GENETICALLY MODIFIED FUNGI AND THEIR USE IN LIPID PRODUCTION

Abstract

The invention refers to fungal cells, and especially to oleaginous fungal cells that have been genetically modified to produce enzymes of the pyruvate dehydrogenase bypass route to enhance their lipid production. Especially the cells are modified to overexpress genes encoding pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and/or acetyl-CoA synthetase (ACS), optionally together with a gene encoding diacylglycerol acyltransferase (DAT), or to express genes encoding PDC together with ALD and/or ACS. Methods of producing lipids, biofuels and lubricants using the modified fungi are also disclosed as well as expression cassettes useful therein. A new enzyme having phosholipid:diacylglycerol acyltransferase (PDAT) activity and a polynucleotide encoding it are also disclosed, which are useful in the lipid production. A recombinant Cryptococcus cell and its construction is described.

Description

[0001] This application is a Divisional of copending application Ser. No. 13/806,281, filed on Dec. 21, 2012, which was filed as PCT International Application No. PCT/FI2011/050594 on Jun. 21, 2011, which claims the benefit under 35 U.S.C. .sctn.119(a) to Patent Application No. 20105733, filed in FINLAND on Jun. 24, 2010, all of which are hereby expressly incorporated by reference into the present application.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

[0002] This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled "0837_0322PUS2_SequenceListing.txt" created on Dec. 21, 2012 and is 58,075 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to fungi, which have been genetically modified to enhance their lipid production. The invention also relates to a method for preparing the fungi, and to expression cassettes for genetic modification of the fungi. The invention further relates to a method of producing lipids by these fungi. The lipids produced are useful in manufacturing biofuels, lubricants and functional fatty acids. The invention thus also provides a method for producing biofuels and lubricants. Still further the invention provides a new enzyme protein that is useful in the methods, and a nucleic acid encoding it. Even further the invention relates to a recombinant Cryptococcus cell, and a method for its construction.

BACKGROUND OF THE INVENTION

[0004] Biofuels are current favorites to be the next generation transportation fuels. They are produced from renewable biological sources such as vegetable oils and animal fats. They are biodegradable, non-toxic and have a low emission profile. Due to the limited sources of biodiesel raw materials such as rape seed oil, soy bean oil or palm oil, it is of importance to expand biodiesel raw materials to non-food materials like microbes. The benefits of using microbes for production of oils are: they are affected neither by seasons nor by climates, they are able to produce high lipid contents, and the oils can be produced from a wide variety of sources with short production times, especially from residues with abundant nutrition. Microbiologically produced lipids may also be used e.g. for the production of functional fatty acids.

[0005] A few fungal species accumulate remarkable amounts of lipid in the cells. It has been observed that lipids accumulate in these so called oleaginous fungi under nitrogen limited conditions, which has resulted in a hypothesis for effective lipid accumulation (Review Ratledge and Wynn 2002 and references thenceforth). Nitrogen limitation causes activation of the AMP deaminase which utilizes AMP to produce NH.sub.4. The decrease in AMP concentration inhibits the activity of mitochondrial isocitrate dehydrogenase (IDH) which is part of the mitochondrial tricarboxylic (TCA) cycle. Reduction in IDH activity results in equilibration of isocitrate to citrate by aconitase. Produced citrate is transferred to the cytosol where it is converted with Coenzyme A (CoA) to acetyl-CoA by ATP:citrate lyase (ACL) with ATP hydrolysis.

[0006] Cytosolic acetyl-CoA can be further used in fatty acid synthesis. A comprehensive review on fatty acid synthesis and elongation in yeast, especially in Saccharomyces cerevisiae, is that of Tehlivets et al., 1997. In the first step of fatty acid synthesis acetyl-CoA is carboxylated by the addition of carbon dioxide to malonyl-CoA by the enzyme acetyl-CoA carboxylase in an ATP demanding reaction. In the following reactions by the fatty acid synthase systems acyl and malonyl moieties from acyl-CoA and malonyl-CoA, respectively, are transferred to acyl carrier proteins (ACPs), after the acyl chain, typically initiated by acetyl-CoA, is condensated with malonyl-ACP followed by reduction of the 3-ketoacyl-ACP to 3-hydroxyacyl-ACP, dehydration to enoyl-ACP, and a second reduction to a saturated acyl-chain that is extended by two carbon atoms. These synthesis steps are usually repeated seven times resulting in palmitoyl ACP (C16:0). Palmitic acid and intermediates of the fatty acid synthesis after hydrolysed to acyl-CoAs by hydrolase/thioesterase, can be further modified by different elongases and desaturases to different length acyl-chains with or without double bonds. In one cycle of fatty acid synthesis two NADPHs are required in the reduction steps. Acyl-CoAs can be further synthesised to triacylglycerols.

[0007] Triacylglycerol synthesis starts from glycerol-3-phosphate or dihydroxyacetone phosphate which is acylated (dihydroxyacetone-phosphate also reduced) to 1-acyl-glycerol-3-phosphate which is further acylated to phosphatidic acid. Phosphatidic acid can be further dephosphorylated to diacylglycerol. Diacylglycerol is further acylated to triacylglycerol mainly by acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) utilizing acyl-CoA or phosphatidylcholine, respectively, as acyl donors. The triacylglycerol pathway in yeast S. cerevisiae is described in more detail in a mini-review of Sorger and Daum 2003.

[0008] Phospholipid:diacylglycerol acyltransferase (PDAT) encoding genes originating from S. cerevisiae and Yarrowia lipolytica have been expressed in yeasts S cerevisiae and Y. lipolytica to enhance their triacylglycerol production (WO00/60095 and WO2005/003322, respectively). WO2009/126890 provides systems for producing engineered oleaginous yeast or fungi that express caroteinoids. Oleaginy is promoted e.g. by increased or heterologous expression of DGAT or PDAT, whereas reducing the activity of PDC is expected to promote oleaginy.

[0009] Methods of manufacturing biodiesel and other oil-based compounds using glycerol as an energy source in fermentation of oil-bearing microorganisms have been described e.g. in US2009/0004715. Methods of producing lipid-based biofuels from cellulose containing feedstock by heterotrophic fermentation of microorganisms have been described in US2009/0064567. Both publications focus on the use of algae as lipid producers. No details are given. WO2007/136762 provides genetically engineered microorganisms that produce desired products from the fatty acid biosynthetic pathway.

[0010] With the above-described triacylglycerol production pathway high triglyceride yields indicated as triglyceride production per used carbon source cannot be achieved or triacylglycerol production per cell biomass cannot be significantly enhanced. In general, lipids especially triglycerides are produced when nitrogen becomes a growth limiting factor at the late logarithmic or early stationary growth phase resulting in a low triglyceride production rate compared e.g. to yeast ethanol production. Additionally, the need of several carbons and reduced cofactors in synthesis of triacylglycerol result in low yield per used carbon. The present invention uses another route for microbial lipid production. In the present invention microbial lipid production rate and yields are enhanced, and the need of reduced cofactors from the outside of the lipid pathway is decreased. The present invention further provides lipid production that is not linked to nitrogen limitation.

SUMMARY OF THE INVENTION

[0011] The present invention is based on the use of a pyruvate dehydrogenase bypass route for producing cytosolic acetyl-CoA. The invention makes use of an active cytosolic pathway for acetyl-CoA production, which proceeds via the enzymatic reaction catalyzed by pyruvate decarboxylase (PDC). This pathway is known in Crabtree-positive yeast S. cerevisiae, where it is essential for cytosolic acetyl-CoA production, but it has not been characterized in oleaginous yeasts and oleaginous filamentous fungi. In oleaginous fungi another cytosolic pathway for acetyl-CoA production, proceeding via the reaction catalyzed by pyruvate dehydrogenase (PDH) is well known and which has been shown to be essential for cytosolic acetyl-CoA production from pyruvate. This pathway operates via mitochondria and in this pathway a higher fraction of carbon is lost than in the pathway via pyruvate decarboxylase. In the pyruvate decarboxylase pathway, exploited in this invention, a higher fraction of carbon is directed to triacylglycerol and it is not dependent on mitochondrial enzyme activities. Further, it produces NAD(P)H which is needed in the following fatty acid synthesis. The pyruvate dehydrogenase bypass route optionally together with an enhanced diacylglycerol acyltransferase activity as provided by the invention removes many bottlenecks in microbial lipid production.

[0012] The invention is directed to genetically modified fungal cells that have been modified to enhance the expression of a nucleic acid encoding PDC, ALD, ACS and/or DAT, and to methods of constructing them.

[0013] In particular the invention is directed to a genetically modified oleaginous fungal cell comprising at least one nucleic acid with enhanced expression encoding an enzyme selected from the group consisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS).

[0014] The invention is further directed to a genetically modified fungal cell comprising:

[0015] a) a nucleic acid with modified expression encoding a pyruvate decarboxylase (PDC) enzyme, and

[0016] b) at least one nucleic acid with modified expression encoding an enzyme selected from the group consisting of acetaldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and diacylglycerol acyltransferase (DAT).

[0017] The invention is also directed to a genetically modified fungal cell comprising:

[0018] a) at least one nucleic acid with modified expression encoding an enzyme selected from the group consisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), and

[0019] b) a nucleic acid with modified expression encoding a diacylglycerol acyltransferase (DAT) enzyme.

[0020] The invention is also directed to a method of producing lipids comprising cultivating the genetically modified fungal cell according to the invention in a medium containing carbon and nitrogen sources, and recovering the lipids produced.

[0021] The invention is further directed to a method of producing biofuel, or lubricant said method comprising cultivating the genetically modified fungal cell according to the invention in a medium containing carbon and nitrogen sources, and recovering the lipids produced, and optionally esterifying said lipids to obtain biodiesel or lubricant, or hydrogenizing the lipids to obtain renewable diesel or lubricant.

[0022] The invention is still further directed to methods of preparing i.e. constructing a genetically modified fungal cell of the invention, said methods comprising transforming a fungal cell with

[0023] at least one nucleic acid with enhanced expression encoding an enzyme selected from the group consisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS); or with

[0024] a) a nucleic acid with enhanced expression encoding a pyruvate decarboxylase (PDC) enzyme, and

[0025] b) at least one nucleic acid with enhanced expression encoding an enzyme selected from the group consisting of acetaldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and diacylglycerol acyltransferase (DAT); or with

[0026] a) at least one nucleic acid with modified expression encoding an enzyme selected from the group consisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), and

[0027] b) a nucleic acid with modified expression encoding a diacylglycerol acyltransferase (DAT) enzyme.

[0028] In addition the invention is directed to an expression cassette comprising

[0029] a) at least one nucleic acid with modified expression encoding an enzyme selected from the group consisting of pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), and

[0030] b) a nucleic acid with modified expression encoding a diacylglycerol acyltransferase (DAT) enzyme.

[0031] Conveniently the genetically modified fungal cells are constructed according to the invention by transforming the fungal cell with the nucleic acid(s) encoding said enzyme(s) resulting in enhanced enzyme activity of said enzyme.

[0032] Still further the invention is directed to an enzyme protein having phosholipid:diacylglycerol acyltransferase (PDAT) activity and at least 40% sequence identity to SEQ ID NO:52, or an enzymatically active fragment or variant thereof, and to an isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid encoding said protein, b) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:53 or SEQ ID NO:93, c) a complementary strand of a) or b), and d) a sequence that is degenerate as a result of the genetic code to anyone of sequences a)-c).

[0033] The invention is additionally directed to a genetically modified fungal cell comprising a nucleic acid with modified expression encoding said PDAT, and to a method of producing lipids, comprising cultivating said genetically modified fungal cell in a medium containing carbon and nitrogen sources, and recovering the lipids produced.

[0034] Still further the invention is directed to the use of a genetically modified fungal cell of the invention for producing lipids, biofuels, biodiesel, renewable diesel or lubricants. The use for producing lipids includes e.g. the use for producing precursors of fatty acids e.g. of functional fatty acids, and for producing the fatty acids or functional fatty acids.

[0035] As a still further aspect, the invention is directed to a genetically modified Cryptococcus cell, which has been modified to enhance the expression of a heterologous nucleic acid, and to a method of constructing the cell, said method comprising transforming a Cryptococcus cell with a nucleic acid encoding a heterologous protein.

[0036] Specific embodiments of the invention are set forth in the dependent claims. Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows probable metabolic routes for cytosolic acetyl-CoA production via the pyruvate dehydrogenase route in grey, and via the pyruvate dehydrogenase bypass route i.e. the pyruvate decarboxylase route in black.

[0038] FIG. 2 shows the metabolic route for triacylglycerol production.

[0039] FIG. 3 is a diagram depicting plasmid pKK81.

[0040] FIG. 4 is a diagram depicting plasmid pKK82.

[0041] FIG. 5 is a diagram depicting plasmid pKK86.

[0042] FIG. 6 is a diagram depicting plasmid pKK95.

[0043] FIG. 7 is a diagram depicting plasmid pKK85.

[0044] FIG. 8 is a diagram depicting plasmid pKK75.

[0045] FIG. 9 is a diagram depicting plasmid pKK94.

[0046] FIG. 10 is a diagram depicting plasmid pKK96.

[0047] FIG. 11 is a diagram depicting plasmid pKK98.

[0048] FIG. 12 is a diagram depicting plasmid pKK101.

[0049] FIG. 13 is a diagram depicting plasmid pKK102.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The presumed pyruvate dehydrogenase (PDH) pathway for producing cytosolic acetyl-CoA is shown in grey in FIG. 1. First cytosolic pyruvate is transported into the mitochondria where it is oxidatively decarboxylated to acetyl-CoA and carbon dioxide by the pyruvate dehydrogenase complex. The resulting acetyl-CoA is then entering the tricarboxylic (TCA) cycle by citrate synthase (CS) which catalyses the condensation reaction of the two-carbon acetate residue from acetyl-CoA and a molecule of four-carbon oxaloacetate (OAA) to form the six-carbon citrate. Citrate is then isomerised to isocitrate which is the substrate for mitochondrial isocitrate dehydrogenase (IDH). Under limited nitrogen supply AMP deaminase activity increases leading to production of IMP and ammonium from AMP. The decrease in the amount of AMP results in decrease in mitochondrial isocitrate dehydrogenase (IDH) activity, whereby the amount of citrate in the mitochondria increases. The mitochondrial citrate is transported into the cytosol, where ATP:citrate lyase (ACL) converts citrate, ATP and CoA into acetyl-CoA and oxaloacetate. The oxaloacetate is degraded by malate dehydrogenase (MDH) to malate, which in turn is converted to pyruvate and carbon dioxide by malic enzyme (MAE) under production of NADPH, which is an important cofactor in fatty acid synthesis. In this invention cytosolic acetyl-CoA is produced via a pyruvate dehydrogenase bypass pathway, which is shown in black in FIG. 1. This pathway is also called the pyruvate decarboxylase pathway. In this pathway pyruvate is decarboxylated to acetaldehyde and carbon dioxide by pyruvate decarboxylase (PDC). Acetaldehyde is further oxidised to acetate by NADP.sup.+ (or NAD.sup.+)-dependent acetaldehyde dehydrogenase (ALD). Acetate is then converted to acetyl-CoA by acetyl-CoA synthetase (ACS) with ATP and Coenzyme A. In this cytosolic pathway [pyruvate+CoA+ATP+NAD(P).sup.+=Acetyl-CoA+CO.sub.2+NAD(P)H+AMP+PPi+H.sup.- +] the overall acetyl-CoA yield is higher than in the pyruvate dehydrogenase-ATP:citrate lyase pathway [pyruvate+CoA+ATP+NAD.sup.+ (in mitochondria)+oxaloacetate (in mitochondria)=Acetyl-CoA+CO.sub.2+ADP+Pi+NAD.sup.+ (in mitochondria)+H.sup.++oxaloacetate (in cytosol)].

[0051] Cytosolic acetyl-CoA is used by NADPH-dependent fatty acid synthase (FAS) and other enzymes for the production of acyl-CoA esters of different length, which then are attached to for example glycerol through the triglyceride metabolic pathway shown in FIG. 2. In the last step of this pathway an acyl group from acyl-CoA or from a phospholipid is attached to the diacylglycerol by acyl-CoA:diacylglycerol acyltransferase (DGAT) or phospholipid:diacylglycerol acyltransferase (PDAT).

[0052] In the present invention triacylglycerol production of fungi will be enhanced by overexpressing at least one gene, which encodes an enzyme involved in the pyruvate decarboxylate pathway for converting pyruvate to acetyl-CoA as shown in FIG. 1, together with a gene, which encodes an enzyme that catalyses the acylation of diacylglycerol to triacylglycerol as shown in FIG. 2.

[0053] Lipid production including triacylglycerol production in the cell is a highly NADPH demanding process. E.g. in production of 1 mole of oleic acid (9-octadecenoic acid) 17 mole of NADPH is needed. NADPH produced by malic enzyme has been proposed to be the main source for NADPH needed in fatty acid synthesis. Said NADPH production occurs totally outside the cytosolic acetyl-CoA production pathway resulting in the consumption of extra carbons in NADPH production, even though the reaction of the malic enzyme is linked to the degradation of the oxaloacetate produced by ATP:citrate lyase. In the present invention fatty acid and further triacylglycerol production is connected directly to NADPH cofactor production by NADP-dependent acetaldehyde dehydrogenase thus reducing the need to produce NADPH outside the triglyceride production pathway resulting in an increased triacylglycerol yield. The NADP.sup.+-dependent acetaldehyde dehydrogenase produces simultaneously one NADPH and one acetate molecule from NADP+ and acetaldehyde resulting in production of one mole of NADPH per one mole of pyruvate. This means that half of the NADPH molecules needed in the fatty acid synthesis are produced simultaneously with the cytosolic acetyl-CoA production. This simultaneous NADPH production with lower carbon loss during production of cytosolic acetyl-CoA results in a better yield in fatty acid production following also better yield in triacylglycerol production. In this invention only one carbon is lost from the carbon skeleton downstream of pyruvate prior to cytosolic acetyl-CoA. Additionally, no side reactions are needed to cleave metabolites further outside the triglyceride production pathway. The production of cytosolic acetyl-CoA via the pyruvate dehydrogenase bypass completes the existing pyruvate dehydrogenase pathway for cytosolic acetyl-CoA production.

[0054] Triacylglycerols and other lipids are naturally produced in fungi via the pyruvate dehydrogenase pathway during growth, but the main triacylglycerol and lipid accumulation occurs when excess citrate will be available after nitrogen limitation in the late stage of cultivation. This triacylglycerol production in late logarithmic or stationary phases of cultivation results in low triacylglycerol production rates, especially at the early stage of cultivation. In this invention expression of the pyruvate dehydrogenase bypass catalyzed by PDC, ALD, and ACS results in a situation where triacylglycerol accumulation is not linked to nitrogen limitation thus allowing enhanced triacylglycerol production during cultivation resulting in a better triacylglycerol production rate. The earlier triacylglycerol production is further enhanced by expressing an acyltransferase such as a phospholipid:diacylglycerol acyltransferase (PDAT) encoding gene e.g. under a constitutive promoter thus increasing triacylglycerol concentration at the expense of phospholipids.

[0055] Contrary to oleaginous yeasts and moulds like Cryptococcus curvatus and Mucor circinelloides, S. cerevisiae lacks the pyruvate dehydrogenase route for acetyl-CoA production. In this Crabtree-positive yeast cytosolic acetyl-CoA, and further fatty acids and triacylglycerols, are produced only via pyruvate dehydrogenase bypass. The essential role of the pyruvate dehydrogenase bypass including the enzymes pyruvate decarboxylate (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS) in cytosolic acetyl-CoA and further in lipid production and the role of ALD in the generation of reducing equivalents (NADH and NADPH) in S. cerevisiae has been described for example in Flikweert et al. 1996, Pronk et al 1996, Saint-Prix et al 2004). In US2009/0053797 expression of endogenous NADP-dependent acetaldehyde dehydrogenase (ALD6) gene and native or modified endogenous ACS1 gene or Salmonella enterica acetyl-CoA synthetase (ACS1) gene in S. cerevisiae resulted in an increased concentration of cytosolic acetyl-CoA in the production of isoprenoids. Shiba et al., 2007 found that overexpression of ALD6 and ACS1 in S. cerevisiae increased cytosolic acetyl-CoA derived amorphadiene overproduction, whereas overexpression of ACS2 with ALD6 did not. The acetyl-CoA synthetase isoforms ACS1 and ACS2 behave differently in S. cerevisiae: ACS1 gene has been shown to be under glucose repression whereas ACS2 gene has been shown to be constitutively expressed and co-regulated with structural genes of fatty acid biosynthesis (van den Berg et al 1996, Hiesinger et al. 1997).

[0056] The essential role of the PDH bypass in S. cerevisiae has been described: Deletion of three structural genes for pyruvate decarboxylase (PDC1, PDC5 and PDC6) results in loss of growth in a defined glucose medium (Flikweert et al 1996, Pronk et al. 1996), and deletion of two acetyl-CoA synthetase encoding genes ACS1 and ACS2 is lethal on all carbon sources (Takahashi et at 2006 and van den Berg et at 1996), indicating that there is no alternative route for cytosolic acetyl-CoA production in S. cerevisiae. The enhanced expression of this pathway has been found to induce cytosolic acetyl-CoA production in S. cerevisiae (e.g. US2009/0053797 and WO2008/080124). However, the existence and functionality of the pyruvate dehydrogenase bypass pathway in oleaginous yeasts or moulds has not been described in the literature.

[0057] PDC has been characterised e.g. from the Rhizopus oryzae, which in addition to lipids produced ethanol (Skory 2003). Also, ALD and ACS have been characterised from some of the oleaginous fungi e.g. A. nidulans (Flipphi et al. 2001, Connerton et al. 1990). Still, it has also been shown that deletion of the only cytoplasmic ACS encoding gene from A. nidulans had no effect on growth on glucose (Sandeman et al. 1989). Instead, it has been shown in several articles that the cytosolic acetyl-CoA for lipid production will be produced via pyruvate dehydrogenase and ATP:citrate lyase (ACL) in oleaginous fungi (Wynn et al 2001, Boulton and Ratledge 1981). ATP:citrate lyase has been shown to be absent from the non-oleaginous yeasts (Boulton and Ratledge 1981). E.g. ACL is absent from the sequenced members of the Saccharomycotina with the exception of Y. lipolytica which is oleaginous yeast. The essential role of ACL for cytosolic acetyl-CoA production has also been shown at a functional level by deteting the acl gene from A. nidulans. This deletion strain could not grow in the absence of external sources of cytoplasmid acetyl-CoA, which strongly suggests that ACL activity is required to generate cytoplasmic acetyl-CoA. This also indicates the absence of any pyruvate dehydrogenase bypass pathway, which could compensate acl deletion (Hynes and Murray 2010). The production of cytosolic acetyl-CoA via ATP:citrate lyase in oleaginous fungi is also suggested in the patent applications WO2005/003322 and US2006/094087, where diacylglycerol transferase encoding genes have been expressed to enhance triacylglycerol production.

[0058] The term "pyruvate dehydrogenase bypass" refers to an alternative route to the pyruvate dehydrogenase reaction for the conversion of pyruvate to acetyl-CoA. The pyruvate dehydrogenase bypass comprises the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS). It has been shown in literature that the pyruvate dehydrogenase bypass is not essential in Crabtree-negative yeasts.

[0059] In the present context the corresponding genes encoding the enzyme proteins are indicated in italics.

[0060] The term "PDC" refers to pyruvate decarboxylase enzyme (EC 4.1.1.1). This enzyme catalyses the thiamine pyrophosphate- and Mg.sup.2+-dependent decarboxylation of pyruvate to acetaldehyde and carbon dioxide. A preferred PDC is one of a Crabtree-positive organism. Preferably the PDC is a fungal PDC, especially of a Crabtree-positive fungus, such as S. cerevisiae e.g. PDC1 of S. cerevisiae (GenBank accession number CAA54522, version number CAA54522.1). According to one embodiment of the invention the PDC1 contains the amino acid sequence of SEQ ID NO:95, and/or is encoded for example by a polynucleotide containing the nucleotide sequence of SEQ ID NO:94, SEQ ID NO:96 or SEQ ID NO:97.

[0061] The term "ALD" refers to acetaldehyde dehydrogenase enzyme (EC 1.2.1.5 and EC 1.2.1.4). This enzyme catalyses the reaction where acetaldehyde is oxidized to acetate, and NAD or NADP-cofactor is reduced to NADH or NADPH, respectively. NADP-specific ALDs are preferred. In the present invention the ALD is preferably a fungal ALD, more preferably of S. cerevisiae, and most preferably it is S. cerevisiae ALD6, which is encoded for example by a polynucleotide of SEQ ID NO:48 or 49, and/or comprises the amino acid sequence of SEQ ID NO:47. Further, the ALD is preferably a cytosolic ALD. ALD6 is cytosolic. Cytosolic ALD can be modified from mitochondrial isoforms of ALD by removing the mitochondrial targeting signal from the originally mitochondrial ALD by genetic engineering. The cleavage site of the mitochondrial targeting signal can be decided e.g. with programs designed for this purpose such as MITOPROT. Examples of mitochondrial ALD, which can be modified to be cytosolic are S. cerevisiae ALD4 and ALD5 encoding genes. Suitable ALD encoding genes can be found from databases e.g. KEGG Enzyme database and Brenda with EC numbers 1.2.1.4 and 1.2.1.5. Table 1 contains examples of NAD(P).sup.+ dependent acetaldehyde dehydrogenases.

TABLE-US-00001 TABLE 1 NAD(P).sup.+ -dependent acetaldehyde dehydrogenase enzymes Accession Version number of the number of the amino acid amino acid Organism sequence sequence Database Saccharomyces cerevisiae AAB68304 AAB68304.1 GenBank Saccharomyces cerevisiae DAA07732 DAA07732.1 GenBank Saccharomyces cerevisiae DAA11133 DAA11133.1 GenBank Aspergillus niger A2QMA4 A2QMA4.1 TrEMBL Aspergillus niger A2QiG1 A2QiG1.1 TrEMBL Aspergillus niger A5AAZ8 A5AAZ8.1 TrEMBL Aspergillus niger A2Q9V7 A2Q9V7.1 TrEMBL Aspergillus fumigatus Q4WQP1 Q4WQP1.1 TrEMBL Pichia angusta Q12648 Q12648 Swiss-Prot Pichia stipitis A3M013 A3M013.2 TrEMBL Candida dubliniensis B9W6J2 B9W6J2.1 TrEMBL Candida glabrata CAG59952 CAG59952.1 GenBank Kluyveromyces lactis CAH00079 CAH00079.1 Genbank Lachancea thermotolerans CAR23570 CAR23570.1 Genbank Burkholderia xenovorans Q13WK4 Q13WK4.1 TrEMBL Vibrio harveyi Q56694 Q56694.1 Swiss-Prot Mus musculus P47739 P47739.1 Swiss-Prot Mus musculus Q80VQ0 Q80VQ0.1 Swiss-Prot Rattus norvegicus P11883 P11883.3 Swiss-Prot Rattus norvegicus Q5XI42 Q5XI42.1 Swiss-Prot Canis lupus familiaris A3RF36 A3RF36.1 Swiss-Prot Bos taurus P30907 P30907.2 Swiss-Prot Bos taurus Q1JPA0 Q1JPA0.1 Swiss-Prot Homo sapiens P30838 P30838.2 Swiss-Prot Homo sapiens P43353 P43353.1 Swiss-Prot

[0062] The term "cytosolic" refers to a fluid component of the cytoplasm excluding organelle and other suspended intracellular structures.

[0063] The term "ACS" refers to acetyl-CoA synthetase enzyme (EC 6.2.1.1). The enzyme catalyses the reaction where acetyl-CoA is formed from acetate and CoA with hydrolysis of ATP. Preferably the ACS is a fungal ACS, more preferably S. cerevisiae ACS, especially S. cerevisiae ACS2. Preferably the ACS encoding gene to be expressed is a gene, which is not under glucose repression and/or which gene product is not subject to post-translational regulation e.g. acetylation, in the original species. In a particular embodiment the ACS contains the sequence of SEQ ID NO:50. Several yeasts such as Candida albicans have genes similar to S. cerevisiae ACS2, which most likely are not under post-translational regulation. This abolishes the need to modify the existing gene. According to one embodiment ACS2 is encoded by a polynucleotide having the sequence of SEQ ID NO:51 or 92.

[0064] The term "DAT" refers to diacylglycerol acyltransferase enzyme (EC 2.3.1.X). The enzyme catalyses a reaction where an acyl group is transferred to 1,2-diacylglycerol to the position sn-3. DAT includes both acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) and phospholipid:diacylglycerol acyltransferase (PDAT). Preferably the DAT is PDAT.

[0065] The term "PDAT" refers to phospholipid:diacylglycerol acyltransferase enzyme (EC 2.3.1.158). The enzyme catalyses a reaction where an acyl group from a phospholipid is transferred to 1,2-diacylglycerol to the position sn-3 via an acyl-CoA-independent mechanism. Preferably the PDAT is of fungal origin, and more preferably of an oleaginous fungus. In one preferred embodiment the PDAT is a Rhizopus oryzae PDAT, and most preferably it is encoded by a polynucleotide that contains the sequence of SEQ ID NO:53 or 93. Preferably the PDAT contains an amino acid sequence having at least 40% identity to SEQ ID NO:52, or an enzymatically active fragment or variant thereof.

[0066] In particular preferred embodiments of the invention the encoded enzyme comprises an amino acid sequence with a sequence identity of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% to SEQ ID NO:95, 47, 50, or 52, or an enzymatically active fragment or variant thereof.

[0067] Percent identity of amino acid sequences can conveniently be computed using BLASTP version 2.2.23 software with default parameters. Sequences having an identities score and a positives score of a given percentage, using the BLASTP version 2.2.23 algorithm with default parameters, are considered to be that percent identical or homologous (Altschul et al. 1997).

[0068] It is well known that deletion, addition or substitution of one or a few amino acids does not necessarily change the catalytic properties of an enzyme protein. Therefore the invention also encompasses variants and fragments of the given amino acid sequences having the stipulated enzyme activity. The term "variant" as used herein refers to a sequence having minor changes in the amino acid sequence as compared to a given sequence. Such a variant may occur naturally e.g. as an allelic variant within the same strain, species or genus, or it may be generated by mutagenesis or other gene modification. It may comprise amino acid substitutions, deletions or insertions, but it still functions in substantially the same manner as the given enzymes, in particular it retains its catalytic function as an enzyme.

[0069] A "fragment" of a given protein sequence means part of that sequence, i.e. a sequence that has been truncated at the N- and/or C-terminal end. It may for example be the mature part of a protein comprising a signal sequence, or it may be only an enzymatically active fragment of the mature protein.

[0070] The term "lipid" refers to a group of organic compounds that are relatively or completely insoluble in water but soluble in nonpolar organic solvents. These properties are a result of long hydrocarbon tails, which are hydrophobic in nature. The term thus encompasses fats, oils, waxes, fatty acids, fatty acid derivatives, like phospholipids, glycolipids, acylglycerids such as monoglycerides, diglycerides, and triglycerides and terpenoids such as carotenoids and steroids.

