genetically converted yeast

EP 2 504 421 Β1 is modified such that its activity and/or expression is increased relatíve to the corresponding native yeast. In certain preferred examples, the genetically converted yeast is modified such that the activity and/or expression of mevalonate kinase (e.g., Y. lipolytica mevalonate kinase (Genolevures YALI0B16038g)) in the genetically converted yeast is increased to at least 1,2-fold; or 1,5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or 10-fold; or 15-fold; or 20-fold; or 50fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than the activity and/or expression ofthe corresponding native yeast.

[0039] In certain preferred examples, an enzyme that is modified in a genetically converted yeast as disclosed herein is glycerol kinase (e.g., Y. lipolytica glycerol kinase (Genolevures YALI0F00484g)). In somé preferred examples, glycerol kinase (e.g., Y .lipolytica glycerol kinase (Genolevures YALI0F00484g)) in a genetically converted yeast is modified such that its activity and/or expression is increased relatíve to the corresponding native yeast. In certain preferred examples, the genetically converted yeast is modified such that the activity and/or expression of mevalonate kinase glycerol kinase (e.g., Y. lipolytica glycerol kinase (Genolevures YALI0F00484g)) in the genetically converted yeast is increased to at least 1,2-fold; or 1,5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or 10-fold; or 15-fold; or 20-fold; or 50fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than the activity and/or expression ofthe corresponding native yeast.

[0040] In certain examples ofthe compositions and methods disclosed herein, the enzyme that is modified in a genetically converted yeast or non-genetically converted yeast is 5-aminolevulinate synthase (e.g., encoded by Saccharomyces cerevisiae HEM1 gene). Preferably, its activity and/or expression is increased relatíve to the corresponding native yeast.

[0041] In certain preferred examples disclosed herein, the converted yeast is a genetically converted yeast; in other preferred examples, the genetically converted yeast is a transgenic yeast. Further examples are a yeast that includes both transgenic and genetic alterations.

[0042] In certain examples, disclosed are compositions that include a yeast (fór example a genetically converted yeast such as disclosed herein óra non-genetically converted yeast) wherein at least 10% ofthe totál lipid content is squalene; or at least 20% ofthe totál lipid content is squalene; or at least 25% ofthe totál lipid content is squalene; or at least 28% ofthe totál lipid content is squalene; or at least 30% ofthe totál lipid content is squalene; or at least 32% ofthe totál lipid content is squalene; or at least 35% of the totál lipid content is squalene; or at least 37% of the totál lipid content is squalene; or at least 38% of the totál lipid content is squalene; or at least 40% of the totál lipid content is squalene; or at least 42% ofthe totál lipid content is squalene; or at least 45% ofthe totál lipid content is squalene; or at least 47% ofthe totál lipid content is squalene; or at least 50% ofthe totál lipid content is squalene; or at least 52% ofthe totál lipid content is squalene; or at least 55% of the totál lipid content is squalene; or at least 57% of the totál lipid content is squalene; or at least 60% or more ofthe totál lipid content is squalene.

[0043] The phrase genetically converted yeast or genetically altered yeast as used herein refers to a yeast having one or more genetic modifications, such as transgenes and/or modified enzymes which contain one or more designed mutation(s). Such designed mutations may result in a modified enzyme having an activity that is different from the native enzyme. Such differences can include differences in substrate specificity or level of activity. As used herein, a transgenic yeast is one type ofa genetically converted yeast.

[0044] The term native yeast as used herein refers to a yeast that is nőt genetically converted (i.e., transgenic or genetically altered). Native yeasts include wild type yeasts as well as yeasts that have been selectively bred to attain particular characteristics.

[0045] The phrase transgenic yeast refers to a yeast having a gene from another yeast species or non-yeast species. Such a gene may be referred to as a transgene.

[0046] As used herein the term target gene refers to the gene encoding the enzyme to be modified.

[0047] The phrase oleaginous yeast refers to a yeast that contains at least about 20% cell dry weight (cdw) lipid extractable from the organism. The capacity to accumulate levels of lipid at least about 20% cdw is nőt confined to a particular genus; greaterthan about 20% cdw lipid has been reported in Lipomyces lipofer, L. starkeyi, L. tetrasporus, Candida lipolytica, C. diddensiae, C. paralipolytica, C. curvata, Cryptococcus albidus, Cryptococcus laumentii, Geotrichum candidum, Rhodotorula graminus, Trichosporon pullulans, Rhodosporidium toruloides, Rhodotorula glutinus, Rhodotorula gracilis, and Yarrowia lipolytica. See, e.g., Tatsumi, etal. U.S. Pat. No. 4,032,405, and Rattray, Microbial Lipids, Vol. 1 (1998).

[0048] The term about as used herein means in quantitative terms plus or minus 10%. Fór example, about 3% would encompass 2.7-3.3% and about 10% would encompass 9-11%.

[0049] Unless otherwise indicated, any percentages stated herein are percent by weight.

[0050] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.

EP 2 504 421 Β1

DETAILED DESCRIPTION OF THE INVENTION

Types of Yeast.

[0051] The compositions and methods as disclosed herein can be based on any of a number of yeast species or strains. In certain examples, the yeast is an oleaginous yeast. For example the yeast may be Cryptococcus curvatus (for example ATCC 20508), Yarrowia lipolytica (for example ATCC 20688 or ATCC 90811), Rhodotorula glutinus (for example ATCC 10788 or ATCC 204091), and Rhorosporidium toruloides. The inventors have discovered that, relatíve to certain other yeast (such as Yarrowia lipolytica), Cryptococcus curvatus and Rhodotorula glutinis grow to very high celi densities on a wide variety of substrates, and produce large amounts of totál lipid under many culture conditions. Accordingly, in certain examples Cyptococcus curvatus and Rhodotorula glutinis may be particularly advantageous for the compositions and methods as disclosed herein. There are many genetic tools (for example, transformation protocols, selectable markers) that are well developed and specific for Yarrowia lipolytica; as such in somé embodiments Yarrowia lipolytica may be particularly advantageous for the compositions and methods as disclosed herein. In the compositions and methods according to the present invention, the genetically converted yeast is a Yarrowia lipolytica strain selected from the group consisting of ATCC 90812, ATCC MYA-2613, or Yeastern polg.

