WO2008000277A2 - Cellules fongiques métaboliquement modifiées à teneur élevée en acides gras polyinsaturés - Google Patents

Cellules fongiques métaboliquement modifiées à teneur élevée en acides gras polyinsaturés Download PDF

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WO2008000277A2
WO2008000277A2 PCT/DK2007/050079 DK2007050079W WO2008000277A2 WO 2008000277 A2 WO2008000277 A2 WO 2008000277A2 DK 2007050079 W DK2007050079 W DK 2007050079W WO 2008000277 A2 WO2008000277 A2 WO 2008000277A2
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delta
desaturase
seq
gene
padhl
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PCT/DK2007/050079
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WO2008000277A3 (fr
WO2008000277A8 (fr
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Jochen FÖRSTER
Nina Katarina Gunnarsson
Malin Inga Elisabeth Svensson
Helga Susana Moreira David
Iben Plate
Sabina De Andrade Pereira Tavares
Jean-Marie Mouillion
Sara Petersen BJØRN
Per MØLLER
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Fluxome Sciences A/S
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/004—Cytochrome-b5 reductase (1.6.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6432—Eicosapentaenoic acids [EPA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis

Definitions

  • This invention relates to the production of fatty acids and particularly to the production of the polyunsaturated fatty acids (PUFAs) gamma-linolenic acid (GLA), arachidonic acid (ARA) and eicosapentaenoic acid (EPA) in genetically engineered fungal cells, and in particular to metabolically engineered Saccharomyces cerevisiae cells with increased content of GLA, ARA and EPA.
  • PUFAs polyunsaturated fatty acids
  • GLA gamma-linolenic acid
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • PL)FAs are polyunsaturated fatty acids with a long hydrocarbon chain composed of 18 or more carbon atoms having two or more double bonds and a terminal carboxylate group.
  • the properties of polyunsaturated fatty acids are highly influenced by the position of the double bond, and one differentiates omega-3 PL)FAs, which have the first double bond at the third position counting from the methyl end of the carbon chain, and omega-6 PL)FAs, which have the first double bond at the sixth position counting from the methyl end of the carbon chain.
  • Eicosapentaenoic acid (EPA; 5, 8, 11, 4, 17-20:5) belongs to the former group, while, arachidonic acid (ARA; 5, 8, 11, 14-20:4) and gamma-linolenic acid (GLA, 6, 9, 12-18:3) belong to the latter group.
  • PL)FAs are essential for humans, and it has been proven that they have many beneficial effects on human health, including proper development of brain and visual functions and prevention of disease, such as cardiovascular disease and cancer.
  • Natural dietary sources of GLA and ARA include animal meats, egg yolks, and shellfish, while the main dietary source of EPA is fish oil.
  • Commercial ARA- containing oils are produced by Martek Biosciences Corporation (Columbia, Md.), while EPA is available commercially as diverse mixtures of docosahexaenoic acid (DHA) and EPA (concentrates), which are produced by distillation of fish oils.
  • DHA docosahexaenoic acid
  • EPA concentrates
  • a recombinant microbial approach of production has many advantages. Most notably, a microbial process for EPA production circumvents certain problems associated with fish-oil derived products, such as bad taste and contamination by environmental pollutants.
  • the fatty acid composition of fish oil may vary during the fishing season.
  • Many naturally ARA- and EPA-producing microbes also contain inherent drawbacks, such as low growth rates and difficult-to-control fermentation behaviour.
  • lack of tools for genetic manipulation of natural producers makes it difficult to alter, e.g., the fatty acid composition in the oil that they produce.
  • the use of a well-characterized microbial host for ARA and EPA production provides solutions for the above-mentioned problems and, importantly, offers possibilities to develop the process further by metabolic engineering.
  • S. cerevisiae is one of the most well-characterized production organisms in biotechnology, and additionally has a long tradition in the manufacturing of food and beverage products.
  • S. cerevisiae was engineered to produce PL)FAs from a non-fatty acid carbon source.
  • S. cerevisiae has previously been used as a platform in which to analyze the function and substrate specificity of various fatty acid desaturases and elongases (e.g. US 6,432,684, PCT/US98/07422, WO 200244320-A, WO 99/27111) by means of adding exogenous fatty acid substrates.
  • Others have utilized S. cerevisiae to reconstitute parts of the pathway towards ARA and EPA with the intention of transferring this technology to plants (e.g. Domergue et al., 2003, Beaudoin et al., 2000, US 2003/0177508), still making use of an exogenous supplementation of desatu rated fatty acid substrate.
  • the authors of said disclosures express a heterologous Acyl-CoA/lysophosphatidic acid acyltransferase, a heterologous diacylglycerol acyltransferase, a heterologous glycerol-3-phosphate acyltransferase and a heterologous phospholipase C in their production host Yarrowia lipolytica.
  • the authors claim a recombinant PUFA-producing Yarrowia lipolytica expressing phospholipase A2, although experimental results on the use of this type of phospholipase are not disclosed.
  • the authors also claim expression of a phospholipid:DAG acyltransferase (PDAT) in Y. lipolytica, and more specifically over-expression of a native PDAT from Y. lipolytica.
  • PDAT phospholipid:DAG acyltransferase
  • acyltransferases that can be used for increasing the yield of PUFAs in different production hosts (e.g. WO2004/076617-A2, WO06052807-A2, WO06052824A2, WO06052814A2).
  • PUFA production pathways have also been introduced into plant production hosts, disclosed e.g. in WO2004/057001 and WO2005/012316.
  • metabolic engineering methods for increasing the yield of heterologoys PUFAs in transgenic plants are disclosed, including expression of a heterologous phospholipase, a ketoacyl-CoA reductase and a dehydratase involved in fatty acid elongation.
  • S. cerevisiae has the advantage of being well characterized, safe and easily manipulated by targeted genetic engineering. It is therefore well suited for yield improvement through metabolic engineering. It has been shown that the lipid content of PUFA-producing S. cerevisiae can be substantially increased by applying metabolic engineering approaches (PCT/DK2005/000372 and US-2006- 0051847-A1). Presently, applicants have introduced further genetic modifications into fungal cells that surprisingly and substantially alter the fatty acid composition and increase the yield of GLA, ARA and EPA in the recombinant yeast.
  • the present invention relates to the construction and engineering of non-plants more particularly microorganisms, such as fungal cells, for improved PL)FA production through overexpression and/or deletion of various endogenous genes, or through expression of heterologous genes.
  • the present invention describes methods for improving production of polyunsaturated fatty acids comprising expression of heterologous genes and/or genetic modifications of native genes in a Saccharomyces cerevisiae.
  • the present invention relates to the construction and engineering of fungal cells for improved production of particular PL)FAs, including gamma- linolenic acid, arachidonic acid, and eicosapentaenoic acid.
  • the present invention particularly relates to improvement of the PL)FA content in the host organism through metabolic engineering, e.g. through over-expression of fatty acid synthases, over-expression of enzymes involved in fatty acid desaturation, over-expression or deletion of regulatory proteins, over-expression of acyltransferases and/or lipases, or expression of corresponding heterologous enzymes.
  • the present invention relates to a method for the production of polyunsaturated fatty acid (PL)FA) in a fungal cell comprising at least two desaturases, said method comprises increasing the in vivo desaturase efficiency in said cell.
  • PL polyunsaturated fatty acid
  • One embodiment discloses that particularly over-expression of MCRl and/or CYB5 increases the in vivo desaturase efficiency.
  • Another aspect present invention relates to fungal cells containing the genetic engineering described in the present invention that yields EPA and ARA in the ratio of at least 1 : 1, preferably at least 2: 1, most preferably 2.6: 1. Such high ratios are important for optimising the commercial potential of large scale PL)FA production.
  • the invention also relates to improvement of the total fatty acid content in the host organism through modification of transcriptional regulation of structural genes involved in phospholipid and fatty acid synthesis.
  • the present invention discloses new approaches for increasing the fatty acid content in particular fungal cells.
  • such an increase in the fatty acid content is achieved by over-expression of at least one gene selected from the group consisting of INO2 and INO4.
  • INO2 and INO4 are selected from the group consisting of INO2 and INO4.
  • OPIl deletion of the gene OPIl.
  • the present inventors have developed improved methods for producing polyunsaturated fatty acids by metabolic engineering of fungal host cells expressing heterologous pathways to GLA, ARA and EPA.
  • production of ARA in S. cerevisiae can be accomplished by simultaneous expression of heterologous genes encoding proteins with the following activities: delta-12 desaturase, delta-6 desaturase, delta-6 elongase and delta-5 desaturase (figure 1).
  • production of ARA can be achieved by simultaneous heterologous expression of genes encoding delta-12 desaturase, delta-9 elongase, delta-8 desaturase and delta-5 desaturase (figure 2).
  • the yield of ARA can be improved by additionally expressing a heterologous delta-9 desaturase and/or applying various metabolic engineering strategies (PCT/DK2005/000372 and US-2006-0051847-A1).
  • production of EPA in S. cerevisiae can be achieved by following the same strategies as described above for ARA, and additionally expressing a heterologous gene encoding an omega-3 desaturase (figure 3 and 4).
  • the present disclosure describes metabolic engineering strategies that further increases the yield of ARA, EPA, and/or intermediates in the pathway to ARA and EPA in a genetically modified fungal host cell, in particular S. cerevisiae, and demonstrates that metabolic engineering can be used to enable commercial production of polyunsaturated fatty acids in S. cerevisiae.
  • the prior art approaches to produce ARA, EPA, and/or intermediates in the pathway to ARA and EPA in fungal cells are comprehended in the present term "expression of a heterologous pathway in a fungal cell".
  • This application relates to further genetic modifications into fungal cells that surprisingly and substantially alter the fatty acid composition and increase the yield of GLA, ARA and EPA in the recombinant yeast comprising both prior art disclosed and potential novel heterologous pathways.