[0071] The term "fatty acid" refers to a compound obtainable via condensation of malonyl coenzyme A units by a fatty acid synthase system. They may be saturated or unsaturated. "Functional fatty acid" refers to a fatty acid compound having at least one functional group e.g. a hydroxyl (--OH) or carboxyl (--COOH) group within the fatty acid and being responsible for the characteristic chemical reactions of those molecules.

[0072] The term "fatty acid derivative" refers to a compound having at least one esterified fatty acyl group. Fatty acid derivatives include e.g. phospholipids, glycolipids and acylglycerides.

[0073] The term "acylglyceride" is synonymous with "acylglycerol" and refers to a compound having a glycerol moiety with one or several hydroxyl groups esterified to a fatty acid.

[0074] The terms "monoglyceride" and "monoacylglycerol" refer to a glyceride where one fatty acid residue has been esterified to a glycerol molecule. The fatty acid residue in the monoacylglycerol can be a short or long chain fatty acid with or without double bonds.

[0075] The terms "diglyceride" and "diacylglycerol" and "DAG" refer to glyceride where two fatty acid residues have been esterified to a glycerol molecule. Fatty acid residues in diacylglycerol can be short or long chain fatty acids with or without double bonds.

[0076] The terms "triglyceride" and "triacylglycerol" and "TAG" refer to a glyceride where three fatty acid residues have been esterified to a glycerol molecule. Fatty acid residues in triacylglycerol can be short or long chain fatty acids with or without double bonds. Triacylglycerol is the major acylglycerol group in oleaginous fungi.

[0077] The term "acyl-CoA" refers to a fatty acid residue, which has been esterified to a CoA molecule. Fatty acid residues in acyl-CoA can be short or long chain fatty acids with or without double bonds.

[0078] The term "phospholipids" refers to any lipid containing a diglyceride combined with a phosphate group and a simple organic molecule such as choline or ethanolamine.

[0079] The term "glycolipid" refers to a lipid attached with a carbohydrate.

[0080] The term "fat" refers to a group of organic compounds that are relatively or completely insoluble in water but soluble in nonpolar organic solvents and which are solids at normal room temperature.

[0081] The term "oil" refers to a group of organic compounds that are relatively or completely insoluble in water but soluble in nonpolar organic solvents and which are liquids at normal room temperature.

[0082] Generally fats and oils are triesters of glycerol and fatty acids.

[0083] The term "wax" refers to a compound that may contain long-chain alkanes, esters, polyesters and hydroxyl esters of long-chain primary alcohols and fatty acids.

[0084] The term "terpenoid" refers to a compound formally derived from hydrocarbon isoprene.

[0085] The term "steroid" refers to a terpenoid lipid compound having a sterane core and additional functional groups. Sterols are special forms of steroids, with a hydroxyl group at the atom C-3 and a skeleton derived from cholestane.

[0086] The term "carotenoids" refers to a compound belonging to the category of tetraterpenoids. Structurally they are in the form of a polyene chain, which is sometimes terminated by rings.

[0087] In the present invention fungal cells are genetically modified to express particular enzymes. The cells can be genetically modified to produce increased levels of lipid by transforming them with nucleic acids that have been modified to enhance the expression of nucleic acids encoding at least one of PDC, ALD and ACS, together with a nucleic acid that has been modified to enhance the expression of a nucleic acid encoding DAT so as to allow overexpression of the enzymes. A "genetically modified" organism or cell is an organism or cell that comprises an expression modified nucleic acid. It may be a recombinant organism or cell, or a host organism or cell, or a mutant.

[0088] "Nucleic acid" is a macromolecule comprising a chain of monomeric nucleotides i.e. a polynucleotide. It can be e.g. DNA such as cDNA or genomic DNA or mRNA, and it can be e.g. recombinantly or synthetically produced, double or single stranded, encompassing both sense and antisense strands.

[0089] "Recombinant" nucleic acid refers to an artificial combination of at least two otherwise separated sequences, i.e. to a not naturally occurring combination of nucleic acids.

[0090] "Nucleic acid with modified expression" as used herein denotes nucleic acids that are foreign or exogenous to the host meaning that they are not naturally found in said host. The term also includes nucleic acids that are endogenous i.e. naturally found in the host, but which are produced in an unnatural amount e.g. as multiple copies, or nucleic acids that differ in sequence from the naturally occurring nucleic acids but encode the same type of protein. Further, the term includes nucleic acids comprising at least two nucleotide sequences that do not occur in the same relationship to each other in nature, such as e.g. an endogenous protein encoding sequence that is operably linked to a transcriptional control element e.g. a promotor and/or terminator in a way that does not occur in nature. Said promotor and/or terminator can be of endogenous or exogenous origin. High copy number plasmids comprising the protein encoding nucleotide sequence are also considered nucleic acids with modified expression. The above mentioned expression modified nucleic acids encompass recombinant nucleic acids, herein also called heterologous nucleic acids. Alternatively the expression modified nucleic acid can be a mutated nucleic acid. "Modified expression" in this context is used only in the meaning of over-expression i.e. "enhanced expression". The enhanced expression results in overproduction of the expressed protein in the modified organism compared to that in an unmodified organism.

[0091] In one embodiment of the invention the nucleic acid with modified expression contains an exogenous gene encoding PDC, ALD, ACS or DAT derived from another organism. The genes encoding PDC, ALD or ACS can be obtained from yeast such as Saccharomyces cerevisiae, Candida glabrata, Dekkera bruxellensis, Kluyvemmyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia angusta, Pichia stipitis, Zygosaccharomyces rouxii, Issatchenkia terricola, Debaryomyces hansenii, Candida angusta and Pichia guilliermondii, and the DAT can be obtained from yeast or filamentous fungi such as Saccharomyces cerevisiae, Candida glabrata, Zygosaccharornyces rouxii, Lachancea thermotolerans, Ashbya gossypii, Cryptococcus curvatus, Cryptococcus albidus, Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula Rhodotorula graminis, Yarrowia lipolytica, Aspergillus nidulans, Aspergillus oryzae, Fusarium oxysporum, Humicola lanuginose, Mortierella alpina, Mortierella vinacea, Mucor circinelloides, Mucor plumbeus, Penicillium spinulosum, and Rhizopus oryzae. In another embodiment the enzyme genes to be expressed are endogenous genes, the promotor of which is replaced with another promotor, preferably a constitutive promotor, or the existing promotor is modified to become a constitutive promotor.

[0092] Proteins or polynucleotides "derived from", "originated from" or "obtained from" a particular organism encompass products isolated from said organism, as well as modifications thereof. A protein derived from a particular organism may be a recombinantly produced product, which is identical to, or a modification of the naturally occurring protein. The protein may also be modified e.g. by glycosylation, phosphorylation or other chemical modification. Products derived from the particular organism also encompass mutants and natural variants of the products, where one or more nucleic acid and/or amino acid is deleted, inserted and/or substituted.

[0093] Expression of any combination of the genes of the pyruvate dehydrogenase bypass route may be linked to expression of the DAT encoding gene. The expression of the DAT encoding gene may for example be combined to the expression of an ALD encoding gene, or an ACS encoding gene, or a PDC encoding gene. In another embodiment both ASC and ALD, or both PDC and ACS, or PDC and ALD, are overexpressed, and in still another all PDC, ALD and ACS are overexpressed together with the DAT encoding gene. In one specific embodiment of the invention the expression of S. cerevisiae ALD encoding gene ALD6 is linked to the expression of a PDAT encoding gene.

[0094] Alternatively expression of PDC may be combined with expression of ACS and/or ALD thus providing the combinations PDC+ALD, PDC+ACS and PDC+ALD+ACS. These combinations may be expressed with or without further expressing a gene encoding DAT, such as PDAT.

[0095] "Fungal" "fungus" and "fungi" as used herein refers to yeast and filamentous fungi i.e. moulds. A genetically modified fungal cell is also referred to as host cell.

[0096] The yeast host cell may be selected for example from the genera Cryptococcus, Candida, Galactomyces, Hansenula, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia. Preferably the yeast host cell is selected from the group consisting of Candida sp., Cryptococcus curvatus, Cryptococcus albidus, Galactomyces geotrichum, Hansenula ciferri, Lipomyces hpofer, Lipomyces ssp., Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica. In one embodiment the yeast is selected from the phylum Basidiomycota, which includes Cryptococcus, Rhodotorula and Rhodosporidium. Most preferably it is Cryptococcus curvatus.

[0097] The filamentous fungus host cell may be selected from the genera Aspergillus, Cunninghamella, Fusarium, Glomus, Humicola, Mortierella, Mucor, Penicillium, Pythium and Rhizopus. Preferably the filamentous fungus is selected from the group consisting of Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus niger, Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius, Humicola lanuginose, Mortierella isabellina, Mortierella pusilla, Mortierella vinacea, Mucor circinelloides, Mucor plumbeus, Mucor ramanniana, Penicillium lilacinum, Penicillium spinulosum, Pythium ultimum and Rhizopus oryzae. According to one embodiment the filamentous fungus belongs to subphylum Mucoromycotina, which includes Mucor and Mortierella. Most preferably it is Mucor circinelloides.

[0098] According to one preferred embodiment the fungal host cell is an oleaginous fungus. The term "Oleaginous fungi" refers to yeasts or filamentous fungi, which accumulate at least 10%, 12.5%, 15%, 17.5%, preferably at least 20% or even at least 25% (w/w) of their biomass as lipid. They may even accumulate at least 30%, 40%, 50%, 60%, 70%, 80% (w/w) or more of their biomass as lipids. The biomass is usually measured as cell dry weight (CDW). Oleaginous fungi are found e.g. in genera Cryptococcus, Candida, Galactomyces, Hanseluna, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia, Aspergillus, Cunninghamella, Fusarium, Glomus, Humicola, Mortierella, Mucor, Penicillium, Pythium and Rhizopus, and especially in species Candida sp., Cryptococcus curvatus, Cryptococcus albidus, Galactomyces geotrichum, Hansenula ciferri, Lipomyces lipofer, Lipomyces ssp., Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans, Yarrowia lipolytica, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus niger, Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius, Humicola lanuginose, Mortierella isabellina, Mortierella pusilla, Mortierella vinacea, Mucor circinelloides, Mucor plumbeus, Mucor ramanniana, Penicillium lilacinum, Penicillium spinulosum, Pythium ultimum and Rhizopus oryzae. In one embodiment it is a Crabtree-negative oleaginous yeast, and in another embodiment it is a Crabtree-positive filamentous fungus. In still another embodiment the filamentous fungus is Crabtree-negative, or the yeast is Crabtree-positive. Saccharomyces yeasts including S. cerevisiae are not oleaginous fungi. A "Crabtree-positive" organism is one that is capable of producing ethanol in the presence of oxygen, whereas a "Crabtree-negative" organism is not.

[0099] According to one preferred embodiment the host cell is a Cryptococcus, and especially Cryptococcus curvatus. Genetically modified Cryptococcus curvatus strains have not been described in literature (Meesters et al. 1997). Routinely, in yeast expression systems S. cerevisiae promoters and terminators, original genes without codon optimisation or codon-optimised for S. cerevisiae are used. In this invention we showed that it is possible to express genes successfully in C. curvatus when using endogenous promoters and terminators i.e. which originate from the species wherein the expression cassette will be transformed, and an expressed gene that is codon-optimised according to codon usage of the species wherein the expression cassette will be transformed, or its close relative, which has a genome that is known at a level where codonoptimisation is possible. Such a close relative is e.g. Ustilago maydis. In a preferred embodiment the promotors are constitutive promotors, especially from the glycolysis pathway.

[0100] Promoters and terminators of oleaginous fungi might contain sites for different regulatory elements and transcription factors than the promoters and terminators of S. cerevisiae due to the different nature of the strains: S. cerevisiae can grow and produce ethanol under anaerobic conditions whereas C. curvatus does not produce ethanol at all, and lipids it produces under aerobic conditions. Also the codon usage differs in S. cerevisiae and in C. curvatus: E.g. S. cerevisiae codons and genome are adenine and thymine rich, whereas the ratio of guanine and cytosine is much higher in C. curvatus (Meesters et al. 1997).

[0101] It is also important to use endogenous promoters and terminators with resistance markers to detect transformants after transformations. Due to the fact that the genome of C. curvatus is not known different procedures described in this invention can be carried out to clone endogenous promoters and terminators. Preferably constitutive promoters are used.

[0102] The fungal cell can be genetically modified by transforming it with a heterologous nucleic acid that encodes a heterologous protein. "Heterologous" in this context means not naturally occurring. In one embodiment, the cell is transformed with a heterologous nucleic acid that encodes at least one of PDC, ALD and ACS, and a heterologous nucleic acid comprising a nucleic acid that encodes DAT, operably linked to allow expression of the genes encoding said enzymes. DNA isolation, enzymatic treatment and genetical modifications may be carried out using standard molecular biology methods as described e.g. Sambrook and Russell (2001). The promoter and terminator regions of the genes of interest can be cloned e.g. from a yeast or filamentous fungus strain of interest by polymerase chain reaction (PCR) using gene specific oligonucleotides designed based on known published gene sequences of strains of the same species or other species or gene sequences of the strain of interest. Oligonucleotides designed based on the sequence of the strain of interest are preferred.

[0103] New gene fragments from yeast and filamentous fungus species or strains with unknown genomic sequences can be cloned by PCR by using degenerative primers. The term "degenerative primer" refers to mixtures of similar kinds of synthesized primers differing from each other by one nucleotide. Degenerative primers for cloning of specific gene fragments from desired species or strains are designed based on known characterised or putative gene sequences.

[0104] A person skilled in art can use known characterised gene sequences as templates in a Blast search to find out other characterised or putative gene sequences. Another possibility is to search specific gene sequences by enzymes names from databases containing genomic sequences of species from different genome sequencing projects. These kinds of databases are found for example, but not excluding, in Broad Institute's Fungal Genome Initiative sequence projects. In the searches of specific gene sequences species that are closely related to the yeast and filamentous fungus species of interest are preferred. After a set of specific gene sequences has been found nucleotide sequence alignments are carried out with appropriate programs e.g. Clustal W, resulting in a consensus sequence, which is used in designing degenerative primers. Designed degenerative primers are used in a PCR reaction with DNA of the yeast or filamentous fungus strain of interest as a template. Resulting PCR fragments are gel isolated and sequenced directly or after being cloned into plasmids. Detected sequences are used in Blast searches to confirm that right gene fragments have been cloned.

[0105] An unknown promoter and/or terminator region of a gene of interest can be cloned by a chromosome walking method. The term "chromosome walking" refers to sequential isolation of clones carrying overlapping sequences of a known gene region and an unknown sequence of an adjacent gene region produced by ligation-mediated PCR amplification method (Mueller and Wold 1989). Gene specific oligonucleotides corresponding to a known sequence of a gene of interest are designed and used in PCR reactions with linker specific oligonucleotides. The known sequence of the gene of interest may originate e.g. from a sequence of a gene fragment generated in a PCR reaction with degenerative oligos or from a gene sequence published in sequence databases. The resulting PCR fragments are gel isolated and sequenced directly or after being cloned into plasmids. Detected sequences are used in Blast searches to confirm that right gene fragments have been cloned. If needed, the chromosome walking experiment will be repeated so many times that desired length of promoter or terminator region has been cloned. The existence of the sequence of the promoter or terminator region of the desired gene is confirmed by usual bioinformatics methods e.g. with multiple sequence alignment.

[0106] A strain specific gene fragment containing the promoter and/or terminator region of the gene of interest can also be cloned by conventional library screening methods described e.g. in Sambrook and Russell (2001). The sequences of the oligonucleotides used in PCR reactions to clone desired promoter or terminator regions can also contain sequences of restriction sites of specific restriction enzymes in addition to the gene specific sequence. The PCR fragment containing the desired promoter or terminator will be cloned into plasmid e.g. pBluescript and sequenced.

[0107] Promoters used in expression cassettes can be promoters of constitutively expressed genes e.g. of 3-phosphoglycerate dehydrogenase (PGK), triose phosphate isomerase (TPI) or enolase (ENO). Alternatively, the promoter used in the expression cassette can be a promoter of a gene, which is expressed under specific cultivation conditions.

[0108] "Expression cassette" as used herein refers to a nucleic acid construct that comprises a transcription initiation or transcription control sequence, e.g. a promotor, operably linked to a coding region for the protein to be transcribed, and preferably a transcription termination region. In addition it conveniently comprises one or more marker regions, i.e. regions encoding a selection marker.

[0109] Genes to be expressed can be cloned directly from the desired species or strains by conventional molecular biology methods e.g. by using PCR. More preferably the genes to be expressed are synthesised using a codon optimised nucleotide sequence based on the known codon usage of the host strain. If the codon usage of the host strain or host species is not known, the gene to be expressed can be codon optimised based on the known codon usage of a closely related species of the host strain. Alternatively, the gene to be expressed can be synthesised according to a known amino acid sequence by translating the amino acid sequence into a DNA sequence, preferably into a codon-optimised DNA sequence. The term "codon optimization" refers to an optimization of a synthetic nucleotide sequence encoding expressed genes to enhance gene product production in a host strain. Codon optimization can occur by replacing existing codons of the original gene by the codons used more often in the host strain. In codon optimisation also internal TATA-boxes, chi-sites, ribosomal entry sites, AT-rich or GC-rich sequence stretches, repeated sequences, RNA secondary structures and cryptic splice donor and acceptor sites can be avoided. Additionally in codon optimisation sequences of restriction sites of specific restriction enzymes can be avoided. Preferably, in a synthesised gene sequence the CTG codon will be avoided due to its different coding in different fungi (leucine or serine).

[0110] A nucleic acid that is "degenerate as a result of the genetic code" to a given sequence, means that it contains one or more different codons, but encodes for the same amino acids. A "polynucleotide" as used herein may be a single or double stranded polynucleic acid. The term encompasses genome DNA, cDNA and RNA.

[0111] A "synthetic gene" or "synthetic nucleotide sequence" is an artificially designed gene or sequence, which has been synthesised into a physical DNA sequence.

[0112] The gene to be expressed is cloned between a promoter and terminator. An expression cassette of the gene to be expressed with promoter and terminator, and a marker gene can be transformed. The marker gene can be under its own promoter and terminator, but preferably it is under a functional promoter and terminator from another species, or more preferably under a functional promoter and terminator of the host strain. Markers to be used can be antibiotic markers like genes for hygromycin, geneticin and cerulenin resistances or other dominant marker like the melibiase gene. Additionally genes of the amino acid synthesis can be used as markers with auxotrophic fungi. The gene to be expressed can be transformed into the yeast or filamentous fungus as a plasmid to produce epitopic transformants, or as a DNA fragment containing the expression cassette to produce transformants with the expression cassette integrated into the genome of the host strain.

[0113] Expression cassettes containing the marker gene and gene to be expressed can be transformed in the same DNA fragment, or the expression cassettes of marker gene and gene to be expressed can be in separate DNA fragments. Transformation methods contain chemical, protoplast, electroporation methods. Transformants can be selected based on their ability to grow on a medium (solid or liquid) containing antibiotics, or a medium lacking some essential component e.g. an amino acid, or transformants can be selected based on different phenotype such as colour reaction of the transformants in specific conditions.

[0114] The DNA level of the transformants can be characterised by PCR or by Southern analysis using conventional molecular biology methods. Additionally enzyme activities of the expressed gene can be assayed as indicated in the example 27 or as described e.g. in Dahlqvist et al. 2000 and Postma et al. 1989,

[0115] The gene to be expressed can be an endogenous gene, whereby the promoter region can be replaced with a promoter of a constitutively expressed gene, or with a promoter of a gene, which is expressed under specific cultivation conditions. Additionally, expression of an endogenous gene can be enhanced by classical mutagenesis.

[0116] The genetically modified fungi of the present invention are capable of producing increased levels of lipids, and especially of triacylglycerols. The increase may be at least a 1.5, 3, 5 or 10 fold increase in lipid or triacylglycerol concentration in transformants compared to the unmodified host strain during cultivation. Alternatively, it may be at least a 1.5, 3, 5 or 10 fold increase in lipid or triacylglycerol yield per used carbon source (e.g. glucose) in transformants compared to the unmodified host strain. It may also refer to a 1.5, 3, 5 or 10 fold increase in lipid or triacylglycerol production rate (mg/l/h) compared to the unmodified host strain. This increase in lipid or triacylglycerol production can be detected either intracellularly or in the amount of lipids and triacylglycerols in culture medium.

[0117] The genetically modified fungi are cultivated in a medium containing appropriate carbon and nitrogen sources together with other optional ingredients like yeast extract, peptone, minerals and vitamins, such as KH.sub.2PO.sub.4, Na.sub.2HPO.sub.4, MgSO.sub.4, CaCl.sub.2, FeCl.sub.3, ZnSO.sub.4, citric acid, MnSO.sub.4, CoCl.sub.2, CuSO.sub.4, Na.sub.2MoO.sub.4, FeSO.sub.4, H.sub.3BO.sub.4, D-biotin, CaPantothenate, nicotinic acid, myoinositol, thiamine, pyridoxine, p-aminobenzoic acid. The invention works at a wide range of C/N ratios about from 20 to 160 under microaerobic (50 ml medium in 250 ml flask with 100 rpm shaking) and aerobic (50 ml medium in 250 ml flask with 250 rpm shaking or 1-2 vvm in bioreactors) conditions from the beginning of the cultivation to the end of cultivation as far as up to at least 8 days. The host cells used are preferably such that are able not only to use hexoses, such as glucose, but also pentoses such as xylose, and arabinose, or even glycerol as carbon source. Preferably the carbon source is a hexose and/or pentose sugars containing material such as cellulose or hemicellulose. The genetically modified host cells are preferably grown on agricultural or industrial waste materials e.g. cellulose or hemicellulose containing materials, which makes the lipid production economically and environmentally beneficial.

[0118] After cultivation, the yeast or filamentous fungal cells are normally separated from the culture medium. Lipids are recovered from the cells typically by using non-polar organic solvents, such as hexane. Prior to extraction, cells can be dried and/or disrupted. Alternatively, the lipids can be recovered directly from the culture medium, which of course is an advantage. This is especially convenient when the host cell is Cryptococcus, such as C. curvatus. Preferably lipid extraction is carried out to obtain lipids which mainly contain triacylglycerol (TAG). The lipid fraction can also contain mono- and diglycerides, free fatty acids, phospholipids, glycolipids and other lipids.

[0119] The lipids, and especially the TAG and the fatty acids, obtained can be used for preparing biofuels and lubricants. Said lipids may be directly used as biofuel or lubricant, but usually they are further processed to e.g. biodiesel or renewable diesel and/or lubricant formulations. "Biofuel" as used herein refers to fuel that has been at least partially biologically produced.

[0120] "Biodiesel" consists essentially of fatty acid methyl esters and is typically produced by transesterification in which the acylglycerides are converted to fatty acid methyl esters. According to EU directive 2003/30/EU biodiesel refers to a methyl-ester produced from vegetable oil or animal oil, of diesel quality to be used as biofuel. More broadly, biodiesel refers to long-chain alkyl esters, such as methyl, ethyl or propyl esters, from vegetable oil or animal fat of diesel quality. In the present content biodiesel can also be produced from fungal lipids.

[0121] "Renewable diesel" refers to fuel which is produced by hydrogen treatment (hydrogenation or hydroprocessing) of lipids of animal, vegetable or fungal origin, or their mixtures. Renewable diesel can be produced also from waxes derived from biomass by gasification and Fischer-Tropsch synthesis. Optionally, in addition to hydrogen treatment, isomerization or other processing steps can be performed. In hydrogen treatment, acylglycerides are converted to corresponding alkanes (paraffins). The alkanes (paraffins) can be further modified by isomerization or by other process alternatives. These processes can also produce hydrocarbons which are suitable for jet fuel or gasoline applications.

[0122] "Lubricant" refers to a substance, such as grease, lipid or oil that reduces friction when applied as a surface coating to moving parts. Two other main functions of a lubricant are heat removal and dissolving impurities. Applications of lubricants include, but are not limited to uses in internal combustion engines as engine oils, additives in fuels, in oil-driven devices such as pumps and hydraulic equipment, or in different types of bearings. Typically lubricants contain 75-100% base oil and the rest is additives. In the present invention at least part of the base oil is of fungal origin. Viscosity index is used to characterise base oil. Typically high viscosity index is preferred.

[0123] Lubricants or at least the base oil for lubricants may be prepared in the same way as described above for the biofuels. Usually conventional additives such as viscosity modifyers, antioxidants, pour point modifyers etc. are added to the base oil to obtain the lubricant.

[0124] The lipids, and especially the TAG and the fatty acids obtained can also be used for precursors for functional fatty acids. Said lipids are further processed to release fatty acyls which can be used in the production of functional fatty acids, like dicarboxylic acids and epoxides, which can be further converted into products like polyesters, polyurethane, coatings and resins.

[0125] The following examples are provided to illustrate the invention, but are not intended to limit the scope thereof. The enzyme names used are based on sequence homology.

Example 1A: Cloning of Cryptococcus curvatus TEF (CcTEF1) Promoter and Terminator Region

[0126] A genomic .about.800 bp fragment of the C. curvatus TEF gene was amplified by PCR with degenerative primers identified as SEQ ID NO:1 (Yeast TEF1), and SEQ ID NO:2 (Yeast TEF4), using C. curvatus (C-01440, VTT Culture Collection) genomic DNA as the template. The degenerative primers were designed based on a consensus sequence of the putative TEF1 genes of Ustilago maydis, Candida guilliermondii and Candida tropicalis. The detected genomic fragment was sequenced.

[0127] Genomic fragments containing the CcTEF1 promoter region were obtained with ligation-mediated PCR amplification (Mueller, P. R. and Wold, B. 1989). A mixture of a linker identified as SEQ ID NO:3 (PCR linker I), and a linker identified as SEQ ID NO:4 (PCR linker II) was ligated to PvuII digested C. curvatus genomic DNA with T4 DNA ligase (New England BioLabs). Samples of the ligation mixtures were used as templates for 50 .mu.l PCR reactions containing 0.1 .mu.M of a primer identified as SEQ ID NO: 3 (PCR linker I), and 1 .mu.M of a primer identified as SEQ ID NO:5 (CC_TEF2). The reaction mixture was heated at 94.degree. C. for 3 minutes after 2 U of Dynazyme EXT was added. The reactions were cycled 30 times as follows: 1 minute at 94.degree. C., 2 minutes at 60.degree. C. and 2 minutes at 72.degree. C., with final extension of 10 minutes at 72.degree. C. A diluted sample of this first PCR-amplification was used as the template in a nested PCR reaction (50 .mu.l) containing 0.05 .mu.M of a primer identified as SEQ ID NO:3 (PCR Linker I), and 0.5 .mu.M of a primer identified as SEQ ID NO:6 (CC_TEF1). The reaction mixture was heated at 94.degree. C. for 3 minutes after 2 U of Dynazyme EXT was added. The reactions were then cycled 30 times as follows: 1 minute at 94.degree. C., 2 minutes at 60.degree. C. and 2 minutes at 72.degree. C., with final extension of 10 minutes at 72.degree. C.

[0128] A .about.800 bp fragment was isolated and sequenced. Nested primers identified as SEQ ID NO:7 (CC_TEF6), and SEQ ID NO:8 (CC_TEF5) were designed and used in a ligation-mediated PCR amplification together with oligonucleotides identified as SEQ ID NO:3 (PCR linker I), and a linker identified as SEQ ID NO:4 (PCR linker II) similarly as above except that NruI-digested C. curvatus DNA was used. A .about.2000 bp PCR fragment was isolated and sequenced.

[0129] The C. curvatus TEF1 promoter was PCR amplified using primers identified as SEQ ID NO:9 (CC_TEF10), and SEQ ID NO:10 (CC_TEF11) and the C. curvatus genomic DNA as the template. A PCR fragment was digested with SacII and XbaI. A 1276 bp fragment was gel isolated and ligated to a SacII and XbaI-digested pBluescript KS-plasmid (Stratagene). The resulting plasmid was designated pKK58. Plasmid pKK58 contains C. curvatus TEF1 promoter.

[0130] A genomic fragment containing the CcTEF1 terminator region was obtained with a ligation-mediated PCR amplification with C. curvatus TEF1 gene specific oligonucleotides identified as SEQ ID NO:11 (CC_TEF3), and SEQ ID NO:12 (CC_TEF4) together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II) similarly as above except that NruI-digested C. curvatus DNA was used. A .about.1600 bp PCR fragment was isolated and sequenced.

[0131] The C. curvatus TEF1 terminator was PCR amplified using primers identified as SEQ ID NO:13 (CC_TEF7) and SEQ ID NO:14 and the C. curvatus genomic DNA as the template. A PCR fragment was digested with XmaI and EcoRI. A 358 bp fragment was gel isolated and ligated to XmaI and EcoRI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK55. Plasmid pKK55 contains the C. curvatus TEF1 terminator.

Example 1B: Cloning of Cryptococcus curvatus TPI (CcTPI1) Promoter and Terminator Region

[0132] A genomic fragment of the C. curvatus TPI1 gene was amplified by PCR from genomic C. curvatus (C-01440, VTT Culture Collection) DNA with degenerative primers identified as SEQ ID NO:15 (Yeast TPI5), and SEQ ID NO:16 (Yeast TPI8). The degenerative primers were designed based on a consensus sequence of the TPI1 genes of Ustilago maydis and Cryptococcus neoformans. A .about.800 bp genomic fragment was isolated and sequenced.

[0133] A genomic fragment containing the CcTPI1 promoter region was obtained with a ligation-mediated PCR amplification with TPI1 gene specific oligonucleotides identified as SEQ ID NO:17 (CC_TPI2), and SEQ ID NO:18 (CC_TPI1), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that EcoRV-digested C. curvatus DNA was used. A .about.1300 bp PCR fragment was isolated and sequenced.

[0134] The C. curvatus TPI1 promoter was PCR amplified by using primers identified as SEQ ID NO:19 (CC_TPI7) and SEQ ID NO:20 (CC_TPI_9) and the C. curvatus DNA as the template. A PCR fragment was digested with BamHI and SbfI. A 851 bp fragment was gel isolated and ligated to a BamHI and PstI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK63. Plasmid pKK63 contains the C. curvatus TPI1 promoter.

[0135] A genomic fragment containing the CcTPI1 terminator region was obtained with a ligation-mediated PCR amplification with TPI1 gene specific oligonucleotides identified as SEQ ID NO:21 (CC_TPI 4) and SEQ ID NO:22 (CC_TPI3), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that EcoRV-digested C. curvatus DNA was used. A .about.1200 bp PCR fragment was isolated and sequenced.