Gene repair oligonucleobases [0052] The methods disclosed herein may be practiced with gene repair oligonucleobases having the conformations and chemistries as described in detail below. The gene repair oligonucleobases include mixed duplex oligonucleotides, non-nucleotide containing molecules, single stranded oligodeoxynucleotides and other gene repair molecules described in the below noted patents and patent publications. The gene repair oligonucleobases have alsó been described in published scientific and patent literature using other names including recombinagenic oligonucleobases; RNA/DNA chimeric oligonucleotides; chimeric oligonucleotides; mixedduplexoligonucleotides(MDONs); RNA DNA oligonucleotides (RDOs); gene targeting oligonucleotides; genoplasts; single stranded modified oligonucleotides; Single stranded oligodeoxynucleotide mutational vectors; duplex mutational vectors; and heteroduplex mutational vectors.

[0053] Oligonucleobases having the conformations and chemistries described in U.S. Pat. No. 5,565,350 by Kmiec (Kmiec I) and U.S. Pat. No. 5,731,181 by Kmiec (Kmiec II) are suitable for use as gene repair oligonucleobases .

[0054] The gene repair oligonucleobases in Kmiec I and/or Kmiec II contain two complementary strands, one of which contains at least one segmentof RNA-type nucleotides (an RNAsegment) that are base paired to DNA-type nucleotides of the other strand.

[0055] Kmiec II discloses that purine and pyrimidine base-containing non-nucleotides can be substituted for nucleotides. Additiönai gene repair molecules that can be used are described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Patent No. PCT/US00/23457; and in International Patent Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789.

[0056] In one example, the gene repair oligonucleobase is a mixed duplex oligonucleotide in which the RNA-type nucleotides ofthe mixed duplex oligonucleotide are made RNase resistant by replacing the 2’-hydroxyl with a fluoro, chloro or bromo functionality or by piacing a substituent on the 2’-O. Suitable substituents include the substituents taught by the Kmiec II. Alternative substituents include the substituents taught by U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin Applications).

[0057] As used herein, a 2’-fluoro, chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a 2’-OH substituted with a substituent described in the Martin Applications or Sproat is termed a 2’-Substituted Ribonucleotide. As used herein the term RNA-type nucleotide means a 2’-hydroxyl or 2’-Substituted Nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester Iinkage or any of the non-natural linkages taught by Kmiec I or Kmiec II. As used herein the term deoxyribo-type nucleotide means a nucleotide having a 2’-H, which can be linked to other nucleotides of a gene repair oligonucleobase by an unsubstituted phosphodiester Iinkage or any of the non-natural linkages taught by Kmiec I or Kmiec II.

[0058] In a particuíar example disclosed herein, the gene repair oligonucleobase is a mixed duplex oligonucleotide that is linked solely by unsubstituted phosphodiester bonds. In alternative examples, the Iinkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus-based linkages as taught by Kmiec II. In yet another example, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2’-Substituted Nucleotide. Particuíar preferred examples of2’-Substituted Ribonucleotidesare2’-fluoro, 2’-methoxy, 2’-propyloxy, 2’-allyloxy, 2’-hydroxylethyloxy, 2’-methoxyethyloxy, 2’-fluoropropyloxy and 2’-trifluoropropyloxy substituted ribonucleotides. More preferred examples of 2’-Substituted Ribonucleotides are 2’-fluoro, 2’-methoxy, 2’-methoxyethyloxy, and 2’-allyloxy substituted nucleotides.

EP 2 504 421 Β1

In another example, the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds.

[0059] Although mixed duplex oligonucleotides having only a single type of 2’-substituted RNA-type nucleotide are more conveniently synthesized, the methods disclosed herein can be practiced with mixed duplex oligonucleotides having two or more types of RNA-type nucleotides. The function ofan RNA segment may nőt be affected by an interruption caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides, accordingly, the term RNA segment encompasses such as interrupted RNA segment. An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative example, an RNA segment can contain alternating RNase-resistant and unsubstituted 2’-OH nucleotides. The mixed duplex oligonucleotides preferably have fewer than 100 nucleotides and more preferably fewer than 85 nucleotides, bút more than 50 nucleotides. The first and second strands are Watson-Crick base paired. In one example, the strands ofthe mixed duplex oligonucleotide are covalently bonded by a linker, such as a single stranded hexa, penta ortetranucleotide so that the first and second strands are segments ofa single oligonucleotide chain having a single 3’ and a single 5’ end. The 3’ and 5’ ends can be protected by the addition of a hairpin cap whereby the 3’ and 5’ terminál nucleotides are Watson-Crick paired to adjacent nucleotides. A second hairpin cap can, additionally, be placed at the junction between the first and second strands distant from the 3’ and 5’ ends, so that the Watson-Crick pairing between the first and second strands is stabilized.

[0060] The first and second strands contain two regions that are homologous with two fragments of the target gene, i.e., have the same sequence as the target gene. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of connecting DNA segment and may alsó contain DNA-type nucleotides that are nőt within the intervening DNA segment. The two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a heterologous region. The heterologous region can contain one, two or three mismatched nucleotides. The mismatched nucleotides can be contiguous or alternatively can be separated by one ortwo nucleotides that are homologous with the target gene. Alternatively, the heterologous region can alsó contain an insertion orone, two, three orof five or fewer nucleotides. Alternatively, the sequence ofthe mixed duplex oligonucleotide may differ from the sequence ofthe target gene only by the deletion of one, two, three, orfive orfewer nucleotides from the mixed duplex oligonucleotide. The length and position ofthe heterologous region is, in this case, deemed to be the length ofthe deletion, even though no nucleotides ofthe mixed duplex oligonucleotide are within the heterologous region. The distance between the fragments ofthe target gene that are complementary to the two homologous regions is ídentically the length of the heterologous region when a substitution or substitutions is intended. When the heterologous region contains an insertion, the homologous regions are thereby separated in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene, and the converse is applicable when the heterologous region encodes a deletion.

[0061] The RNA segments ofthe mixed duplex oligonucleotides are each a part ofa homologous region, i.e., a region that is identical in sequence to a fragment ofthe target gene, which segments together preferably contain at least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-type nucleotides or yet more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides. In one example, RNA segments of the homology regions are separated by and adjacent to, i.e., connected by an intervening DNA segment. In one example, each nucleotide ofthe heterologous region is a nucleotide of the intervening DNA segment. An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a mutator segment.