  • the present invention relates to a method for the production of polyunsaturated fatty acid (PL)FA) in a fungal cell comprising at least two desaturases, said method comprises increasing the in vivo desaturase efficiency in said cell.
  • the in vivo desaturation efficiency is related to the desaturation complex within the cells, and by increasing the effect of the desaturation complex higher yields of PL)FAs may be achieved as disclosed in the examples below. This may for example be achieved by either over-expression or heterologous expression of at least one of the genes selected from the group consisting of MCRl and CYB5.
  • the at least two desaturases are selected from the group consisting of delta-9 desaturase, delta-6 desaturase, delta-12 desaturase, delta-5 desaturase, omega-3 desaturase, delta-4 desaturase and delta-8- desaturase.
  • the present invention also relates to a method for producing polyunsaturated fatty acids comprising expression of a heterologous pathway in a fungal cell, wherein the heterologous expression further comprises over-expression of at least one of the genes selected from the group consisting of FAS2, LROl, SPO14, INO2, INO4, MCRl and CYB5.
  • the present inventors here presents a method for the production of polyunsaturated fatty acid (PL)FA) in a fungal cell comprising at least two desaturases, said method comprising increasing the fatty acid content in said cell.
  • PL polyunsaturated fatty acid
  • Increasing the fatty acid content in cells may be achieved by over- expression of the at least one gene selected from the group consisting of INO2 and INO4.
  • the skilled artisan would be able to obtain such over expression both by increasing the endogenous expression of INO2 and INO4 by tools available in the art and by e.g. heterologous expression of the genes.
  • the present invention relates to a method for producing polyunsaturated fatty acids comprising expression of a heterologous pathway in a fungal cell, wherein the heterologous expression further comprises heterologous expression of at least one of the nucleotide sequences selected from the group consisting of nucleotide sequences encoding cytochrome b5, cytochrome b5 reductase, FAS alpha subunit, FAS beta subunit and FAS (both subunits).
  • the present invention relates to a method for producing polyunsaturated fatty acids comprising expression of a heterologous pathway in a fungal cell, wherein the heterologous expression further comprises deletion of OPIl. Deletion of OPIl also generates metabolic engineered cells that have increased fatty acid content.
  • polyunsaturated fatty acid relates to a long hydrocarbon chain composed of 18 or more carbon atoms having at least 2 double bonds and a terminal carboxylate group.
  • a fatty acid may be esterified to form triglycerides and/or phospholipids as well as sphingolipids.
  • the present invention also relates to such esterified products.
  • the fatty acid product of the present invention can be free fatty acids.
  • Free fatty acids have a free carboxyl group, are not chemically connected to any other compound including triacylglycerides, phospholipids or sphingolipids, and can be present freely in any compartment of the cell.
  • these intermediate products could be oleic acid, linoleic acid, gamma-linolenic acid, dihomo-gamma-linoleic acid, eicosadienoic acid, particularly, eicosadienoic acid with double bonds in position 11 and 14, eicosatrienoic acid, particularly, eicosatrienoic acid with double bonds in position 11, 14, and 17, arachidonic acid, alpha-linoleic acid, stearidonic acid, eicosatetraenoic acid, particularly eicosatetraenoic acid with double bonds in position 8, 11, 14 and 17, eicosapentaenoic acid, particularly eicosapentaenoic acid with double bonds in position 5, 8, 11, 14 and 17.
  • the polyunsaturated fatty acid comprises at least 3 double bonds, such as 3 double bonds, such as 4 double bonds, such as 5 double bonds or such as
  • polyunsaturated fatty acid is selected from the group consisting of gamma-linolenic acid, arachidonic acid and eicosapentaenoic acid.
  • said polyunsaturated fatty acid is gamma-linolenic acid.
  • said polyunsaturated fatty acid is arachidonic acid.
  • said polyunsaturated fatty acid is eicosapentaenoic acid.
  • An oxygen-requiring pathway shall mean that at least one of the enzymes in the pathway requires oxygen to function.
  • the expression of nucleic acids coding for desaturase usually leads to a pathway that requires oxygen for activity as desaturases usually are oxygen-requiring enzymes.
  • genes natively present in the host organism including genes in the pathway to fatty acids, including the pathway to any desirable PL)FA in a cell of the present invention, are in the present context termed endogenous genes.
  • the technology described within the present invention relates to genetically engineered fungal host cells that produce PL)FAs through expression of a heterologous pathway, and that have been further genetically engineered to produce increased amounts of PL)FAs.
  • any heterologous gene with a specific function mentioned in the present application one or more endogenous genes with the same or similar function may be present in the chosen fungal host cell.
  • the chosen fungal cell in question may contain endogenous genes that have satisfactory expression for PL)FA production at commercially viable yields without the need for heterologous expression of a PL)FA pathway.
  • the technology described within the present invention also relates to such fungal cells, which contain an endogenous pathway for PL)FA production.
  • the genetically transformed cells particularly harbour a heterologous pathway from stearic acid to PL)FAs by expression of the following heterologous enzymes delta-9 desaturase, delta-12 desaturase, delta-9 elongase, delta-8 desaturase omega-3 desaturase, delta-6 desaturase, delta-6 elongase, delta-5 desaturase, or subsets hereof.
  • heterologous genes in the pathway to PL)FAs can be chosen among a wide range of described and published sequences, or can be isolated from any living organism, including fungi, plants, animals, algae and marine protists, amoeba and bacteria, that harbours pathways to oleic acid, linoleic acid, alpha-linolenic, gamma-linoleic acids, dihomo-gamma-linolenic acid, arachidonic acid, stearidonic acid, eicosatetraenoic acid or EPA.
  • the present invention utilizes the simultaneous heterologous expression of genes encoding delta-12 desaturase, delta-6 desaturase, delta-6 elongase and delta-5 desaturase in a microorganism, which leads to the production of PL)FAs, and in particular production of arachidonic acid.
  • expression it is meant the production of a functional polypeptide through the transcription of a nucleic acid segment into mRNA and translation of the mRNA into a protein.
  • heterologous expression it is generally meant that a nucleic acid, not naturally present in the host genome, is present in the host cell and is operably linked to promoter and terminator nucleic acid sequences in a way so it is expressed in the host cell.
  • heterologous expression further relates to the presence of a nucleic acid with a similar function to a naturally present nucleic acid, wherein the expression of said heterologous nucleic acid product changes the fatty acid composition.
  • yeast of a fungal delta-9 desaturase with different substrate specificity than the native yeast delta-9 desaturase changes the fatty acid composition of yeast.
  • Said nucleic acid may be contained on an extra chromosomal nucleic acid construct or may be integrated in the host genome. Methods for isolation of nucleic acids for heterologous expression and preferred embodiments of heterologous expression are further described in details below.
  • heterologous expression of a pathway is meant that several genes are expressed heterologously, whose gene products constitute steps in a pathway, not naturally present in the host.
  • the present invention relates to a method for producing polyunsaturated fatty acids comprising expression of a heterologous pathway in a fungal cell, wherein the heterologous expression further comprises heterologous expression of nucleotide sequences encoding at least one of the enzymes selected from the group consisting cytochrome b5, cytochrome b5 reductase, FAS alpha subunit, FAS beta subunit and FAS (both subunits).
  • heterologous genes allows not only the production of omega-6 fatty acids, but also the production of omega-3 fatty acids, simultaneously or not, in host cells that endogenously only produce fatty acids of up to 18 carbon atoms of length with up to one double bond.
  • the expression of said heterologous genes allows the production of eicosapentaenoic acid in said host cells.
  • heterologous genes generally improves the production of eicosapentaenoic acid and/or one or more of its intermediate precursors, including arachidonic acid, in a host cell.
  • a general advantage of this method is that it allows the use of non-fatty acid substrates, such as sugars.
  • fatty acid-containing substrates such as oils derived from, for example, plants, animals or microorganisms, can also be used.
  • heterologous pathways described herein generally improves the production of polyunsaturated fatty acids such as arachidonic acid in a host cell and can also lead to improved production of one or more of its intermediate precursors.
  • a general advantage of this method is that it allows the use of non- fatty acid substrates, such as sugars.
  • fatty acid-containing substrates such as oils derived from, for example, plants, animals or microorganisms, can also be used.
  • the fermentation substrate for the production of PL)FAs maybe any complex medium or defined medium e.g. containing sugars, such as glucose, mannose, fructose, sucrose, galactose, lactose, erythrose, threose, ribose, glyceraldehyde, dihydroxyacetone, ribulose, cellobiose, starch, glycogen, trehalose, maltose, maltotriose, xylose, arabinose, stachyose, raffinose, or non-fermentable carbon sources, such as ethanol, acetate, lactate, or glycerol, or oils, such as oils derived from plants, animals or microorganisms or fatty acids, such as butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, o
  • sugars
  • the present invention relates to a method according to the present application, wherein the fungal cell is grown on a non fatty acid substrate.
  • a non-fatty acid substrate is the exclusive carbon source.
  • the fungal cell is cultivated in a myo-inositol deficient medium.
  • a "non-fatty acid substrate” relates to any substrate, but not a fatty acid, with two or more carbon atoms, such as but not limited to sugars, such as glucose, mannose, fructose, sucrose, galactose, lactose, erythrose, threose, ribose, glyceraldehyde, dihydroxyacetone, ribulose, cellobiose, starch, glycogen, trehalose, maltose, maltotriose, xylose, arabinose, stachyose, raffinose, or non-fermentable carbon source, such as but not limited to ethanol, lactate, acetate and glycerol.
  • sugars such as glucose, mannose, fructose, sucrose, galactose, lactose, erythrose, threose, ribose, glyceraldehyde, dihydroxyacetone, ribulose
  • a living organism needs a supply of many or all of the macroelements such as carbon, sulphur, phosphor, nitrogen, oxygen or hydrogen.