[0136] The C. curvatus TPI1 terminator was PCR amplified by using primers identified as SEQ ID NO:23 (CC_TPI5) and SEQ ID NO:24 (CC_TPI6) and the C. curvatus genomic DNA as the template. A PCR fragment was digested with XbaI and BamHI. A 361 bp fragment was gel isolated and ligated to a XbaI and BamHI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK61. Plasmid pKK61 contains C. curvatus TPI1 terminator.

Example 1C: Cloning of Cryptococcus curvatus ENO (CcENO1) Promoter and Terminator Region

[0137] A genomic fragment of the C. curvatus ENO1 gene was amplified by PCR from genomic C. curvatus (C-01440, VTT Culture Collection) DNA with degenerative primers identified as SEQ ID NO:25 (YeastENO5) and SEQ ID NO:26 (YeastENO10). The degenerative primers were designed based on a consensus sequence of ENO1 genes of Ustilago maydis and Cryptococcus neoformans. A .about.1000 bp genomic fragment was isolated and sequenced.

[0138] A genomic fragment containing the CcENO1 promoter region was obtained with a ligation-mediated PCR amplification with ENO1 gene specific oligonucleotides identified as SEQ ID NO:27 (CC_ENO2) and SEQ ID NO:28 (CC_ENO1), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that PvuII-digested C. curvatus DNA was used.

[0139] A .about.600 bp fragment was isolated and sequenced. Nested primers identified as SEQ ID NO:29 (CC_ENO5) and SEQ ID NO:30 (CC_ENO6) were designed and used in a ligation-mediated PCR amplification together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II) similarly as above except that SspI-digested C. curvatus DNA was used. A 2000 bp PCR fragment was isolated and sequenced.

[0140] The C. curvatus ENO1 promoter was PCR amplified by using primers identified as SEQ ID NO:31 (CC_ENO9) and SEQ ID NO:32 (CC_ENO10) and the C. curvatus genomic DNA as the template. A PCR fragment was digested with EcoRI. A 1214 bp fragment was gel isolated and ligated to a EcoRI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK74. Plasmid pKK74 contains the C. curvatus ENO1 promoter.

[0141] A genomic fragment containing the CcENO1 terminator region was obtained with a ligation-mediated PCR amplification with ENO1 gene specific oligonucleotides identified as SEQ ID NO:33 (CC_ENO4) and SEQ ID NO:34 (CC_ENO3), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that NruI-digested C. curvatus DNA was used. A .about.1100 bp PCR fragment was isolated and sequenced.

[0142] The C. curvatus ENO1 terminator was PCR amplified by using primers identified as SEQ ID NO:35 (CC_ENO7) and SEQ ID NO:36 (CC_ENO8), and the C. curvatus genomic DNA as the template. A PCR fragment was digested with HindIII. A 375 bp fragment was gel isolated and ligated to a HindIII-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK60. Plasmid pKK60 contains the C. curvatus ENO1 terminator.

Example 1D: Cloning of C. curvatus GPD (CcGPD1) Terminator Region

[0143] A genomic fragment containing the CcGPD1 terminator region was obtained with a ligation-mediated PCR amplification with GPD1 gene specific oligonucleotides identified as SEQ ID NO:37 (CC_GPD3) and SEQ ID NO:38 (CC_GPD4), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that SspI-digested C. curvatus DNA was used. A .about.1800 bp fragment was isolated and partially sequenced. GPD1 gene specific oligonucleotides were designed according to the C. curvatus GPD1 gene (GenBank Accession number AF126158, version number AF126158.1) sequence.

[0144] The C. curvatus GPD1 terminator was PCR amplified by using primers identified as SEQ ID NO:39 (CC_GPD6) and SEQ ID NO:40 (CC_GPD7), and the C. curvatus genomic DNA as the template. A PCR fragment was digested with XbaI and BamHI. A 336 bp fragment was gel isolated and ligated to a XbaI and BamHI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK54. Plasmid pKK54 contains C. curvatus GPD1 terminator.

Example 2A: Cloning of the E. coli Hygromycin Resistance Gene; Construction of a Plasmid (pKK76) Having the E. Con Hygromycin Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator

[0145] The E. coli hygromycin (hph) gene, that confers resistance to hygromycin B, was PCR amplified using primers identified as SEQ ID NO:41 (Hph 5) and SEQ ID NO:42 (Hph 3), and the plasmid pRLMEX30 (Mach et al. 1994) DNA as the template. A PCR fragment was digested with SpeI. A 1048 bp fragment was gel isolated and ligated to SpeI-digested pBluescript KS-plasmid and sequenced. The resulting plasmid was designated pKK52.

[0146] Plasmid pKK58 was digested with SacII and SbfI. A 1272 bp fragment was gel isolated. Plasmid pKK52 was digested with SbfI. A 1034 bp fragment was gel isolated. Plasmid pKK58 contains the C. curvatus TEF1 promoter and plasmid pKK61 contains the C. curvatus TPI1 terminator. The 1272 bp fragment originating from plasmid pKK58 and the 1034 bp fragment originating from the pKK52 plasmid were ligated to a 3285 bp fragment obtained by digesting a plasmid designated as pKK61 with SacII and SbfI. The resulting plasmid was designated pKK76. Plasmid pKK76 contains the E. coli hygromycin gene under the control of the C. curvatus TEF1 promoter and the C. curvatus TPI1 terminator.

Example 2B: Cloning of the E. coli G418 Resistance Gene; Construction of a Plasmid (pKK67) Having the E. coli Geneticin Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator

[0147] The E. coli G418 resistance gene was PCR amplified using primers identified as SEQ ID NO:43 (Kan 5) and SEQ ID NO:44 (Kan 3), and the plasmid pPIC9K (Invitrogen) DNA as the template. A PCR fragment was digested with SpeI. A 838 bp fragment was gel isolated and ligated to SpeI-digested pBluescript KS-plasmid and sequenced. The resulting plasmid was designated pKK51.

[0148] Plasmid pKK58 was digested with SacII and SbfI. A 1272 bp fragment was gel isolated. Plasmid pKK51 was digested with SbfI. A 824 bp fragment was gel isolated. The 1272 bp fragment originating from pKK58 plasmid and the 824 bp fragment originating from pKK51 plasmid were ligated to a 3285 bp fragment obtained by digesting a plasmid designated pKK61 with SacII and SbfI. Plasmid pKK58 contains the C. curvatus TEF1 promoter and plasmid pKK61 contains the C. curvatus TPI1 terminator. The resulting plasmid was designated pKK67. Plasmid pKK67 contains the E. coli G418 resistance gene under the control of the C. curvatus TEF1 promoter and the C. curvatus TPI1 terminator.

Example 2C: Cloning of S. cerevisiae Cerulenin Resistance Gene; Construction of a Plasmid (pKK91) Having the S. cerevisiae Cerulenin Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator

[0149] The S. cerevisiae cerulenin resistance gene was PCR amplified using primers identified as SEQ ID NO:45 (CERR 5) and SEQ ID NO:46 (CERR 3), and the plasmid pSH47Y DNA as the template. Plasmid pSH47Y contains the cerulenin resistance gene from the plasmid pCR1 (Nakazawa et al. 1993). A PCR fragment was digested with SbfI. A 1685 bp fragment was gel isolated and ligated to PstI-digested pBluescript KS-plasmid and sequenced. The resulting plasmid was designated pKK80.

[0150] Plasmid pKK80 was digested with PstI. A 1685 bp fragment was gel isolated and ligated to a 4557 bp fragment obtained by digesting a plasmid designated as pKK67 with SbfI. Plasmid pKK67 contains the E. coli G418 resistance gene linked to a C. curvatus TEF1 promoter and C. curvatus TEF1 terminator. The resulting plasmid was designated pKK91. Plasmid pKK91 contains the S. cerevisiae cerulenin resistance gene under the control of the C. curvatus TEF1 promoter and the C. curvatus TPI1 terminator.

Example 3A. Construction of a Plasmid (pKK81, FIG. 3) Containing the Hygromycin Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under the Control of the CcENO1 Promoter and the CcTEF1 Terminator

[0151] The plasmid UmALD (Geneart AG, Germany) contains a S. cerevisiae ALD6 (SEQ ID NO:47) encoding gene which has been codon optimized according to Ustilago maydis yeast codon usage (SEQ ID NO:48) with flanking SbfI restriction sites. The plasmid RoALD (Geneart AG, Germany) contains a S. cerevisiae ALD6 (SEQ ID NO:47) encoding gene which has been codon optimized according to Rhizopus oryzae filamentous fungus codon usage (SEQ ID NO:49) with flanking SbfI restriction sites and an E. coli kanamycin resistance gene. Plasmid UmALD was digested with SfiI. A 1554 bp fragment was gel isolated and ligated to a 2258 bp fragment obtained by digesting a plasmid RoALD with SfiI. The resulting plasmid was designated as pKK50. The plasmid pKK50 contains a S. cerevisiae ALD6 (SEQ ID NO:47) encoding gene which has been codon optimized according to Ustilago maydis yeast codon usage (SEQ ID NO:48) with flanking SbfI restriction sites and an E. coli kanamycin resistance gene.

[0152] Plasmid pKK74 was digested with EcoRI and SbfI. A 1204 bp fragment was gel isolated. Plasmid pKK55 was digested with EcoRI and SbfI. A 347 bp fragment was gel isolated. The 1204 bp fragment originating from plasmid pKK74 and the 347 bp fragment originating from plasmid pKK55 were ligated to a 2961 bp fragment obtained by digesting a plasmid designated pKK74 with EcoRI. Plasmid pKK74 contains C. curvatus ENO1 promoter and plasmid pKK55 contains C. curvatus TEF1 terminator. The resulting plasmid was designated as pKK77pre.

[0153] Plasmid pKK50 was digested with SbfI. A 1514 bp fragment was gel isolated and ligated to a 4517 bp fragment obtained by digesting plasmid pKK77pre with SbfI. The resulting plasmid was designated as pKK77. The plasmid pKK77 contains the S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator.

[0154] Plasmid pKK76 was digested with BamHI and XmnI. A 2652 bp fragment was gel isolated and ligated to a 6031 bp fragment obtained by digesting plasmid pKK77 with BamHI. The plasmid pKK76 contains the E. coli hygromycin resistance gene under the control of the CcTEF1 promoter and the CcTPI1 terminator. The resulting plasmid was designated as pKK81 (FIG. 3).

Example 3B. Generation of a Genetically Modified C. curvatus (Y23/81) with an Integrated ALD6 Encoding Gene and Hygromycin Resistance Gene by Transforming Wild-Type C. curvatus with Digested Plasmid pKK81 (FIG. 3, Ex. 3A)

[0155] Plasmid pKK81 was restricted with NotI and PspOMI, and the resulting linear DNA was used to transform a wild-type C. curvatus strain ATCC 20509 designated as Y23 by electroporation using a standard electroporation method.

[0156] The transformed cells were screened for hygromycin resistance. Several hygromycin-resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of C. curvatus with NotI and PspOMI cut pKK81 and containing a S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator were designated as Y23/81-8, Y23/81-51, Y23/81-59, Y23/81-66 and Y23/81-69.

Example 4A. Construction of a Plasmid (pKK82, FIG. 4) Containing the G418 Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under the Control of the CcENO1 Promoter and the CcTEF1 Terminator

[0157] Plasmid pKK67 was digested with BamHI and XmnI. A 2442 bp fragment was gel isolated and ligated to a 6031 bp fragment obtained by digesting a plasmid pKK77 with BamHI. The plasmid pKK67 contains the E. coli G418 resistance gene under the control of the CcTEF1 promoter and the CcTPI1 terminator and the plasmid pKK77 contains S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator. The resulting plasmid was designated as pKK82 (FIG. 4).

Example 4B. Generation of a Genetically Modified C. curvatus (Y23/82) with an Integrated ALD6 Encoding Gene and G418 Resistance Gene by Transforming Wild-type C. curvatus with Digested Plasmid pKK82 (FIG. 4, Ex. 4A)

[0158] Plasmid pKK82 was restricted with NotI and PspOMI, and the resulting linear DNA was used to transform wild-type C. curvatus strain ATCC 20509 designated as Y23 by electroporation. The transformed cells were screened for G418 resistance. Several G418-resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of C. curvatus with NotI and PspOM1 cut pKK82 and containing S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator were designated as Y23/82-1, Y23/82-2, Y23/82-4 and Y23/82-13.

Example 5A. Construction of a Plasmid (pKK86, FIG. 5) Containing the Hygromycin Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator and the S. cerevisiae ACS2 Encoding Gene Under the Control of the CcTPI1 Promoter and the CcENO1 Terminator

[0159] Plasmid pKK63 was digested with BamHI and SbfI. A 851 bp fragment was gel isolated and ligated to a 3295 bp fragment obtained by digesting plasmid pKK60 with Ban-II and SbfI. The plasmid pKK63 contains the CcTPI1 promoter and the plasmid pKK60 contains the CcENO1 terminator. The resulting plasmid was designated as pKK78pre.

[0160] The plasmid UmACS contains the S. cerevisiae ACS2 (SEQ ID NO:50) encoding gene which has been codon optimized according to Ustilago maydis yeast codon usage (SEQ ID NO:51) with flanking SbfI restriction sites. Plasmid UmACS was digested with SbfI and DraI. A 2060 bp fragment was gel isolated and ligated to a 4146 bp fragment obtained by digesting plasmid pKK78pre with SbfI. The resulting plasmid was designated as pKK78.

[0161] Plasmid pKK76 was digested with BamHI and DraI. A 2652 bp fragment was gel isolated and ligated to a 6206 bp fragment obtained by digesting plasmid pKK78 with BamHI. The plasmid pKK76 contains the E. coli hygromycin resistance gene under the control of the CcTEF1 promoter and the CcTPI1 terminator. The resulting plasmid was designated as pKK86 (FIG. 5).

Example 5B. Generation of a Genetically Modified C. curvatus (Y23/86) with an Integrated ACS2 Encoding Gene and Hygromycin Resistance Gene by Transforming Wild-Type C. curvatus with Digested Plasmid pKK86 (FIG. 5, Ex. 5A)

[0162] Plasmid pKK86 was restricted with NotI and PspOMI, and the resulting linear DNA was used to transform a wild-type C. curvatus strain ATCC 20509 designated as Y23 by electroporation. The transformed cells were screened for hygromycin resistance. Several hygromycin-resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of C. curvatus with Moil and PspOMI cut pKK86 and containing S. cerevisiae ACS2 encoding gene under the control of the CcTPI1 promoter and the CcENO1 terminator were designated as Y23/86-86, Y23/86-92, Y23/86-93, Y23/86-98 and Y23/86-100.

Example 6A. Construction of a Plasmid (pKK95, FIG. 6) Containing the Cerulenin Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator and the R. oryzae PDAT Encoding Gene Under the Control of the CcTPI1 Promoter and the CcGPD1 Terminator

[0163] Plasmid pKK54 was digested with KpnI and SbfI. A 394 bp fragment was gel isolated and ligated to a 3742 bp fragment obtained by digesting plasmid designated as pKK63 with KpnI and SOL The plasmid pKK54 contains the CcGPD1 terminator and the plasmid pKK63 contains the CcTPI1 promoter. The resulting plasmid was designated as pKK93pre. Plasmid UmPDAT was digested with SbfI. A 1844 bp fragment was gel isolated and ligated to a 4136 bp fragment obtained by digesting plasmid pKK93pre with SbfI. The plasmid UmPDAT contains a R. oryzae PDAT (SEQ ID NO:52) encoding gene which has been codon optimized according to U. maydis yeast codon usage (SEQ ID NO:53) with flanking SbfI restriction sites. The resulting plasmid was designated as pKK93.

[0164] The plasmid pKK93 was digested with EcoRI and DraI. A 3029 bp fragment was gel isolated and ligated to a 6242 bp fragment obtained by digesting plasmid pKK91 with EcoRI. The plasmid pKK91 contains the S. cerevisiae cerulenin resistance gene under the control of the CcTEF1 promoter and the CcTPI1 terminator. The resulting plasmid was designated as pKK95 (FIG. 6).

Example 6B. Generation of a Genetically Modified C. curvatus (Y23/95) with an Integrated PDAT Encoding Gene and Cerulenin Resistance Gene by Transforming Wild-Type C. curvatus with Digested Plasmid pKK95 (FIG. 6, Ex. 6A)

[0165] Plasmid pKK95 was restricted with EcoRV, and the resulting linear DNA was used to transform a wild-type C. curvatus strain ATCC 20509 designated as Y23 by electroporation. The transformed cells were screened for cerulenin resistance. Several cerulenin-resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of C. curvatus with EcoRV cut pKK95 and containing the R. oryzae PDAT encoding gene under the control of the CcTPI1 promoter and the CcGPD1 terminator were designated as Y23/95-87, Y23/95-98, Y23/95-99, Y23/95-104 and Y23/95-109.

Example 7A. Construction of a Plasmid (pKK85, FIG. 7) Containing the Hygromycin Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under the Control of the CcENO1 Promoter and the CcTEF1 Terminator and the S. cerevisiae ACS2 Encoding Gene Under the Control of the CcTPI1 Promoter and the CcENO1 Terminator

[0166] Plasmid pKK77 was digested with EcoRI and XmnI. A 3073 bp fragment was gel isolated and ligated to a 6206 bp fragment obtained by digesting plasmid pKK78 with EcoRI. The plasmid pKK77 contains the S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator and the plasmid pKK78 contains the S. cerevisiae ACS2 encoding gene under the control of the CcTPI1 promoter and the CcENO1 terminator. The resulting plasmid was designated as pKK84. The plasmid pKK76 was digested with BamHI and XmnI. A 2652 bp fragment was gel isolated and ligated to a 9279 bp fragment obtained by digesting pKK84 with BamHI. The plasmid pKK76 contains the hygromycin resistance gene under the control of the CcTEF1 promoter and the CcTPI1 terminator. The resulting plasmid was designated as pKK85 (FIG. 7).

Example 7B. Generation of a Genetically Modified C. curvatus (Y23/85) with an Integrated ALD6 Encoding and ACS2 Encoding Genes and Hygromycin Resistance Gene by Transforming Wild-Type C. curvatus with Digested Plasmid pKK85 (FIG. 7, Ex. 7A)

[0167] Plasmid pKK85 was restricted with NotI and PspOMI, and the resulting linear DNA was used to transform wild-type C. curvatus strain ATCC 20509 designated as Y23 by electroporation. The transformed cells were screened for hygromycin resistance. Several hygromycin-resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of C. curvatus with NotI and PspOMI cut pKK85 and containing the S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator and the S. cerevisiae ACS2 encoding gene under the control of the CcTPI1 promoter and the CcENO1 terminator were designated as Y23/85-119, Y23/85-125, Y23/85-128 Y23/85-129 and Y23/85-139.

Example 8. Generation of a Genetically Modified C. curvatus (Y23/81/95) with an Integrated ALD6 Encoding and PDAT Encoding Genes and Hygromycin and Cerulenin Resistance Genes by Transforming Genetically Modified Strain Y23/81-51 (Ex. 3B) with Plasmid pKK95 (FIG. 6, Ex. 6A)

[0168] Plasmid pKK95 was restricted with EcoRV and the resulting linear DNA was used to transform a genetically modified strain Y23/81-51 by electroporation. The transformed cells were screened for cerulenin and hygromycin resistance. Several cerulenin and hygromycin resistance colonies were analysed at DNA level by PCR. The transformants originating from the transformation of genetically modified strain Y23/81-51 with EcoRV cut pKK95 and containing the S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator and the R. oryzae PDAT encoding gene under the control of the CcTPI1 promoter and the CcGPD1 terminator were designated as Y23/81/95-18 and Y23/81/95-42.

Example 9. Generation of a Genetically Modified C. curvatus (Y23/85/95) with an Integrated ALD6, ACS2 and PDAT Encoding Genes and Hygromycin and Cerulenin Resistance Genes by Transforming Genetically Modified Strain Y23/85-128 (Ex. 7B) with Plasmid pKK95 (FIG. 6, Ex. 6A)

[0169] Plasmid pKK95 was restricted with EcoRV and the resulting linear DNA was used to transform a genetically modified strain Y23/85-128 by electroporation. The transformed cells were screened for cerulenin and hygromycin resistance. Several cerulenin and hygromycin resistance colonies were analysed at DNA level by PCR. The transformants originating from the transformation of genetically modified strain Y23/85-128 with EcoRV cut pKK95 and containing the S. cerevisiae ALD6 encoding gene under the control of the CcENO1 promoter and the CcTEF1 terminator, the S. cerevisiae ACS2 encoding gene under the control of the CcTPI1 promoter and the CcENO1 terminator and R. oryzae PDAT encoding gene under the control of the CcTPI1 promoter and the CcGPD1 terminator were designated as Y23/85/95-4 and Y23/85/95-68.

Example 10. Cloning of Mucor circinelloides TPI (McTPI1) Promoter and Terminator Region

[0170] A genomic fragment of the M. circinelloides TPI1 gene was amplified by PCR from genomic M. circinelloides f. griseocyanus (D-82202, VTT Culture Collection) DNA with degenerative primers identified as SEQ ID NO:54 (Mould TPI1) and SEQ ID NO:55 (Mould TPI3). The degenerative primers were designed based on a consensus sequence of TPI1 genes of Rhizopus oryzae, Fusarium oxysporum, Aspergillus fumigatus, A. terreus and A. nidulans. A .about.400 bp genomic fragment was isolated and sequenced.

[0171] A genomic fragment containing the McTPI1 promoter region was obtained with a ligation-mediated PCR amplification with TPI1 gene specific oligonucleotides identified as SEQ ID NO:56 (MC_TPI2) and SEQ ID NO:57 (MC_TPI1), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that SspI-digested M. circinelloides DNA was used. A .about.1500 bp PCR fragment was isolated and sequenced.

[0172] The M. circinelloides TPI1 promoter was PCR amplified by using primers identified as SEQ ID NO:58 (MC_TPI1) and SEQ ID NO:59 (MC_TPI_8), and the M. circinelloides genomic DNA as the template. A PCR fragment was digested with PstI and BamHI. A 1251 bp fragment was gel isolated and ligated to a PstI and BamHI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK56. Plasmid pKK56 contains the M. circinelloides TPI1 promoter.

[0173] A genomic fragment containing the McTPI1 terminator region was obtained with a ligation-mediated PCR amplification with TPI1 gene specific oligonucleotides identified as SEQ ID NO:60 (MC_TPI4) and SEQ ID NO:61 (MC_TPI3), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that NruI-digested M. circinelloides DNA was used. A .about.1500 bp PCR fragment was isolated and partially sequenced.

[0174] The M. circinelloides TPI1 terminator was PCR amplified by using primers identified as SEQ ID NO:62 (MC_TPI5) and SEQ ID NO:63 (MC_TPI6), and the M. circinelloides genomic DNA as the template. A PCR fragment was digested with XbaI and BamHI. A 347 bp fragment was gel isolated and ligated to a XbaI and BamHI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK57. Plasmid pKK57 contains the M. circinelloides TPI1 terminator.

Example 11. Cloning of Mucor circinelloides TEF (McTEF1) Promoter and Terminator Region

[0175] A genomic fragment of the M. circinelloides TEF1 gene was amplified by PCR from genomic M. circinelloides f. griseocyanus (D-82202, VTT Culture Collection) DNA with degenerative primers identified as SEQ ID NO:64 (Mould TEF1) and SEQ ID NO:65 (Mould TEF4). The degenerative primers were designed based on a consensus sequence of TEF1 genes of Rhizopus oryzae, Fusarium oxysporum, Aspergillus terreus and A. nidulans. A .about.600 bp genomic fragment was isolated and sequenced.

[0176] A genomic fragment containing the McTEF1 promoter region was obtained with a ligation-mediated PCR amplification with TEF1 gene specific oligonucleotides identified as SEQ ID NO:66 (MC_TEF2) and SEQ ID NO:67 (MC_TEF1), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that SspI-digested M. circinelloides DNA was used. A .about.500 bp PCR fragment was isolated and sequenced. Nested primers identified as SEQ ID NO:68 (MC_TEF6) and SEQ ID NO:69 (MC_TEF5) were designed and used in a ligation-mediated PCR amplification together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as above except that HaeIII-digested M. circinelloides DNA was used. A .about.1500 bp PCR fragment was isolated and sequenced.

[0177] The M. circinelloides TEF1 promoter was PCR amplified by using primers identified as SEQ ID NO:70 (MC_TEF9) and SEQ ID NO:71 (MC_TEF10), and the M. circinelloides genomic DNA as the template. A PCR fragment was digested with HindIII and PstI and a 1387 bp fragment was gel isolated and ligated to a HindIII and PstI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK64. Plasmid pKK64 contains the M. circinelloides TEF1 promoter.

[0178] A genomic fragment containing the McTEF1 terminator region was obtained with a ligation-mediated PCR amplification with TPI1 gene specific oligonucleotides identified as SEQ ID NO:72 (MC_TEF4) and SEQ ID NO:73 (MC_TEF3), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that SspI-digested M. circinelloides DNA was used. A .about.1100 bp PCR fragment was isolated and sequenced. Nested primers identified as SEQ ID NO:74 (MC_TEF8) and SEQ ID NO:75 (MC_TEF7) were designed and used in a ligation-mediated PCR amplification together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as above except that HpaI-digested M. circinelloides DNA was used. A .about.1200 bp PCR fragment was isolated and sequenced.

[0179] The M. circinelloides TEF1 terminator was PCR amplified by using primers identified as SEQ ID NO:76 (MC_TEF11) and SEQ ID NO:77 (MC_TEF12), and the M. circinelloides genomic DNA as the template. A PCR fragment was digested with XmaI and EcoRI and a 389 bp fragment was gel isolated and ligated to a XmaI and EcoRI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK65. Plasmid pKK65 contains the M. circinelloides TEF1 terminator.

Example 12. Cloning of Mucor circinelloides PGK (McPGK1) Promoter Region

[0180] A genomic fragment of the M. circinelloides PGK1 gene was amplified by PCR from genomic M. circinelloides griseocyanus (D-82202, VTT Culture Collection) DNA with degenerative primers identified as SEQ ID NO:78 (Mould PGK4) and SEQ ID NO:79 (Mould PGK2). The degenerative primers were designed based on a consensus sequence of PGK1 genes of Rhizopus oryzae, Fusarium oxysporum, Aspergillus fumigatus, A. oryzae and A. nidulans. A .about.250 bp genomic fragment was isolated and sequenced.

[0181] A genomic fragment containing the McPGK1 promoter region was obtained with a ligation-mediated PCR amplification with PGK1 gene specific oligonucleotides identified as SEQ ID NO:80 (MC_PGK2) and SEQ ID NO:81 (MC_PGK1), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that SspI-digested M. circinelloides DNA was used. A .about.1300 bp PCR fragment was isolated and sequenced. Nested primers identified as SEQ ID NO:82 (MC_PGK4) and SEQ ID NO:83 (MC_PGK3) were designed and used in a ligation-mediated PCR amplification together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as above except that HaeIII-digested M. circinelloides DNA was used. A .about.600 bp PCR fragment was isolated and sequenced.

[0182] The M. circinelloides PGK1 promoter was PCR amplified by using primers identified as SEQ ID NO:84 (MC_PGK5) and SEQ ID NO:85 (MC_PGK6), and the M. circinelloides genomic DNA as the template. A PCR fragment was digested with SacII and XbaI and a 1291 bp fragment was gel isolated and ligated to a SacII and XbaI-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK62. Plasmid pKK62 contains the M. circinelloides PGK1 promoter.

Example 13. Cloning of Mucor circinelloides GPD (McGPD1) Promoter Region

[0183] A genomic fragment containing the McGPD1 promoter region was obtained with a ligation-mediated PCR amplification with Mucor circinelloides (Syn. racemosus) GPD1 gene (GenBank accession number AJ293012, version number AJ293012.1) specific oligonucleotides identified as SEQ ID NO:86 (MC_GPD2) and SEQ ID NO:87 (MC_GPD1), together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as in Example 1A except that EcoRV-digested M. circinelloides (D-82202, VTT Culture Collection) DNA was used. A .about.500 bp PCR fragment was isolated and sequenced. Nested primers identified as SEQ ID NO:88 (MC_GPD10) and SEQ ID NO:89 (MC_GPD9) were designed and used in a ligation-mediated PCR amplification together with oligonucleotides identified as SEQ ID NO:3 (PCR Linker I) and SEQ ID NO:4 (PCR Linker II), similarly as above except that StuI-digested M. circinelloides DNA was used. A .about.1400 bp PCR fragment was isolated and sequenced.

[0184] The M. circinelloides GPD1 promoter was PCR amplified by using primers identified as SEQ ID NO:90 (MC_GPD11) and SEQ ID NO:91 (MC_GPD12), and the M. circinelloides genomic DNA as the template. A PCR fragment was digested with EcoRI and HindIII and a 1440 bp fragment was gel isolated and ligated to a EcoRI and HindIII-digested pBluescript KS-plasmid. The resulting plasmid was designated pKK59. Plasmid pKK59 contains the M. circinelloides GPD1 promoter.

Example 14A: Cloning of E. coli Hygromycin Resistance Gene; Construction of a Plasmid (pKK69) Having the E. coli Hygromycin Resistance Gene Under the Control of the McPGK1 Promoter and the McTPI1 Terminator

[0185] Plasmid pKK62 was digested with SacII and SbfI. A 1286 bp fragment was gel isolated. Plasmid pKK52 was digested with SbfI. A 1034 bp fragment was gel isolated. The 1286 bp fragment originated from plasmid pKK62 and the 1034 bp fragment originated from plasmid pKK52 were ligated to a 3268 bp fragment obtained by digesting plasmid pKK57 with SacII and W. Plasmid pKK52 contains the E. coli hygromycin resistance gene, plasmid pKK62 contains the M. circinelloides PGK1 promoter and plasmid pKK57 contains the M. circinelloides TPI1 terminator. The resulting plasmid was designated pKK69. Plasmid pKK69 contains the E. coli hygromycin resistance gene under the control of the M. circinelloides PGK1 promoter and the M. circinelloides TPI1 terminator.

Example 14B: Cloning of S. cerevisiae Cerulenin Resistance Gene; Construction of a Plasmid (pKK92) Having the S. cerevisiae Cerulenin Resistance Gene Under the Control of the McPGK1 Promoter and the McTPI1 Terminator

[0186] Plasmid pKK80 was digested with PstI. A 1685 bp fragment was gel isolated. Plasmid pKK80 contains the S. cerevisiae cerulenin resistance gene. The 1685 bp fragment was ligated to a 4554 bp fragment obtained by digesting plasmid pKK69 with SbfI. Plasmid pKK69 contains the M. circinelloides PGK1 promoter and the M circinelloides TRI1 terminator. The resulting plasmid was designated pKK92. Plasmid pKK92 contains the S. cerevisiae cerulenin resistance gene under the control of the M. circinelloides PGK1 promoter and the M. circinelloides TPI1 terminator.