[0062] In another example disclosed herein, the gene repair oligonucleobase is a single stranded oligodeoxynucleotide mutational vector (SSOMV), which is disclosed in International Patent Application PCT/US00/23457, U.S. Pat. Nos. 6,271,360, 6,479,292, and 7,060,500 . The sequence ofthe SSOMV is based on the same principles as the mutational vectors deseribed in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789. The sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired genetic alteration termed the mutator region. The mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, bút having a different sequence. Such a mutator region can cause a substitution. Alternatively, the homologous regions in the SSOMV can be contiguous to each other, while the regions in the target gene having the same sequence are separated by one, two or more nucleotides. Such a SSOMV causes a deletion from the target gene of the nucleotides that are absent from the SSOMV. Lastly, the sequence of the target gene that is identical to the homologous regions may be adjacent in the target gene bút separated by one two or more nucleotides in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of target gene.

[0063] The nucleotides ofthe SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3’ terminál and/or 5’ terminál internucleotide linkage or alternatively the two 3’ terminál and/or 5’ terminál internucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an internucleotide linkage is the linkage between nucleotides ofthe SSOMV and does nőt include the linkage between the 3’ end nucleotide or 5’ end nucleotide and a blocking substituent, see supra. In a specific example the length ofthe SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a totál length of at least 20 deoxynucle9

EP 2 504 421 Β1 otides and at least two homology regions shouid each have lengths of at least 8 deoxynucleotides.

[0064] The SSOMV can be designed to be complementary to either the coding or the non-coding strand ofthe target gene. When the desired mutation is a substitution of a single base, it is preferred that both the mutator nucleotide be a pyrimidine. To the extent that is consistent with achieving the desired functional result it is preferred that both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines. Particularly preferred are SSOMV that encode transversion mutations, i.e., a C orT mutator nucleotide is mismatched, respectively, with a C orT nucleotide in the complementary strand.

[0065] In addition to the oligodeoxynucleotide the SSOMV can contain a 5’ blocking substituent that is attached to the 5’ terminál carbons through a linker. The chemistry ofthe linker is nőt critical other than its length, which shouid preferably be at least 6 atoms long and that the linker shouid be flexible. Avarietyof non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used. Particularly preferred as reagents to make SSOMV are the reagents sold asCy3™ and Cy5™ by Glen Research, Sterling Va., which are blocked phosphoroamidites that upon incorporation intő an oligonucleotide yield 3,3,3’,3’-tetramethyl Ν,Ν’-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is the most preferred. When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5’ terminál ofthe oligodeoxynucleotide through as a phosphodiester with a 5’ terminál phosphate. The chemistry of the dye linker between the dye and the oligodeoxynucleotide is nőt critical and is chosen fór synthetic convenience. When the commercially available Cy3 phosphoramidite is used as directed the resulting 5’ modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, N’phosphatidylpropyl 3,3,3’,3’-tetramethyl indomonocarbocyanine.

[0066] In one preferred example, the indocarbocyanine dye is tetra substituted at the 3 and 3’ positions ofthe indole rings. Without limitations as to theory these substitutions prevent the dye from being an intercalating dye. The identity ofthe substituents as these positions are nőt critical. The SSOMV can in addition have a 3’ blocking substituent. Again the chemistry ofthe 3’ blocking substituent is nőt critical.

Heterologous Expression [0067] In certain examples, heterologous expression is used to express foreign genes or extra copies of endogenous genes in yeast (fór example, Yarrowia lipolytica). Heterologous expression in yeast can be performed using methods well known in the art. Expression of foreign genes or extra copies of endogenous genes in yeast using heterologous expression may involve use of a vector that includes (a) promoter sequences fór transcriptional initiation, (b) terminátor sequences fór termination of transcription, and (c) a selectable marker. Heterologous expression and expression vectors may be as described, fór example, in Madzak, C., Gaillardin, C., and Beckerich, J-M., 2004 Heterologous Protein Expression and Secretion in the Non-Conventional Yeast Yarrowia lipolytica: a review, Journal of Biotechnology 109:63-81. In certain examples of the compositions and methods disclosed herein, the vector is pYLEXI (Yeastern). A non-limiting list of selectable marker genes that may be used includes ura3, Iys5, trp1, Ieu2, ade1, E.coli hph encoding hygromycin resistance, and SUC2 from Saccharomyces cerevisiae. A non-limiting list of promoters that may be used includes pLEU2, pXPR2, pPOX2, pPOT1,plCL1, pG3P, pMTP, pTEF, and pRPS7. In certain examples, the promoter is the hp4d promoter, which is a strong, constitutive hybrid promoter (U.S. Patent 6,083,717 issued Jul.4,2000). A nonlimiting list of terminátor sequences that may be used includes XPR2t, LIP2t, and PHO5t.

[0068] Incertain examples, oneormoreot Yarrowia lipolytica LYS1 (Geno/evures YALI0B15444g), TRP1 (Genolevures YALIOB07667g), and ADE1 (Genolevures YALI0E33033g) genes are used as selectable markers. In certain examples, one or more of Yarrowia lipolytica URA3 (GenBank: U40564.1) or LEU2 (Genoluveres YALI0C00407) genes are used as selectable markers.

[0069] In certain examples, an integrative expression vector includes one or more promoters and/or terminátor sequences selected from the group consisting of Yarrowia lipolytica glycolytic pathway genes, alkane or glycerol utilization genes, XPR2, ACC1, HMG1, ERG1, and ERG9.

[0070] In certain examples of one or both subunits of Yarrowia lipolytica ATP citrate lyase (Genoleveres YALI0D24431 g and YALI0E34793g) in Yarrowia lipolytica are overexpressed.

Modified enzymes [0071] A modified or mutated enzyme of the present disclosure can be modified or mutated by base pair changes, insertions, substitutions, and the Iike.

[0072] The genes encoding enzymes involved in the fatty acid biosynthesis pathway and isoprenoid biosynthesis pathway are the preferred targets fór mutation. In somé examples, the target gene encodes an acyl CoA carboxylase. In other examples, the target gene encodes an HMG-CoA reductase. In other examples, the target gene encodes a squalene epoxidase. In other examples, the target gene encodes a squalene synthase. In certain examples, the target gene encodes ATP citrate lyase. Mutations can be designed that reduce or eliminate the activity of an enzyme, enhance

EP 2 504 421 Β1 the activity of an enzyme, or that altér the activity of the enzyme (e.g., change the substrate selectivity).