  • An organism may grow on mixtures of different carbon sources, such as a fatty acid substrate and a non-fatty acid substrate. If a substrate or substrates is referred to an exclusive carbon or exclusive carbon sources, it is only that substrate or those substrates that is supplied to the organism as a carbon source. This shall not exclude other macroelements or other nutritional requirements, such as requirements for example for trace elements and vitamins. For example, if a non- fatty acid substrate is exclusively supplied as a carbon source. This means, it is only that non-fatty acid that is supplied as a carbon source without supplying another carbon source.
  • An advantage of using for example a sugar, such as glucose or sucrose, or glycerol exclusively as a carbon source can be at least one of the following reasons: lower raw material cost, easier control of the fermentation process, better and constant quality of the produced oil.
  • said non-fatty acid substrate is the exclusive carbon source.
  • a preferred embodiment of the present invention relates to a method according to the present invention, wherein said host cell, such as Saccharomyces cerevisiae, is cultivated in a myo-inositol deficient medium.
  • said host cell such as Saccharomyces cerevisiae
  • PL)FA containing yeast may be produced using well-known fermentation processes, such as batch, fed-batch or chemostat fermentation.
  • a fed- batch or chemostat fermentation process is preferred over a batch process, as the high initial glucose concentration in a batch process may cause overflow metabolism and alcoholic fermentation and thereby a low biomass yield.
  • Any process that results in high biomass concentrations is suitable for production of PUFA-containing yeast, since the PUFA-containing lipid is incorporated in the membranes and lipid bodies of the yeast.
  • Fed-batch processes are often used for production of baker's yeast and may be suitable for production of PL)FA containing yeast.
  • a growth substrate is continuously fed into the reactor at a rate which optimally allows full respiratory growth of the yeast cells.
  • the feeding rate can be optimized to allow the cells to grow at a maximal or a high specific growth rate without significant accumulation of by-products such as ethanol, glycerol, acetate, succinate, lactate, or pyruvate.
  • an exponential feed-profile is applied in order to achieve high biomass productivity.
  • the feeding rate can be pre-set as a linear or exponential rate, or a combination of a linear and exponential rate.
  • the feeding rate may be controlled on-line by one or several parameters, such as the concentration of ethanol, glycerol, acetate, succinate, lactate or pyruvate in the fermentation broth, the concentration and rate of formation of biomass in the fermentation broth, the oxygen tension in the fermentation broth, pH of the fermentation broth or the rate of acid or base addition for pH control.
  • the feeding rate may be controlled by on-line measurements of one or more of these or other parameters, using the on-line measurements for feed-back control of the feeding rate.
  • the feed-back control may be in the form of an algorithm that takes into account the predicted response of the culture to increased feeding rates.
  • a chemostat process may also suitable for the production of PL)FA containing yeast.
  • a chemostat process the growth substrate is continuously fed into the reactor, while the working volume of the reactor is kept constant by continuously removing culture broth from the reactor at the same rate as the feeding rate.
  • the culture thereby reaches a steady state, where the concentrations of biomass, substrates and products in the reactor are constant over time.
  • Chemostat processes have the advantages that the process can keep running for a long time (up to several hundred hours) at a high production rate and that the product can be harvested continuously.
  • chemostats comprise the advantage that the conditions in the reactor can be precisely controlled and are unchanging over time, such that optimal conditions for biomass and PL)FA can be maintained without making use of advanced on-line feed control.
  • any fermentation process for production of yeast biomass it is important to maintain a sufficiently high aeration rate and oxygen mass transfer to allow unrestricted respiration and fully aerobic metabolism.
  • sufficient oxygen supply is important for the production of PL)FAs, since oxygen is a direct substrate in the fatty acid desaturation reactions.
  • Suitably high aeration rates may be achieved for example by sparging the fermentor with air or pure oxygen or a mix of air and pure oxygen at suitable flow rates.
  • Mass transfer may for example be improved by altering the shape, size and number of impellers or by using different size, shape and designs of the fermentation reactor. For example, an airlift reactor design may be used.
  • the ratio of carbon to nitrogen in the growth medium of fed-batch and continuous fermentation may be an important parameter for production of fatty acids. Normally, a higher yield of fatty acids on biomass is achieved when the cells are limited for nitrogen than during carbon-limitation. Usually a molar C/N ratio of less than 6 may result in clear carbon-limitation, while a molar C/N ration of more than 40 may result in clear nitrogen-limitation. Generally, a nitrogen limited medium composition is desired for production of fatty acids. However, it is also desired not to have a high residual concentration of the carbon source, especially glucose, in the reactor. High residual glucose concentration may result in fermentative metabolism and formation of by-products such as ethanol.
  • a molar C/N ratio in the medium in between 6 and 40, preferably between 10 and 25, such as a C/N ratio of around 15, is presently preferred.
  • the presently preferred nitrogen sources for production of polyunsaturated fatty acids are easily assimilated sources such as ammonium salts, while the presently preferred carbon source is glucose.
  • Other, less easily assimilated nitrogen sources such as urea or amino acids may also be beneficial for production of polyunsaturated fatty acids, since their uptake rate is lower and their use as nitrogen sources therefore may result in a similar metabolism as during nitrogen limitation by an easily assimilated nitrogen source.
  • the presently preferred carbon-source for production of polyunsaturated fatty acids is glucose, as yeasts grow at a high specific growth rate and with a high biomass yield using this carbon source.
  • the carbon source may also be other sugars, such as sugars selected from the group consisting of mannose, fructose, sucrose, galactose, lactose, erythrose, threose, ribose, glyceraldehyde, dihydroxyacetone, ribulose, cellobiose, starch, glycogen, trehalose, maltose, maltotriose, xylose, arabinose, stachyose, raffinose, and non-fermentable carbon sources, such as but not limited to ethanol, lactate, acetate and glycerol.
  • Non- fermentable carbon sources have the advantage that they may be easily channelled into the fatty acid bio-synthetic pathway in the form of acetyl-CoA and malonyl-CoA, which are the precursors for fatty acid synthesis.
  • the biomass yield on non-fermentable carbon-sources is lower than on sugars.
  • the fermentation process may also utilize several carbon-sources.
  • the fatty acids are preferably in the form of a vegetable oil and are supplied in addition to the carbon source or carbon sources used for growth. Fatty acids from the supplied oil can be taken up and used as substrates in the polyunsaturated fatty acid biosynthetic pathway, resulting in increased polyunsaturated fatty acid percentage in total fatty acid. Furthermore, oil supplementation is likely to increase the total content of fatty acids in the biomass. Fatty acids from the oil supplementation can be taken up by the cell and be incorporated into the biomass, mainly in the form of phospholipids or triacylglycerols. Presently preferred supplementation oils include Sunflower oil, Safflower oil, Peanut oil, Soybean oil, Olive oil, Cottonseed oil and Canola oil.
  • host cell relates to host cells selected from the group consisting of micro-organisms, animals, fungi, bacteria, invertea (insects), plants or protozoa.
  • host cells selected from the group consisting of micro-organisms, animals, fungi, bacteria, invertea (insects), plants or protozoa.
  • microscopic organisms including bacteria, viruses, unicellular algae, protozoans and microscopic fungi including yeast.
  • the host cell is a non-plant host cell.
  • the microorganism may be a fungus, and more specifically a filamentous fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belong to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis K. marxianus var. marxianus, K.
  • Aspergillus e.g. A. niger, A. awamori, A. oryzae, A. nidulans
  • a yeast belong to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri,
  • thermotolerans a yeast belonging to the genus Candida, e.g. C. utilis, C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, or other yeast genus, e.g. Cryptococcus, Debaromyces, Hansenula, Pichia, Yarrowia,
  • bacteria a non-exhaustive list of suitable bacteria is given as follows: a species belonging to Bacillus, a species belonging to the genus Escherichia, a species belonging to the genus Lactobacillus, a species belonging to the genus Corynebacterium, a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas, etc., that are well known in the art.
  • the preferred microorganisms of the invention may be S. cerevisiae, A. niger, Escherichia coli or Bacillus subtilis.
  • the constructed and engineered microorganism can be cultivated using commonly known processes, including chemostat, batch, fed-batch cultivations, etc.
  • the present invention relates to a method for producing a polyunsaturated fatty acid comprising combining heterologous expression of genes encoding various desaturases and elongases in a host cell as described herein, wherein said host cell is selected from the group consisting of plants, micro-organisms, animals, fungi, bacteria, invertea (insects) or protozoa.
  • the present invention relates to a method for producing a polyunsaturated fatty acid comprising combining heterologous expression of genes encoding various desaturases and elongases in a host cell as described herein, wherein said host cell is a fungus, and preferably, wherein said fungus is a filamentous fungus or a yeast.
  • said yeast is selected from the group of the genus Saccharomyces, Kluyveromyces, Candida, Pichia, Cryptococcus, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces Schizosaccharomyces, Lipomyces.
  • yeast is Saccharomyces cerevisiae.
  • said filamentous fungus is selected from the group of the genus Aspergillus, Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella or Trichoderma.
  • said Aspergillus is selected from the species Aspergillus niger, Aspergillus awamori, Aspergillus oryzae or Aspergillus nidulans. And in a presently most preferred embodiment, said host is Aspergillus niger.
  • the present invention relates to a method for producing a polyunsaturated fatty acid comprising combining heterologous expression of genes encoding various desaturases and elongases in a host cell as described herein, wherein said host is a bacterium.
  • said bacterium is selected from the group of Bacillus, Escherichia, Lactobacillus, Corynebacterium, Acetobacter, Acinetobacter, or Pseudomonas
  • said bacterium is Bacillus subtilis.
  • said one host cell is Escherichia coli.