Example 15A. Construction of a Plasmid (pKK75, FIG. 8) Containing the Hygromycin Resistance Gene Under the Control of the McPGK1 Promoter and the McTPI1 Terminator and the S. cerevisiae ALD6 Gene Under the Control of the McTPI1 Promoter and the McTEF1 Terminator

[0187] Plasmid pKK56 was digested with BamHI and PstI. A 1251 bp fragment was gel isolated. Plasmid pKK56 contains the McTPI1 promoter. Plasmid RoALD was digested with SbfI. A 1514 bp fragment was gel isolated. The plasmid RoALD contains the S. cerevisiae ALD6 (SEQ ID NO:47) encoding gene which has been codon optimized according to Rhizopus oryzae filamentous fungus codon usage (SEQ ID NO:49) with flanking SbfI restriction sites. The 1251 bp fragment originating from the plasmid pKK56 and the 1514 bp fragment originating from the plasmid RoALD were ligated to a 3321 bp fragment obtained by digesting plasmid pKK65 with BamHI and SbfI. Plasmid pKK65 contains the McTEF1 terminator. The resulting plasmid was designated as pKK73. Plasmid pKK73 contains the S. cerevisiae ALD6 encoding gene under the control of the M. circinelloides TPI1 promoter and the M. circinelloides TEF1 terminator.

[0188] Plasmid pKK69 was digested with BamHI and XmnI. A 2652 bp fragment was gel isolated and ligated to a 6086 bp fragment obtained by digesting plasmid pKK73 with BamHI. Plasmid pKK69 contains the E. coli hygromycin resistance gene under the control of the M. circinelloides PGK1 promoter and the M. circinelloides TPI1 terminator. The resulting plasmid was designated as pKK75 (FIG. 8).

Example 15B. Generation of a Genetically Modified Mucor circinelloides (M22/75) with an Integrated ALD6 Encoding Gene and a Hygromycin Resistance Gene by Transforming Wild-Type M. circinelloides with Digested Plasmid pKK75 (FIG. 8, Ex. 15A)

[0189] Plasmid pKK75 was restricted with KpnI and NotI. A 5866 bp fragment was gel isolated and used to transform a wild-type M. circinelloides strain (D-82202, VTT Culture Collection) designated as M22, using a Mucor protoplast transformation method (Wolff et. al. 2002). The transformed cells were screened for hygromycin resistance. Several hygromycin-resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of the wild-type M. circinelloides strain with KpnI and NotI cut pKK75 and containing the S. cerevisiae ALD6 encoding gene under the control of the McTPI1 promoter and the McTEF1 terminator were designated as M22/75-80 and M22/75-86.

Example 16A. Construction of a Plasmid (pKK94, FIG. 9) Containing the Hygromycin Resistance Gene Under the Control of the McPGK1 Promoter and the McTPI1 Terminator and the S. cerevisiae ALD6 Encoding Gene Under the Control of the McTPI1 Promoter and the McTEF1 Terminator and the S. cerevisiae ACS2 Encoding Gene Under the Control of the McTEF1 Promoter and the McTPI1 Terminator

[0190] Plasmid pKK57 was digested with PstI. A 351 bp fragment was gel isolated and ligated to a 4326 bp fragment obtained by digesting plasmid pKK64 with PstI. The plasmid pKK57 contains the McTPI terminator and the plasmid pKK64 contains the McTEF promoter. The resulting plasmid was designated pKK90Pre. Plasmid RoACS was digested with SbfI. A 2060 bp fragment was gel isolated and ligated to a 4677 bp fragment obtained by digesting plasmid pKK90Pre with SbfI. The plasmid RoACS contains the S. cerevisiae ACS2 (SEQ ID NO:50) encoding gene which has been codon optimized according to Rhizopus oryzae filamentous fungus codon usage (SEQ ID NO:92) with flanking SbfI restriction sites. The resulting plasmid was designated as pKK90.

[0191] Plasmid pKK75 was digested with KpnI followed by removal of the 3' overhangs by T4 DNA polymerase and each of the 4 dNTPs. KpnI (blunt)-digested plasmid pKK75 was digested with NotI and a 5866 bp fragment was gel isolated. The plasmid pKK75 contains the E. coli hygromycin resistance gene under the control of the M. circinelloides PGK1 promoter and the M. circinelloides TPI1 terminator and S. cerevisiae ALD6 encoding gene under the control of the M. circinelloides TPI1 promotor and the M. circinelloides TEF1 terminator. The 5866 bp fragment originating from plasmid pKK75 was ligated to a 6698 bp fragment obtained by digesting plasmid pKK90 with SmaI and NotI. The resulting plasmid was designated as pKK94 (FIG. 9).

Example 16B. Generation of a Genetically Modified Mucor circinelloides (M22/94) with an Integrated ALD6 and ACS2 Encoding Genes and a Hygromycin Resistance Gene by Transforming Wild-Type M. circinelloides with Digested Plasmid pKK94 (FIG. 9, Ex. 16A)

[0192] Plasmid pKK94 was restricted with KpnI and SacI. A 9701 bp fragment was gel isolated and used to transform a wild-type M. circinelloides strain (D-82202, VTT Culture Collection) designated as M22, using the transformation method described in Example 15B. The transformed cells were screened for hygromycin resistance. Several hygromycin resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of a wild-type M. circinelloides strain with KpnI and SacI cut pKK94 and containing the S. cerevisiae ALD6 encoding gene under the control of the McTPI1 promoter and the McTEF1 terminator and S. cerevisiae ACS2 encoding gene under the control of the McTEF1 promoter and the McTPI1 terminator were designated as M22/94-12, M22/94-16 and M22/94-24.

Example 17A. Construction of a Plasmid (pKK96, FIG. 10) Containing the Hygromycin Gene Under the Control of the McPGK1 Promoter and the McTPI1 Terminator and the S. cerevisiae ACS2 Encoding Gene Under the Control of the McTEF1 Promoter and the McTPI1 Terminator

[0193] Plasmid pKK94 was digested with BsrGI and a 9683 bp fragment was gel isolated and self ligated. The resulting plasmid was designated as pKK96 (FIG. 10). The plasmid pKK96D contains the S. cerevisiae ACS2 encoding gene under the control of the McTEF1 promoter and the McTPI1 terminator.

Example 17B. Generation of a Genetically Modified Mucor circinelloides (M22/96) with an Integrated ACS2 Encoding Gene and Hygromycin Resistance Gene by Transforming Wild-Type M. circinelloides with Digested Plasmid pKK96 (FIG. 10, Ex. 17A)

[0194] Plasmid pKK96 was restricted with KpnI and SacI. A 6824 bp fragment was gel isolated and used to transform the wild-type M. circinelloides strain (D-82202, VTT Culture Collection) designated as M22, using the transformation method described in Example 15B. The transformed cells were screened for hygromycin resistance. Several hygromycin resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of the wild-type M. circinelloides strain with KpnI and SacI cut pKK96D and containing the S. cerevisiae ACS2 encoding gene under the control of the McTEF1 promoter and the McTPI1 terminator were designated as M22/96-1 and M22/96-6.

Example 18A. Construction of a Plasmid (pKK98) Containing the Cerulenin Resistance Gene Under the Control of the McPGK1 Promoter and the McTPI1 Terminator and the R. oryzae PDAT Gene Under the Control of the McGPD1 Promoter and the McTEF1 Terminator

[0195] Plasmid pKK59 was digested with SbfI and XbaI. A 1467 bp fragment was gel isolated and ligated to a 3309 bp fragment obtained by digesting plasmid pKK65 with SbfI and XbaI. The plasmid pKK59 contains the McGPD1 promoter and the plasmid pKK65 contains the McTEF1 terminator. The resulting plasmid was designated as pKK88. Plasmid RoPDAT was digested with SbfI. A 1844 bp fragment was gel isolated and ligated to a 4776 bp fragment obtained by digesting plasmid pKK88 with SbfI. The plasmid RoPDAT contains the R. oryzae PDAT (SEQ ID NO:52) encoding gene, which has been codon optimized according to R. oryzae codon usage (SEQ. ID. NO 93) with flanking SbfI restriction sites. The resulting plasmid was designated as pKK97.

[0196] Plasmid pKK97 was digested with EcoRI. A 3659 bp fragment was gel isolated and ligated to a 6239 bp fragment obtained by digesting plasmid pKK92 with EcoRI. The plasmid pKK92 contains the S. cerevisiae cerulenin resistance gene under the control of the McPGK1 promoter and the McTPI1 terminator. The resulting plasmid was designated as pKK98 (FIG. 11).

Example 18B. Generation of a Genetically Modified Mucor circinelloides (M22/98) with an Integrated PDAT Encoding Gene and Cerulenin Resistance Gene by Transforming Wild-Type M. circinelloides with Digested Plasmid pKK98 (FIG. 11, Ex. 18A)

[0197] Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp fragment was gel isolated and used to transform the wild-type M. circinelloides strain (D-82202, VTT Culture Collection) designated as M22, using the transformation method described in Example 15B. The transformed cells were screened for cerulenin resistance. Several cerulenin resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of the wild-type M. circinelloides strain with ApaI and SacII cut pKK98 and containing the R. oryzae PDAT encoding gene under the control of the McGPD1 promoter and the McTEF1 terminator were designated as M22/98-16.

Example 19. Generation of a Genetically Modified Mucor circinelloides (M22/75/98) with Integrated ALD6 and PDAT Encoding Genes and Hygromycin and Cerulenin Resistance Genes by Transforming Genetically Modified Strain M22/75-86 (Ex 15B) with Digested Plasmid pKK98 (FIG. 11, Ex. 18A)

[0198] Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp fragment was gel isolated and used to transform the genetically modified strain M22/75-86, using the transformation method described in Example 15B. The transformed cells were screened for cerulenin resistance. Several cerulenin resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of the recombinant M22/75-86 strain with ApaI and SacII cut pKK98 and containing the R. oryzae PDAT encoding gene under the control of the McGPD1 promoter and the McTEF1 terminator and the S. cerevisiae ALD6 encoding gene under the control of the McTPI1 promoter and the McTEF1 terminator were designated as M22/75/98-7 and M22/75/98-9.

Example 20. Generation of a Genetically Modified Mucor circinelloides (M22/94/98) with Integrated ALD6, ACS2 and PDAT Encoding Genes and Hygromycin and Cerulenin Resistance Genes by Transforming Genetically Modified Strain M22/94-31 (Ex 16B) with Digested Plasmid pKK98 (FIG. 11, Ex. 18A)

[0199] Plasmid pKK98 was restricted with ApaI and SacII. A 7029 bp fragment was gel isolated and used to transform the genetically modified strain M22/94-31, using the transformation method described in Example 15B. The transformed cells were screened for cerulenin resistance. Several cerulenin resistant colonies were analysed at DNA level by PCR. The transformants originating from the transformation of the recombinant M22/94-31 strain with ApaI and SacII cut pKK98 and containing the R. oryzae PDAT encoding gene under the control of the McGPD1 promoter and the McTEF1 terminator, the S. cerevisiae ALD6 encoding gene under the control of the McTPI1 promoter and the McTEF1 terminator and the S. cerevisiae ACS2 encoding gene under the control of the McTEF1 promoter and the McTPI1 terminator were designated as M22/94/98-19 and M22/94/98-22.

Example 21. Lipid Extraction and Total Lipid and Triglyceride Concentration Measurements

[0200] A lipid extraction method was modified from the protocol of Folch et al., 1957. 0.5 to 2 ml of cell culture was taken into an Eppendorf tube. The sample was centrifuged and the supernatant discarded. The pellet was placed rapidly in liquid nitrogen and stored at -80.degree. C. Alternatively, filamentous fungal cells of 2 to 12 ml culture broth were collected by vacuum filtration through disks of glass microfiber filters (Whatman, England). After washing twice with distilled water, biomass was removed from the filter using a clean spatula and put into 2 ml microfuge tubes, which were placed rapidly in liquid nitrogen and stored at -80.degree. C. In a homogenisation step the frozen pellet was suspended in 500 .mu.l of ice-cold methanol with 0.1% BHT (2,6-Di-tertbutyl-4-methylphenol) and homogenised with a Mixer Mill homogenizator with 5-mm zirconium oxide and 3-mm yttrium stabilized zirconium oxide balls (Retsch) at 25 Hz for 5 min. After homogenisation 1000 .mu.l of chloroform was added and homogenisation repeated. After re-homogenisation 300 .mu.l of 20 mM acetic acid was added and the sample vortexed for 10 min. After vortexing the sample was centrifuged 13 000 rpm for 5 min at RT. The lower phase was recovered and 1000 .mu.l of chloroform was added to the remaining phase, vortexed and recentrifuged. The lower phases were combined into pre-weighed 2 ml microfuge tubes, and dried, after which the total lipid content of the sample was determined by gravimetry. Then the lipid sample was redissolved in 1.5 ml of chloroform:methanol (2:1)+0.1% BHT and stored at -20.degree. C. For triacylglycerol analysis 100 to 1500 .mu.l of chloroform:methanol extracted lipids were evaporated and re-dissolved in 200-1000 .mu.l of isopropanol. Triacylglycerols were measured enzymatically from the samples by using the Konelab Triglycerides Kit (Thermo Scientific, Finland) and Cobas Mira automated analyser (Roche) or a microtitre-plate reader (Varioskan, Thermo Electron Corporation). This lipid extraction and total lipid and triglyceride concentration measurements methods were used in the following examples if not otherwise indicated.

Example 22. Microaerobic Shake Flask Characterization of Strains Y23/81-51 and Y23/81-66 (Ex. 3B), Y23/86-86 and Y23/86-92 (Ex. 5B), Y23/95-87 and Y23/95-109 (Ex. 6B) and Y23/85/95-4 (Ex. 9), in Glucose Medium with C/N Ratio of 20

[0201] Transformants were separately cultivated in 50 ml of culture medium "glucose CN20" (pH 5.5, 20 g glucose, 0.3 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 100 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control.

[0202] Lipid extraction and triacylglycerol concentration measurement were carried out as described in Example 21. Cell dry weight was determined by centrifuging 1 ml of the culture broth in pre-dried, pre-weighed Eppendorf tubes. After washing with 1 ml of distilled water the cell pellet was dried at 100.degree. C. for 24 hours and weighed again. HPLC analyses for sugars were conducted with a Waters 2690 Separation Module and Water System Interfase Module liquid chromatography coupled with a Waters 2414 differential refractometer and Waters 2487 dual absorbance detector. The liquid chromatography columns were a 100.times.7.8 mm Fast Acid Analysis column from Bio-Rad and a 300.times.7.8 mm Aminex HPX-87H column from Bio-Rad. The columns were equilibrated with 2.5 mM H.sub.2SO.sub.4 in water at 55.degree. C. and samples were eluted with 2.5 mM H.sub.2SO.sub.4 in water at 0.5 ml/min flow rate. Data acquisition was done using Waters Millennium software. This HPLC method was used in all appropriate Examples.

[0203] After 48 hours cultivation (Table 2A), when 4 to 8 g/l glucose was left, Y23/81, Y23/86, Y23/95 and Y23/85/95 transformants produced 12, 22, 15 and 24% more triacylglycerols with higher rate, respectively, than the control strain in glucose medium. Triacylglycerol yields per used glucose were also better up to 10% with the transformants compared to the control strain. Additionally Y23/85/95 transformant had 13% higher triacylglycerol yield on biomass than the control strain (12.9 and 11.4% TAG yield on biomass, respectively). In particular, after 24 hours of cultivation (Table 2B), the strains expressing ALD or ACS alone, or ALD and ACS together, had enhanced production of triacylglycerols, measured as concentration (g/l) and as yield per biomass and used glucose, with higher rate (mg/l/h) compared to the control strain.

TABLE-US-00002 TABLE 2A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) and yield (%) per used glucose after 48 hours microaerobic cultivation in glucose medium with C/N ratio of 20 Yield TAG TAG (% used TAG Strain (g/l) glucose) mg/l/h Control 0.72 5.88 15.0 Y23/81 (ALD) 0.81 5.90 16.8 Y23/86 (ACS) 0.88 6.00 18.3 Y23/95 (PDAT) 0.82 6.02 17.2 Y23/85/95 0.89 6.46 18.5 (ALD + ACS + PDAT)

TABLE-US-00003 TABLE 2B Triacylglycerol (TAG) concentration (g/l) and yield (%) per biomass (CDW) and used glucose after 24 hours microaerobic cultivation in glucose medium with C/N ratio of 20 TAG Yield TAG Yield TAG TAG Strain (g/l) (% CDW) (% used glucose) mg/l/h Control 0.22 5.15 4.29 9.1 Y23/81-66 (ALD) 0.26 6.09 4.86 10.8 Y23/85-125 (ALD + 0.31 7.01 5.49 12.8 ACS) Y23/86-86 (ACS) 0.30 6.76 5.26 12.5

[0204] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and rates of production, and triacylglycerol yields from used glucose or per dry weight in cultivation with low C/N ratios.

Example 23. Aerobic Shake Flask Characterization of Strains Y23/81-8, 51, 59, 66 and 69 (Ex. 3B), Y23/85-119, 125, 128, 129 and 139 (Ex. 7B), Y23/86-86, 92, 93, 98 and 100 (Ex. 5B), Y23/95-99 and 104 (Ex. 6B), Y23/81/95-42 (Ex. 8) and Y23/85/95-4 and 68 (Ex. 9), in Glucose Medium with C/N Ratio of 65

[0205] Transformants were separately cultivated in 50 ml of Yeast culture medium II (pH 5.5, 20 g glucose, 0.3 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.6 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22. Lipid extraction and triacylglycerol measurements as described in Example 21.

[0206] After 44 hours cultivation (Table 3A) transformants Y23/81, Y23/85, Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had 4, 3, 6, 1, 12 and 4% better triacylglycerol yields per used glucose, respectively, than the control strain. Additionally, transformants Y23/81/95 and Y23/85/95 had 7-8% higher triacylglycerol yield on biomass than the control strain (42.8, 43.0 and 40.0% TAG yield [/CDW], respectively). After 25 hours cultivation (Table 3B), the strains expressing ALD or ACS alone, or ALD and ACS together, had enhanced production of triacylglycerols, measured as concentration (g/l) and as yield per biomass and used glucose.

TABLE-US-00004 TABLE 3A Maximal triacylglycerol (TAG) yield (%) per used glucose after 44 hours cultivation in glucose medium with C/N ratio of 65 Yield TAG Strain (% used glucose) Control 16.6 Y23/81 (ALD) 17.3 Y23/85 (ALD + ACS) 17.2 Y23/86 (ACS) 17.6 Y23/95 (PDAT) 16.9 Y23/81/95 (ALD + PDAT) 18.6 Y23/85/95 (ALD + ACS + PDAT) 17.4

TABLE-US-00005 TABLE 3B Triacylglycerol (TAG) concentration (g/l) and yield (%) per biomass (CDW) and used glucose after 25 hours cultivation in glucose medium with C/N ratio of 65 TAG Yield TAG Yield TAG TAG Strain (g/l) (% CDW) (% used glucose) mg/l/h Control 1.01 21.7 10.3 40.5 Y23/81-51, 66 (ALD) 1.15 26.4 12.5 46.0 Y23/85-125, 129 1.14 27.2 13.3 45.4 (ALD + ACS) Y23/86-92, 100 (ACS) 1.18 22.4 12.0 47.1

[0207] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and rates of production and triacylglycerol yields from used glucose or per dry weight.

Example 24. Aerobic Shake Flask Characterization of Strains Y23/81-66 (Ex. 3B), Y23/85-125 (Ex. 7B), Y23/86-92 (Ex. 5B), Y23/95-98 (Ex. 6B) and Y23/81/95-18 (Ex. 8) in Glucose Medium with C/N Ratio of 103

[0208] Transformants were separately cultivated in 50 ml of Yeast culture medium IV (pH 5.5, 20 g glucose, 0.15 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22. Lipid extraction and triacylglycerol measurements as described in Example 21.

[0209] After 35 hours cultivation, when 6-8 g/l glucose was left, transformants Y23/81-66, Y23/85-125, Y23/86-92, Y23/95-98 and Y23/81/95-18 had 3, 6, 9, 11 and 11% higher triacylglycerol titre and rate than the control strain, respectively. The transformants Y23/81-66, Y23/85-125, Y23/86-92, Y23/95-98 and Y23/81/95-18 had also 11, 19, 7, 20 and 30% better triacylglycerol yields per used glucose, respectively, than the control strain. Additionally Y23/81/95-18 had 24% higher yield on biomass than the control strain (49.4 and 40.0% TAG yields on biomass, respectively).

TABLE-US-00006 TABLE 4 Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) and yield (%) per used glucose after 35 hours cultivation in glucose medium with C/N ratio of 103 Yield TAG (% used Strain TAG (g/l) glucose) TAG mg/l/h Control 2.00 16.2 57.1 Y23/81-66 (ALD) 2.05 18.0 58.7 Y23/85-125 (ALD + ACS) 2.11 19.3 60.3 Y23/86-92 (ACS) 2.17 17.4 61.9 Y23/95-98 (PDAT) 2.22 19.4 63.5 Y23/81/95-18 (ALD + PDAT) 2.22 21.1 63.5

[0210] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and rates of production and triacylglycerol yield from used glucose or per dry weight with high C/N ratios.

Example 25. Aerobic Shake Flask Characterization of Strains M22/75-86 (Ex. 15B), M22/96-1 (Ex. 17B), M22/94-24 (Ex. 16B), M22/75/98-9 (Ex. 19) and M22/94/98-19 (Ex. 20), in Glucose Medium with C/N Ratio of 40

[0211] Transformants were separately cultivated in 50 ml of mould C/N 40 medium (pH 5.5, 20 g glucose, 1.4 g yeast extract, 2.5 g KH.sub.2PO.sub.4, 0.3 g (NH.sub.4).sub.2SO.sub.4, 10 mg ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO.sub.4, 0.5 g MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg FeCl.sub.3*6H.sub.2O per litre). Each flask (250 ml) was inoculated with 1*10.sup.7 spores. The cultivations were maintained at a temperature of 25.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Mucor circinelloides wild type strain M22 was used as a control. Cell dry weight was determined by vacuum filtration through disks of glass microfiber filters (Whatman, England) of 2 to 24 ml culture broth. After washing twice with distilled water, biomass was removed from the cloth using clean spatula and transferred to pre-dried, pre-weighed 2 ml microfuge tubes in which the mycelia were dried at 100.degree. C. for 48 hours and weighed after cooling in a dessicator. HPLC analysis was carried out as described in Example 22. Lipid extraction and total lipid and triacylglycerol measurements as described in Example 21.

[0212] After 46 hours cultivation (Table 5) transformants M22/75-86, M22/96-1, M22/94-24, M22/75/98-9 and M22/94/98-19 produced 21, 9, 18, 91 and 55% more triacylglycerol with higher rate than the control strain, respectively. The transformant M22/75-86, M22/96-1., M22/94-24, M22/75/98-9 and M22/94/98-19 had also 39 (20), 23 (10), 18 (13), 127 (57) and 80 (25)% higher triacylglycerol yield on used glucose (on biomass) than the control strain, respectively. Additionally the transformants M22/75/98-9 and M22/94/98-19 had higher total lipid concentration (0.93-1.09 g/l) and rate (20-24 mg/l/h) with yields on biomass (26.1-31.4%) and on used glucose (6.29-7.52%) than the control strain (0.71 g/l, 15 mg/l/h, 24.6% and 4.09%, respectively).

TABLE-US-00007 TABLE 5 Triacylglycerol (TAG) and total lipid concentrations (g/l), rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) and used glucose after 46 hours cultivation in glucose medium with C/N ratio of 40 Yield Yield TAG TAG (% used TAG Strain TAG g/l (% CDW) glucose) mg/l/h Control 0.33 11.5 1.92 7.2 M22/75-86 (ALD) 0.40 13.8 2.67 8.8 M22/96-1 (ACS) 0.36 12.6 2.36 7.9 M22/94-24 (ALD + ACS) 0.39 13.0 2.27 8.5 M22/75/98-9 (ALD + 0.63 18.1 4.35 13.7 PDAT) M22/94/98-19 0.51 14.4 3.46 11.1 (ALD + ACS + PDAT) Yield lipid Yield lipid (% used Lipid Strain Lipid g/l (% CDW) glucose) mg/l/h Control 0.71 24.6 4.09 15 M22/75-86 (ALD) 0.76 26.1 5.04 17 M22/96-1 (ACS) 0.70 24.2 4.53 15 M22/94-24 (ALD + ACS) 0.71 23.5 4.10 15 M22/75/98-9 (ALD + 1.09 31.4 7.52 24 PDAT) M22/94/98-19 (ALD + 0.93 26.1 6.29 20 ACS + PDAT)

[0213] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol and total lipid concentrations and rates of production and triacylglycerol and total lipid yields from used glucose or per dry weight.

Example 26. Aerobic Shake Flask Characterization of Strains M22/75-86 (Ex. 15B), M22/96-1 (Ex. 17B), M22/94-24 (Ex. 16B), M22/75/98-9 (Ex. 19) and M22/94/98-19 (Ex. 20), in Glucose Medium with C/N Ratio of 66

[0214] Transformants were separately cultivated in 50 ml of mould C/N 66 medium (pH 5.5, 20 g glucose, 1.0 g yeast extract, 2.5 g KH.sub.2PO.sub.4, 0.1 g (NH.sub.4).sub.2SO.sub.4, 10 mg ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO.sub.4, 0.5 g MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg FeCl.sub.3*6H.sub.2O per litre). Each flask (250 ml) was inoculated with 1*10.sup.7 spores. The cultivations were maintained at a temperature of 25.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction, enzyme activity measurement and HPLC analysis were withdrawn periodically during cultivation. Mucor circinelloides wild type strain M22 was used as a control. Cell dry weight was determined as described in Example 25 and HPLC analysis was carried out as described in Example 22. Lipid extraction and total lipid and triacylglycerol measurements as described in Example 21.

[0215] After 93 hours cultivation, when 2-6 g/l glucose was left, transformants M22/75-86, M22/96-1, M22/94-24, M22/75+98-9 and M22/94+98-19 had produced 34, 30, 45, 186 and 214% more triacylglycerols with higher rate than the control strain. The transformants M22/75-86, M22/96-1, M22/94-24, M22/75+98-9 and M22/94+98-19 also had 63 (24), 68 (30), 62 (43), 229 (72) and 102 (104)% higher triacylglycerol yield on used glucose (on biomass) than the control strain. Additionally, the transformants produced more lipids (0.89-2.29 g/l) with higher rates (9.6-24.6 mg/l/h) and with higher yields on used glucose (6.90-17.68%) and on biomass (37.2-57.7%) than the control strain (0.71 g/l, 7.6 mg/l/h, 4.82% and 30.2%, respectively).

TABLE-US-00008 TABLE 6 Triacylglycerol (TAG) and total lipid concentrations (g/l), rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) and used glucose after 93 hours cultivation in glucose medium with C/N ratio of 66. Yield Yield TAG TAG TAG (% used TAG Strain g/l (% CDW) glucose) mg/l/h Control 0.44 18.6 2.97 4.7 M22/75-86 (ALD) 0.59 23.0 4.84 6.4 M22/96-1 (ACS) 0.57 24.1 4.99 6.1 M22/94-24 (ALD + ACS) 0.64 26.6 4.82 6.9 M22/75/98-9 1.26 31.9 9.76 13.6 (ALD + PDAT) M22/94/98-19 1.38 37.9 9.00 14.8 (ALD + ACS + PDAT) Yield lipid Yield lipid (% used Lipid Strain Lipid g/l (% CDW) glucose) mg/l/h Control 0.71 30.2 4.8 7.6 M22/75-86 (ALD) 0.96 37.2 7.8 10.3 M22/96-1 (ACS) 0.89 37.3 7.7 9.6 M22/94-24 (ALD + ACS) 0.91 37.6 6.9 9.8 M22/75/98-9 (ALD + 2.29 57.7 17.7 24.6 PDAT) M22/94/98-19 2.00 55.1 13.1 21.5 (ALD + ACS + PDAT)

[0216] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol and total lipid concentrations and rates of production and triacylglycerol and total lipid yields from used glucose or per dry weight with high C/N ratios.

Example 27. Microaerobic Shake Flask Characterization of Strains Y23/81-51 and Y23/81-66 (Ex. 3B), Y23/85-125 and Y23/85-128 (Ex. 7B), Y23/86-86 and Y23/86-92 (Ex. 5B), Y23/95-87 and Y23/95-109 (Ex. 6B) and Y23/85/95-4 (Ex. 9), in Xylose Medium with C/N Ratio of 20

[0217] Transformants were separately cultivated in 50 ml of culture medium "xylose CN20" (pH 5.5, 20 g xylose, 0.3 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 100 rpm. Samples for cell dry weight measurement, lipid extraction, enzyme activity measurement and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control.

[0218] Lipid extraction and triacylglycerol concentration measurements were carried out as described in Example 21. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22.

[0219] At the end of cultivation samples for acetaldehyde dehydrogenase and acetyl-CoA synthetase activity measurements were taken. Cells were harvested by centrifugation and washed once with 100 mM Tris-HCl pH 7.0. Washed cells were stored at -20.degree. C. Frozen cells were melted and washed once with 100 mM Tris-HCl pH 7.0 and suspended in 100 mM Tris-HCl pH 7.0, 1 mM DTT buffer containing EDTA-free protease inhibitors (Roche, USA). Cell disruption was carried out with 0.5 mm diameter glass beads (Sigma Chemicals Co, USA) in a Fast Prep homogenizer (Thermo Scientific, USA). Cell debris was removed by centrifugation and supernatant was used in enzyme activity measurements. Acetaldehyde dehydrogenase and acetyl-CoA synthetase activity measurements were carried out with a Konelab Arena automatic analyzer (Thermo Scientific, Finland). The acetaldehyde dehydrogenase reaction mixture contained (final concentration) 50 mM potassium phosphate pH 7.0, 15 mM pyrazole, 0.4 mM DTT, 10 mM MgCl.sub.2, 0.4 mM NADP and cell extract. The reaction was started with 0.1 mM acetaldehyde. The formation of NADPH was followed at 340 nm. One unit was defined as the amount of formation of 1 .mu.mol of NADPH per min. The acetyl-CoA synthetase reaction mixture contained (final concentration) 100 mM Tris-HCl pH 7.5, 10 mM L-malate pH 7.5, 0.2 mM Coenzyme A, 8 mM ATP, 1 mM NAD, 10 mM MgCl.sub.2, 3 U/ml malate dehydrogenase, 0.4 U/ml citrate synthase and cell extract. The reaction was started with 100 mM potassium acetate. The formation of NADH was followed at 340 nm. One unit was defined as the amount of formation of 1 .mu.mol of NADH per min.

[0220] After 24 hours cultivation the transformants Y23/81, Y23/85, Y23/86, Y23/95 and Y23/85/95 produced 13-31% more triacylglycerol with 5-38% higher yield on biomass than the control strain. The transformants Y23/81, Y23/86, Y23/95 and Y23/85/95 also had 4-12% higher yields on used xylose than the control strain. The transformants having ALD (ACS) encoding gene expressed had 21.5 to 42.7 (1.5 to 1.9) times higher ALD (ACS) activity than the control strain.