[0073] In wild-type oleaginous yeast, acetyl-CoA is extensively channeled into fatty acid biosynthesis via acetyl-CoA carboxylase (ACCase). Thus in orderto increase the amountofacetyl-CoA available for squalene synthesis, it is desirable to reduce the enzymatic expression or specific activity of ACCase. An exemplary gene sequence for ACCase is the ACC1 gene in Saccharomyces cerevisiae as shown in accession number Z71631. Accordingly in certain examples, reduced intracellular activities of ACCase, the enzyme at the branch point between mevalonate biosynthesis and triglyceride biosynthesis will decrease the amount of acetyl-CoA partitioned for oil synthesis, thereby increasing its availa bility to the isoprene pathway.

[0074] HMG-CoA reductase activity is the rate-limiting enzyme for isoprene biosynthesis. Exemplary gene sequences for HMG-CoA reductase include the HMG1 and HMG1 genes in Sacchanomyces cerevisiae as shown in accession numbers NC_001145 and NC_001144, respectfully. Accordingly, in certain examples, HMG-CoA reductase activity will be increased by modifying the HMGR gene to increase transcription, stabilize the resultant protein, and/or reduce product feedback inhibition.

[0075] Decreasing ACCase activity and/or increasing HMG-CoA reductase activity in a yeast can create a core isoprenoid production organism capable of producing a number of related isoprenoid products by the manipulation of subsequent enzymes in the pathway.

[0076] Squalene epoxidase catalyzes the first committed step of sterol biosynthesis. An exemplary gene sequence for Squalene epoxidase is the ERG1 gene in Saccharomyces cerevisiae as shown in accession number NC_001139. Accordingly, in certain examples, squalene epoxidase activity, sensitivity to inhibitors and/or expression will be attenuated in a yeast, for example by catalytically important residues in the enzyme’s amino acid sequence.

[0077] Squalene synthase catalyzes the synthesis of squalene by condensing two c15 isoprene precursors (farnesyl diphosphate (FPP)). An exemplary gene sequence for squalene synthase is the ERG9gene in Saccharomyces cerevisiae as shown in accession number NC_001140. Accordingly, in certain examples, squalene synthase activity and/or expression will be increased in a yeast.

[0078] ATP citrate lyase (E.C. 4.1.3.8) catalytically cleaves citrate to produce acetyl CoA and oxaloacetate. Acetyl CoA can be used by ACCase for fatty acid biosynthesis orby acetyl CoA acetyl transferase for the production of isoprenes and derivatives such as squalene.

[0079] Mevalonate kinase is the first enzyme after HMG-CoA Reductase in the mevalonate pathway, and catalyzes the conversion of Mevalonate to Mevalonate-5-phosphate. Accordingly, in certain examples, mevalonate kinase activity and/or expression levels will be increased in yeast, for example, by changing catalytically important residues in the enzyme’s amino acid sequence or increasing its gene dosage or transcript levels.

[0080] Glycerol kinase catalyzes the transfer of a phosphate from ATP to glycerol to form glycerol phosphate. Accordingly, in certain examples, glycerol kinase activity and/or expression levels will be increased in yeast, for example by changing catalytically important residues in the enzyme’s amino acid sequence or increasing its gene dosage or transcript levels.

[0081] The result ofthe metabolic changes in certain examples will be to channel carbon from acetyl-CoAto squalene, and attenuate major com petitive pathways for this carbon stream, resulting in a significant increase of squalene produced.

Delivery of gene repair oligonucleobases into yeast cells [0082] Any commonly known method can be used in the methods disclosed herein to transform a yeast cell with a gene repair oligonucleobase. Exemplary methods include the use of electroporation, LiOAc, biolistics, spheroplasting, and/or Agrobacterium (see, for example, McClelland, C.M., Chang, Y.C., and KwonChung, K.J. (2005) Fungal Genetics and Biology 42:904-913).

[0083] In certain examples, a gene repair oligonucleobase is introduced into a yeast cell by electroporation. In somé examples, a gene repair oligonucleobase is introduced into a yeast cell that has been chemically treated with PEG (3350 or4000 mw) and/or Lithium Acetate by electroporation. In certain examples, a gene repair oligonucleobase is introduced into a yeast cell using PEG (3350 or 4000 mw) and/or Lithium Acetate.

[0084] Specific conditions for using microcarriers in the methods disclosed herein are described in International Publication WO 99/07865, US09/129,298. For example, ice cold microcarriers (60 mg/mL), mixed duplex oligonucleotide (60 mg/mL), 2.5 M CaCI₂ and 0.1 M spermidine are added in that order; the mixture gently agitated, e.g., by vortexing, for 10 minutes and let stand at room temperature for 10 minutes, whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol. Exemplary concentrations ofthe components in the adhering solution include 8-10 μg/μL microcarriers, 14-17 μg/μL mixed duplex oligonucleotide, 1.1-1.4 M CaCI₂ and 18-22 mM spermidine. In one example, the component concentrations are 8 μg/μL microcarriers, 16.5 μg/μL mixed duplex oligonucleotide, 1.3 M CaCI₂ and 21 mM spermidine.

[0085] In somé examples, gene repair oligonucleobases can be delivered to the yeast cell by electroporation, according to techniques well known to those skilled in the art. (See, e.g. Becker, D. M., and Guarente, L. High Efficiency Trans11

EP 2 504 421 Β1 formation ofYeast by Electroporation. Methods in Enzymology, vol. 194, section [12] pp. 182-186. 1991. Elsevier Academic Press, London.

Selection of yeast having the desired modified enzyme [0086] Yeast expressing the modified enzyme can be identified through any of a number of means. In one method, a co-conversion strategy using gene repair oligonucleobases (GRONs) to target both a selectable conversion (i.e., a marker) and a non-selectable conversion (e.g., a target gene of interest) in the same experiment. In this way, the cells to which GRONs were nőt delivered or were unable to transmit the conversions specified by the GRON would be eliminated. Since delivery of GRONs targeting unrelated genes is nőt expected to be selective, at somé frequency, a colony with a successfully selected conversion would alsó be expected to have a conversion in one ofthe other targeted genes. Conversion events would be resolved by single nucleotide polymorphism (SNP) analysis.