  • the present invention relates to a genetically modified Saccharomyces cerevisiae capable of producing polyunsaturated fatty acids with four or more double bonds when grown on a non- fatty acid substrate.
  • the present invention relates to a genetically modified Saccharomyces cerevisiae according to the invention, wherein said Saccharomyces cerevisiae is capable of producing polyunsaturated fatty acids with four or more double bonds when grown on a non-fatty acid substrate as the exclusive carbon source.
  • Saccharomyces cerevisiae is capable of producing polyunsaturated fatty acids with four or more double bonds when grown on a non-fatty acid substrate as the exclusive carbon source.
  • Metabolic engineering is a term that is used for rational modification of the metabolism of cells with the objective of altering metabolic fluxes, often with the aim to increase yield of a specific product.
  • the concept of metabolic engineering includes analysis of the metabolism at several levels as well as development of hypotheses that can be tested experimentally.
  • Microbial metabolism and in particular the regulation of microbial metabolism is far from being fully understood at present, even in a well-characterized microorganism such as S. cerevisiae. In particular, this applies to alteration of the magnitude of metabolic fluxes.
  • a certain change e.g. deletion of a native gene or expression of a heterologous gene, has a positive or negative effect on the metabolic flux through a specific pathway, and thereby on the resulting yield of the desired product.
  • fatty acids are desaturated by NADH and 02 -dependent membrane-bound multiprotein enzyme complexes (figure 5). These complexes consist of three necessary protein components; a desaturase enzyme, an electron donor such as cytochrome b5 or ferredoxin, and an NADH (or NADPH) -dependent reductase enzyme.
  • the total desaturation reaction (exemplified by desaturation of Stearoyl-CoA (18:0) to form oleoyl-CoA (18: 1)) can be written as shown below:
  • the introduction of a double bond in the fatty acid chain thus involves oxidation of NADH and reduction of molecular oxygen to form NAD+ one molecule of water.
  • the formation of the double bond involves an intricate system of electron transport through the desaturation complex: electrons are transferred from NADH to the FAD moiety of cytochrome b5 reductase, and further to the heme iron atom of 2 molecules of cytochrome b5 which is then reduced to its ferrous (Fe2+) form. Electrons from the ferrous cytochrome b5 molecules are transported to the non- heme iron atoms of the desaturase enzyme which then are reduced to their ferrous forms. When in its ferrous form, the desaturase binds and activates molecular oxygen and creates a double bond in the fatty acid substrate, with the simultaneous formation of H2O.
  • Desaturases binds and activates molecular oxygen and creates a double bond in the fatty acid substrate, with the simultaneous formation of H2O.
  • Fatty acid desaturase enzymes exist in many varieties.
  • the biochemical best characterized desaturase is the soluble delta-9 desaturases from castor (Ricinus communis).
  • This enzyme has been purified and subjected to resonance Raman spectroscopy analysis (Fox et al. 1994) which allowed the authors to identify the presence of an oxo-bridged diiron cluster in the enzyme and propose a structure for the diiron site along with a molecular mechanism of action for oxygen activation.
  • the two iron atoms are bound at the active site by several histidine and glutamate residues and are in the ferric form connected by an oxo-bridge.
  • the enzyme is in the resting state when the iron atoms are in the ferric form.
  • membrane-associated desaturases are membrane-associated and this makes purification and biochemical characterization difficult.
  • membrane-bound desaturases are iron-chelating enzymes that have an iron-driven mechanism of action similar to the soluble fatty acid desaturases.
  • the best characterized membrane-associated desaturase is the rat liver delta-9 desaturase. This desaturase exhibits an absorption spectrum that is characteristic of an oxo-bridged diiron cluster.
  • the present invention relates to a method for the production of PL)FA in a fungal cell comprising at least two desaturases selected from the group consisting of delta-9 desaturase, delta-6 desaturase, delta-12 desaturase, delta-5 desaturase, omega-3 desaturase, delta-4 desaturase and delta-8- desaturase.
  • the desaturases are delta-9 desaturase and delta-12 desaturase.
  • the desaturases is delta-9 desaturase, delta-12 desaturase and delta-6 desaturase.
  • the desaturases are delta-9 desaturase, delta-12 desaturase, delta-5 desaturase and delta-6 desaturase.
  • the desaturases are delta-9 desaturase, delta-12 desaturase, delta-5 desaturase, delta-6 desaturase and omega-3 desaturase.
  • Cytochrome b5 as electron donor in the desaturation complex Cytochrome b5 acts as the electron donor in most eukaryotic desaturation complexes.
  • the main exception to this rule is the plastid and chloroplast desaturases of higher plants, which typically utilize ferredoxin rather than cytochrome b5.
  • Cytochrome b5 binds the iron-carrying prosthetic group heme, which is a central component in the electron transport chain of the desaturation reaction.
  • the importance of cytochrome b5 in the desaturation complex is underscored by the fact that several desaturase enzymes contain a cytochrome b5 subdomain; in these enzymes the desaturase is thus physically linked to cytochrome b5.
  • the cytochrome b5 domain can either be located at the C- terminus or the N-terminus of the desaturase sequence.
  • the positioning of the domain at the C-terminus or N-terminus seems related to the substrate specificity of the enzymes in that the so called front-end desaturases, which create a double bond between the carboxylated carbon and a pre-existing double bond in the fatty acid, typically contain N-terminal cytochrome b5 domains, while other desaturases contain no cytochrome b5 domain or a C-terminal cytochrome b5 domain.
  • the desaturases that do not contain a cytochrome b5 domain utilize the free form of cytochrome b5; this interaction seems to be rather universal since desaturases that are expressed in heterologous hosts usually can function well together with the cytochrome b5 and cytochrome b5 reductase of the host organism. While it has been shown for several cytochrome b5-containing desaturases that the cytochrome b5 domain is essential for the function of the enzyme (Mitchell et al. 1995, Sayanova et al. 1999), it is not clear to what extent the cytochrome b5- containing desaturases also utilize the free form of cytochrome b5.
  • cytochrome b5 activity is either accessed in the form of a separately encoded cytochrome b5, or incorporated in the desaturase itself in the form of a cytochrome b5 domain.
  • delta-12 desaturases and omega-3 desaturases typically do not contain a cytochrome b5 domain
  • front-end desaturases including delta- 9 desaturases, delta-6 desaturase, delta-5 desaturase and delta-8 desaturase
  • heme-binding cytochrome b5 domain either at the N-terminal or the C- terminal part of the protein.
  • Cytochrome b5 reductase is as well as cytochrome b5 an integral part of the desaturation complex, however, in contrast to cytochrome b5, no desaturases have yet been isolated that contain a cytochrome b5 reductase domain. It is therefore likely that a separate cytochrome b5 reductase is essential for any desaturation reaction. In vivo desaturase efficiency
  • the distribution of fat between PL)FA and saturated fatty acids does not only depend on the amount of desaturase present in the cell. It also depends on how efficient the desaturases work e.g. a certain level of PL)FA can be obtained by either increasing the amount of desaturase or by increasing the efficiency of the desaturases present.
  • In vivo desaturase efficiency is a measure of how good a certain amount of desaturase works. The higher efficiency the more PL)FA is created with the same amount of desaturase. In vivo desaturase efficiency can be estimated by relating the actual in vivo desaturation activity to the concentration of desaturase in the cell.
  • Actual in vivo desaturation activity can be measured by analyzing the fatty acid content and composition of the cell, since the cellular content of unsaturated fatty acids directly reflects the actual in vivo desaturation activity.
  • the desaturation activity can be expressed as mg unsaturated fatty acids per mg cell dry-weight.
  • the concentration of a specific desaturase in the cell can be measured indirectly by performing Northern blot analysis or real-time RT PCR. By these methods, it is possible to estimate the mRNA copy number of the desaturase per mg cell dry- weight.
  • the desaturase protein concentration can be measured directly by Western blot.
  • This method is more cumbersome, especially since antibodies for PL)FA desaturases are generally not commercially available.
  • the preferred method of measuring the level of desaturases in the cell is therefore to use real-time RT PCR to estimate the mRNA copy number per mg cell dry-weight for each desaturase present in the cell.
  • the two strains express the same set of desaturases, and each of these desaturases are present in the same gene-copy number, and furthermore are expressed using the same promoter in both strains, it can be considered that the two strains contain an equal number of desaturase mRNA copy number and also the same desaturase protein concentration.
  • the efficiency can be used to assess whether all desaturases are actually actively performing fatty acid desaturation.
  • the present invention discloses a method wherein the increased in vivo desaturase efficiency can be measured by the following steps:
  • -identifying a fungal cell population as having an increased in vivo desaturase efficiency if the second cell population compared to the first cell population show an increase in the PL)FA fraction (% of PL)FA of total fatty acid) by at least 0,5%, such as at least 1%, e.g. at least 1.5%, such as at least 5%, e.g. at least 25%, such as at least 40%, e.g. at least 50%.
  • fungal cell population is to be understood as one or more fungal cells.
  • a high total rate of fatty acid desaturation in the cell For production of polyunsaturated fatty acids at high yields, it is desirable to have a high total rate of fatty acid desaturation in the cell. To achieve this, it is of importance to maintain a high level of the relevant desaturase enzymes, which typically may be achieved by increasing the expression of the genes encoding these relevant desaturases, for example by introducing multiple gene copies and/or by applying strong promoters.
  • the molar concentration of desaturase enzymes increases above the molar concentration of available cytochrome b5 and cytochrome b5 reductase in the cell, this results in a situation where not all desaturase molecules can find partners to form a desaturation complex and where excess desaturase enzymes will accumulate. Therefore, in order to increase the in vivo desaturation efficiency, the level of cytochrome b5 and cytochrome b5 reductase of the desaturation complex needs to be increased.