TABLE-US-00009 TABLE 7 Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) and used xylose after 24 hours microaerobic cultivation in xylose medium with C/N ratio of 20 TAG Yield TAG Yield TAG (% TAG Strain (g/l) (% CDW) used xylose) mg/l/h Control 0.16 4.33 3.46 6.8 Y23/81 (ALD) 0.20 5.98 3.61 8.2 Y23/85 (ALD + ACS) 0.18 4.54 3.46 7.3 Y23/86 (ACS) 0.21 5.33 3.86 8.9 Y23/95 (PDAT) 0.20 5.55 3.68 8.4 Y23/85/95 0.20 5.55 3.72 8.2 (ALD + ACS + PDAT)

TABLE-US-00010 TABLE 8 Relative acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS) activities in microaerobic cultivation in xylose with C/N ratio of 20 compared to the control strain ALD and ACS activities Strain ALD ACS Control 1 1 Y23/81 (ALD) 42.7 1.2 Y23/85 (ALD + ACS) 27.3 1.9 Y23/86 (ACS) 4.3 1.5 Y23/95 (PDAT) 2.3 1.2 Y23/85/95 (ALD + ACS + PDAT) 21.5 1.5

[0221] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and rates of production and triacylglycerol yields from used xylose or per dry weight with low C/N ratios. Additionally, the example shows that acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS) enzymes are expressed in active forms.

Example 28. Aerobic Shake Flask Characterization of Strains Y23/81-8 and 59 (Ex. 3B), Y23/85-119 and 128 (Ex. 7B), Y23/86-86 and 92 (Ex. 5B), Y23/95-99 and 109 (Ex. 6B), Y23/81/95-18 and 42 (Ex. 8) and Y23/85/95-4 and 68 (Ex. 9), in Xylose Medium with C/N Ratio of 103

[0222] Transformants were separately cultivated in 50 ml of Yeast xylose culture medium IV (pH 5.5, 20 g xylose, 0.15 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22. Lipid extraction and triacylglycerol measurements as described in Example 21.

[0223] After 52 hours cultivation the transformants Y23/81, Y23/85, Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had produced 23, 19, 50, 52, 40 and 75% more triacylglycerol than the control strain. The transformants Y23/81, Y23/85, Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had also 35, 47, 59, 60, 103 and 111% higher triacylglycerol yield on biomass and 11, 11, 15, 28, 66 and 63% higher triacylglycerol yield on used xylose than the control strain. Additionally the transformants Y23/81, Y23/85, Y23/86, Y23/95, Y23/81/95 and Y23/85/95 had higher lipid concentration (2.40-2.90 g/l) and rate (46-56 mg/l/h) with higher yield on biomass (56.5-78.8%) than the control strain (2.35 g/l, 45 mg/l/h and 52.2%, respectively). The transformants Y23/81/95 and Y23/85/95 had also higher lipid yield on used xylose (29.0-31.6%) than the control strain (25.6%).

TABLE-US-00011 TABLE 9 Triacylglycerol (TAG) and total lipid concentrations (g/l), rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) after 52 hours cultivation in xylose medium with C/N ratio of 103. Triacylglycerol yield (%) from used xylose is also indicated. Yield TAG Yield TAG (% used TAG Strain TAG (g/l) (% CDW) xylose) mg/l/h Control 0.60 13.3 6.52 19.0 Y23/81 (ALD) 0.83 17.9 7.23 26.4 Y23/85 (ALD + ACS) 0.79 19.6 7.26 25.0 Y23/86 (ACS) 0.90 21.1 7.52 28.5 Y23/95 (PDAT) 0.91 21.3 8.37 28.8 Y23/81/95 0.84 27.0 10.8 26.6 (ALD + PDAT) Y23/85/95 1.05 28.0 10.7 33.3 (ALD + ACS + PDAT) Yield lipid Strain Lipid (g/l) (% CDW) Lipid mg/l/h Control 2.35 52.2 45 Y23/81 (ALD) 2.90 62.3 56 Y23/85 (ALD + ACS) 2.53 62.9 49 Y23/86 (ACS) 2.58 60.6 50 Y23/95 (PDAT) 2.40 56.5 46 Y23/81/95 (ALD + PDAT) 2.45 78.8 47 Y23/85/95 (ALD + ACS + PDAT) 2.85 76.0 55

[0224] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol and total lipid concentrations and rates of production and triacylglycerol and total lipid yields per dry weight and triacylglycerol yields from used xylose with high C/N ratios.

Example 29. Aerobic Shake Flask Characterization of Strains M22/75-80 (Ex. 15B), M22/96-6 (Ex. 17B), M22/98-16 (Ex. 18B), M22/94-16 (Ex. 16B), M22/75/98-7 (Ex. 19) and M22/94/98-22 (Ex. 20), in Xylose Medium with C/N Ratio of 66

[0225] Transformants were separately cultivated in 50 ml of mould xylose C/N 66 medium (pH 5.5, 20 g xylose, 1.0 g yeast extract, 2.5 g KH.sub.2PO.sub.4, 0.1 g (NH.sub.4).sub.2SO.sub.4, 10 mg ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO4, 0.5 g MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg FeCl.sub.3*6H.sub.2O per litre). Each flask (250 ml) was inoculated with 1*10.sup.7 spores. The cultivations were maintained at a temperature of 25.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction, enzyme activity measurement and HPLC analysis were withdrawn periodically during cultivation. Mucor circinelloides wild type strain M22 was used as a control. Cell dry weight was determined as described in Example 25, and HPLC analysis was carried out as described in Example 22. Lipid extraction and total lipid and triacylglycerol measurements as described in Example 21.

[0226] After 143 hours cultivation the transformants M22/75-80, M22/96-6, M22/98-16, M22/75/98-7 and M22/94/98-22 had higher TAG concentration (0.33-0.48 g/l TAG) compared to the control strain (0.28 g/l TAG). The all transformants had also higher TAG yield (%) per biomass (13.71-17.78%) than the control strain (12.15%) and the transformants M22/96-6, M22/98-16, M22/75/98-7 and M22/94/98-22 had also higher TAG yield (%) per used xylose (4.83-6.23%) than the control strain (3.97%). Additionally the transformants M22/75/98-7 and M22/94/98-22 had higher lipid concentration (0.79-1.13 g/l), rate (9.2-10.8 mg/l/h) and lipid yields on biomass (48.9-55.6%) and on used xylose (17.1-18.3%) than the control strain (0.92 g/l, 6.4 mg/l/h, 40.0% and 13.0%, respectively).

TABLE-US-00012 TABLE 10 Triacylglycerol (TAG) and total lipid concentrations (g/l), rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) and used xylose after 143 hours cultivation in xylose medium with C/N ratio of 66 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) xylose) mg/l/h Control 0.28 12.2 3.97 1.9 M22/75-80 (ALD) 0.33 14.8 3.45 2.3 M22/96-6 (ACS) 0.33 15.3 5.40 2.3 M22/94-16 (ALD + ACS) 0.27 13.7 3.67 1.9 M22/98-16 (PDAT) 0.48 13.7 4.83 3.3 M22/75/98-7 0.48 17.8 6.23 3.3 (ALD + PDAT) M22/94/98-22 0.45 16.1 5.31 3.1 (ALD + ACS + PDAT) Yield lipid Lipid Yield lipid (% used Lipid Strain (g/l) (% CDW) xylose) mg/l/h Control 0.92 40.0 13.0 6.4 M22/75-80 (ALD) 1.00 44.8 10.5 7.0 M22/96-6 (ACS) 0.90 41.5 14.7 6.3 M22/94-16 (ALD + ACS) 0.82 41.6 11.1 5.7 M22/98-16 (PDAT) 1.62 46.4 16.4 11.3 M22/75/98-7 (ALD + PDAT) 1.32 48.9 17.1 9.2 M22/94/98-22 (ALD + 1.54 55.6 18.3 10.8 ACS + PDAT)

[0227] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol and total lipid concentrations and rates of production and triacylglycerol and total lipid yields from used xylose or per dry weight with high C/N ratios.

Example 30. Production of Triacylglycerol or Lipid by Strains of C. curvatus Modified by Addition of Genes Encoding ALD and PDAT (Y23/81/95-18, Ex. 8) or ALD and PDAT and ACS (Y23/85/95-4 Ex. 9) in High Cell Density Cultures Grown on Glucose with C/N Ratio of 80

[0228] Transformants (Y23/85/95-4 and Y23/81/95-18) were separately cultivated in Multifors bioreactors (max. working volume 500 ml, Infors HT, Switzerland) at pH 4.0, 30.degree. C., in 500 ml medium containing 90 to 96 g glucose, 2.56 g (NH.sub.4).sub.2SO.sub.4, 1.2 g KH.sub.2PO.sub.4, 0.3 g Na.sub.2HPO.sub.4.2H.sub.2O, 1.5 g MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.6H.sub.2O, 5.26 mg citric acid.H.sub.2O, 5.26 mg ZnSO.sub.4.7H.sub.2O, 0.1 mg MnSO.sub.4.4H.sub.2O, 0.5 mg CoCl.sub.2.6H.sub.2O, 0.26 mg CuSO.sub.4.5H.sub.2O, 0.1 mg Na.sub.2MoO.sub.4.2H.sub.2O, 1.4 mg FeSO.sub.4.7H.sub.2O, 0.1 mg H.sub.3BO.sub.4, 0.05 mg D-biotin, 1.0 mg CaPantothenate, 5.0 mg nicotinic acid, 25 mg myoinositol, 1.0 mg thiamine.HCl, 1.0 mg pyridoxine.HCl and 0.2 mg p-aminobenzoic acid per litre. The pH was maintained constant by addition of 1 M KOH or 1 M H.sub.2PO.sub.4. Cultures were agitated at 1000 rpm (2 Rushton turbine impellors) and aerated at 2 volumes air per volume culture per minute (vvm). Clerol FBA 3107 antifoaming agent (Cognis, SaintFargeau-Ponthierry Cedex France, 1 ml 1.sup.-1) was added to prevent foam accumulation. Bioreactors were inoculated to initial OD.sub.600 of 0.5 to 4.0 with cells grown in the same medium (substituting 1.5 g urea per litre for (NH.sub.4).sub.2SO.sub.4 and omitting the CaCl.sub.2.6H.sub.2O) in 50 ml volumes in 250 ml flasks at 30.degree. C. with shaking at 200 rpm for 24 to 42 h. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as the control. For measurement of the yield of triacylglycerol on glucose or biomass, some control cultures contained 58 to 134 g glucose 1.sup.-1.

[0229] Lipid extraction and triacylglycerol concentration measurements were carried out as described in Example 21. Cell dry weight was determined by centrifuging 0.5 to 2.0 ml culture broth in pre-dried, pre-weighed 2 ml microfuge tubes. After washing twice with 1.8 ml distilled water, the cell pellet was dried at 100.degree. C. for 48 h and weighed after cooling in a dessicator. HPLC analyses were carried out as described in Example 22.

[0230] Table 11 shows that a transformant containing the genes for ALD and PDAT produced 23% more triacylglycerol than Y23, with a 24% increase in the yield on glucose consumed when cells were cultivated to high cell density in bioreactor cultures. A transformant containing the genes for ALD, ACS and PDAT produced 15% more triacylglycerol than Y23, with a 12% increase in the yield on glucose consumed.

TABLE-US-00013 TABLE 11 Triacylglycerol produced in pH controlled bioreactor culture of Y23 and transformants of Y23 expressing ALD + PDAT or ALD + ACS + PDAT, with glucose as carbon source and C/N 80. Data is the average of 2 (transformants) or 4 to 9 (Y23) cultures .+-. standard error of the mean. Percentage increase is shown in parenthesis Yield TAG Strain TAG (g/l) (% glucose consumed) Y23 17.3 .+-. 0.3 18.8 .+-. 1.0 Y23/81/95-18 (ALD + PDAT) 21.3 .+-. 0.8 23.3 .+-. 1.0 (23%) (24%) Y23/85/95-4 19.9 .+-. 0.1 21.1 .+-. 0.1 (ALD + ACS + PDAT) (15%) (12%)

[0231] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and triacylglycerol yields from used glucose in high cell density cultures.

Example 31. Production of Triacylglycerol or Lipid by Strains of C. curvatus Modified by Addition of Genes Encoding ALD and PDAT (Y23/81/95-18, Ex. 8) or ALD and PDAT and ACS (Y23/85/95-4, Ex. 9) in High Cell Density Cultures Grown on Xylose with C/N Ratio of 80

[0232] Transformants (Y23/85/95-4 and Y23/81/95-18) were separately cultivated in Multifors bioreactors as described in Example 30. The medium contained 92 to 118 g xylose per litre, instead of glucose. Bioreactors were inoculated to initial OD.sub.600 of 17 to 24 with cells grown in low nitrogen medium with glucose as carbon source in the Multifors bioreactors at 30.degree. C., as described for lipid production in Example 30. Alternatively, some cultures of Y23 were inoculated with cells grown in flasks, as described in Example 30, to initial OD.sub.600 0.2 to 0.5. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as the control.

[0233] Lipid extraction and total lipid and triacylglycerol concentration measurements were carried out as described in Example 21. Cell dry weight was determined as described in Example 30. HPLC analyses were carried out as described in Example 22.

[0234] Table 12 shows that a transformant containing the genes for ALD and PDAT produced 7% more triacylglycerol than Y23, with a 17% increase in the yield on xylose consumed and a 3% increase in the yield on biomass when cells were cultivated to high cell density in bioreactor cultures. A transformant containing the genes for ALD, ACS and PDAT produced only 1% more triacylglycerol than Y23, but showed 13% increase in the yield on xylose consumed and 4% increase in yield on biomass.

TABLE-US-00014 TABLE 12 Triacylglycerol produced in pH controlled bioreactor culture of Y23 and transformants of Y23 expressing ALD + PDAT or ALD + ACS + PDAT, with lose as carbon source and C/N 80. Data is the average of 2 (transformants) or 4 (Y23) cultures, .+-. standard error of the mean. Percentage increase is shown in parenthesis. Yield TAG Yield TAG (% Strain TAG (g/l) (% CDW) xylose consumed) Y23 17.9 .+-. 1.4 46.9 .+-. 4.0 17.2 .+-. 1.2 Y23/81/95-18 19.1 .+-. 0.3 48.4 .+-. 5.7 20.2 .+-. 0.5 (ALD + PDAT) (7%) (3%) (17%) Y23/85/95-4 18.1 .+-. 0.3 49.0 .+-. 3.4 19.5 .+-. 0.0 (ALD + ACS + PDAT) (1%) (4%) (13%)

[0235] Table 13 shows that a transformant containing the genes for ALD and PDAT produced 10% more lipid than Y23, with a 21% increase in the yield on xylose consumed and a 2% increase in the yield on biomass when cells were cultivated to high cell density in bioreactor cultures. A transformant containing the genes for ALD, ACS and PDAT produced 3% more lipid than Y23, with 16% higher yield on xylose consumed and a 3% increase in yield on biomass.

TABLE-US-00015 TABLE 13 Lipid produced in pH controlled bioreactor culture of Y23 and transformants of Y23 expressing ALD + PDAT or ALD + ACS + PDAT, with xylose as carbon source and C/N 80. Data is the average of 2 (transformants) or 4 (Y23) cultures, .+-. standard error of the mean. Percentage increase is shown in parenthesis. Yield lipid Yield lipid (% Strain Lipid (g/l) (% CDW) xylose consumed) Y23 20.5 .+-. 1.3 55.9 .+-. 7.2 19.8 .+-. 2.5 Y23/81/95-18 (ALD + PDAT) 22.5 .+-. 0.6 57.0 .+-. 1.9 23.8 .+-. 0.8 (10%) (2%) (21%) Y23/85/95-4 21.2 .+-. 0.1 57.5 .+-. 1.0 22.9 .+-. 0.3 (ALD + ACS + PDAT) (3%) (3%) (16%)

[0236] This example shows that expression of ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol and total lipid concentrations and triacylglycerol and total lipid yields from used xylose or per dry weight in high cell density cultures.

Example 32. Production of Triacylglycerol or Lipid by Strain of M. circinelloides Modified by Addition of Genes Encoding ALD and PDAT and ACS (M22/94/98-19, Ex. 20) in pH Controlled Bioreactor Cultures Grown on Glucose with C/N Ratio of 60

[0237] Transformant (M22/94/98-19) was cultivated in Braun Biostat.RTM. CT bioreactors (2.5 max working volume, B. Braun Biotech International, Sartorius AG, Germany) at pH 5.0, 30.degree. C., in 1.0 to 1.2 l medium containing 53 g glucose, 1.57 g (NH.sub.4).sub.2SO.sub.4, 2.5 g KH.sub.2PO.sub.4, 0.2 g MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.6H.sub.2O, 5.26 mg citric acid.H.sub.2O, 5.26 mg ZnSO.sub.4.7H.sub.2O, 0.1 mg MnSO.sub.4.4H.sub.2O, 0.5 mg CoCl.sub.2.6H.sub.2O, 0.26 mg CuSO.sub.4.5H.sub.2O, 0.1 mg Na.sub.2MoO.sub.4.2H.sub.2O, 1.4 mg FeSO.sub.4.7H.sub.2O, 0.1 mg H.sub.3BO.sub.4, 0.005 mg D-biotin, and 0.05 mg thiamine.HCl per litre. The pH was maintained constant by addition of 1 M KOH or 1 M H.sub.2PO.sub.4. Cultures were agitated at 600 rpm (2 Rushton turbine impellors) and aerated at 1 volume air per volume culture per minute (vvm). Polypropylene glycol (mixed molecular weights containing Fluka P1200, Fluka P2000 and Henkel Performance Chemicals Foamaster in a ratio of 4:4; 1, 1 ml 1.sup.-1) was added to prevent foam accumulation. Bioreactors were inoculated to an initial biomass concentration of approximately 100 mg 1.sup.-1 with mycelia grown in the same medium with the following modifications: 15 g glucose 1.sup.-1, enough (NH.sub.4).sub.2SO.sub.4 to provide a C/N ratio of 16.2, and additionally 4.0 g agar 1.sup.-1. Pre-cultures were grown in 50 ml volumes in 250 ml flasks at 30.degree. C. with shaking at 200 rpm for 42 to 72 h. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Biomass was separated from the culture supernatant by filtration under vacuum. Mucor circinelloides wild type strain M22 was used as the control.

[0238] Lipid extraction and total lipid and triacylglycerol concentration measurements were carried out as described in Example 21. Cell dry weight was determined as described in Example 25, except that disposable cleaning cloth (X-tra, 100% viscose household cleaning cloth, Inex Partners Oy, Helsinki) was used in vacuum filtration. HPLC analyses were carried out as described in Example 22.

[0239] Table 14 shows that a transformant containing the genes for ALD, ACS and PDAT produced 17% more triacylglycerol than Y23, with 8% increase in the yield on glucose consumed, when cells were cultivated in bioreactor cultures.

TABLE-US-00016 TABLE 14 Triacylglycerol produced in pH controlled bioreactor cultures of M22 and transformant of M22 expressing ALD + ACS + PDAT, with glucose as carbon source and C/N 60. Percentage increase is shown in parenthesis. Yield TAG (% Strain TAG (g/l) glucose consumed) M22 9.8 21.9 M22/94/98-19 11.5 (17%) 23.6 (8%) (ALD + ACS + PDAT)

[0240] Table 15 shows that a transformant containing the genes for ALD, ACS and PDAT produced 44% more lipid than M22, with 55% higher yield on glucose consumed and 26% increase in yield of lipid on biomass, when cells were cultivated in bioreactor cultures.

TABLE-US-00017 TABLE 15 Lipid produced in pH controlled bioreactor cultures of M22 and transformant of M22 expressing ALD + ACS + PDAT, with glucose as carbon source and C/N 60. Percentage increase is shown in parenthesis. Yield lipid Yield lipid (% Strain Lipid (g/l) (% CDW) glucose consumed) M22 10.4 59.4 .+-. 2.8 19.7 M22/94/98-19 14.9 (44%) 75.4 .+-. 3.5 30.5 (55%) (ALD + ACS + PDAT) (26%)

[0241] This example shows that expression of ALD6, ACS2 and PDAT genes enhanced triacylglycerol and total lipid concentrations and triacylglycerol and total lipid yields from used glucose or total lipid yield per dry weight in high cell density cultures.

Example 33. Production of Triacylglycerol or Lipid by Strain of M. circinelloides Modified by Addition of Genes Encoding ALD and PDAT and ACS (M22/94/98-19, Ex. 20) in pH Controlled Bioreactor Cultures Grown on Xylose with C/N Ratio of 60

[0242] Transformant (M22/94/98-19) was cultivated in Braun Biostat CT bioreactors as described in Example 32. The medium contained 44 to 56 g xylose per litre, instead of glucose cultures were supplemented with 1 g peptone per litre and the (NH.sub.4).sub.2SO.sub.4 concentration was reduced to 1.12 g per litre. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Mucor circinelloides wild type strain M22 was used as the control.

[0243] Lipid extraction and total lipid and triacylglycerol concentration measurements were carried out as described in Example 21. Cell dry weight was determined as described in Example 32. HPLC analyses were carried out as described in Example 22.

[0244] Table 16 shows that a transformant containing genes for ALD, ACS and PDAT produced 11% more triacylglycerol than M22, with 10% increased yield on xylose consumed and 9% increased yield on biomass in pH controlled bioreactor cultures.

TABLE-US-00018 TABLE 16 Triacylglycerol produced in pH controlled bioreactor cultures of M22 and transformant of M22 expressing ALD + ACS + PDAT, with xylose as carbon source and C/N 60. Percentage increase is shown in parenthesis. Yield TAG TAG Yield TAG (% xylose Strain (g/l) (% CDW) consumed) M22 5.6 46.5 .+-. 2.7 22.9 M22/94/98-19 6.2 50.9 .+-. 1.4 25.1 (ALD + ACS + PDAT) (11%) (9%) (10%)

[0245] Table 17 shows that a transformant containing the genes for ALD and ACS and PDAT produced 24% more lipid than M22 and the yield of lipid on biomass was 22% higher than in M22, when mycelia were grown on xylose. The yield of lipid on xylose consumed was increased 18%.

TABLE-US-00019 TABLE 17 Lipid produced in pH controlled bioreactor culture of M22 and transformant of M22 expressing ALD + ACS + PDAT, with xylose as carbon source and C/N 60. Percentage increase is shown in parenthesis. Yield lipid Lipid Yield lipid (% xylose Strain (g/l) (% CDW) consumed) M22 5.8 49.2 .+-. 4.6 24.1 M22/94/98-19 7.1 58.2 .+-. 0.6 28.7 (ALD + ACS + PDAT) (22%) (18%) (19%)

[0246] This example shows that expression of ALD6, ACS2 and PDAT genes enhanced triacylglycerol and total lipid concentrations and triacylglycerol and total lipid yields from used xylose or per dry weight in high cell density cultures.

Example 34. Construction of a Plasmid Containing a Marker Gene Under the Control of an Endogenous Promoter and Terminator and a Pyruvate Decarboxylase (PDC) Encoding Gene Under the Control of an Endogenous Promoter and Terminator

[0247] A pyruvate decarboxylase (PDC) encoding gene, such as PDC1 from S. cerevisiae (SEQ ID NO:94) which encodes the amino acid sequence of SEQ ID NO:95 is codon optimised. The codon optimised PDC encoding gene with flanking SbfI restriction sites is digested with SbfI and ligated to a plasmid containing an endogenous promoter and terminator, such as plasmid pKK77pre. The resulting plasmid which contains the PDC encoding gene under the control of the endogenous promoter and terminator will be linearised e.g. with BamHI and ligated with a fragment containing the marker gene under the control of the endogenous promoter and terminator. Such fragment can be obtained e.g. by digesting a plasmid pKK67 with BamHI and XmnI. The resulting plasmid contains the PDC encoding gene under the control of the endogenous promoter and terminator and the marker gene under the control of the endogenous promoter and terminator.

Example 35. Generation of Genetically Modified Strain with an Integrated PDC Together with ALD6 and/or ACS2 Encoding Genes and Marker Genes by Transforming Genetically Modified Strains with Plasmid Containing PDC Encoding Gene (Ex 34)

[0248] The plasmid containing the PDC encoding gene (Ex. 34) is restricted e.g. with NotI and PspOMI, and the resulting linear DNA containing the PDC encoding gene under the control of the endogenous promoter and terminator and the marker gene under the control of the endogenous promoter and terminator is used to transform e.g. a genetically modified strain Y23/81-51 (Ex. 3B), Y23/86-92 (Ex. 5B) or Y23/85-128 (Ex. 7B) by electroporation or a genetically modified strain M22/75-86 (Ex. 15B), M22/96-1 (Ex. 17B) or M22/94-31 (Ex 16B) using the transformation method described in Example 15B. The transformed cells are screened e.g. for antibiotic resistance. Several transformed colonies are analysed at DNA level by PCR.

Example 36. Aerobic Shake Flask Characterization of Strains Harbouring PDC Together with ALD6 and/or ACS2 Encoding Genes and Marker Genes in Glucose or Xylose Medium with Different C/N Ratios

[0249] Transformants are separately cultivated in 50 ml of culture medium such as described in Examples 22, 23, 24, 27 or 28. Each flask (250 ml) is inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations are maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis are withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 is used as a control. Cell dry weight is determined and HPLC analysis is carried out as described in Example 22. Lipid extraction and triacylglycerol measurements as described in Example 21. Alternatively transformants are separately cultivated in 50 ml of culture medium such as described in Examples 25, 26 or 29. Each flask (250 ml) is inoculated with 1*10.sup.7 spores. The cultivations are maintained at a temperature of 25.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis are withdrawn periodically during cultivation. Mucor circinelloides wild type strain M22 is used as a control. Cell dry weight is determined as described in Example 25 and HPLC analysis is carried out as described in Example 22. Lipid extraction and triacylglycerol measurements as described in Example 21.

[0250] The transformants harbouring PDC together with ALD6 and/or ACS2 encoding genes produce more triacylglycerol than the control strain. Additionally the transformants harbouring PDC together with ALD6 and/or ACS2 encoding gene have higher triacylglycerol yield on used carbon than the control strain.

Example 37A. Construction of a Plasmid (pKK101) Containing the G418 Resistance Gene Under the Control of the CcTEF1 Promoter and the CcTPI1 Terminator and the S. cerevisiae PDC1 Encoding Gene Under the Control of the CcTPI1 Promoter and the CcTEF1 Terminator

[0251] The plasmid Y4_TPIp-PDC-TEFt (Geneart AG, Germany) contains a S. cerevisiae PDC1 (SEQ ID NO:95) encoding gene which has been codon optimized according to Ustilago maydis yeast codon usage (SEQ ID NO:96) with the CcTPI1 promoter and the CcTEF1 terminator. Plasmid Y4_TPIp-PDC-TEFt was digested with EcoRI. A 2893 bp fragment was gel isolated and ligated to a 5381 bp fragment obtained by digesting a plasmid designated pKK67 (Ex. 2B) with EcoRI. The resulting plasmid was designated as pKK101 (FIG. 12). The plasmid pKK101 contains a S. cerevisiae PDC1 encoding gene under the control of the CcTPI1 promoter and the CcTEF1 terminator and the E. coli G418 resistance gene under the control of the CcTEF1 promoter and the CcTPI1 terminator.

Example 37B. Generation of Genetically Modified Strains with an Integrated PDC Encoding Gene and G418 Resistance Gene by Transforming Wild-Type C. curvatus and Genetically Modified Strains Y23/81-66 (Ex. 3B), Y23/85-125 (Ex. 7B), Y23/86-86 (Ex. 5B), Y23/95-99 (Ex. 6B), Y23/81/95-18 (Ex. 8) and Y23/85/95-4 (Ex. 9) with Digested Plasmid pKK101 (FIG. 12, Ex. 37A)

[0252] Plasmid pKK101 was restricted with SacII and PspOMI, and the resulting linear DNA was used to transform wild-type C. curvatus strain ATCC20509 designated as Y23 and genetically modified strains Y23/81-66, Y23/85-125, Y23/86-86, Y23/95-99, Y23/81/95-18 and Y23/85/95-4 by electroporation. The transformed cells were screened for G418 resistance. Several G418 resistance colonies were analysed at DNA level by PCR. The transformants originating from the transformation of wild-type C. curvatus strain ATCC20509 designated as Y23 and genetically modified strains Y23/81-66, Y23/85-125, Y23/86-86, Y23/95-99, Y23/81/95-18 and Y23/85/95-4 with SacII and PspOMI cut pKK101 and containing the S. cerevisiae PDC1 encoding gene under the control of the CcTPI1 promoter and the CcTEF1 terminator were designated as Y23/101-55, Y23/101-57, Y23/101-59, Y23/81/101-4, Y23/85/101-13, Y23/85/101-14, Y23/85/101-19, Y23/86/101-23, Y23/95/101-1, Y23/95/101-2, Y23/81/95/101-20, Y23/85/95/101-7 and Y23/85/95/101-8.

Example 38A. Construction of a Plasmid (pKK102) Containing the Cerulenin Resistance Gene Under the Control of the McPGK1 Promoter and the McTPI1 Terminator and the S. cerevisiae PDC1 Encoding Gene Under the Control of the McPGK1 Promoter and the McTEF1 Terminator

[0253] The plasmid M22_PGKp-PDC-TEFt (Geneart AG, Germany) contains a S. cerevisiae PDC1 (SEQ ID NO:95) encoding gene which has been codon optimized according to Rhizopus oryzae filamentous fungus codon usage (SEQ ID NO:97) with the McPGK1 promoter and the McTEF1 terminator. Plasmid M22_PGKp-PDC-TEFt was digested with SalI. A 3363 bp fragment was gel isolated and ligated to a 6239 bp fragment obtained by digesting a plasmid designated pKK92 (Ex. 14B) with SalI. The resulting plasmid was designated as pKK102 (FIG. 13). The plasmid pKK102 contains a S. cerevisiae PDC1 encoding gene under the control of the McPGK1 promoter and the McTEF1 terminator and the g cerevisiae cerulenin resistance gene under the control of the McPGK1 promoter and the McTPI1 terminator.

Example 38B. Generation of a Genetically Modified Mucor Circinelloides (M22/94/102) with Integrated ALD6, ACS1 and PDC1 Encoding Genes and Hygromycin and Cerulenin Resistance Genes by Transforming Genetically Modified Strain M22/94-12 (Ex. 16B) with Digested Plasmid pKK102 (FIG. 13, Ex. 38A)

[0254] Plasmid pKK102 was restricted with SacII and PspOMI, and the resulting linear DNA was used to transform the genetically modified strain M22/94-12, using the transformation method described in Example 15B. The transformed cells were screened for cerulenin resistance. Several cerulenin resistant colonies were analysed at DNA level by PCR. The transformant originating from the transformation of the genetically modified strain M22/94-12 with SacII and PspOMI cut pKK102 and containing the S. cerevisiae PDC1 encoding gene under the control of the McPGK1 promoter and the McTEF1 terminator was designated as M22/94/102-31.