[0087] Thus, genomic DNA is extracted from yeast and sereening ofthe individual DNA samples using a SNP detection technology, e.g., allele-specific Polymerase Chain Reaction (ASPCR), for each target. To independently confirm the sequence change in positive yeast, the appropriate region ofthe target gene may be PCR amplified and the resulting amplicon either sequenced directly or cloned and multiple inserts sequenced.

[0088] Alternatively, the incorporation ofthe mutation intő the gene of interest can be identified by any of a number of molecular biology techniques designed to detect single nucleotide mutations in extracted nucleic acid (e.g., amplification methods such as PCR and single nucleotide primer extension analysis). Larger mutations can be detected by amplification and sequencing of the region of the target gene to be mutated.

[0089] Alternatively, yeast or yeast cells containing the modified enzyme can be identified by, for example, analysis ofthe composition of isoprenoids produced by the yeast. Thus, the yeast can be grown and oils extracted and analyzed using methods known in the art (e.g., gas chromatography or HPLC).

EXAMPLES

Example 1. Cryptococcus curvatus and Rhodotorula glutinis transformation systems. (nőt according to the present invention) [0090] To create a Cryptococcus curvatus (ATCC strain 20508) and Rhodotorula glutinis (ATCC strains 10788 and 204091) transformation system, a KANMX expression cassette (promoter-gene-terminator) which confers kanamycin resistance to S. cerevisiae is used as a selectable marker to convert the strains from kanamycin sensitivity to resistance (See e.g., Baudin, A., et al. (1993) Nucleic Acids Research (21) 3329-3330). The strains are transformed with the expression cassette alone, as well as KANMX ligated to restriction fragments of a plasmid reported in R. glutinus (See e.g Óloké, J.K., and Glick, B.R. (2006) African Journal of Biotechnology 5(4):327-332) containing DNA origins of replication. DNA is introduced intő C. curvatus and R. glutinis by electroporation, LiOAc, biolistics, spheroplasting, and/or Agrobacterium (McClelland, C.M., Chang, Y.C., and Kwon-Chung, K.J. (2005) Fungal Genetics and Biology 42:904-913).

Example 2. Selectable Markers. (nőt according to the present invention) [0091] To generate uracil auxotrophic mutants in Cyptococcus curvatus and Rhodotorula glutinis, cells were grown in minimál média containing anti-metabolite 5-fluoroorotic acid to selectfor resistant mutants with lesions in the ura3 or ura5 genes. 33 stable 5-FOA^R colonies of Cryptococcus curvatus and 20 stable 5-FOA^R colonies oí Rhodotorula glutinis were banked. Wild type URA3 genes from both Cryptococcus curvatus and Rhodotorula glutinis are cloned and the mutant ura3 genes in the 5-FOA^R isolates are sequenced.

[0092] Other auxotrophic markers are cloned by functional complementation in Saccharomyces cerevisiae (See Ho, Y.R., and Chang, M.C. (1988) Chinese Journal of Microbiology and Immunology 21(1):1-8). Genomic and/or cDNA libraries are constructed from Cryptococcus curvatus and Rhodotorula glutinis for ligation intő a uracil-selectable Saccharomyces expression vector for transformation intő strain YPH500 (MATo, ura3-52 Iys2-8O1 ade2-101 trp1-\63 hits3Δ200 Ieu2-\1) to selectfor lysine, adenine, tryptophan, histidine, and leucine prototrophs. From these prototrophs, the corresponding genes for LYS2, ADE2, TRP1, HIS3, and LEU2 are sequenced from the genomic or cDNA insert.

Example 2. Gene Manipulation in Yeast using RTDS technology. (nőt according to the present invention) [0093] The alleles of the Ieu2, Iys5 and ura3 genes from Yarrowia lipolytica strain ATCC 90811 (leu2-35 Iys5-12 ura3-18 XPR2B) were cloned by PCR and their sequences compared to the wild type alleles to identify differences. [0094] For ura3, differences were found at positions 1365 (A^G mutation, resulting in a silent change of AAA—>AAG coding for lysine), 1503 (AAGAA extra sequences in ATCC 90811 which results in a frame change, bút which comes

EP 2 504 421 Β1 back in frame at 1511 resulting in 7 additional amino acids, after which the sequence continues as the YL URA3 in GenBank), 1511 (extra T in ATCC 90811), and 1978 (C^T mutation, leading to a stop mutation truncating the protein 24 amino acids short ofthe carboxy terminus). A GRON oligonucleotide was designed to restore prototrophy by converting STOP(TGA)^R (CGA) to yield 264R based on YIUra3 - YLU40564 amino acid numbering. The GRONs used are YIUra31264/C/40/5’Cy3/3’idC, which has the sequence

VCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGGH, and

YIUra31264/NC/40/5’Cy3/3’idC, which has the sequence

VCCTCCTCAATAGGATCTCGGTTCTGGCCGTACAGACCTCGH,where V=CY3;

H=3’DMT dC CPG. 10, 30, and 50 μg of each of the GRONs were transformed intő Yarrowia lipolytica strain ATCC 90811 using a Lithium acetate-based method, and plated onto ura- 2% glucose. A totál of 82 ura+ colonies were obtained with the GRON designed using the coding strand and 6 colonies with the GRON designed using the non-coding strand, demonstrating the strand bias common in transforming with gap-repairoligonucleotides. Sequencing of 18 ofthe codingstrand transformants demonstrated the intended change in 17 ofthe clones.

[0095] For LEU2 differences were found at positions 1710 (extra C absent leading to a frame shift and premature protein termination); 1896 (extra T); 2036 (T—>A mutation, located after the stop codon); 2177 (extra T in missing, located after stop codon).

[0096] For LEU2 differences were found at positions 1092 (G^A TCG^TCA, a conservative substitution (Serine)); 1278 (G^A CAG^CAA, a conservative substitution (Glutamine)); 1279 (G^A GGT^ATT, changing V^l).

[0097] Accordingly, the mutations can be used for various purposes, for example to convert prototrophic yeast to become auxotrophic and vice versa.

[0098] A similar strategy for demonstrating the effectiveness of RTDS technology in Cyptococcus curvatus and Rhodotorula glutinis is performed as described for Yarrowia lipolytica in which ura3 mutations are corrected to restore prototrophy.