  • cytochrome b5 reductase Overexpression of cytochrome b5 reductase in a cell that has an increased level of cytochrome b5 increases the efficiency of desaturation even further.
  • both cytochrome b5 and cytochrome b5 reductase are overexpressed in a PUFA-producing cell.
  • Cytochrome b5 is encoded by CYB5 and Cytochrome b5 reductase is encoded by MCRl.
  • expression of a heterologous delta-12 desaturase or omega-3 desaturase in S. cerevisiae requires the activity of the cytochrome b5 encoded by CYB5, since these enzymes do not contain cytochrome b5 domains.
  • delta-9 desaturase and the heterologous fatty acid desaturases in the pathway to ARA and EPA require the activity of a separately encoded cytochrome b5 reductase.
  • cytochrome b5 and cytochrome b5 reductase are not sufficiently active to achieve a high flux through the desaturation pathway. Therefore, the genes encoding these proteins can be overexpressed using a strong constitutive promoter such as, e.g., the ADHl promoter, the TDH3 promoter, the TPIl promoter or the HXT7 promoter, or a strong inducible promoter such as the GALl promoter or the GALlO promoter.
  • a strong constitutive promoter such as, e.g., the ADHl promoter, the TDH3 promoter, the TPIl promoter or the HXT7 promoter, or a strong inducible promoter such as the GALl promoter or the GALlO promoter.
  • heterologous desaturases do not function as efficiently in conjunction with the endogenous cytochrome b5 and cytochrome b5 reductase as they would in conjunction with the corresponding proteins from the donor organism.
  • the delta-9 desaturase, delta-12 desaturase, delta-6 desaturase and delta-5 desaturase from Mortierella alpina are expressed in a fungal host cell, it may be beneficial to also express the cytochrome b5 and the cytochrome b5 reductase from M. alpina.
  • cytochrome b5 and cytochrome b5 reductase A non-exhaustive list of heterologous genes encoding cytochrome b5 and cytochrome b5 reductase that could be beneficial for GLA, ARA and EPA production in a fungal host cell, particularly S. cerevisiae, is given in Table 1. Table 1. Cytochrome b5 and cytochrome b5 reductase genes useful for increasing GLA, ARA and EPA yield in a recombinant fungal cell.
  • sequences encoding cytochrome b5 and cytochrome b5 reductase can be obtained from other sources.
  • Useful cytochrome b5 and cytochrome b5 reductase sequences can be derived from any source, for example, isolated from organisms containing highly unsaturated fatty acids.
  • Genes encoding cytochrome b5 and cytochrome b5 reductase can also be identified in available sequence data using bioinformatics.
  • the sequences can be isolated and expressed in the fungal host cell using conventional DNA amplification and cloning techniques, or they can be chemically synthesized de novo.
  • the sequences can be codon-optimized for expression in the host cell.
  • S. cerevisiae MCRl (SEQ ID NO: 19) is over-expressed in a S. cerevisiae cell expressing a heterologous pathway to polyunsaturated fatty acids such as gamma-linolenic acid, arachidonic acid and/or eicosapentaenoic acid.
  • MCRl overexpression results in increased arachidonic acid content to more than 4% of the total fatty acid.
  • S. cerevisiae CYB5 (SEQ ID NO: 18) is over- expressed in a S. cerevisiae cell expressing a heterologous pathway to polyunsaturated fatty acids such as gamma-linolenic acid, arachidonic acid and/or eicosapentaenoic acid.
  • CYB5 overexpression results in increased gamma-linolenic acid content to more than 13% of the total fatty acid.
  • Cytochrome b5 is involved in the sterol and lipid biosynthesis pathways and is required in fatty acid desaturation. It catalyses the reaction from reduced cytochrome b5 and Fe3+ to oxidized cytochrome b5 and Fe2+ ( Figure 5) More details can be found above under fatty acid desaturation..
  • MCRl is defined as a gene encoding cytochrome b5 reductase (EC. 1.6.2.2). This enzyme catalyzes the reaction from NADH and oxidized cytochrome b5 to reduced cytochrome b5 and NAD+ ( Figure 5). More details can be found above under fatty acid desaturation.
  • Fatty acid synthase catalyzes the synthesis of long-chain saturated fatty acids from acetyl-CoA and malonyl-CoA.
  • FAS consist of a heterohexamer ( ⁇ 6 ⁇ 6) of two multifunctional ⁇ and ⁇ subunits that are encoded by FAS2 and FASl genes, respectively.
  • the FASl gene encodes four functional domains- acetyl transferase, enoyl reductase, dehydratase and malonyl/palmitoyl transferase.
  • the FAS2 gene product contains three domains- ⁇ -ketoacyl synthase, ⁇ -ketoacyl reductase and acyl carrier protein. S.
  • cerevisiae reduces the amount of C16 fatty acids and increases the amount of C18 fatty acids (Oura et al. 2006 Curr Genet 49: 393-402). It is therefore possible that expression of this gene in S. cerevisiae can increase the flux towards PUFA-production.
  • the organisms M. alpina and Aspergillus nidulans are also known to produce more C18 fatty acids than C16 fatty acids.
  • A. nidulans contains two functionally distinct FASs: one required for primary fatty acid metabolism and the other required for secondary metabolism.
  • the FAS involved in primary fatty acid metabolism is interesting for heterologus expression in S. cerevisiae since it is possible that the flux towards PUFA-production can be increased by expression of FAS genes from organisms producing more C18 than C16 fatty acids.
  • sequences encoding FAS ⁇ and ⁇ subunits can be obtained from other sources.
  • Useful FAS encoding sequences can be derived from any source, for example, isolated from organisms containing highly unsaturated fatty acids. Genes encoding FAS ⁇ and ⁇ subunits can also be identified in available sequence data using bioinformatics.
  • the sequences can be isolated and expressed in the fungal host cell using conventional DNA amplification and cloning techniques, or they can be chemically synthesized de novo. Furthermore, the sequences can be codon-optimized for expression in the fungal host.
  • FESl is defined as a gene encoding fatty acid synthase, beta unit
  • FES2 is defined as a gene encoding fatty acid synthase, alpha unit
  • Glycerol-3 phosphate G3P
  • DHAP di- hydroxy acetone phosphate
  • a fatty acid chain can be added to either G3P or DHAP by the action of the acyltransferases encoded by GATl and GAT2.
  • the products of these reactions are l-acyl-G3P and 1-acyl-DHAP, respectively.
  • 1-acyl-DHAP can be converted into 1- acyl-G3P by the enzyme encoded by AYRl.
  • a second fatty acid chain is then added to this carbon backbone to form the intermediate phosphatidic acid (PA), which serves as the precursor both for phospholipids via the CDP-DAG pathway and triacylglycerol (figure 13).
  • PA intermediate phosphatidic acid
  • Phosphatidic acid is de-phosphorylated to form diacylglycerol via the action of the phosphatases encoded by LPPl and DPPl.
  • Diacylglycerol in turn, can be further acylated to form triacylglycerol (TAG) or can enter the Kennedy pathway towards aminoglycerophospholipids.
  • the final acylation of diacylglycerol to form TAG can be carried out by several enzymes, including the enzymes encoded by DGAl, AREl, ARE2 and LROl. While Dgalp, Arelp and Are2p transfer fatty acid chains from acyl-CoA molecules to diacylglycerol, the acyltransferase encoded by LROl transfers the fatty acid from the sn-2 acyl group of the phospholipid phosphatidylcholine (PC). It has been shown (e.g. Domergue et al.
  • LROl is overexpressed in a fungal cell expressing a heterologous pathway to polyunsaturated fatty acids such as gamma-linolenic acid, arachidonic acid and/or eicosapentaenoic acid.
  • LROl is defined as a gene encoding a phospholipid- diacylglycerol acyltransferase (EC 2.3.1.158), which catalyzes the transfer of an acyl chain from phosphatidylcholine to diacylglycerol, resulting in formation of TAG.
  • Phospholipase D is defined as a gene encoding a phospholipid- diacylglycerol acyltransferase (EC 2.3.1.158), which catalyzes the transfer of an acyl chain from phosphatidylcholine to diacylglycerol, resulting in formation of TAG.
  • fatty acid elongases generally act on CoA-bound fatty acids, and the full desaturation and elongation pathway towards polyunsaturated fatty acids such as gamma-linolenic acid, arachidonic acid and eicosapentaenoic acid therefore involves fatty acid substrates in different lipid pools. It is contemplated that increased exchange of fatty acids between the phospholipid, TAG, and acyl-CoA pool will enable higher flux through the pathway to polyunsaturated fatty acids.
  • Phospholipase D encoded by SPO14
  • SPO14 will increase the flux to polyunsaturated fatty acids.
  • Phospholipase D hydrolyses PC into PA and choline, whereby the PA originating from PC can be converted into the storage compound TAG.
  • SPO14 is over-expressed in a in a fungal cell expressing a heterologous pathway to polyunsaturated fatty acids such as gamma-linolenic acid, arachidonic acid and/or eicosapentaenoic acid.
  • SPO14 is a gene encoding Phospholipase D (EC. 3.1.4.4) Regulation of lipid synthesis
  • INOl gene encodes inositol 1-phosphate synthase, which converts glucose 6-phosphate to inositol 1-phosphate.
  • the reaction is fundamental for synthesis of inositol containing phospholipids when inositol is absent from the growth medium.
  • INOl gene transcription is repressed when inositol is in the medium.
  • PI phosphatidylinositol
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • Transcriptional regulation includes the positive regulators of phospholipid biosynthesis INO2 and INO4 as well as the negative regulator OPIl.
  • the gene products exert their effect by controlling expression of transcription of the INOl and CHOI structural genes as well as INO2 and OPIl.
  • the heterodimeric complex consisting of Ino2 and Ino4 bind the inositol-choline responsive element (ICRE) to activate transcription of INOl, INO2 and OPIl.