Example 39. Aerobic Shake Flask Characterization of Strains Y23/101-59 (Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-14 (Ex. 37B), Y23/86/101-23 (Ex. 37B), Y23/95/101-2 (Ex. 37B), Y23/81/95/101-20 (Ex. 37B) and Y23/85/95/101-9 (Ex. 37B) in Glucose Medium with C/N Ratio of 153

[0255] Transformants were separately cultivated in 50 ml of Glucose-CN153 medium (pH 5.5, 30 g glucose, 0.15 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22. Lipid extraction and triacylglycerol measurements as described in Example 21. Lipid extractions from the culture medium i.e. from the supernatant samples recovered after centrifugation of the cell lipid extraction samples or dry weight measurement samples were carried out as follows. To 150 .mu.l of supernatant sample 150 .mu.l of 0.9% NaCl and 150-1500 .mu.l of chloroform:methanol (2:1) was added. The sample was vortexed 2 minutes and sample was incubated at room temperature at 30 min. After incubation sample was centrifuged 10 000 rpm for 3 min at RT. The lower phase was recovered into microfuge tubes, dried and redissolved in 1.5 ml of chloroform:methanol (2:1) and stored at -20.degree. C. prior triacylglycerol measurements as described in Example 21. Alternatively, dried lipid pellet was redissolved directly in isopropanol and triacylglycerol was measured as described in Example 21.

[0256] After 47 hours cultivation (Table 18), when 17 to 18 g/l glucose was left, Y23/101-59, Y23/85/101-14, Y23/86/101-23, Y23/95/101-2, Y23/81/95/101-20 and Y23/85/95/101-8 transformants produced 52, 78, 9, 27, 32 and 48% more triacylglycerol with higher rate, respectively, than the control strain in glucose medium. Also triacylglycerol yields on biomass and per used glucose were 33 to 83% and 35 to 85% higher in transformants Y23/101-59, Y23/85/101-14, Y23/86/101-23, Y23/95/101-2, Y23/81/95/101-20 and Y23/85/95/101-8 than the control strain, respectively.

TABLE-US-00020 TABLE 18 Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) and yield (%) per biomass (CDW) and used glucose in the yeast cells after 47 hours cultivation in glucose medium with C/N ratio of 153 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) glucose) mg/l/h Control 1.30 22.5 10.9 28 Y23/101-59 (PDC) 1.98 37.1 16.3 42 Y23/85/101-14 2.31 41.1 20.2 49 (PDC + ALD + ACS) Y23/86/101-23 1.42 29.8 15.4 30 (PDC + ACS) Y23/95/101-2 1.65 30.9 14.7 35 (PDC + PDAT) Y23/81/95/101-20 1.72 36.4 16.4 37 (PDC + ALD + PDAT) Y23/85/95/101-8 1.93 36.6 18.1 41 (PDC + ALD + ACS + PDAT)

[0257] After 94 hours cultivation (Table 19A), when 6 to 11 g/l glucose was left, Y23/101-59, Y23/85/101-14, Y23/81/95/101-20 and Y23/85/95/101-8 transformants produced 5, 27, 8 and 12% more triacylglycerol with higher rate, respectively, than the control strain in glucose medium. Also triacylglycerol yields on biomass and per used glucose were 9 to 33% and 7 to 38% higher in transformants Y23/101-59, Y23/81/101-4, Y23/85/101-14, Y23/86/101-23, Y23/95/101-2, Y23/81/95/101-20 and Y23/85/95/101-8 than the control strain, respectively. Additionally, triacylglycerol concentration in the culture medium in the cultivations with the transformants Y23/81/101-4 and Y23/86/101-23 was 525 and 350% higher, respectively, than in the cultivation with the control strain (Table 19B). Additionally, the total triacylglycerol yields per used glucose calculated from the intracellular triacylglycerol concentration and the triacylglycerol concentration detected from the culture medium were 13 to 38% higher with the transformants than with the control strain.

TABLE-US-00021 TABLE 19A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) and yield (%) per biomass (CDW) and used glucose in the yeast cells after 94 hours cultivation in glucose medium with C/N ratio of 153 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) glucose) mg/l/h Control 4.06 43.4 17.4 43 Y23/101-59 (PDC) 4.28 51.6 19.9 46 Y23/81/101-4 3.39 55.7 18.6 36 (PDC + ALD Y23/85/101-14 5.17 57.9 22.7 55 (PDC + ALD + ACS) Y23/86/101-23 4.00 53.4 21.1 43 (PDC + ACS) Y23/95/101-2 3.50 47.5 20.1 37 (PDC + PDAT) Y23/81/95/101-20 4.38 62.0 24.0 47 (PDC + ALD + PDAT) Y23/85/95/101-8 4.55 54.4 21.5 48 (PDC + ALD + ACS + PDAT)

TABLE-US-00022 TABLE 19B Triacylglycerol (TAG) concentration (g/l) in the culture medium and calculated total TAG concentration (g/l), rate (mg/l/h) and yield (%) per used glucose in cultivation after 94 hours cultivation in glucose medium with C/N ratio of 153 Yield total TAG TAG Total TAG (% used total TAG Strain (g/l) (g/l) glucose) mg/l/h Control 0.04 4.10 17.6 44 Y23/101-59 (PDC) 0.01 4.29 20.0 46 Y23/81/101-4 0.25 3.63 19.9 39 (PDC + ALD Y23/85/101-14 0.03 5.20 22.8 55 (PDC + ALD + ACS) Y23/86/101-23 0.18 4.18 22.1 44 (PDC + ACS) Y23/95/101-2 0.01 3.51 20.2 37 (PDC + PDAT) Y23/81/95/101-20 0.03 4.42 24.2 47 (PDC + ALD + PDAT) Y23/85/95/101-8 0.04 4.6 21.7 49 (PDC + ALD + ACS + PDAT)

[0258] This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and rates of production and triacylglycerol yields from used glucose or per dry weight in the yeast cell and/or in the culture medium with high C/N ratio in different stages of the cultivation.

Example 40. Aerobic Shake Flask Characterization of Strains Y23/101-55 (Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-19 (Ex. 37B), Y23/86/101-23 (Ex. 37B), Y23/95/101-2 (Ex. 37B) and Y23/85/95/101-7 (Ex. 37B) in Glucose Medium with C/N Ratio of 28

[0259] Transformants were separately cultivated in 50 ml of Glucose-CN28 medium (pH 5.5, 30 g glucose, 0.3 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus glucose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22. Lipid extraction from the yeast cells and triacylglycerol measurements were carried out as described in Example 21 and lipid extraction from the culture medium as described in Example 39.

[0260] After 46 hours cultivation (Table 20A), when 12 to 14 g/l glucose was left, Y23/101-55, Y23/81/101-4, Y23/85/101-19, Y23/86/101-23, Y23/95/101-2 and Y23/85/95/101-7 transformants produced 18, 17, 20, 24, 7 and 17% more triacylglycerol with higher rate, respectively, than the control strain in glucose medium. Also triacylglycerol yields on biomass and per used glucose were 10 to 35% and 7 to 26% higher with the transformants than the control strain, respectively. After 72 and 94 hours cultivation triacylglycerol concentration in the culture medium with the transformants Y23/81/101-4 and Y23/86/101-23 was 195 and 56% and 35 and 30% higher, respectively, than in the cultivations with the control strain (Table 20B). Additionally, yield of triacylglycerol detected in culture medium per used glucose was 57 to 206% and 36 to 56% higher with the transformants Y23/81/101-4 and Y23/86/101-23 than with the control after 72 and 94 hours cultivation, respectively.

TABLE-US-00023 TABLE 20A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) and yield (%) per biomass (CDW) and used glucose in the yeast cells after 46 hours cultivation in glucose medium with C/N ratio of 28 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) glucose) mg/l/h Control 3.10 46.6 20.6 67 Y23/101-55 (PDC) 3.67 51.2 24.4 80 Y23/81/101-4 3.64 53.5 22.1 79 (PDC + ALD Y23/85/101-19 3.71 53.8 23.2 81 (PDC + ALD + ACS) Y23/86/101-23 3.84 63.1 26.0 83 (PDC + ACS) Y23/95/101-2 3.31 54.7 23.3 72 (PDC + PDAT) Y23/85/95/101-7 3.64 58.5 26.3 79 (PDC + ALD + ACS + PDAT)

TABLE-US-00024 TABLE 20B Triacylglycerol (TAG) concentration (g/l) and yield (%) per used glucose in the culture medium after 72 and 94 hours cultivation in glucose medium with C/N ratio of 28 72 h Yield TAG % 94 h Yield TAG TAG (/used TAG (% used Strain (g/l) glucose) (g/l) glucose) Control 0.85 3.19 2.79 9.84 Y23/81/101-4 2.51 9.75 4.35 15.4 (PDC + ALD) Y23/86/101-23 1.15 5.00 3.62 13.4 (PDC + ACS)

[0261] This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and rates of production and triacylglycerol yields from used glucose or per dry weight in the yeast cell and/or in the culture medium with low C/N ratio at different stages of the cultivation.

Example 41. Aerobic Shake Flask Characterization of Strains Y23/101-57 (Ex. 37B), Y23/81/101-4 (Ex. 37B), Y23/85/101-13 (Ex. 37B), Y23/86/101-23 (Ex. 37B), Y23/95/101-1 (Ex. 37B), Y23/81/95/101-20 (Ex. 37B) and Y23/85/95/101-8 (Ex. 37B) in Xylose Medium with C/N Ratio of 103

[0262] Transformants were separately cultivated in 50 ml of Yeast culture medium IV (pH 5.5, 20 g xylose, 0.15 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2 H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 0.45 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus xylose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22. Lipid extraction from the yeast cells and triacylglycerol measurements were carried out as described in Example 21 and lipid extraction from the culture medium as described in Example 39.

[0263] After 47 hours cultivation (Table 21) Y23/101-57, Y23/81/101-4, Y23/85/101-13 and Y23/95/101-1 transformants produced 12, 18, 4 and 5% more triacylglycerol with higher rate, respectively, than the control strain in xylose medium. Also triacylglycerol yields on biomass and per used xylose were up to 28% and 16% higher with the transformants Y23/101-57, Y23/81/101-4, Y23/85/101-13, Y23/86/101-23 and Y23/95/101-1 than the control strain, respectively. Also total lipid concentration, rate and yields on biomass and per used xylose were 8 to 29%, 8 to 29%, 19 to 44% and 11 to 33% higher with the transformants than the control strain, respectively, after 47 hours cultivation in xylose medium.

TABLE-US-00025 TABLE 21 Triacylglycerol (TAG) and total lipid concentrations (g/l), rates (mg/l/h) and yields (%) per biomass (CDW) and used xylose in the yeast cells after 47 hours cultivation in xylose medium with C/N ratio of 103 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) xylose) mg/l/h Control 1.90 38.5 16.0 41 Y23/101-57 (PDC) 2.12 44.7 18.0 45 Y23/81/101-4 2.24 49.2 18.6 48 (PDC + ALD Y23/85/101-13 1.98 44.3 18.1 42 (PDC + ALD + ACS) Y23/86/101-23 1.85 47.7 18.3 39 (PDC + ACS) Y23/95/101-1 2.00 38.5 16.5 43 (PDC + PDAT) Yield lipid Lipid Yield lipid (% used Lipid Strain (g/l) (% CDW) xylose) mg/l/h Control 2.40 48.5 20.2 51 Y23/101-57 (PDC) 2.75 57.9 23.3 59 Y23/81/101-4 2.70 59.3 22.4 57 (PDC + ALD Y23/85/101-13 2.60 58.1 23.7 55 (PDC + ALD + ACS) Y23/86/101-23 2.70 69.7 26.8 57 (PDC + ACS) Y23/95/101-1 3.10 59.6 25.6 66 (PDC + PDAT)

[0264] After 94 hours cultivation (Table 22A) Y23/101-57, Y23/81/101-4, Y23/85/101-13, Y23/86/101-23, Y23/95/101-1, Y23/81/95/101-20 and Y23/85/95/101-8 transformants produced 7, 9, 8, 7, 6, 6 and 4% more triacylglycerol with higher rate, respectively, than the control strain in xylose medium. Also triacylglycerol yields on biomass and per used xylose were 4 to 25% and 5 to 17% higher with the transformants than the control strain, respectively. Triacylglycerol concentration in the culture medium with the transformants Y23/81/101-4 and Y23/86/101-23 was 222 and 156% higher, respectively, than in the cultivations with the control strain (Table 22B). Additionally, the total triacylglycerol yields per used xylose calculated from the intracellular triacylglycerol concentration and the triacylglycerol concentration detected from the culture medium were 7 to 17% higher with the transformants than with the control strain.

TABLE-US-00026 TABLE 22A Triacylglycerol (TAG) concentration (g/l), rate (mg/l/h) and yield (%) per biomass (CDW) and used xylose in the yeast cells after 94 hours cultivation in xylose medium with C/N ratio of 103 Yield TAG TAG Yield TAG (% used TAG Strain (g/l) (% CDW) xylose) mg/l/h Control 4.50 72.0 23.9 48 Y23/101-57 (PDC) 4.82 79.4 25.6 51 Y23/81/101-4 4.92 84.2 26.1 52 (PDC + ALD Y23/85/101-13 4.85 75.1 25.7 52 (PDC + ALD + ACS) Y23/86/101-23 4.82 88.5 25.6 51 (PDC + ACS) Y23/95/101-1 4.79 79.8 25.7 51 (PDC + PDAT) Y23/81/95/101-20 4.78 89.7 27.9 51 (PDC + ALD + PDAT) Y23/85/95/101-8 4.66 80.4 25.2 50 (PDC + ALD + ACS + PDAT)

TABLE-US-00027 TABLE 22B Triacylglycerol (TAG) concentration (g/l) in the culture medium and calculated total TAG concentration (g/l), rate (mg/l/h) and yield (%) per used xylose in cultivation after 94 hours cultivation in xylose medium with C/N ratio of 103 Yield total TAG TAG Total TAG (% used total TAG Strain (g/l) (g/l) xylose) mg/l/h Control 0.09 4.59 24.3 49 Y23/101-57 (PDC) 0.07 4.89 26.0 52 Y23/81/101-4 0.29 5.22 27.7 55 (PDC + ALD Y23/85/101-13 0.10 4.94 26.2 53 (PDC + ALD + ACS) Y23/86/101-23 0.23 5.05 26.8 54 (PDC + ACS) Y23/95/101-1 0.07 4.86 26.1 52 (PDC + PDAT) Y23/81/95/101-20 0.09 4.86 28.4 52 (PDC + ALD + PDAT) Y23/85/95/101-8 0.10 4.76 25.7 51 (PDC + ALD + ACS + PDAT)

[0265] This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol and total lipid concentrations and rates of production and triacylglycerol and total lipid yields from used xylose or per dry weight in the yeast cell and/or in the culture medium with high C/N ratio in different stages of the cultivation.

Example 42. Aerobic Shake Flask Characterization of Strains Y23/101-57 (Ex. 37B), Y23/85/101-13 (Ex. 37B) and Y23/81/95/101-20 (Ex. 37B) in Xylose Medium with C/N Ratio of 20

[0266] Transformants were separately cultivated in 50 ml of Xylose-CN20 medium (pH 5.5, 20 g xylose, 0.3 g (NH.sub.4).sub.2SO.sub.4, 7.0 g KH.sub.2PO.sub.4, 2.5 g Na.sub.2HPO.sub.4*2H.sub.2O, 1.5 g MgSO.sub.4*7H.sub.2O, 4.0 g Yeast extract, 51 mg CaCl.sub.2, 8 mg FeCl.sub.3*6H.sub.2O and 0.1 mg ZnSO.sub.4*7H.sub.2O per litre). Each flask (250 ml) was inoculated to an OD.sub.600 of 0.3 with cells grown on yeast peptone plus xylose plates. The cultivations were maintained at a temperature of 30.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as a control. Cell dry weight was determined and HPLC analysis was carried out as described in Example 22. Lipid extraction from the yeast cells and triacylglycerol measurements were carried out as described in Example 21 and lipid extraction from the culture medium as described in Example 39.

[0267] After 94 hours cultivation (Table 23A) transformants Y23/101-57 and Y23/85/101-13 produced 9 and 5% more triacylglycerol with higher rate, respectively, than the control strain in xylose medium. Also triacylglycerol yields on biomass and per used xylose were 2 to 10% and 4 to 8% higher with the transformants than the control strain, respectively. Triacylglycerol concentration in the culture medium with the transformants Y23/101-57, Y23/85/101-13 and Y23/81/95/101-20 was 163, 163 and 188% higher, respectively, than in the cultivations with the control strain (Table 21B). Additionally, the total triacylglycerol yields per used xylose calculated from the intracellular triacylglycerol concentration and the triacylglycerol concentration detected from the culture medium were 4 to 12% higher with the transformants than with the control strain.

TABLE-US-00028 TABLE 23A Triacylglycerol (TAG) concentration (g/l) and yield (%) per biomass (CDW) and used xylose in the yeast cells after 94 hours cultivation in xylose medium with C/N ratio of 20 Yield TAG TAG Yield TAG (% used Strain (g/l) (% CDW) xylose) Control 4.21 68.7 21.1 Y23/101-57 (PDC) 4.57 75.8 22.8 Y23/85/101-13 4.41 69.9 22.0 (PDC + ALD + ACS)

TABLE-US-00029 TABLE 23B Triacylglycerol (TAG) concentration (g/l) in the culture medium and calculated total TAG concentration (g/l), rate (mg/l/h) and yield (%) per used xylose in cultivation after 94 hours cultivation in xylose medium with C/N ratio of 20 Yield total TAG TAG Total TAG (% used total TAG Strain (g/l) (g/l) xylose) mg/l/h Control 0.08 4.28 21.4 46 Y23/101-57 (PDC) 0.21 4.78 23.9 51 Y23/85/101-13 0.21 4.61 23.1 49 (PDC + ALD + ACS) Y23/81/95/101-20 0.23 4.45 22.3 47 (PDC + ALD + PDAT)

[0268] This example shows that expression of PDC1, ALD6, ACS2 and PDAT genes in different combinations enhanced triacylglycerol concentrations and rates of production and triacylglycerol yields from used xylose or per dry weight in the yeast cell and/or in the culture medium with low C/N ratio.

Example 43. Aerobic Shake Flask Characterization of Strains M22/94-16 (Ex. 16B) and M22/94/102-31 (Ex. 38B), in Xylose Medium with C/N Ratio of 21

[0269] Transformants were separately cultivated in 50 ml of mould C/N 21 medium (pH 5.5, 10 g glucose, 1.4 g yeast extract, 2.5 g KH.sub.2PO.sub.4, 0.3 g (NH.sub.4).sub.2SO.sub.4, 10 mg ZnSO.sub.4*7H.sub.2O, 2 mg CuSO.sub.4*5H.sub.2O, 10 mg MnSO.sub.4, 0.5 g MgSO.sub.4*7H.sub.2O, 0.1 g CaCl.sub.2, 20 mg FeCl.sub.3*6H.sub.2O per litre). Each flask (250 ml) was inoculated with 1*10.sup.7 spores. The cultivations were maintained at a temperature of 28.degree. C. with shaking at 250 rpm. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Mucor circinelloides wild type strain M22 was used as a control. Cell dry weight was determined as described in Example 25 and HPLC analysis was carried out as described in Example 22. Lipid extraction and total lipid and triacylglycerol measurements as described in Example 21.

[0270] After 96 hours cultivation (Table 24) transformants M22/94-16 and M22/94/102-31 produced 18 and 30% more triacylglycerol with higher rate, respectively, than the control strain in xylose medium. Also triacylglycerol yields on biomass and per used xylose were 10 to 34% and 1 to 24% higher with the transformants than the control strain, respectively. Also total lipid concentration and yield on biomass were 15 to 16% and 6 to 33% higher in the transformants than in the control.

TABLE-US-00030 TABLE 24 Triacylglycerol (TAG) and total lipid concentrations (g/l), rates (mg/l/h) and yields (%) per biomass (CDW; cell dry weight) and used xylose after 96 hours cultivation in xylose medium with C/N ratio of 21 Yield TAG TAG Yield TAG (% used TAG Strain g/l (% CDW) xylose mg/l/h Control 0.60 34.9 13.9 6 M22/94-16 0.71 38.4 14.0 7 (ALD + ACS) M22/94/102-31 0.78 53.2 17.2 8 (PDC + ALD + ACS) Yield lipid Lipid Yield lipid (% used Lipid Strain g/l (% CDW) xylose mg/l/h Control 0.81 47.6 18.9 8 M22/94-16 0.94 50.5 18.5 10 (ALD + ACS) M22/94/102-31 0.93 63.5 20.5 10 (PDC + ALD + ACS)

[0271] This example shows that expression of PDC1, ALD6 and ACS2 genes in different combinations enhanced triacylglycerol and total lipid concentrations and rates of production and triacylglycerol and total lipid yields from used xylose or per dry weight in the yeast cell culture me with low C/N ratio.

Example 44. Production of Triacylglycerol or Lipid by Strains of C. curvatus Modified by Addition of Genes Encoding PDC and ALD and PDAT and ACS (Y23/85/95/101-8, Ex. 37B) in High Cell Density Cultures Grown on Glucose with C/N Ratio of 28

[0272] Transformant (Y23/85/95/101-8) was cultivated in Multifors bioreactors (max. working volume 500 ml, Infors HT, Switzerland) at pH 4.0, 30.degree. C., in 500 ml medium containing 90 to 96 g glucose, 6.74 g (NH.sub.4).sub.2SO.sub.4, 1.2 g KH.sub.2PO.sub.4, 0.3 g Na.sub.2HPO.sub.4.2H.sub.2O, 1.5 g MgSO.sub.4.7H.sub.2O, 0.1 g CaCl.sub.2.6H.sub.2O, 5.26 mg citric acid.H.sub.2O, 5.26 mg ZnSO.sub.4.7H.sub.2O, 0.1 mg MnSO.sub.4.4H.sub.2O, 0.5 mg CoCl.sub.2.6H.sub.2O, 0.26 mg CuSO.sub.4.5H.sub.2O, 0.1 mg Na.sub.2MoO.sub.4.2H.sub.2O, 1.4 mg FeSO.sub.4.7H.sub.2O, 0.1 mg H.sub.3BO.sub.4, 0.05 mg D-biotin, 1.0 mg CaPantothenate, 5.0 mg nicotinic acid, 25 mg myoinositol, 1.0 mg thiamine.HCl, 1.0 mg pyridoxine.HCl and 0.2 mg p-aminobenzoic acid per litre. The pH was maintained constant by addition of 1 M KOH or 1 M H.sub.2PO.sub.4. Cultures were agitated at 1000 rpm (2 Rushton turbine impellors) and aerated at 2 volumes air per volume culture per minute (vvm). Clerol FBA 3107 antifoaming agent (Cognis, SaintFargeau-Ponthierry Cedex France, 1 ml l.sup.-1) was added to prevent foam accumulation. Bioreactors were inoculated to initial OD.sub.600 of 0.5 to 4.0 with cells grown in the same medium (substituting 1.5 g urea per litre for (NH.sub.4).sub.2SO.sub.4 and omitting the CaCl.sub.2.6H.sub.2O) in 50 ml volumes in 250 ml flasks at 30.degree. C. with shaking at 200 rpm for 24 to 42 h. Samples for cell dry weight measurement, lipid extraction and HPLC analysis were withdrawn periodically during cultivation. Cryptococcus curvatus wild type strain Y23 was used as the control.

[0273] Lipid extraction and triacylglycerol concentration measurements were carried out as described in Example 21. Lipid extraction from the culture medium was carried out as described in Example 39. Cell dry weight was determined as described in Example 30. HPLC analyses were carried out as described in Example 22.

[0274] Table 25A shows that a transformant containing the genes PDC, ALD, ACS and PDAT produced 58% more triacylglycerol than Y23, with a 67% and 185% increase in the yields on glucose consumed and on biomass, respectively. Additionally, the transformant containing the genes PDC, ALD, ACS and PDAT produced 20% more triacylglycerol than Y23, with a 111% increase in the yield on glucose consumed in the culture medium (Table 25B).

TABLE-US-00031 TABLE 25A Triacylglycerol produced in pH controlled bioreactor culture of Y23 and transformant of Y23 expressing PDC + ALD + ACS + PDAT, with glucose as carbon source and C/N 28. Data is the average of 2 cultures .+-. standard error of the mean. Percentage increase is shown in parenthesis. Yield TAG TAG (% glucose Yield TAG Strain (g/l) consumed) (% per CDW) Y23 1.30 .+-. 0.06 1.22 .+-. 0.01 2.67 .+-. 0.14 Y23/85/95/101-8 2.05 .+-. 0.34 2.04 .+-. 0.22 4.95 .+-. 0.33 (PDC + ALD + ACS + (+58%) (+67%) (+185%) PDAT)

TABLE-US-00032 TABLE 25B Triacylglycerol produced in the culture medium in pH controlled bioreactor culture of Y23 and transformant of Y23 expressing PDC + ALD + ACS + PDAT, with glucose as carbon source and C/N 28. Data is the average of 2 cultures .+-. standard error of the mean. Percentage increase is shown in parenthesis. Yield TAG TAG (% glucose Strain (g/l) consumed) Y23 0.15 .+-. 0.04 0.18 .+-. 0.03 Y23/85/95/101-8 0.18 .+-. 0.00 0.38 .+-. 0.07 (PDC + ALD + ACS + (+20%) (+111%) PDAT)

[0275] This example shows that expression of PDC1, ALD6, ACS1 and PDAT enhanced triacylglycerol concentrations and triacylglycerol yields from used glucose or per dry weight in high cell density cultures in the yeast cell and/or in the culture medium.

Sequences used:

[0276] SEQ ID NOs: 1 and 2 correspond to primers YeastTEF1 and YeastTEF4, respectively, used to isolate genomic fragment of the C. curvatus TEF gene.

[0277] SEQ ID NOs: 3 and 4 correspond to primers PCR linker I and PCR linker II, respectively, used in chromosome walk experiments.

[0278] SEQ ID NOs: 5, 6, 7 and 8 correspond to primers CC_TEF2, CC_TEF1, CC_TEF6 and CC_TEF5 respectively, used to isolate genomic fragments of the C. curvatus TEF promoter region in chromosome walk experiments.

[0279] SEQ ID NOs: 9 and 10 correspond to primers CC_TEF10 and CC_TEF11, respectively, used to isolate promoter of the C. curvatus TEF gene.

[0280] SEQ ID NOs: 11 and 12 correspond to primers CC_TEF3 and CC_TEF4, respectively, used to isolate genomic fragment of the C. curvatus TEF terminator region in chromosome walk experiments.

[0281] SEQ ID NOs: 13 and 14 correspond to primers CC_TEF7 and CC_TEF8, respectively, used to isolate terminator of the C. curvatus TEF gene.

[0282] SEQ ID NOs: 15 and 16 correspond to primers Yeast TPI5 and Yeast TPI8, respectively, used to isolate genomic fragment of the C. curvatus TPI gene.

[0283] SEQ ID NOs: 17 and 18 correspond to primers CC_TPI2 and CC_TPI1, respectively, used to isolate genomic fragment of the C. curvatus TPI promoter region in chromosome walk experiments.

[0284] SEQ ID NOs: 19 and 20 correspond to primers CC_TPI7 and CC_TPI9, respectively, used to isolate promoter of the C. curvatus TPI gene.

[0285] SEQ ID NOs: 21 and 22 correspond to primers CC_TPI4 and CC_TPI3, respectively, used to isolate genomic fragment of the C. curvatus TPI terminator region in chromosome walk experiments.

[0286] SEQ ID NOs: 23 and 24 correspond to primers CC_TPI5 and CC_TPI6, respectively, used to isolate terminator of the C. curvatus TPI gene.

[0287] SEQ ID NOs: 25 and 26 correspond to primers YeastENO5 and YeastENO10, respectively, used to isolate genomic fragment of the C. curvatus ENO gene.

[0288] SEQ ID NOs: 27, 28, 29 and 30 correspond to primers CC_ENO2, CC_ENO1, CC_ENO5 and CC_ENO6, respectively, used to isolate genomic fragments of the C. curvatus ENO promoter region in chromosome walk experiments.

[0289] SEQ ID NOs: 31 and 32 correspond to primers CC_ENO9 and CC_ENO10, respectively, used to isolate promoter of the C. curvatus ENO gene.

[0290] SEQ ID NOs: 33 and 34 correspond to primers CC_ENO4 and CC_ENO3, respectively, used to isolate genomic fragment of the C. curvatus ENO terminator region in chromosome walk experiments.

[0291] SEQ ID NOs: 35 and 36 correspond to primers CC_ENO7 and CC_ENO8, respectively, used to isolate terminator of the C. curvatus ENO gene.

[0292] SEQ ID NOs: 37 and 38 correspond to primers CC_GPD3 and CC_GPD4, respectively, used to isolate genomic fragment of the C. curvatus GPD terminator region in chromosome walk experiments.

[0293] SEQ ID NOs: 39 and 40 correspond to primers CC_GPD6 and CC_GPD7, respectively, used to isolate terminator of the C. curvatus GPD gene.

[0294] SEQ ID NOs: 41 and 42 correspond to primers Hph 5 and Hph 3, respectively, used to isolate E. coli hygromycin gene.

[0295] SEQ ID NOs: 43 and 44 correspond to primers Kan 5 and Kan 3, respectively, used to isolate E. coli G418 resistance gene.

[0296] SEQ ID NOs: 45 and 46 correspond to primers CERR 5 and CERR 3, respectively, used to isolate S. cerevisiae cerulenin resistance gene.

[0297] SEQ ID NO: 47 corresponds to the amino acid sequence of the S. cerevisiae ALD6 gene, with GenBank accession number AAB68304 (version number AAB68304.1).

[0298] SEQ ID NO: 48 corresponds to S. cerevisiae ALD6 protein encoding DNA codon optimized according to Ustilago maydis-fungus codon usage.

[0299] SEQ ID NO: 49 corresponds to S. cerevisiae ALD6 protein encoding DNA codon optimized according to Rhizopus oryzae-filamentous fungus codon usage.

[0300] SEQ ID NO: 50 corresponds to the amino acid sequence of the S. cerevisiae ACS2 gene, with GenBank accession number CAA97725 (version number CAA97725.1.

[0301] SEQ ID NO: 51 corresponds to S. cerevisiae ACS2 protein encoding DNA codon optimized according to Ustilago maydis-fungus codon usage.

[0302] SEQ ID NO: 52 corresponds to the amino acid sequence of the Rhizopus oryzae PDAT gene, encoded by gene with locus number RO3G_07851.3 in Broad Institute Rhizopus oryzae database.