[0099] In certain examples, the effectiveness of RTDS in Y. lipolytica may be demonstrated by integrating a mutated version ofthe E. co//hygromycin gene intő its genome. This version ofthe gene, which harbors a point mutation at G34T, encodes an E12STOP change such that the natural hygromycin sensitivity of Y. lipolytica is nőt affected. Transformation with a GRON correcting this mutation will confer resistance of the Y. lipolytica strain, for example, up to 1000 ug/ml of hygromycin. Double mutations in the hygromycin resistance (HGH) gene are alsó constructed, comprising of G34T A37T (E12ASTOP K13STOP) which may be corrected by a single GRON, and G34T T149G (E12STOP Y46 STOP) which may be corrected by 2 GRONS.

[0100] To testing GRON activity in Yarrowia, the natural sensitivity of wild-type Yarrowia lipolytica to the aminoglycoside antibiotic hygromycin B was used. Hygromycin B (hmB) is an aminocyclitol antibiotic produced by Streptomyces hygroscopicus which inhibits protein synthesis in both procaryotes and eucaryotes by interfering with ribosomal translocation and with aminoacyltRNA recognition. Resistance can be conferred by introduction of the hph gene (alsó known as aph(4)) from E. co//(GÉNBANK V01499) which encodes an aminocyclitol phoshotransferase that inactivates hygromycin B by covalent addition of a phosphate group to the 4-position of the cyclitol ring. Yarrowia lipolytica strain Polg (Mát a ura3-302: :URA3 Ieu2-27O xpr2-322 axp-2 from Yeastern) was transformed with the E. coli hph gene containing either a single (E12stopfrom G34T) or double mutation E12stopK13stop (G34T.A37T) mutations cloned intő vector pyLEX1-2uura3-13, putting the gene under control ofthe hpd4 promoter and XPR2 terminator. The linearized vector was integrated intő the genome upon selection for restoration of prototrophy conferred by the LEU2 marker. The resultant strains harbor disabled versions ofthe hygromycin phosphotransferase gene (hence hygromycin sensitive), and were converted with the following GRONs restoring either G34T or G34T.A37T to wild type (hygromycin resistant).

GRONs for restoring E12stop to wild type (T34G)

HPH2/C/42/5’Cy3/3’idC

5’Cy3-GAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAG-3’idC Η Ρ Η 2/N Cl42/5’ Cy 3/3’ id C

5’Cy3-HCTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAGTTC-3’idC

GRONs for restoring E12stopK13stop to wild type (T34G T37A)

HPH3/C/43/5’Cy3/3’idC

5’Cy3-CTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCG-3’idC

HPH3/NC/43/5’Cy3/3’idC

5’Cy3-CGAACTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAG-3’idC [0101] 3C^g of the indieated GRON was used to convert the single- or double- hph mutant strain in replicate (x6),

EP 2 504 421 Β1 pooled, and an aliquot plated onto YEPD plates containing 100-1000 μg/ml hygromycin to optimize the signal-to-noise ratio. With both strains, significant numbers of putatively converted colonies were obtained at any given hygromycin concentration above the ’No DNA’ control, with a strong bias toward the non-coding GRON strand in both cases. Taken together, these resuits suggest GRON conversion ofthe hygromycin phosphotransferase gene target in Yarrowia lipolytica, and further that conversion of two mutations (T34G T37A) can be accomplished using a single GRON. DNA sequencing is performed to confirm restoration ofthe wild-type genotype.

EP 2 504 421 Β1

<td> Colonies on 1000 μς/ιτιΙ Hygromycin</td><td> o</td><td> o</td><td> 00</td><td> o</td><td> CM</td><td> 00</td>

<td> Colonies on 800 μg/ml Hygromycin</td><td> o</td><td> -</td><td> o</td><td> o</td><td> O</td><td> 00</td>

<td> Colonies on 600 μg/ml Hygromycin</td><td> -</td><td> -</td><td> CM</td><td> -</td><td> CM</td><td></td>

<td> Colonies on 400 μg/ml Hygromycin</td><td> -</td><td> CD</td><td> CM</td><td> ’xl’</td><td> -</td><td> in</td>

<td> Colonies on 200 μg/ml Hygromycin</td><td> o</td><td> -</td><td> CM</td><td> o</td><td> CM</td><td> co</td>

<td> Colonies on 100 μg/ml Hygromycin</td><td> o</td><td> o</td><td> 20</td><td> o</td><td> CM</td><td> in</td>

<td> DNA</td><td> No DNA</td><td> 30μg coding strand</td><td> 1 C O Ό c c c cn ro 2 á- 8 ω CO</td><td> No DNA</td><td> 30μg coding strand</td><td> 1 C O Ό ccc cn ro 2 á- 8 ω CO</td>

<td> Strain</td><td> E12stop</td><td> E12stop</td><td> E12stop</td><td> E12stopK13stop</td><td> E12stopK13stop</td><td> E12stopK13stop</td>

EP 2 504 421 Β1

Example 3. Cioning of target genes. (nőt according to the present invention) [0102] The sequences for ACCase, HMGR, squalene synthase and squalene epoxidase, available in the NCBI database from Saccharomyces and other yeasts, are used as a source of PCR primers and the corresponding genes arc cloned from Cryptococcus curvatus and Rhodotorula glutinis along with their corresponding regulatory regions (promoters, terminators). To identify ’up’ and ’down’ promoter mutations that increase or decrease transeription, respectively, the promoters for these four genes are cloned with a relatively error-prone DNA polymerase to generate point mutations in the promoters, and these fragments are cloned intő plasmids fused with Green Fluorescent Protein (GFP) or betagalactosidase reporter genes for testing in vitro in S. cerevisiae or E. coli. Promoter up mutations are reintroduced intő the HMGR and squalene synthase genomic sequences by RTDS, while down promoter mutations are being made in the genomic ACCase and squalene epoxidase sequences. The promoters from essential genes (e.g. GAPDH, actin) in R. glutinis and C. curvatus are cloned for use in heterologous gene expression. Primers for PCR cioning are designed from homology to these genes in S. cerevisiae.

Example 4. Manipulation of target for increased squalene production.

[0103] ACCase. The number of copies of the ACCase gene is determined in R. glutinis and C. curvatus and other yeasts. RTDS is utilized to reduce ACCase expression by introducing stop codons immediately after the translational start site in any extra copies.