  • ICRE inositol-choline responsive element
  • the structural gene ACCl encoding acetyl-CoA carboxylase which catalyzes the initial and rate-limiting step in fatty acid synthesis, is also under this coordinated transcriptional control.
  • transcript levels are repressed in ino2/ino4 mutant strains and derepressed in opil mutant strains.
  • Ino2 and Ino4 act as positive regulators of lipid synthesis it is possible that overexpression of the genes encoding these proteins will increase the total lipid synthesis in the strains. Likewise, Opil acts as a negative regulator of lipid synthesis and a deletion of the gene encoding Opil might result in a higher lipid yield of the mutated strain.
  • over-expression of INO2 and INO4 in addition to deletion of OPIl is likely to result in increased phospholid content in relation to the TAG content, which is likely to increase the content of polyunsaturated fatty acids such as gamma-linolenic acid, arachidonic acid and eicosapentaenoic acid in the cell.
  • polyunsaturated fatty acids such as gamma-linolenic acid, arachidonic acid and eicosapentaenoic acid in the cell.
  • INOl is a gene encoding inositol 1-phosphate synthase (EC. 5.5.1.4), which converts glucose 6-phosphate to inositol 1-phosphate.
  • CHOI is a gene encoding phosphatidylserine synthase (EC. 2.7.8.8).
  • INO2 is a gene encoding a protein which forms one part of a heterodimeric complex that binds to the inositol-choline responsive element.
  • INO4 is a gene encoding a protein which forms one part of a heterodimeric complex that binds to the inositol-choline responsive element.
  • OPIl is a gene encoding a negative regulator of transcription of the INOl, CHOI and INO2 genes.
  • the present inventors here presents a method for the production of polyunsaturated fatty acid (PL)FA) in a fungal cell comprising increasing the fatty acid content by over-expression of INO2 and/or INO4 and/or deletion of OPI.
  • PL polyunsaturated fatty acid
  • inositol-responsive genes have in common a repeated regulatory element with the consensus sequence 5'-CATGTGAAAT-3' in their promoter sequences, usually referred to as the inositol-sensitive upstream activation sequence (UASINO).
  • UASINO inositol-sensitive upstream activation sequence
  • inositol is the main effector in transcriptional regulation of the L)ASINO containing genes (Jesch et al. 2005)
  • another lipid precursor, choline has additional effects on the transcription of inositol-regulated genes and the L)ASINO sequence is therefore also sometimes referred to as the inositol/choline-responsive element (ICRE).
  • ICRE inositol/choline-responsive element
  • the transcriptional factors Ino2 and Ino4 are positive transcriptional activators, which together form a complex that binds to the L)ASINO element (Greenberg and Lopes 1996, Ambroziak and Henry 1994). Ino2 and Ino4 are therefore thought to control synchronized expression of structural enzymes in phospholipid biosynthesis. In addition to the positive regulators Ino2 and Ino4, many structural genes encoding phospholipid biosynthetic enzymes are regulated by the negative transcriptional regulator Opil. Even though Opil does not directly interact with the L)ASINO element, it has been shown that the L)ASINO element is required for repression by Opil (Bachhawat et al. 1995).
  • the Ino2-Ino4 complex binds to the promoter sequences of at least 32 genes in S. cerevisiae (Harbison et al. 2004), while it has been shown that 24 genes are more than 2-fold upregulated in an opil-deleted strain (Jesch et al. 2005). Among these genes, many are regulated both by the Ino2-Ino4 complex and Opil.
  • Genes that are regulated both by the Ino2-Ino4 complex and Opil and encode structural enzymes in phospholipid biosynthesis include CDSl, CHOI, INOl, ITRl, OPI3, PSDl and RMD12, while genes that are regulated by the Ino2-Ino4 complex but are unaffected by Opil include ADOl, CKIl, HNMl, OLEl and SAHl.
  • Ino2-Ino4 and Opil regulate transcription of structural genes in fatty acid synthesis.
  • One example of this is the transcriptional regulation of ACCl (Hasslacher et al. 1993), encoding acetyl-CoA carboxylase, which is one of the key structural enzymes in fatty acid synthesis.
  • Acetyl-CoA carboxylase catalyzes the caroxylation of acetyl-CoA to form malonyl-CoA, which is the elongation unit used by the fatty acid synthase for synthesis of fatty acids.
  • Accl is the only source of malonyl-CoA in the cell, and is therefore a key enzyme in fatty acid synthesis.
  • Accl is activated by citrate and inhibited in the presence of fatty acids.
  • Opil repress many structural genes in phospholipid biosynthesis it is anticipated that deleting Opil in a PUFA-producing yeast will result in increased lipid content and thereby increased PL)FA content.
  • Ino2 and Ino4 activate transcription of many genes in phospholipid biosynthesis, it is anticipated that overexpression of these transcription factors in a PUFA-producing yeast will result in increased lipid content and thereby increased PL)FA content.
  • both Opil and the Ino2-Ino4 complex regulate the transcription of many L)ASINO containing genes. Therefore it is preferred that Opil deletion and overexpression of INO2 and INO4 is combined in a PUFA-producing yeast.
  • S. cerevisiae Genetic modification of S. cerevisiae can be carried out using well-established methods. Thus, various methods are available for the person skilled in the art, allowing deletion and overexpression of native genes, expression of heterologous genes, as well as different types of genetic modifications such as point mutations and gene fusions. Methods for performing the above mentioned modifications in S. cerevisiae are described in, e.g., Erdeniz et al. (1997) Genome Res 7, 1174-83; Wach et al. (1994) Yeast 10, 1793-1808; Longtine et al. 1998 Yeast 14, 953-961.
  • heterologous gene such as a heterologous gene encoding cytochrome b5, cytochrome b5 reductase or FAS
  • expression from plasmids can be beneficial because it is a versatile system that can be used in different genetic backgrounds.
  • it is often possible to achieve high expression of the heterologous gene by using a high-copy, 2 ⁇ origin- based plasmid.
  • Expression by genomic integration on the other hand, often results in a more stable strain that does not easily loose the heterologous gene.
  • heterologous gene integrated into the genome
  • several copies of the heterologous gene can be integrated and strong yeast promoters can be used to control expression.
  • the position in the genome chosen for integration also influences the expression. Since some regions of the genome are less transcribed than others, it is anticipated that a heterologous gene can be differentially transcribed depending on its location in the genome.
  • heterologous sequences usually contain a different codon usage than S. cerevisiae sequences, which can impair efficient translation of the gene transcript. Optimization of the codon-usage in a heterologous gene can therefore improve expression of the gene.
  • Codon-optimization is done by employing the host-preferred codons, as determined from codons of the highest frequency in highly expressed proteins of the host of interest.
  • the codon-optimized sequence coding for a heterologous polypeptide can then be chemically synthesized in the form of oligonucleotides and be assembled by PCR-techniques.
  • Native yeast genes such as OPI
  • OPI can be deleted using established methods, for example gene deletion or gene disruption using a bi-partite gene targeting substrate Erdeniz et al. (1997) Genome Res 7, 1174-83.
  • reducing the transcription of the gene by, e.g. promoter replacement with a weaker promoter
  • introducing mutations that destroys or modifies the function of the gene product, or other methods of reducing or abolishing the activity of the gene product would give similar effects as deletion of the gene.
  • the heterologous pathway to polyunsaturated fatty acids can be expressed in S. cerevisiae using expression from vectors or integration into the genome.
  • the expression of a heterologous pathway to PL)FAs in S. cerevisiae further comprises expression of one or several heterologous genes encoding cytochrome b5, cytochrome b5 reductase and FAS by integration into the genome under the control of strong yeast promoters, such as, but not limited to, the ADHl promoter, the TDH3 promoter, the TPIl promoter or the HXT7 promoter.
  • strong yeast promoters such as, but not limited to, the ADHl promoter, the TDH3 promoter, the TPIl promoter or the HXT7 promoter.
  • the described additional expression of heterologous genes is anticipated to result in an increased content of EPA, such that the percentage of EPA in total fatty acid is at least 3%, for example 4% EPA of total fatty acid, 6% EPA of total fatty acid, 8% EPA of total fatty acid, 10% EPA of total fatty acid, 12% EPA of total fatty acid, 14% EPA of total fatty acid, or more.
  • the expression of a heterologous pathway to PL)FAs in S. cerevisiae further comprises over-expression of one or several native genes such as LROl, SPO14, INO2, INO4, CYB5 and MCRl by replacing the native promoter in the genome with a stronger promoter, such as, but not limited to, the ADHl promoter, the TDH3 promoter, the TPIl promoter or the HXT7 promoter.
  • native genes such as LROl, SPO14, INO2, INO4, CYB5 and MCRl by replacing the native promoter in the genome with a stronger promoter, such as, but not limited to, the ADHl promoter, the TDH3 promoter, the TPIl promoter or the HXT7 promoter.
  • Over-expression of one ore more of the described native genes increases the percentage of ARA in the recombinant S. cerevisiae to at least 4% of the total fatty acid. Furthermore, combining several over-expressions in a single S. cerevisiae strain is anticipated to increase the percentage of ARA even more, resulting in 6% ARA of total fatty acid, 8% ARA of total fatty acid, 10% ARA of total fatty acid, 12% ARA of total fatty acid, 14% ARA of total fatty acid, or more.
  • the described over-expression of LROl, SPO14, INO2, INO4, CYB5 and/or MCRl is anticipated to result in an increased content of EPA, such that the percentage of EPA in total fatty acid is at least 3%. Furthermore, combining over-expression of several of these genes is anticipated to result in even higher EPA content, such as 4% EPA of total fatty acid, 6% EPA of total fatty acid, 8% EPA of total fatty acid, 10% EPA of total fatty acid, 12% EPA of total fatty acid, 14% EPA of total fatty acid, or more.