[0303] SEQ ID NO: 53 corresponds to Rhizopus oryzae PDAT protein encoding DNA codon optimized according to Ustilago maydis-fungus codon usage.

[0304] SEQ ID NOs: 54 and 55 correspond to primers Mould TPI1 and mould TPI3, respectively, used to isolate genomic fragment of the Mucor circinelloides TPI gene.

[0305] SEQ ID NOs: 56 and 57 correspond to primers MC_TPI2 and MC_TPI1, respectively, used to isolate genomic fragment of the Mucor circinelloides TPI promoter region in chromosome walk experiments.

[0306] SEQ ID NOs: 58 and 59 correspond to primers MC_TPI7 and MC_TPI8, respectively, used to clone promoter of the Mucor circinelloides TPI gene.

[0307] SEQ ID NOs: 60 and 61 correspond to primers MC_TPI4 and MC_TPI3, respectively, used to isolate genomic fragment of the Mucor circinelloides TPI terminator region in chromosome walk experiments.

[0308] SEQ ID NOs: 62 and 63 correspond to primers MC_TPI5 and MC_TPI6, respectively, used to clone terminator of the Mucor circinelloides TPI gene.

[0309] SEQ ID NOs: 64 and 65 correspond to primers Mould TEF1 and Mould TEF4, respectively, used to isolate genomic fragment of the Mucor circinelloides TEF gene.

[0310] SEQ ID NOs: 66, 67, 68 and 69 correspond to primers MC_TEF2, MC_TEF1, MC_TEF6 and MC_TEF5, respectively, used to isolate genomic fragments of the Mucor circinelloides TEF promoter region in chromosome walk experiments.

[0311] SEQ ID NOs: 70 and 71 correspond to primers MC_TEF9 and MC_TEF10, respectively, used to clone promoter of the Mucor circinelloides TEF gene.

[0312] SEQ ID NOs: 72, 73, 74 and 75 correspond to primers MC_TEF4, MC_TEF3, MC_TEF8 and MC_TEF7, respectively, used to isolate genomic fragments of the Mucor circinelloides TEF terminator region in chromosome walk experiments.

[0313] SEQ ID NOs: 76 and 77 correspond to primers MC_TEF11 and MC_TEF12, respectively, used to clone terminator of the Mucor circinelloides TEF gene.

[0314] SEQ ID NOs: 78 and 79 correspond to primers Mould PGK4 and Mould PGK2, respectively, used to isolate genomic fragment of the Mucor circinelloides PGK gene.

[0315] SEQ ID NOs: 80, 81, 82 and 83 correspond to primers MC_PGK2, MC_PGK1, MC_PGK4 and MC_PGK3, respectively, used to isolate genomic fragments of the Mucor circinelloides PGK promoter region in chromosome walk experiments.

[0316] SEQ ID NOs: 84 and 85 correspond to primers MC_PGK5 and MC_PGK6, respectively, used to clone promoter of the Mucor circinelloides PGK gene.

[0317] SEQ ID NOs: 86, 87, 88 and 89 correspond to primers MC_GPD2, MC_GPD1, MC_GPD10 and MC_GPD9, respectively, used to isolate genomic fragment of the Mucor circinelloides GPD promoter region in chromosome walk experiments.

[0318] SEQ ID NOs: 90 and 91 correspond to primers MC_GPD11 and MC_GPD12, respectively, used to clone promoter of the Mucor circinelloides GPD gene.

[0319] SEQ ID NO: 92 corresponds to S. cerevisiae ACS2 protein encoding DNA codon optimized according to Rhizopus oryzae-fungus codon usage.

[0320] SEQ ID NO: 93 corresponds to Rhizopus oryzae PDAT protein encoding DNA codon optimised according to Rhizopus oryzae-fungus codon usage.

[0321] SEQ ID NO: 94 corresponds to S. cerevisiae PDC1 protein encoding DNA, with GenBank accession number X77316 (version number X77316.1).

[0322] SEQ ID NO: 95 corresponds to the amino acid sequence of the S. cerevisiae PDC1 gene, with GenBank accession number CAA54522 (version number CAA54522.1.

[0323] SEQ ID NO: 96 corresponds to S. cerevisiae PDC1 protein encoding DNA codon optimized according to Ustilago maydis-fungus codon usage.

[0324] SEQ ID NO: 97 corresponds to S. cerevisiae PDC1 protein encoding DNA codon optimized according to Rhizopus oryzae-fungus codon usage.

REFERENCES

[0325] Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. 1997. Nucleic Acids Res. 25:3389-3402.

[0326] Boulton, C. A. and Ratledge, C. 1981. J. Gen. Microbiol. 127:169-176.

[0327] Folch J., Lees M. and Stqanley, G. H. S. J. Biol. Chem 226:497-509 (1957)

[0328] van den Berg, M. A., de Jong-Gubbels, P., Steensma, H. Y., van Dijken, J. P. and Pronk, J. T. 1996. J. Biol. Chem. 271:28953-28959.

[0329] Connerton, I. F., Fincham, J. R. S., Sandeman, R. A. and Hynes, M. J. 1990. Mol. Microbiol. 4:451-460.

[0330] Dahlqvist, A., Stahl, U., Lenman, M., Banas, A., Lee, M., Sandager, L., Ronne, H. and Stymne, S. 2000. PNAS 97:6487-6492

[0331] Flikweert, M. T., Van der Zanden, L., Janssen, W. M. TH. M., Steensma, H. Y., Van Dijken, J. P. and Pronk, J. T. 1996. Yeast 12:247-257.

[0332] Flipphi, M., Mathieu, M., Cirpus, I., Panozzo, C. and Felenbok, B. 2001. J. Biol. Chem. 276:6950-6958.

[0333] Hiesinger, M., Wagner, C. and Schuller, H.-J. 1997. FEBS Lett. 415:16-20. Hynes, M. J. and Murray, S. L. 2010. Euk. Cell 9:1039-1048.

[0334] Mach, R. L., Schindler, M. and Kubicek, C. P. 1994. Curr Genet. 25, 567-570 Meesters, P. A. E. P., Springer, J. and Eggink, G. 1997. Appl. Microbiol. Biotechnol. 47:663-667.

[0335] Mueller, P. R. and Wold, B. 1989, "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR." Science 246:780-786.

[0336] Nakazawa, N., Hashimoto, H., Harashima, S. and Oshima, Y. 1993. J. Ferm. Bioeng. 76:60-63.

[0337] Postma, E., Verduyn, C., Scheffers, W. A. and van Dijken, J. P. 1989. Appl. Environ. Microbiol. 55:468-477.

[0338] Pronk, J. T., Steensma, H. Y. and Van Dijken, J. P. 1996. Yeast 12:1607-1633.

[0339] Ratledge, C. and Wynn, J. P. 2002. Adv. Appl. Microbiol. 51:1-51.

[0340] Saint-Prix, F., Bonquist, L. and Dequin, S. 2004. Microbiol. 150:2209-2220.

[0341] Sambrook, J. and Russell, D. W. 2001. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York, US.

[0342] Shiba, Y, Paradise, E M, Kirby J, Ro, D-K and Keasling J D. 2007. Metabolic Engineering 9; 160-168.

[0343] Skory, C. D. 2003. Curr. Microbiol. 47:59-64.

[0344] Sorger D. and Daum G. (2003) Appl. Microbiol. Biotechnol 61:289-299.

[0345] Takahashi, H., McCaffery, J. M., Irizarry, R. A. and Boeke, J. D. 2006. Mol. Cell 23:207-217.

[0346] Tehlivets, O., Scheuringer K. and Kohlwein S. D. (1997) Biocim Biophys Acta 1771:255-270.

[0347] Wolff, A. M., Appel, K. F., Petersen, J. B., Poulsen, U. and Arnau, J. 2002 FEMS Yeast Res. 2:203-213

Sequence CWU 1

1

97124DNAArtificial Sequenceprimer 1tacaagtgyg gtggtatyga caag 24223DNAArtificial Sequenceprimer 2tcwacggayt tgacttcagt ggt 23325DNAArtificial Sequenceprimer 3gcggtgaccc gggagatctg aattc 25412DNAArtificial Sequenceprimer 4gaattcagat ct 12517DNAArtificial Sequenceprimer 5cacgctcacg ctcggcc 17615DNAArtificial Sequenceprimer 6ccgaggtcgg cggcc 15719DNAArtificial Sequenceprimer 7ggcaggcgca aagctggac 19826DNAArtificial Sequenceprimer 8gcagtcactg tcattgtcgc actacc 26932DNAArtificial Sequenceprimer 9tccccgcggg gatccatcac gcctgcccgt cc 321047DNAArtificial Sequenceprimer 10gctctagagc ctgcaggttt ttataggttc tgcgaatggt tagtacg 471125DNAArtificial Sequenceprimer 11cctccaggac gtctacaaga tcggc 251216DNAArtificial Sequenceprimer 12cccgtcggcc gtgtcg 161338DNAArtificial Sequenceprimer 13tccccccggg cctgcaggtt gtagagccct cggttctg 381431DNAArtificial Sequenceprimer 14ggaattccgg aggcttgtca tcatacgaga c 311523DNAArtificial Sequenceprimer 15ggtaactkka agatgaacgg ctc 231623DNAArtificial Sequenceprimer 16gckccrccga craggaawcc rtc 231720DNAArtificial Sequenceprimer 17gggaagttgg cgctgtggac 201817DNAArtificial Sequenceprimer 18cgctgagctt ggcgtcg 171929DNAArtificial Sequenceprimer 19cgggatcccg gaattcctga ccacccgcg 292039DNAArtificial Sequenceprimer 20tgtgtgcctg caggcttgga tatgctgttt taggtttgg 392119DNAArtificial Sequenceprimer 21aagcgcgtct cgcagaagg 192217DNAArtificial Sequenceprimer 22acggcggctc cgtcaac 172335DNAArtificial Sequenceprimer 23gctctagagc ctgcagggat gaggcgtggc atagg 352426DNAArtificial Sequenceprimer 24cgggatcccg cagctgacga caggct 262525DNAArtificial Sequenceprimer 25cccgtcacct cycagaagga gattg 252619DNAArtificial Sequenceprimer 26cggtctcacc ggatcggtg 192716DNAArtificial Sequenceprimer 27ttggcagcgg cctcgg 162818DNAArtificial Sequenceprimer 28ccaaggatgg cgttggcg 182919DNAArtificial Sequenceprimer 29cgtgctcctg cccaggagg 193020DNAArtificial Sequenceprimer 30cgatgctctc ggcagttgcg 203129DNAArtificial Sequenceprimer 31cggaattctg tctgtacgag tctgtacac 293238DNAArtificial Sequenceprimer 32ggaattcctg caggtttgag gtgaggttgt tgttttgg 383317DNAArtificial Sequenceprimer 33ggcgtgcaac gccctcc 173421DNAArtificial Sequenceprimer 34ccatccaggc gtgggtactg a 213532DNAArtificial Sequenceprimer 35cccaagcttc ctgcagggtg cgcgtagtgc gc 323629DNAArtificial Sequenceprimer 36cccaagcttg ggacgccgag gagcatctc 293721DNAArtificial Sequenceprimer 37cgtcggtctt tgacgccaag g 213823DNAArtificial Sequenceprimer 38cgtggtacga caacgagtac ggc 233943DNAArtificial Sequenceprimer 39gctctagagc ctgcaggatc ccttcgagga tgtagttagg ttg 434031DNAArtificial Sequenceprimer 40cgggatcccg tggaggtgtc tgtgatgacg a 314138DNAArtificial Sequenceprimer 41ggactagtcc tgcaggatga aaaagcctga actcaccg 384237DNAArtificial Sequenceprimer 42ggactagtcc tgcaggctat tcctttgccc tcggacg 374335DNAArtificial Sequenceprimer 43ggactagtcc tgcaggatga gccatattca acggg 354439DNAArtificial Sequenceprimer 44ggactagtcc tgcaggttag aaaaactcat cgagcatca 394538DNAArtificial Sequenceprimer 45acacaccctg caggatgagt gtgtctaccg ccaagagg 384634DNAArtificial Sequenceprimer 46gtgtgtcctg caggttaatt tgcggccggt accg 3447500PRTSaccharomyces cerevisiae 47Met Thr Lys Leu His Phe Asp Thr Ala Glu Pro Val Lys Ile Thr Leu 1 5 10 15 Pro Asn Gly Leu Thr Tyr Glu Gln Pro Thr Gly Leu Phe Ile Asn Asn 20 25 30 Lys Phe Met Lys Ala Gln Asp Gly Lys Thr Tyr Pro Val Glu Asp Pro 35 40 45 Ser Thr Glu Asn Thr Val Cys Glu Val Ser Ser Ala Thr Thr Glu Asp 50 55 60 Val Glu Tyr Ala Ile Glu Cys Ala Asp Arg Ala Phe His Asp Thr Glu 65 70 75 80 Trp Ala Thr Gln Asp Pro Arg Glu Arg Gly Arg Leu Leu Ser Lys Leu 85 90 95 Ala Asp Glu Leu Glu Ser Gln Ile Asp Leu Val Ser Ser Ile Glu Ala 100 105 110 Leu Asp Asn Gly Lys Thr Leu Ala Leu Ala Arg Gly Asp Val Thr Ile 115 120 125 Ala Ile Asn Cys Leu Arg Asp Ala Ala Ala Tyr Ala Asp Lys Val Asn 130 135 140 Gly Arg Thr Ile Asn Thr Gly Asp Gly Tyr Met Asn Phe Thr Thr Leu 145 150 155 160 Glu Pro Ile Gly Val Cys Gly Gln Ile Ile Pro Trp Asn Phe Pro Ile 165 170 175 Met Met Leu Ala Trp Lys Ile Ala Pro Ala Leu Ala Met Gly Asn Val 180 185 190 Cys Ile Leu Lys Pro Ala Ala Val Thr Pro Leu Asn Ala Leu Tyr Phe 195 200 205 Ala Ser Leu Cys Lys Lys Val Gly Ile Pro Ala Gly Val Val Asn Ile 210 215 220 Val Pro Gly Pro Gly Arg Thr Val Gly Ala Ala Leu Thr Asn Asp Pro 225 230 235 240 Arg Ile Arg Lys Leu Ala Phe Thr Gly Ser Thr Glu Val Gly Lys Ser 245 250 255 Val Ala Val Asp Ser Ser Glu Ser Asn Leu Lys Lys Ile Thr Leu Glu 260 265 270 Leu Gly Gly Lys Ser Ala His Leu Val Phe Asp Asp Ala Asn Ile Lys 275 280 285 Lys Thr Leu Pro Asn Leu Val Asn Gly Ile Phe Lys Asn Ala Gly Gln 290 295 300 Ile Cys Ser Ser Gly Ser Arg Ile Tyr Val Gln Glu Gly Ile Tyr Asp 305 310 315 320 Glu Leu Leu Ala Ala Phe Lys Ala Tyr Leu Glu Thr Glu Ile Lys Val 325 330 335 Gly Asn Pro Phe Asp Lys Ala Asn Phe Gln Gly Ala Ile Thr Asn Arg 340 345 350 Gln Gln Phe Asp Thr Ile Met Asn Tyr Ile Asp Ile Gly Lys Lys Glu 355 360 365 Gly Ala Lys Ile Leu Thr Gly Gly Glu Lys Val Gly Asp Lys Gly Tyr 370 375 380 Phe Ile Arg Pro Thr Val Phe Tyr Asp Val Asn Glu Asp Met Arg Ile 385 390 395 400 Val Lys Glu Glu Ile Phe Gly Pro Val Val Thr Val Ala Lys Phe Lys 405 410 415 Thr Leu Glu Glu Gly Val Glu Met Ala Asn Ser Ser Glu Phe Gly Leu 420 425 430 Gly Ser Gly Ile Glu Thr Glu Ser Leu Ser Thr Gly Leu Lys Val Ala 435 440 445 Lys Met Leu Lys Ala Gly Thr Val Trp Ile Asn Thr Tyr Asn Asp Phe 450 455 460 Asp Ser Arg Val Pro Phe Gly Gly Val Lys Gln Ser Gly Tyr Gly Arg 465 470 475 480 Glu Met Gly Glu Glu Val Tyr His Ala Tyr Thr Glu Val Lys Ala Val 485 490 495 Arg Ile Lys Leu 500 481506DNAArtificial SequenceS. cerevisiae codon optimized to Ustilago maydis 48atgaccaagc tccacttcga caccgccgag cccgtcaaga tcaccctccc caacggcctc 60acctacgagc agcccaccgg cctcttcatc aacaacaagt tcatgaaggc ccaggacggc 120aagacctacc ccgtcgagga cccctcgacc gagaacaccg tctgcgaggt cagctcggcc 180accaccgagg acgtggagta cgccatcgag tgcgccgacc gcgccttcca cgacaccgag 240tgggccaccc aggaccctcg cgagcgcggt cgcctcctct ccaaactcgc cgacgaactc 300gagtcgcaga tcgacctcgt cagctcgatc gaggccctcg acaacggcaa gaccctcgcc 360ctcgctcgcg gcgacgtgac gatcgccatc aactgcctcc gcgacgccgc tgcctacgcc 420gacaaggtca acggccgcac catcaacacc ggcgacggct acatgaactt caccaccctc 480gagcccatcg gcgtctgcgg ccagatcatc ccctggaact tccccatcat gatgctcgcc 540tggaagatcg cccctgccct cgccatgggc aacgtctgca tcctcaagcc tgccgccgtc 600acccccctca acgccctcta cttcgcctcg ctctgcaaga aggtcggcat ccctgccggc 660gtcgtcaaca tcgtccctgg ccctggccgc accgtcggcg ctgccctcac caacgacccc 720cgcatccgca agctcgcctt caccggctcg accgaggtcg gcaagtcggt cgccgtcgac 780tcgtcggagt cgaacctcaa gaagatcacg ctcgaactcg gcggcaagtc ggcccacctc 840gtgtttgacg acgccaacat caagaaaacc ctccctaacc tcgtcaacgg catcttcaag 900aacgccggcc agatctgctc gtcgggctcg cgcatctacg tccaggaagg catctacgac 960gaactcctcg ccgccttcaa ggcctacctc gaaaccgaga tcaaggtcgg caaccccttc 1020gacaaggcca acttccaggg cgccatcacc aaccgccagc agttcgacac catcatgaac 1080tacatcgaca tcggcaagaa ggaaggcgct aagatcctca cgggcggtga aaaggtcggc 1140gacaagggct acttcatccg ccccaccgtc ttttacgacg tgaacgagga catgcgcatc 1200gtcaaggaag agatcttcgg ccccgtcgtc accgtcgcca agttcaagac cctcgaagag 1260ggcgtcgaga tggctaactc gagcgaattt ggcctcggct cgggcatcga aaccgagtcg 1320ctctcgaccg gcctcaaggt cgccaagatg ctcaaggccg gcaccgtctg gatcaacacc 1380tacaacgact tcgactcgcg cgtccccttc ggcggcgtca agcagtcggg ctacggccgc 1440gagatgggcg aggaagtcta ccacgcctac accgaggtca aggccgtccg catcaagctc 1500tgatga 1506491506DNAArtificial SequenceS. cerevisiae codon optimized to Rhizopus oryzae 49atgacaaaac ttcattttga tacagctgaa ccagttaaaa tcacacttcc aaatggtctt 60acttatgaac aaccaacagg tctttttatc aacaacaaat ttatgaaagc tcaggatggt 120aaaacatatc cagttgaaga tccatcaaca gaaaatacag tttgtgaagt ttcatcagct 180acaacagagg atgttgaata tgctattgaa tgtgctgatc gagcttttca tgatacagaa 240tgggctacac aagatccacg agaacgaggt cgacttcttt caaaacttgc tgatgaactt 300gaatcacaaa ttgatcttgt ttcatcaatc gaagctcttg ataatggtaa aacacttgct 360cttgctcgag gtgatgttac aattgctatc aattgtttgc gagatgctgc tgcttatgct 420gataaagtta atggtcgaac aattaataca ggtgatggtt atatgaattt tacaacgctt 480gaaccaattg gtgtttgtgg tcaaattatt ccatggaact ttccaattat gatgcttgct 540tggaaaattg ctccagctct tgctatgggt aatgtttgta ttcttaaacc agctgctgtt 600acaccactta atgcacttta ttttgcttca ctttgtaaaa aagttggtat tccagctggt 660gttgttaata ttgttccagg tccaggtcga acagttggtg ctgctcttac aaatgatcca 720cgtattcgaa aacttgcttt tacaggttca acagaagttg gtaaatcagt tgctgttgat 780tcatcagagt caaacttgaa aaaaatcact cttgaacttg gtggtaaatc agctcatctt 840gtctttgatg atgctaacat caaaaaaaca cttccaaacc ttgtcaatgg tatctttaaa 900aacgctggtc aaatttgttc atcaggttca cgaatttatg tccaagaggg tatctatgat 960gaacttcttg ctgcttttaa agcatatctt gagacagaaa ttaaagtcgg taacccattt 1020gataaagcta attttcaagg tgctatcaca aatcgacaac aatttgatac gatcatgaac 1080tatattgata tcggtaaaaa agaaggtgct aaaattctta caggtggtga aaaagttggt 1140gataaaggtt attttatccg accaacagtc ttttatgatg tcaatgaaga tatgcgaatt 1200gtcaaagaag aaatttttgg tccagttgtt acagttgcta aattcaagac acttgaagag 1260ggtgttgaaa tggctaattc atcagaattt ggtcttggtt caggtattga aacagaatca 1320ctttcaacag gtcttaaagt cgctaaaatg cttaaagctg gtacagtttg gattaacacg 1380tataacgatt ttgattcacg agttccattt ggtggtgtta aacaatcagg ttatggtcga 1440gaaatgggtg aagaagttta tcacgcttat acagaagtta aagctgtccg aatcaaactt 1500taataa 150650683PRTSaccharomyces cerevisiae 50Met Thr Ile Lys Glu His Lys Val Val Tyr Glu Ala His Asn Val Lys 1 5 10 15 Ala Leu Lys Ala Pro Gln His Phe Tyr Asn Ser Gln Pro Gly Lys Gly 20 25 30 Tyr Val Thr Asp Met Gln His Tyr Gln Glu Met Tyr Gln Gln Ser Ile 35 40 45 Asn Glu Pro Glu Lys Phe Phe Asp Lys Met Ala Lys Glu Tyr Leu His 50 55 60 Trp Asp Ala Pro Tyr Thr Lys Val Gln Ser Gly Ser Leu Asn Asn Gly 65 70 75 80 Asp Val Ala Trp Phe Leu Asn Gly Lys Leu Asn Ala Ser Tyr Asn Cys 85 90 95 Val Asp Arg His Ala Phe Ala Asn Pro Asp Lys Pro Ala Leu Ile Tyr 100 105 110 Glu Ala Asp Asp Glu Ser Asp Asn Lys Ile Ile Thr Phe Gly Glu Leu 115 120 125 Leu Arg Lys Val Ser Gln Ile Ala Gly Val Leu Lys Ser Trp Gly Val 130 135 140 Lys Lys Gly Asp Thr Val Ala Ile Tyr Leu Pro Met Ile Pro Glu Ala 145 150 155 160 Val Ile Ala Met Leu Ala Val Ala Arg Ile Gly Ala Ile His Ser Val 165 170 175 Val Phe Ala Gly Phe Ser Ala Gly Ser Leu Lys Asp Arg Val Val Asp 180 185 190 Ala Asn Ser Lys Val Val Ile Thr Cys Asp Glu Gly Lys Arg Gly Gly 195 200 205 Lys Thr Ile Asn Thr Lys Lys Ile Val Asp Glu Gly Leu Asn Gly Val 210 215 220 Asp Leu Val Ser Arg Ile Leu Val Phe Gln Arg Thr Gly Thr Glu Gly 225 230 235 240 Ile Pro Met Lys Ala Gly Arg Asp Tyr Trp Trp His Glu Glu Ala Ala 245 250 255 Lys Gln Arg Thr Tyr Leu Pro Pro Val Ser Cys Asp Ala Glu Asp Pro 260 265 270 Leu Phe Leu Leu Tyr Thr Ser Gly Ser Thr Gly Ser Pro Lys Gly Val 275 280 285 Val His Thr Thr Gly Gly Tyr Leu Leu Gly Ala Ala Leu Thr Thr Arg 290 295 300 Tyr Val Phe Asp Ile His Pro Glu Asp Val Leu Phe Thr Ala Gly Asp 305 310 315 320 Val Gly Trp Ile Thr Gly His Thr Tyr Ala Leu Tyr Gly Pro Leu Thr 325 330 335 Leu Gly Thr Ala Ser Ile Ile Phe Glu Ser Thr Pro Ala Tyr Pro Asp 340 345 350 Tyr Gly Arg Tyr Trp Arg Ile Ile Gln Arg His Lys Ala Thr His Phe 355 360 365 Tyr Val Ala Pro Thr Ala Leu Arg Leu Ile Lys Arg Val Gly Glu Ala 370 375 380 Glu Ile Ala Lys Tyr Asp Thr Ser Ser Leu Arg Val Leu Gly Ser Val 385 390 395 400 Gly Glu Pro Ile Ser Pro Asp Leu Trp Glu Trp Tyr His Glu Lys Val 405 410 415 Gly Asn Lys Asn Cys Val Ile Cys Asp Thr Met Trp Gln Thr Glu Ser 420 425 430 Gly Ser His Leu Ile Ala Pro Leu Ala Gly Ala Val Pro Thr Lys Pro 435 440 445 Gly Ser Ala Thr Val Pro Phe Phe Gly Ile Asn Ala Cys Ile Ile Asp 450 455 460 Pro Val Thr Gly Val Glu Leu Glu Gly Asn Asp Val Glu Gly Val Leu 465 470 475 480 Ala Val Lys Ser Pro Trp Pro Ser Met Ala Arg Ser Val Trp Asn His 485 490 495 His Asp Arg Tyr Met Asp Thr Tyr Leu Lys Pro Tyr Pro Gly His Tyr 500 505 510 Phe Thr Gly Asp Gly Ala Gly Arg Asp His Asp Gly Tyr Tyr Trp Ile 515 520 525 Arg Gly Arg Val Asp Asp Val Val Asn Val Ser Gly His Arg Leu Ser 530 535 540 Thr Ser Glu Ile Glu Ala Ser Ile Ser Asn His Glu Asn Val Ser Glu 545 550 555 560 Ala Ala Val Val Gly Ile Pro Asp Glu Leu Thr Gly Gln Thr Val Val 565 570 575 Ala Tyr Val Ser Leu Lys Asp Gly Tyr Leu Gln Asn Asn Ala Thr Glu 580 585 590 Gly Asp Ala Glu His Ile Thr Pro Asp Asn Leu Arg Arg Glu Leu Ile 595 600 605 Leu Gln Val Arg Gly Glu Ile Gly Pro Phe Ala Ser Pro Lys Thr Ile 610 615 620 Ile Leu Val Arg Asp Leu Pro Arg Thr Arg Ser Gly Lys Ile Met Arg 625 630 635 640 Arg Val Leu Arg Lys Val Ala Ser Asn Glu Ala Glu Gln Leu Gly Asp 645 650 655 Leu Thr Thr Leu Ala Asn Pro Glu Val Val Pro Ala Ile Ile Ser