[0104] Squalene Epoxidase. Similarly, an increase in squalene accumulation in S. cerevisiae has been achieved by disruption ofone copy ofthe squalene epoxidase in the diploid. Kamimura, N., Hidaka, M., Masaki, H., and Uozumi, T. (1994) Appl. Microb. Biotech. 42: 353-357. The number of copies of squalene epoxidase in R. glutinis and C. curvatus and other yeasts is determined, and RTDS is used to create or insert a stop codon immediately after the translational start site in extra copies beyond the first one.

[0105] In somé examples, Squalene epoxidase activity is attenuated by addition of terbinafine (an inhibitor of Squalene epoxidase) to the média. In certain examples, amino acid changes to the Squalene epoxidase are made to increase the sensitivity of Squalene epoxidase to terbinafine (for example amino acid changes homologous to G30S, L37P, and R269G mapped on Saccharomyces ERG1). In somé examples, the amino acid changes are made by gene synthesis and replacement of the wild-type gene with the mutant version by homologous recombination. In other examples, the changes are introduced intő the wild-type gene by RTDS.

[0106] HMGR. Both Saccharomyces cerevisiae and mammalian HMGR enzymes contain amino acid sequences in their linker regions which are present in many short-lived proteins that are subject to rapid intracellular turnover in eukaryotes (see Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234: 364-368; and Chun, K.T., and Simoni, R.D. (1991) J. Bioi. Chem. 267(6): 4236-4246). Similar sequences, if present, are identified in the HMGR genes in Y. lipolytica, R. glutinis and/or C. curvatus, and eliminated using RTDS to reduce HMGR protein turnover. Such similar sequences have been found in the S. cerevisiae squalene synthase gene, and it is alsó determined ifsuch sequences are present in the squalene synthase genes in Y. lipolytica, R. glutinis and/or C. curvatus. The sequences, if present in Y. lipolytica, R. glutinis and/or C. curvatus squalene synthase, are alsó eliminated using RTDS to reduce protein turnover. [0107] HMGR in S. cerevisiae comprises two highly conserved domains, of which the N-terminal 552 amino aeids are responsible for membráné association. Overexpression ofthe truncated HMG1 protein containing oniy the C-terminal catalytic portion led a 40-fold increase of HMG-CoA activity in S. cerevisiae with an increased accumulation of squalene to 5.5% of dry matter (Polakowski, T., Stahl, U., and Láng, C. (1998) Appl. Microbiol. Biotech. 49:66-71). It is determined if Y, lipolytica, R. glutinis and C. curvatus HMGR proteins have a similar structure, and, if so, fragments having oniy the soluble catalytic domain may be expressed.

[0108] The protein structure and DNA sequence of HMGR is highly conserved between eukaryotes from fungi to mammals, with a membrane-associated N-terminal domain and catalytic C-terminal domain. The boundary between the two domains can be mapped to a region of amino aeids 500-600 in the Yarrowia lipolytica HMG1 gene (Genelouvres Yarrowia lipolytica YALI0E04807g) where the hydrophobicity plot transitions from hydrophobic to hydrophilic. Resides 548 and 544 are chosen from evaluation ofthe hydrophobicity plot of Yarrowia lipolytica HMG1, and its homology to the N-termini ofthe truncated Saccharomyces cerevisiae (Donald, K.A.G., etal, 1997. Appl. Environ. Micro. 63(9): 3341-3344) and Candida uf///s (Shimada, H. et al, 1998. Appl. Environ. Micro. 64(7):2676-2680) proteins. Accordingly, in one example, amino aeids 548-1000 ofthe C-terminal domain of Yarrowia lipolytica HMG11 is expressed; in a second example amino aeids 544-1000 ofthe C-terminal domain of Yarrowia lipolytica HMG11 is expressed. In related examples, amino aeids 543-1000 ofthe C-terminal domain of Yarrowia lipolytica HMG1 / is expressed; or amino aeids 545-1000 ofthe C-terminal domains of Yarrowia lipolytica HMG1 I is expressed; or amino aeids 546-1000 ofthe C-terminal domains of Yarrowia lipolytica HMG1 I is expressed; or amino aeids 547-1000 of the C-terminal domains of Yarrowia lipolytica HMG1 I is expressed; or amino aeids 549-1000 ofthe C-terminal domains of Yarrowia lipolytica HMG1 I is expressed.

[0109] Expression ofthe 457 amino-acid C-terminal catalytic domain of HMGR (residues 543-1000) in Y. lipolytica

EP 2 504 421 Β1 strain Polg yielded 2% squalene/total lipid compared to 0% in the control strain containing the vector alone in experiments using shakeflasks. The process is repeated and expanded using fermenters.

[0110] In Syrian hamsters, activity of the HMGR catalytic domain is down-modulated by phosphorylation by an AMPdependent kinase (Omkumar, R.V., Darnay, B.G., and Rodwell, V.W. (1994) J. Bioi. Chem. 269:6810-6814), and a similar mode of regulation has been described in S. cerevisiae. It is determined if the HMGR proteins in R. glutinis, C. curvatus and other yeasts are similarly regulated, and if so, RTDS is employed to eliminate the phosphorylation site. [0111] Squalene synthase. Squalene synthase in mammalian systems is coordinately regulated on the transcriptional level along with HMG-CoA synthase and farnesyl diphosphate synthase by SREBPs (sterol regulatory element binding proteins) (Szkopinsda, A., Swiezewska, E., and Karst, F (2000) Biochem. Biophys. Rés. Comm. 267:473-477). SREBPs exist in three forms, of which one binds the squalene synthase promoter. It is determined if such transcription factors and/or binding sites are present on the squalene synthase promoter in R. glutinis, C.curvatus and other yeasts, and, if present, RTDS is used to make changes to such transcription factors and/or binding sites that enhance transcription of squalene synthase.

[0112] Overexpression ofthe Y. lipolytica Squalene Synthase in Y. lipolytica strain Polg yielded 2% squalene/total lipid compared to 0% in the control strain containing the vector alone using shakeflasks. The process is repeated and expanded using fermenters.

Example 5. Growth Conditions fór Cryptococcus curvatus. (nőt according to the present invention) [0113] Cryptococcus curvatus growth was evaluated to determine the best carbon sources to maximize its cell mass in culture. In a Yeast Extract-based rich média (10 g/L yeast extract, 20 g/L peptone), C. curvatus grew well in 2-20% w/v glucose, achieving a maximai level of 55 g/L cell dry weight (CDW) at 16% w/v glucose and above after 4 days. Similarly, C. curvatus grew in the same média with 3-12% w/v glycerol, achieving a CDW of 40 g/L in 12% w/v glycerol after 5 days. C. curvatus was alsó grown in Biodiesel glycerol (Imperial Western Products, Coachella, CA) up to 3.5% w/v, resulting in 23 g/L CDW.