  • Over-expression of one or more of the described native genes is also anticipated to increase the percentage of intermediates in the pathway to ARA and EPA.
  • over-expression of CYB5 results in increased content of gamma- linolenic acid to at least 13 % of the total fatty acid.
  • Combining CYB5 over- expression with additional over-expression of the described native or heterologous genes is anticipated to increase the GLA content even more, so that GLA constitutes 15% of total fatty acid, 18% of total fatty acid, 20% of total fatty acid, 25% of total fatty acid, or more.
  • the described modifications can increase the lipid content of the recombinant S. cerevisiae, and thereby increase the yield of PL)FA. It is therefore anticipated that the described heterologous expression of genes encoding cytochrome b5, cytochrome b5 reductase and/or FAS, over- expression of the native genes LROl, SPO14, INO2, INO4, CYB5 and/or MCRl, and/or deletion of OPIl, can increase the percentage of PL)FA in the yeast dry- weight to more than 0.2 %.
  • the present invention demonstrates how metabolic engineering can be used in S. cerevisiae to increase fatty acid content and fatty acid desaturation.
  • the invention may as well be used in yeast species in general e.g. such as yeast species selected from the group consisting of Y. lipolytica, K. lactis, S. pombe and S. kluyvery, or other yeasts such as yeasts selected from the genera Kluyveromyces, Candida, Pichia, Cryptococcus, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces Schizosaccharomyces, Lipomyces.
  • suitable endogenous target genes When incorporating the invention into other yeast types suitable endogenous target genes must be identified as well as a suitable constitutive promoter.
  • the constitutive promoter may be identified by looking into references such as (http://cbi.labri.fr/Genolevures, WO2006125000: WO2006055322)).
  • a regulatory promoter region from a gene with constitutive expression can be cloned and inserted in front of the endogenous target gene to replace the native promoter and mediate stable over-expression (promoter replacement is described in Example T).
  • promoter replacement is described in Example T).
  • Y. lipolytica common promoter regions to use for over- expression are TEF, GPD, GPDIN, GPM, FBA and YAT.
  • DNA substrates can be amplified by using the described PCR-based gene targeting method (Erdeniz et al, 1997). If the yeast host strain is devoid of markers, genetic modifications can be obtained by using an antibiotic-based technique to isolate and identify transformant cells. Both methods are based on conventional DNA amplification and cloning techniques.
  • the DNA substrates could be designed specifically for each individual host. The targeted integration of substrates is performed by means of homologous recombination and thus substrates will normally be designed to harbour homology to the endogenous DNA target.
  • auxotrophy-based gene targeting For auxotrophy-based gene-targeting, linear bi-partite PCR-generated gene- targeting substrates are preferentially used.
  • the marker gene is preferentially counter-selectable, allowing for pop-in pop-out recombination, for example the URA3 marker or the TRPl marker.
  • the counter-selectable URA3 marker that is used for gene targeting in S. cerevisiae can also be used for selection of positive transformants in Y. lipolytica.
  • Gene targeting in prototrophic yeast strains can be mediated by using antibiotic markers such as the kan r gene, which renders the yeast transformants resistant to the aminoglycoside antibiotic kanamycin G418.
  • antibiotic markers for identification and isolation of transformants are well known (C. Michels, Genetic Techniques for biological research. Wiley 2002).
  • Targeted integration allowing multiple inserts by re-using a marker system is possible using a system referred to as the loxP-kanMX-loxP system (G ⁇ ldener et al., 1996).
  • kan r is flanked by loxP repeats, which again is flanked by sequences homologous to the genetic locus to be disrupted. Positive transformants are isolated on G418-plates.
  • Transformants are furthermore transformed with a plasmid containing the Cre recombinase.
  • Cre-mediated recovery of the kan r marker is performed upon expression of the Cre recombinase (Wach et al., 1994).
  • the gene of interest is fused to a loxP-kanMX-loxP gene disruption cassette by PCR.
  • the present invention demonstrates methods to increase desaturation of fatty acids in baker's yeast, S. cerevisiae.
  • Desaturation requires the activity of a fatty acid desaturase, cytochrome b5 and cytochrome b5 reductase.
  • NADH-cytochrome b5 reductase is encoded by MCRl and cytochrome b5 is encoded by CYB5.
  • Recombinant S. cerevisiae cells expressing a heterologous pathway to polyunsaturated fatty acids show increased production of arachidonic acid and gamma-linolenic acid when over-expressing S. cerevisiae CYB5 and MCRl, respectively.
  • Y lipolytica contain both endogenous YALI0D12122g, which is similar to CYB5 and endogenous YALI0D11330g which is similar to MCRl (http://cbi.labh.fr/Genolevures).
  • endogenous proteins in Y. lipolytica is not specifically described, it is highly likely that they function as cytochrome b5 and cytochrome b5 reductase in desaturation of fatty acids as Y. lipolytica contain both 18:2 and 18:3 fatty acids.
  • Y. lipolytica can be increased by over-expression.
  • Endogenous YALI0D12122g and YALIODl 133Og can be put under the control of a constitutive promoter for over-expression.
  • heterologous CYB5 and MCRl DNA sequences can be isolated and expressed in Y. lipolytica.
  • CYB5 and MCRl donor sequences can be obtained from sources such as S. cerevisiae and M. alpina.
  • the genes can be amplified from DNA or cDNA and be introduced into the genome of Y. lipolytica together with a suitable promoter using said methods. Alternatively, they can be chemically synthesized de novo.
  • the sequences can be codon-optimized for optimal expression in the Y. lipolytica host cell.
  • Y. lipolytica will most likely display increased levels of desaturation if
  • YALI0D12122g and YALIODl 133Og are over-expressed by constitutive promoters as demonstrated in Saccharomyces.
  • Stable genetic integration of promoter sequences and/or heterologous cytochrome b5 and cytochrome b5 reductase can be conducted by the above mentioned auxotrophy-based method where the DNA substrate is cloned by PCR and inserted into the yeast strain by transformation and subsequent homologous recombination integration.
  • stable genetic integration can be conducted by the use of the above described antibiotic resistance-based method.
  • the present invention demonstrates methods to improve the PL)FA content in the host organism through metabolic engineering, e.g. through over-expression of fatty acid synthases, or over-expression of an acyltransferase.
  • LROl encodes for an acyltransferase that mediates the esterification of diacylglycerol using phosphatidylcholine as acyl donor.
  • a significant increase in triglyceride production is expected by over-expressing LROl.
  • FAS2 encodes the FAS alpha-subunit, and over-expressing FAS2 of the fatty acid synthase complex FAS1/FAS2 increases the content of C18 fatty acids over C16 fatty acids.
  • Y. lipolytica In case it is desirable to introduce this aspect of the invention into Y lipolytica it is recognizable that sequence homologues for LROl and FAS2 are found in Y. lipolytica as YALI0E16797g and YALI0B19382g respectively (http://cbi.labri.fr/Genolevures). Over-expressing the native lipolytica proteins can possibly increase the protein activity of the corresponding proteins. Consequently, endogenous Y. lipolytica genes can be put under the control of a constitutive promoter for over-expression. Furthermore, heterologous DNA sequences can be isolated and expressed in Y. lipolytica. FAS2 donor sequences can be obtained from other sources such as S. cerevisiae, S.
  • sequences can be amplified from DNA or cDNA and be introduced into the genome of Y. lipolytica under the control of a suitable promoter using said methods. Alternatively, they can be chemically synthesized de novo. In addition, the sequences can be codon-optimized for optimal expression in the Y. lipolytica host cell.
  • LROl or FAS2 may lead to elevated PL)FA levels in Y. lipolytica.
  • both gene targeting methods might be used for the over-expression of LROl and FAS2.
  • the DNA substrates harbouring the promoter sequences can be isolated from Y. lipolytica and inserted at the desired locus in Y. lipolytica by using either of the two gene targeting methods described above.
  • heterologous LROl and FAS2 can be amplified from other yeasts and integrated in Y. lipolytica by said methods.
  • the present invention demonstrates that metabolic engineering of the regulation mechanism of fatty synthesis increase the fatty acid content in S. cerevisiae. Lipid synthesis is transcriptionally regulated by the positive regulators Ino2 and Ino4 as well as the negative regulator Opil. Thus, over-expression of the native IN02 and IN04 genes and deletion of the native yeast gene OPIl is expected to result in a higher lipid yield of the mutated strain. Y. lipolytica
  • Y. lipolytica exhibit the same type of fatty acid regulation as S. cerevisiae, which corresponds well with the fact that homologues of the regulators in S. cerevisiae can be found in Y. lipolytica (http://cbi.labri.fr/Genolevures).
  • the regulators can also be conserved between the yeast species in respect to function. Consequently, one can transfer the same genetic changes to Y. lipolytica by performing genetic engineering on the genes involved in fatty acid and lipid synthesis regulation.
  • the homologue of S. cerevisiae INO4 in Y. lipolytica is YALI0C02387g.
  • INO2 and INO4 can be amplified from S. cerevisiae where their function is described and introduced into Y. lipolytica by the methods already described.
  • endogenous YALI0C02387g can be over-expressed by inserting a strong constitutive promoter in front of the gene.
  • Donor sequences can be obtained from sources such as the yeasts S. cerevisiae and M. alpina, or they can be chemically synthesized de novo.
  • Regulation of fatty acid synthesis can be altered in a Y. lipolytica strain by for instance over-expressing the positive regulator INO4.
  • the substrates for over- expression can be amplified from Y. lipolytica or from another yeast species. Integration of the promoter substrates can be obtained by using the above mentioned auxotrophy-based gene targeting method or by using an antibiotic resistance-based method.
  • plasmid systems designed for Saccharomyce cerevisiae are functional in other similar yeast species.