Ala 660 665 670 Val Glu Asn Gln Phe Phe Ser Gln Lys Lys Lys 675 680 512052DNAArtificial SequenceS.cerevisiae codon optimized to Ustilago maydis 51atgaccatca aggagcacaa ggtcgtctac gaggcccaca acgtcaaggc cctcaaggct 60ccccagcact tctacaactc gcagcccggc aagggctacg tcaccgacat gcagcactac 120caggagatgt accagcagtc gatcaacgag cccgagaagt tcttcgacaa gatggccaag 180gagtacctcc actgggacgc cccctacacc aaggtccagt cgggctcgct caacaacggc 240gatgtcgcct ggttcctcaa cggcaagctc aacgcctcgt acaactgcgt cgaccgccac 300gccttcgcca accccgacaa gcccgccctc atctacgagg ccgacgacga gtcggacaac 360aagatcatca ccttcggcga actcctccgc aaggtctcgc agatcgccgg cgtcctcaag 420tcgtggggcg tcaagaaggg cgacaccgtc gccatctacc tccccatgat ccccgaggcc 480gtcatcgcca tgctcgccgt cgcccgcatc ggcgccatcc actcggtcgt ctttgccggc 540ttctcggccg gctcgctcaa ggaccgcgtc gtcgatgcca actcgaaggt cgtcatcacc 600tgcgacgagg gcaagcgcgg tggcaagacc atcaacacca agaagatcgt cgacgaaggc 660ctcaacggcg tcgacctcgt ctcgcgcatc ctcgtctttc agcgcaccgg caccgagggc 720atccccatga aggctggccg cgactactgg tggcacgagg aggccgccaa gcagcgcacc 780tacctccccc ctgtctcgtg cgacgccgag gaccccctct tcctcctcta cacctcgggc 840tcgaccggca gccctaaggg cgtcgtccat acgaccggcg gctacctcct cggcgctgcc 900ctcaccaccc gctacgtctt tgacatccac cccgaggacg tgctcttcac cgctggcgac 960gtgggctgga tcaccggcca cacctacgcc ctctacggcc ccctcaccct cggcaccgcc 1020tcgatcatct tcgagtcgac ccccgcctac cccgactacg gccgctactg gcgcatcatc 1080cagcgccaca aggccaccca cttctacgtc gcccccaccg ccctccgcct catcaagcgc 1140gtcggcgagg ccgagatcgc caagtacgac acctcgtcgc tccgcgtcct cggctcggtc 1200ggcgagccca tctcgcccga cctctgggag tggtatcacg agaaggtcgg caacaagaac 1260tgcgtcatct gcgacaccat gtggcagacc gagtcgggtt cgcacctcat cgcccccctc 1320gctggcgccg tccctaccaa gcctggctcg gccaccgtcc ctttcttcgg catcaacgcc 1380tgcatcatcg accccgtcac cggcgtcgaa ctcgagggca acgacgtgga gggcgtcctc 1440gccgtcaagt cgccctggcc ctcgatggcc cgcagcgtct ggaaccacca cgaccgctac 1500atggacacct acctcaagcc ctaccccggc cactacttca ccggcgacgg cgctggtcgc 1560gatcacgatg gctactactg gattcgcggt cgcgtcgacg atgtcgtcaa cgtctcgggc 1620caccgcctct cgacctcgga gatcgaggcc agcatcagca accacgagaa cgtctcggag 1680gccgccgtcg tcggcatccc cgacgaactc accggccaga ccgtcgtcgc ctacgtctcg 1740ctcaaggacg gctacctcca gaacaacgcc accgagggcg acgccgagca catcaccccc 1800gacaacctcc gccgcgaact catcctccag gtccgcggcg agatcggccc cttcgcctcg 1860cccaagacca tcatcctcgt ccgcgacctc cctcgcaccc gctcgggcaa gatcatgcgc 1920cgcgtcctcc gcaaggtcgc ctcgaacgag gccgagcagc ttggtgacct caccaccctc 1980gccaaccctg aggtcgtccc cgccatcatc tcggccgtcg agaaccagtt cttctcgcaa 2040aagaagaagt ga 205252611PRTRhizopus oryzae 52Met Ser Lys Leu Arg Arg Arg Lys Gln Glu Lys Thr Thr Lys Thr Asn 1 5 10 15 Asp Gln Glu Leu Pro Glu Asp Ile Met Ala Asp Ser Ala Ala Asn Val 20 25 30 Phe Lys Glu Lys Arg Pro Phe Trp Gly Arg Lys Arg Phe Asn Phe Ile 35 40 45 Val Gly Leu Ser Val Gly Leu Leu Ala Met Tyr Ala Ala Ser Thr Thr 50 55 60 Pro Val Ala Gln Ser His Ile Asn Ser Leu Gln His Tyr Leu Leu Leu 65 70 75 80 Gln Leu Ala Asp Ile Asp Leu Ala Ser Ile Leu Pro Ala Thr Glu Met 85 90 95 Val Asp Glu Phe Leu Gly Asn Phe Thr Asn Leu Ile Thr Pro Thr Pro 100 105 110 Ala Thr Glu Met Ser Phe Met Pro Ala Leu Glu Tyr Lys Glu Ser Leu 115 120 125 Asp Leu Lys Pro Gln Phe Pro Val Val Met Ile Pro Ala Met Val Arg 130 135 140 Ser Val Leu Leu Asp Lys Glu Ser Trp Thr Glu His Ile Met Leu Asp 145 150 155 160 Pro Glu Thr Gly Leu Asp Pro Pro Gly Tyr Lys Val Arg Ala Val His 165 170 175 Glu Lys Lys Gly Val Glu Ala Ala Asp Tyr Phe Ile Thr Gly Tyr Trp 180 185 190 Val Trp Ala Lys Val Ile Glu Asn Leu Ala Thr Ile Gly Tyr Asp Thr 195 200 205 Asn Asn Met Tyr Phe Ala Ser Tyr Asp Trp Arg Leu Ser Phe Ser Asn 210 215 220 Leu Glu Val Arg Asp Gly Tyr Phe Ser Lys Leu Lys His Thr Ile Glu 225 230 235 240 Leu Ser Lys Lys Gln Ser Gly Gln Lys Ser Val Ile Ile Thr His Ser 245 250 255 Met Gly Gly Thr Met Phe Pro Tyr Phe Leu Lys Trp Val Glu Ser Lys 260 265 270 Gly His Gly Gln Gly Gly Gln Lys Trp Val Asp Glu His Ile Glu Ser 275 280 285 Phe Val Asn Ile Ala Ala Pro Leu Val Gly Val Pro Lys Ala Val Thr 290 295 300 Ser Leu Leu Ser Gly Glu Thr Arg Asp Thr Met Ala Leu Gly Ser Phe 305 310 315 320 Gly Ala Tyr Val Leu Glu Lys Phe Phe Ser Arg Arg Glu Arg Ala Lys 325 330 335 Leu Met Arg Ser Trp Met Gly Gly Ala Ser Met Leu Pro Lys Gly Gly 340 345 350 Glu Ala Ile Trp Gly Arg Gly Gly Asn Ala Pro Asp Asp Glu Glu Asp 355 360 365 Glu Lys Tyr Gln Ser Phe Gly Asn Met Ile Ser Phe Val Pro Arg Pro 370 375 380 Glu Gly Phe Asn Glu Asn Ser Thr Asp Ile Pro Ser Asn Ser Gly Asp 385 390 395 400 Pro Leu Val Arg Asn Tyr Thr Val Gln Gly Ser Ile Gln Leu Leu Thr 405 410 415 Lys Asn Ala Asp Ile Lys Phe Gly Lys Gln Leu Tyr Ala Asn Tyr Ser 420 425 430 Phe Gly Leu Thr Thr Ser Ser Lys Gln Leu Lys Arg Asn Glu Asn Asp 435 440 445 Pro Thr Lys Trp Ser Asn Pro Leu Glu Ser Arg Leu Pro Asn Ala Pro 450 455 460 Asn Met Lys Ile Tyr Cys Phe Tyr Gly Ile Glu Val Pro Thr Glu Arg 465 470 475 480 Ser Tyr Tyr Tyr Ala Ile Leu Asn Glu Asn Met Asp Gln Glu Cys Gly 485 490 495 His Ser Asn Ser Thr Ala Glu Cys Thr Thr Glu Gln Asn Ala Glu Pro 500 505 510 Asn Ser Ser Pro Ala Val Ala Lys Thr Ser Ser Ala Ala Phe Pro Asp 515 520 525 Lys Thr Pro Ser Leu His Ile Asp Ala Ser Ile Asn Asp Pro Val Gln 530 535 540 Arg Ile Glu Thr Gly Ile Arg Phe Ser Asn Gly Asp Gly Thr Val Pro 545 550 555 560 Leu Leu Ser Leu Gly Tyr Met Cys Ala Pro Ser Gly Gly Trp Arg Lys 565 570 575 His Ala Asp Leu Tyr Asn Pro Gly His Ser Pro Val Val Leu Arg Glu 580 585 590 Tyr Lys His Glu Val Ser Thr Ser Lys Leu Asp Val Arg Gly Gly Trp 595 600 605 Ile Ser Tyr 610 531836DNAArtificial SequenceRhizopus oryzae codon optimized to Ustilago maydis 53atgtcgaagc tccgccgtcg caagcaggaa aagaccacca agaccaacga ccaggaactc 60cccgaggaca tcatggctga ctcggccgcc aacgtcttta aggagaagcg ccccttctgg 120ggccgcaagc gcttcaactt catcgtcggc ctcagcgtcg gcctcctcgc catgtacgcc 180gccagcacca cccctgtcgc ccagtcgcac atcaactcgc tccagcacta cctcctcctc 240caactcgccg acatcgacct cgcctcgatc ctccccgcca ccgagatggt cgacgagttc 300ctcggcaact tcaccaacct catcaccccc acccctgcta cggagatgtc gttcatgccc 360gccctcgagt acaaggagtc gctcgacctc aagccccagt tccccgtcgt catgatcccc 420gctatggtcc gctcggtcct cctcgacaag gaaagctgga ccgagcacat catgctcgac 480cccgaaacgg gcctcgaccc acccggctac aaggtccgcg ccgtccacga gaagaagggc 540gtcgaggccg ccgactactt catcaccggc tactgggtct gggccaaggt catcgagaac 600ctcgccacca tcggctacga caccaacaac atgtacttcg cctcgtacga ctggcgcctc 660tcgttctcga acctcgaagt ccgcgacggc tacttctcga agctcaagca caccatcgaa 720ctctcgaaga agcagtcggg ccagaagtcg gtcatcatca cccactcgat gggcggcacc 780atgttccctt actttctcaa gtgggtcgag tcgaagggcc acggccaggg cggtcagaag 840tgggtcgacg agcacatcga gtcgttcgtc aacatcgctg cccccctcgt cggcgtcccc 900aaggccgtca cctcgctcct ctcgggcgag acccgcgaca ccatggccct cggctcgttc 960ggcgcctacg tcctcgagaa gttcttctcg cgtcgagagc gcgccaagct catgcgctcg 1020tggatgggcg gtgcctcgat gctccccaag ggcggcgagg ctatctgggg tcgcggcggt 1080aacgcccccg acgacgagga ggacgagaag taccaatcgt tcggtaacat gatctcgttc 1140gtccctcgcc ccgagggctt caacgagaac tcgaccgaca tcccctcgaa ctcgggcgac 1200cccctcgtcc gcaactacac cgtccagggc tcgatccagc tcctcaccaa gaacgccgac 1260atcaagttcg gcaagcagct ctacgccaac tactcgttcg gcctcaccac ctcgtcgaag 1320cagctcaagc gcaacgagaa cgaccccacc aagtggtcga accccctcga gtcgcgcctc 1380cccaacgccc ccaacatgaa gatctactgc ttctacggca tcgaggtccc caccgaacgc 1440tcgtactact acgccatcct caacgagaac atggaccagg agtgcggcca ctcgaactcg 1500accgccgagt gcaccaccga gcagaacgcc gagcccaact cgtcgcctgc tgtcgccaag 1560acctcgtcgg ccgccttccc cgacaagacc ccttcgctcc acatcgacgc ctcgatcaac 1620gaccctgtcc agcgcatcga gaccggcatc cgcttctcga acggcgacgg caccgtcccg 1680ctcctctcgc tcggctacat gtgcgccccc tcgggcggtt ggcgcaagca cgccgacctc 1740tacaaccccg gccactcgcc cgtcgtcctc cgcgagtaca agcacgaggt ctcgacctcg 1800aagctcgatg tccgcggtgg ctggatctcg tactga 18365422DNAArtificial Sequenceprimer 54gaggtygtcg tykcycctcc yg 225525DNAArtificial Sequenceprimer 55gcgaccttrc crgtrccgat rgccc 255625DNAArtificial Sequenceprimer 56aacaacccaa tcaacaccca tatcc 255724DNAArtificial Sequenceprimer 57ttttgagcag caaccttgat ttcc 245842DNAArtificial Sequenceprimer 58cgggatcccg gaattccagc ctctctaaac gagagttatc cc 425950DNAArtificial Sequenceprimer 59tccccccggg cctgcagggt tgaatatata aagttttttt ttagaaaaaa 506025DNAArtificial Sequenceprimer 60ggtgtctccg tcattgcctg tattg 256125DNAArtificial Sequenceprimer 61gtcgctcgtc aaatgaaggc tattg 256236DNAArtificial Sequenceprimer 62gctctagagc ctgcagggtt gcttcgctcc cctccc 366333DNAArtificial Sequenceprimer 63cgggatcccg cactgccaga ctaaacgcca gag 336424DNAArtificial Sequenceprimer 64cggtaagggt tcyttcaagt acgc 246519DNAArtificial Sequenceprimer 65ggaagacgga grggcttgt 196625DNAArtificial Sequenceprimer 66ttgtacttgg gggtctcgaa cttcc 256722DNAArtificial Sequenceprimer 67gataccacgc tcagcttcag cc 226826DNAArtificial Sequenceprimer 68ggatggatgg atggatgcat agtatg 266927DNAArtificial Sequenceprimer 69cacaacactt ctccaaatct gagaagc 277033DNAArtificial Sequenceprimer 70cccaagcttg ggagcaatgc taaaaaagcc tgg 337153DNAArtificial Sequenceprimer 71tgtgtgcctg caggtttgaa taactatagt atagattttt agtacatcga tgg 537223DNAArtificial Sequenceprimer 72gggatggaac aaggagacca agg 237323DNAArtificial Sequenceprimer 73gactctcctc gaagccatcg atg 237414DNAArtificial Sequenceprimer 74ccaaggccgc cgcc 147525DNAArtificial Sequenceprimer 75gcccttgtat aaagtgtgct tttgg 257642DNAArtificial Sequenceprimer 76tccccccggg cctgcaggat tgctacctgc tagttttttc tt 427732DNAArtificial Sequenceprimer 77ggaattccaa aagtcttttt gggtgtcttt ag 327822DNAArtificial Sequenceprimer 78tcaccaacaa cmancgtaty gt 227923DNAArtificial Sequenceprimer 79tggaaacgma ggttytcnar aag 238019DNAArtificial Sequenceprimer 80caacagcctc accgttggg 198120DNAArtificial Sequenceprimer 81gcagcacctt gttcaagggc 208229DNAArtificial Sequenceprimer 82gacactgttt tgaaatgcct aacccatac 298324DNAArtificial Sequenceprimer 83gtacggaaaa cagagtcatg gtgc 248435DNAArtificial Sequenceprimer 84tccccgcggg gatcccaagg cactaccact acttc 358548DNAArtificial Sequenceprimer 85gctctagagc ctgcagggat taattatatg attcaatgat gaagataa 488621DNAArtificial Sequenceprimer 86ctcgacggaa ccatcgaaac g 218726DNAArtificial Sequenceprimer 87gaaggacaat acgaccaata cgaccg 268832DNAArtificial Sequenceprimer 88ggcttctttc cttggtgaat aaagtgagtt ac 328928DNAArtificial Sequenceprimer 89cggaaattat ccgttcaaac tatcgccc 289020DNAArtificial Sequenceprimer 90agccaaaagt tgaattcgac 209159DNAArtificial Sequenceprimer 91cccaagcttc ctgcaggttt tagaatttat gaaatatata tatataaaga tataatatg 59922052DNAArtificial SequenceS.cerevisiae codon optimized to Rhizopus oryzae 92atgacaatta aagaacataa agtcgtttat gaagctcata atgttaaagc tcttaaagca 60ccacaacatt tctataattc acaaccaggt aaaggttatg tcacagatat gcagcattat 120caagaaatgt atcaacaatc aatcaacgaa ccagagaaat ttttcgataa gatggctaaa 180gaatatcttc attgggatgc tccatataca aaagttcaat caggttcact taacaatggt 240gatgttgctt ggtttcttaa tggtaaactt aacgcttcat ataattgtgt tgatcgacat 300gcttttgcta atccagataa accagctctt atctatgaag ctgatgatga atcagataac 360aaaatcatca catttggtga acttttgcga aaagtttcac aaattgctgg tgttcttaaa 420tcatggggtg ttaaaaaagg tgatacagtt gctatctatc ttccaatgat tcctgaagct 480gttattgcta tgcttgctgt tgctcgaatt ggtgctattc attcagttgt ttttgctggt 540ttttcagctg gttcacttaa agatcgagtt gttgatgcta attcaaaagt tgttatcaca 600tgtgatgaag gtaaacgagg tggtaaaaca attaacacga aaaaaatcgt tgatgaaggt 660cttaatggtg ttgatcttgt ttcacgtatt cttgtttttc aacgaacagg tactgaaggt 720attccaatga aagctggtcg agattattgg tggcatgaag aagctgctaa acaacgaaca 780tatcttccac cagtttcatg tgatgctgaa gatccacttt ttcttttgta tacatctggt 840tcaacaggtt ctccaaaagg tgttgttcat acaacaggtg gttatcttct tggtgctgct 900cttacaacac gatatgtctt tgatattcat ccagaagatg ttctttttac agcaggtgat 960gttggttgga ttacaggtca tacttatgca ctttatggtc cacttacact tggtacagct 1020tcaattatct ttgaatcaac gccagcttat ccagattatg gtcgatattg gcgaattatt 1080caacgacata aagctacaca tttttatgtc gctccaacag cacttcgact tattaaacga 1140gttggtgaag ctgaaattgc aaaatatgat acatcatcac ttcgagttct tggttcagtt 1200ggtgaaccaa tttcaccaga tctttgggaa tggtatcatg aaaaagttgg taacaaaaat 1260tgtgttatct gtgatacaat gtggcaaaca gaatcaggtt ctcatcttat tgctccactt 1320gctggtgctg ttccaacaaa accaggttca gctacagttc cattttttgg tatcaatgct 1380tgtattattg atccagttac aggtgttgaa cttgaaggta atgatgttga aggtgttctt 1440gctgttaaat caccatggcc atcaatggct cgatcagttt ggaatcatca tgatcgttat 1500atggatacgt atcttaaacc atatccaggt cactatttta caggtgatgg tgcaggtcga 1560gatcatgatg gttattattg gattcgaggt cgagttgatg atgttgttaa tgtttcaggt 1620catcgacttt caacatcaga aattgaagca tcaatttcaa accatgaaaa tgtttcagaa 1680gctgctgttg ttggtattcc agatgaactt acaggtcaaa cagttgttgc ttatgtctct 1740cttaaagatg gttatcttca aaacaatgct actgaaggtg atgctgaaca tattacacca 1800gataatcttc gacgagaact tattcttcaa gttcgaggtg aaattggtcc atttgcttca 1860ccaaaaacaa ttattcttgt tcgagatctt ccacgaacac gatcaggtaa aatcatgcga 1920cgagttcttc gaaaagttgc ttcaaatgaa gctgaacaac ttggtgatct tacaacactt 1980gcaaatccag aagttgttcc agctattatt tcagctgtcg aaaaccagtt tttctcacag 2040aaaaagaagt aa 2052931836DNAArtificial SequenceRhizopus oryzae codon optimized to Rhizopus oryzae 93atgtcaaaac ttcgacgacg aaaacaagaa aaaacaacga aaacaaacga tcaagaactt 60cctgaagata ttatggctga ttcagctgct aatgtcttta aagagaaacg accattttgg 120ggtcgaaaac gatttaactt tatcgttggt ctttcagttg gtcttttggc aatgtatgct 180gcttcaacaa caccagttgc tcaatcacat attaactcac ttcagcacta tcttttgctt 240caacttgctg atattgatct tgcttcaatt cttccagcta cagaaatggt tgatgaattt 300cttggtaact ttacaaatct tatcacacca acaccagcaa cagaaatgtc atttatgcca 360gctcttgaat ataaagagtc acttgatctt aaaccacaat ttccagttgt tatgattcca 420gctatggttc gatcagttct tcttgataaa gaatcatgga cagaacatat tatgcttgat 480ccagaaacag gtcttgatcc accaggttat aaagttcgag ctgtccatga aaaaaaaggt 540gttgaagctg ctgattattt cattacaggt tattgggttt gggctaaagt tattgaaaat 600cttgctacga ttggttatga tacgaacaac atgtattttg cttcatatga ttggcgactt 660tcattttcaa atcttgaagt ccgagatggt tatttttcaa aacttaaaca cacgatcgag 720ctttcaaaaa aacaatcagg tcagaaatca gtcattatca cacattcaat gggtggtaca 780atgtttccat attttcttaa atgggtcgaa tcaaaaggtc atggtcaagg tggtcaaaaa 840tgggttgatg aacatattga atcatttgtc aatattgctg ctccacttgt tggtgttcca 900aaagctgtta catcacttct ttcaggtgaa acacgagata caatggctct tggttcattt 960ggtgcttatg tccttgagaa attcttttca cgacgagaac gagctaaact tatgcgatca 1020tggatgggtg gtgcatcaat gcttccaaaa ggtggtgaag caatttgggg tcgaggtggt 1080aatgctccag atgatgaaga agatgagaaa tatcagtcat

ttggtaacat gatttcattt 1140gttccacgac cagaaggttt taacgaaaat tcaacggata ttccatcaaa ttcaggtgat 1200ccacttgttc gaaattatac agtccaaggt tcaattcaac ttcttacgaa aaacgctgat 1260atcaaatttg gtaaacagct ttatgctaac tattcatttg gtcttacaac atcatcaaaa 1320cagcttaaac gaaatgaaaa cgatccaaca aaatggtcaa atccacttga atcacgactt 1380ccaaatgctc caaacatgaa aatctattgt ttttatggta ttgaagttcc aacagagcga 1440tcatattatt atgctatcct taacgaaaat atggatcaag aatgtggtca ttcaaattca 1500acagctgaat gtacaacaga acaaaatgct gaaccaaatt catcaccagc tgttgctaaa 1560acatcatcag ctgcttttcc agataaaaca ccatcacttc atattgatgc ttcaattaat 1620gatccagtcc aacgaattga aacaggtatt cgattttcaa atggtgatgg tacagttcca 1680cttctttcac ttggttatat gtgtgctcca tcaggtggtt ggcgaaaaca tgctgatttg 1740tataatccag gtcattcacc agttgttctt cgagaatata aacatgaagt ctcaacgtca 1800aaacttgatg ttcgaggtgg ttggatttca tattaa 1836941701DNASaccharomyces cerevisiae 94atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120gaaggtatga gatgggctgg taacgccaac gaattgaacg ctcgttacgc cgctgatggt 180tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300gtcccatcca tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420gacatctgta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600attgacacca tcttggtctt ggctaaggat gctaagaacc cagttatctt ggctgatgct 660tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720ccagctttcg tcaccccaat gggtaagggt tccattagcg aacaacaccc aagatacggt 780ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960ttcccaggtg tccaaatgaa attcgttttg caaaagttgt tgaccaatat tgctgacgcc 1020gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140ggtgatgttg tcattgctga aaccggtacc tccgctttcg gtatcaacca aaccactttc 1200ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500actttcggtg ctaaggacta cgaaacccac agagtcgcta ccaccggtga atgggacaag 1560ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga ggttatgttg 1620ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680gctaagcaat aagcgattta a 170195563PRTSaccharomyces cerevisiae 95Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Arg Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Ile Ser Ser Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Cys Thr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Val Leu Ala 195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Ser Glu Gln His 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Asn 325 330 335 Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350 Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355 360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val 370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Val Met Leu Pro Val Phe Asp 530 535 540 Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln 961692DNASaccharomyces cerevisiae 96atgtcggaga tcaccctcgg caagtacctc ttcgagcgcc tcaagcaggt caacgtcaac 60accgtctttg gcctccccgg cgacttcaac ctctcgctcc tcgacaagat ctacgaggtc 120gagggcatgc gctgggccgg caacgccaac gaactcaacg ccgcctacgc cgccgacggc 180tacgcccgca tcaagggcat gtcgtgcatc atcaccacct tcggtgtcgg cgaactctcg 240gccctcaacg gtatcgccgg ctcgtacgct gagcacgtcg gcgtcctcca cgtcgtcggc 300gtcccttcga tctcggccca ggccaagcag ctcctcctcc accacaccct cggtaacggc 360gacttcaccg tctttcaccg catgtcggcc aacatctcgg aaaccaccgc catgatcacc 420gatatcgcca ccgcccctgc cgagatcgac cgctgcatcc gcaccaccta cgtcacccag 480cgccccgtct acctcggcct ccccgccaac ctcgtcgacc tcaacgtccc cgccaagctc 540ctccagaccc ccatcgacat gtcgctcaag cccaacgacg ccgagtcgga gaaggaagtc 600atcgacacca tcctcgccct cgtcaaggac gccaagaacc ccgtcatcct cgccgacgcc 660tgctgctcgc gccacgacgt caaggccgaa acgaagaagc tcatcgatct cacccagttc 720cccgccttcg tcacccccat gggcaagggc tcgatcgacg agcagcaccc ccgctacggc 780ggcgtctacg tcggcaccct ctcgaagccc gaggtcaagg aagccgtcga gtcggccgac 840ctcatcctct cggtcggcgc actcctctcg gacttcaaca ccggctcgtt ctcgtactcg 900tacaagacca agaacatcgt cgagttccac tcggaccaca tgaagatccg caacgccacc 960ttccccggcg tccagatgaa gttcgtcctc cagaagctcc tcaccacgat cgccgacgcc 1020gccaagggct acaagcccgt cgccgtccct gcccgcacac cagccaacgc agccgtccct 1080gcctcgaccc ccctcaagca agagtggatg tggaaccagc tcggcaactt cctccaagag 1140ggcgacgtcg tgatcgccga aaccggcacc tcggccttcg gcatcaacca gaccaccttc 1200cccaacaaca cctacggcat ctcgcaggtc ctctggggct cgatcggctt caccaccggc 1260gccaccctcg gcgctgcctt cgcagctgag gaaatcgacc ccaagaagcg tgtcatcctc 1320ttcatcggcg acggctcgct ccagctcacc gtccaagaga tctcgaccat gatccgctgg 1380ggcctcaagc cctacctctt cgtcctcaac aacgacggct acaccatcga gaagctcatc 1440cacggcccca aggcccagta caacgagatc cagggctggg accacctctc gctcctccct 1500accttcggcg ccaaggacta cgaaacccac cgcgtcgcca caaccggcga gtgggacaag 1560ctcacccagg acaagtcgtt caacgacaac tcgaagatcc gcatgatcga gatcatgctc 1620cccgtctttg acgcccccca gaacctcgtc gagcaggcca agctcaccgc cgccaccaac 1680gccaagcagt ga 1692971692DNASaccharomyces cerevisiae 97atgtcagaaa tcacgcttgg taaatatctt ttcgagcgac ttaaacaggt caatgtcaat 60actgtctttg gtttgccagg tgatttcaac ctttcacttc ttgacaagat ctatgaagtc 120gaaggtatgc gttgggcagg taatgcaaat gaacttaatg cagcatatgc agcagatggt 180tatgcacgaa tcaaaggtat gtcatgtatc atcacaacgt ttggtgtcgg tgaactttca 240gcacttaatg gtattgcagg ttcatatgca gaacatgttg gtgttcttca tgttgttggt 300gttccatcaa tttcagctca agcaaaacag cttcttcttc atcatacact tggtaacggt 360gattttacgg tctttcatcg aatgtcagca aacatctcag aaacaacagc aatgatcaca 420gatattgcaa cagcaccagc agaaattgat cgatgtatcc gaacaacata tgtcacacaa 480cgaccagttt atttgggttt gccagcaaat cttgtcgatc ttaatgtccc agcaaaactt 540cttcagacac caattgacat gtcacttaaa cctaacgatg ccgaatcaga aaaagaagtc 600atcgatacaa tccttgcact tgtcaaagat gcaaaaaacc cagttatcct tgcagatgca 660tgttgttcac gacatgatgt caaagcagaa acgaagaaac ttatcgatct tacacagttt 720ccagcatttg ttacgccaat gggtaaaggt tcaatcgatg aacaacatcc acgatatggt 780ggtgtttatg ttggtacact ttcaaaacca gaggtcaaag aagcagttga atcagcagat 840cttatccttt cagttggtgc acttctttca gattttaaca cgggttcatt ctcatattct 900tataagacga agaacatcgt cgagtttcac tcagatcata tgaagatccg aaacgcaaca 960tttccaggtg tccaaatgaa gtttgtcctt cagaaacttc ttacgacaat tgcagatgca 1020gccaaaggtt ataagccagt tgcagttcca gcacgaacac cagcaaatgc agcagttcca 1080gcttcaacac cacttaaaca agaatggatg tggaatcagc ttggtaactt tcttcaagag 1140ggtgatgttg ttatcgcaga aacaggtaca tcagcatttg gtatcaacca gacaacgttt 1200ccaaacaaca cgtatggtat ctcacaagtt ctttggggtt caatcggttt tacaacaggt 1260gcaacacttg gtgcagcatt tgctgcagaa gaaatcgatc caaaaaagcg agtcatcctt 1320tttatcggtg atggttcact tcagcttaca gttcaagaaa tctctacaat gatccgatgg 1380ggtcttaagc catatctttt tgtccttaac aacgacggtt atacgatcga aaaacttatc 1440catggtccaa aggcacagta taacgaaatt caaggttggg atcacctttc acttttgcca 1500acatttggtg caaaggatta tgagacacat cgagttgcaa caacaggtga atgggacaaa 1560cttacacagg ataagtcatt caacgacaac tcaaagatcc gaatgatcga aattatgctt 1620ccagtctttg atgcaccaca aaatcttgtt gagcaggcaa aacttacagc agcaacaaat 1680gccaaacagt aa 1692

Claims

1. A genetically modified oleaginous fungal cell comprising: a) a nucleic acid with enhanced expression encoding an acetaldehyde dehydrogenase (ALD), and b) a nucleic acid with enhanced expression encoding a diacylglycerol acyltransferase (DAT), wherein said genetically modified oleaginous fungal cell has enhanced lipid production in an aerobic bioreactor compared to a genetically unmodified oleaginous fungal cell.

2. The genetically modified oleaginous fungal cell of claim 1, wherein the encoded ALD is a cytosolic ALD.

3. The genetically modified oleaginous fungal cell of claim 2, wherein the encoded ALD is a fungal ALD, preferably Saccharomyces cerevisiae ALD6.

4. The genetically modified oleaginous fungal cell of claim 1, wherein the DAT encoding nucleic acid encodes a phosholipid:diacylglycerol acyltransferase (PDAT).

5. The genetically modified oleaginous fungal cell of claim 4, wherein the encoded DAT is a fungal DAT, preferably of Rhizopus oryzae, and most preferably it encodes a phosholipid:diacylglycerol acyltransferase (PDAT) having at least 40% sequence identity to SEQ ID NO:52, or an enzymatically active fragment or variant thereof.

6. The genetically modified oleaginous fungal cell of claim 1, further comprising a nucleic acid with enhanced expression encoding acetyl-CoA synthetase (ACS).

7. The genetically modified oleaginous fungal cell of claim 6, wherein the nucleic acid encoding ACS is a gene that is not under glucose repression or its gene product is not subject to post-translational regulation.

8. The genetically modified oleaginous fungal cell of claim 7, wherein the encoded ACS is a fungal ACS, preferably Saccharomyces cerevisiae ACS2.

9. The genetically modified oleaginous fungal cell of claim 1, which is a yeast cell selected from the genera Cryptococcus, Candida, Galactomyces, Hansenula, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia, preferably from the group consisting of Candida sp., Cryptococcus curvatus, Cryptococcus albidus, Galactomyces geotrichum, Hansenula ciferri, Lipomyces lipofer, Lipomyces ssp., Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica, or a filamentous fungal cell selected from the genera Aspergillus, Cunninghamella, Fusarium, Glomus, Humicola, Mortierella, Mucor, Penicillium, Pythium and Rhizopus, preferably from the group consisting of Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus niger, Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius, Humicola lanuginose, Mortierella isabellina, Mortierella pusilla, Mortierella vinacea, Mucor circinelloides, Mucor plumbeus, Mucor ramanniana, Penicillium lilacinum, Penicillium spinulosum, Pythium ultimum and Rhizopus oryzae.

10. The genetically modified oleaginous fungal cell of claim 9, which is from the genera Cryptococcus or Mucor, preferably it is a cell of Cryptococcus curvatus or Mucor circinelloides.

11. A method of producing lipids, comprising cultivating a genetically modified oleaginous fungal cell according to claim 1 in a medium containing carbon and nitrogen sources, and recovering the lipids produced.

12. The method of claim 11, wherein the lipids are recovered from the culture medium.

13. The method of claim 11, wherein lipids comprising acylglycerols, preferably triacylglycerols (TAG) are produced.

14. The method of claim 11, wherein the carbon source is a hexose or pentose sugars containing material.

15. The method of claim 11, comprising producing precursors for functional fatty acids.

16. A method of producing biofuel, or lubricant, said method comprising cultivating a genetically modified oleaginous fungal cell according to claim 1 in a medium containing carbon and nitrogen sources, and recovering the lipids produced, and optionally esterifying said lipids to obtain biodiesel or lubricant, or hydrogenizing the lipids to obtain renewable diesel or lubricant.

17. A method of preparing an oleaginous fungal cell of claim 1, said method comprising transforming a fungal cell with a) a nucleic acid with enhanced expression encoding an acetaldehyde dehydrogenase (ALD) enzyme, and b) a nucleic acid with enhanced expression encoding a diacylglycerol acyltransferase (DAT).

18. Use of a genetically modified fungal cell of claim 1 for producing lipids, precursors of functional fatty acids, functional fatty acids, biofuels, biodiesel, renewable diesel or lubricants.

Images