Example 6. Environmental manipulation of target genes fon increased squalene production.

[0114] Environmental manipulations are tested to increase the net yield of squalene. These include (a) inhibiting ACCase expression and/or activity with oleic acid, olive or other vegetabie oil(s), inositol, choline, soraphen, fluazifop, and clethodim or other ACCase inhibiting herbicides, (b) inhibiting squalene epoxidase expression and/or activity with terbinafine, tolnaftate, and ergosterol or other squalene epoxidase inhibiting fungicides, (c) manipulating the C/N ratio in glycerol-based média (in the starting média or by add-ins), (d) varying the nitrogén source in the média (organíc vs. inorganic vs. simple/complex), (e) varying carbon addition regimes (e.g. batch vs. feeding), (f) examining the effect of depleting nutrients other than carbon source, (g) varying the carbon source to include mixtures of sugars, sugár alcohols, alcohols, polyalcohols, and organíc acids, (h) selecting fór growth on HMGR-inhibitory compounds such as lovastatin or other statin-type inhibitors, and (i) selecting fór high oil production in culture using lipophillic dyes or stains and/or by analyzing fór extractable lipids using, fór example, gravimetric or gas chromatographic methods.

[0115] Fór example, Yarrowia lipolytica ATCC 90904 was cultivated in high Carbon/Nitrogen ratio média (C/N = 420, Li, Y-H., Liu, B., Zhao, Z-B., and Bai, F-W. 2006 Optimized Culture Médium and Fermentation Conditions fór Lipid Production by Rhodosporidium toruloides Chinese Journal of Bíotechnology 22(4): 650-656) (hereinafter CYM001 Media) supplemented with 0 to 50 μg/ml terbinafine at 30’C, 300 rpm fór 120 h. Concentrations of 12.5jj_g/ml or higher of terbinafine resulted in up to 18.5% of totál lipid as squalene.

[0116] Various Yarrowia lipolytica strains are used fór lipid and squalene production including ATCC 20688, ATCC 90811, ATCC 90904, ATCC 90812, ATCC MYA-2613, and Yeastern polg. Fór example, Yarrowia lipolytica strain polg (Yeastern) was cultivated in high Carbon/Nitrogen ratio média (C/N = 420, Li, Y-H., Liu, B., Zhao, Z-B., and Bai, F-W. 2006 Optimized Culture Médium and Fermentation Conditions fór Lipid Production by Rhodosporidium toruloides Chinese Journal of Bíotechnology 22(4): 650-656) (hereinafter CYM001 Media) supplemented with 0 to 50 μg/ml terbinafine at 30’C, 300 rpm fór 120 h. Concentrations of 12.5|jLg/ml or higher of terbinafine resulted in up to 38 % of totál lipid as squalene and Values of totál lipid/Cell Dry weight of up to 51 % were achieved.

[0117] In another example, Yarrowia lipolytica ATCC 90904 was cultivated in CYM001 média supplemented with 0 to 50 μg/ml Oleic acid at 30’C, 300 rpm fór 120 h. Supplementation with ΙΟμΙ/ml Oleic acid was found to improve lipid accumulation 10-fold in lipid/CDW (cell dry weight) over no supplementation.

[0118] In a further example, Yarrowia lipolytica ATCC 90904 was cultivated in CYM001 média supplemented with 0 to 200 μΜ clethodim at 30’C, 300 rpm fór 120 h. Supplementation of 200 μΜ clethodim resulted in a 60-fold increase in the yield (mg) of squalene per 60-ml flask.

[0119] Increased oxygen has been shown to cause the differential regulation of HMG1 and HMG2 in S. cerevisiae, resulting in rapid degradation of HMG2 and increased expression of HMG1 under aerobic conditions (Casey, W.M.,

EP 2 504 421 Β1

Keesler, G.A., Parks, L.W. (1992) J. Bact. 174:7283-7288). It is determined ifthe numberof HMGR genes inouroleaginous yeasts is affected by oxygen and, if so, their expression and activity is manipulated in the fermenter by altering oxygen levels.

[0120] Starting with CYM001 Media (Li, Y-H., Liu, B., Zhao, Z-B., and Bai, F-W. (2006) Chinese Journal of Biotechnology 22(4):650-656), various components and concentrations of components are changed (including the addition of new components) to improve cell growth, percent totál lipid content/unit mass of cells, and percent squalene/total lipid. Media components that are evaluated include: carbon sources: glycerol, glucose, nitrogén sources: ammonium compounds, nitrates, amino acids, mineral salts: potassium, magnesium, sodium, iron, manganese, zinc, calcium, copper, yeast extract, lipid precursors and lipid synthesis affectors: terbinafine, clethodim, oleic acid, palmitoleic acid, linoleic acid, linolenic acid and antifoaming agents. Other factors that are evaluated include: percent inoculum, elapsed fermentation time, temperature, pH, back pressure, dissolved oxygen (DO), feed composition, feed strategy and agitation strategy.

Example 7. Strain Selection. (nőt according to the present invention) [0121] Traditional strain selection methods are used in oleaginous yeasts to increase their net squalene productivity. Strains mutagenized by UV, nitrosoguanidine, or ethane methyl sulfonate are screened and/or selected for increased squalene accumulation. Strains are alsó subjected to iterative selection pressure, such as repeated passage on YEP (15 g/L yeast extract, 5 g/L peptone) média containing 3% glycerol or média containing lovastatin and other known HMGR inhibitors. Strains are alsó subjected to repeated passage on CYM001 Media containing varying amounts of glycerol and/or glucose or média containing lovastatin and/or other known HMGR inhibitors, and/or squalene synthase inhibitors to obtain spontaneous mutants with increased HMGR and/or squalene synthase activity. Such mutations may be in HMGR, squalene synthase, or other genes (secondary site mutations).

[0122] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0123] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, nőt specifically disclosed herein. Thus, for example, the terms comprising, including, containing, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and nőt of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, bút it is recognized that various modifications are possible within the scope of the invention claimed.

[0124] Thus, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation ofthe inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are nőt intended as limitations on the scope ofthe invention.