  • a plasmid system derived from pARpl that contains the AMAl initiating replication sequence from Aspergillus nidulans allows plasmid replication in several different species of Aspergillus (Gems et al., 1991).
  • This shuttle vector containing the replication sequence of Escherichia coli can be used for expression of genes in different species of Aspergillus. Production of PUFA-containing fungal cells
  • Recombinant fungal cells containing heterologous pathways to PL)FAs can be grown in batch, fed batch or chemostat cultivation in order to produce high amounts of PUFA-containing biomass.
  • PUFA-containing biomass can be used as a functional food ingredient, for example as bakers yeast, yeast extract or as a flavour enhancer.
  • the PUFA-containing biomass can also be used directly as a functional food, for example in tablets as an alternative to fish oil capsules.
  • the present invention also relates to food products, such as functional food products, wherein said food product has an increased content of polyunsaturated fatty acids when compared to a product produced by a cell, which is not modified for heterologous expression according to the present invention.
  • Codon usage and optimization Codon usage usually differs among different species.
  • Codon usage and optimization Codon usage usually differs among different species.
  • a preferred embodiment of the present invention relates to a method according to the present invention, wherein said heterologous nucleotide sequences are codon optimized for expression in Saccharomyces cerevisiae. PUFA content
  • the present invention relates to methods, cells, and compositions relating to an improved polyunsaturated fatty acid content, wherein said heterologous expression, over-expression or deletion increases the content of each individual specific polyunsaturated fatty acid, particularly ARA and EPA, to more than 3 % of the total fatty acid content, such as 3% of the total fatty acid content, 4% of the total fatty acid content, 5% of the total fatty acid content, 6% of the total fatty acid content, 7% of the total fatty acid content, 8% of the total fatty acid content, 9% of the total fatty acid content, 10% of the total fatty acid, 15% of the total fatty acid content or more.
  • the method of the invention discloses metabolic engineering which increases the content of arachidonic acid to more than 4 % of the total fatty acid content in the genetically modified fungal cell described herein.
  • the method of the invention discloses metabolic engineering which increases the content of eicosapentaenoic acid to more than 3 % of the total fatty acid content in the genetically modified fungal cell described herein.
  • the method of the invention discloses metabolic engineering which increases the content of gamma-linolenic acid to more than 13 % of the total fatty acid content in the genetically modified fungal cell described herein.
  • the present invention relates methods, cells, and compositions relating to an improved polyunsaturated fatty acid content, wherein said heterologous expression increases the content of each individual specific polyunsaturated fatty acid to more than 0.2% of the biomass dry weight, such as 0.2% of the biomass dry weight, 0.3% of the biomass dry weight, 0.4% of the biomass dry weight, 0.5% of the biomass dry weight, 0.6% of the biomass dry weight, 0.7% of the biomass dry weight, 0.8% of the biomass dry weight, 0.9% of the biomass dry weight, 1% of the biomass dry weight, 2% of the biomass dry weight, 3 of the biomass dry weight, 4% of the biomass dry weight, 5% of the biomass dry weight or more.
  • the present invention relates to a Saccharomyces cerevisiae comprising EPA and ARA in the ratio of at least 1: 1, preferably at least 2: 1, most preferably 2.6: 1.
  • Saccharomyces cerevisiae of the present invention comprises at least 0.4 mg EPA pr. gram dry weight cell, such as at least 0.5 mg EPA pr. gram dry weight cell, at least 0,6 mg EPA pr. gram dry weight cell, at least 0.7 mg EPA pr. gram dry weight cell, at least 0,8 mg EPA pr. gram dry weight cell, at least 0.9 mg EPA pr. gram dry weight cell, at least 10.0 mg EPA pr. gram dry weight cell or more,
  • the metabolic engineered Saccharomyces cerevisiae of the present invention comprises at least 1,5 mg ARA pr. gram dry weight cell, such as at least 1.6 mg ARA pr. gram dry weight cell, at least 1.7 mg ARA pr. gram dry weight cell, at least 1.8 mg ARA pr. gram dry weight cell, at least 1.9 mg ARA pr. gram dry weight cell, at least 2.0 mg ARA pr. gram dry weight cell, at least 2.1 mg ARA pr. gram dry weight cell or more,
  • the metabolic engineered Saccharomyces cerevisiae of the present invention comprises at least 8.0 mg GLA pr. gram dry weight cell, such as at least 8.1 mg GLA pr. gram dry weight cell, at least 8.2 mg GLA pr. gram dry weight cell, at least 8.3 mg GLA pr. gram dry weight cell, at least 8.4 mg GLA pr. gram dry weight cell, at least 8.5 mg GLA pr. gram dry weight cell, at least 8.6 mg GLA pr. gram dry weight cell or more,
  • the metabolic engineered Saccharomyces cerevisiae of the present invention comprises at least 24 mg. PL)FA pr. gram dry weight cell, such as at least 25 mg PL)FA pr. gram dry weight cell, at least 26 mg PL)FA pr. gram dry weight cell, at least 27 mg PL)FA pr. gram dry weight cell, at least 28 mg PL)FA pr. gram dry weight cell, at least 29 mg PL)FA pr. gram dry weight cell, at least 30 mg PL)FA pr. gram dry weight cell or more,
  • Heterologous genes can be expressed in the host organism from extra chromosomal elements.
  • high copy number plasmids are preferred.
  • Other yeast vectors include yeast replicating plasmids (YRps), such as the 2 ⁇ plasmid, which have a chromosomally derived replicating sequence and are propagated in medium copy-number (20 to 40 copies per cell), and yeast centromere plasmids (Ycps; also known as CEN plasmids), which have both a replication origin and a centromere sequence, ensuring stable segregation.
  • yeast expression vectors with differing selection markers can be used in combination when the purpose is to express several heterologous genes.
  • heterologous genes can be expressed from the same plasmid, for example using the pESC vectors (Stratagene), which permit simultaneous, inducible expression from the divergent GAL1/GAL10 promoter sequence.
  • pESC vectors Stratagene
  • a variety of prokaryotic expression systems can be used to express PUFA-synthesizing desaturases and elongases, including the pBR322 plasmid, the pUC plasmids and derivatives thereof.
  • the heterologous genes can be assembled in an artificial operon, meaning that a single promoter sequence controls the expression of a cluster of genes.
  • Several genes encoding PUFA-synthesizing desaturases and elongases can be fused by PCR and subsequently be subcloned into a bacterial expression vector using standard techniques.
  • heterologous genes encoding e.g. cytochrome b5, cytochrome b5 reductase and FAS alpha-subunit, can be expressed by means of vectors as described above.
  • nucleotide sequences can be expressed either from a single vector or from separate vectors.
  • the skilled artisan is also well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention.
  • the heterologous genes are expressed from several vectors. It can also be advantageous to express one or several heterologous genes in the PL)FA pathway from a genomic location.
  • the vector or vectors Once the vector or vectors have been constructed, it may then be introduced into the host cell of choice by methods known to those of ordinary skill in the art including, for example, transfection, transformation and electroporation.
  • the host cell is then cultured under suitable conditions permitting expression of the genes leading to the improved production of the desired PL)FA, which is then recovered and purified.
  • the present invention relates to a genetically modified cell comprising genomic modifications, a vector or several vectors according to the present invention.
  • a further embodiment of the present invention relates to a genetically modified cell, wherein expression of said isolated nucleotide sequences from said vector results in said cell producing a polyunsaturated fatty acid that is not produced in a wild-type of said host cell.
  • Yeast vector for expression of genes encoding delta-6 elongase and delta-5 desaturase Yeast vector for expression of genes encoding delta-6 elongase and delta-5 desaturase.
  • Figure 12 Yeast vector for expression of genes encoding delta-12 desaturase and delta-6 desaturase.
  • Figure 14 Gene-targeting strategy for integration of heterologous genes together with a suitable promoter in the genome of S. cerevisiae.
  • Figure 15 Gene-targeting strategy for integration of heterologous genes together with a suitable promoter in the genome of S. cerevisiae.
  • SEQ ID NO: 1 is a nucleotide sequence of from Mortierella alpina encoding a cytochrome b5
  • SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1
  • SEQ ID NO: 3 is a nucleotide sequence of from Mortierella alpina encoding a cytochrome b5 reductase
  • SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3
  • SEQ ID NO: 5 is a Mortierella alpina amino acid sequence of a cytochrome b5 reductase
  • SEQ ID NO: 6 is a nucleotide sequence of from Saccharomyces kluyveri encoding a FAS alpha-subunit
  • SEQ ID NO: 7 is the amino acid sequence encoded by SEQ ID NO: 6
  • SEQ ID NO: 8 is a nucleotide sequence of from Candida albicans encoding a FAS alpha-subunit
  • SEQ ID NO: 9 is the amino acid sequence encoded by SEQ ID NO: 8
  • SEQ ID NO: 10 is a nucleotide sequence of from Candida albicans encoding a FAS beta-subunit
  • SEQ ID NO: 11 is the amino acid sequence encoded by SEQ ID NO: 10
  • SEQ ID NO: 12 is a nucleotide sequence of from Aspergillus nidulans encoding a
  • SEQ ID NO: 13 is the amino acid sequence encoded by SEQ ID NO: 12
  • SEQ ID NO: 14 is a nucleotide sequence of from Aspergillus nidulans encoding a
  • SEQ ID NO: 15 is the amino acid sequence encoded by SEQ ID NO: 14
  • SEQ ID NO: 16 is a nucleotide sequence of from Yarrowia lipolytica encoding a
  • SEQ ID NO: 17 is the amino acid sequence encoded by SEQ ID NO: 16
  • SEQ ID NO: 18 is the S. cerevisiae CYB5 gene, encoding cytochrome b5
  • SEQ ID NO: 19 is the S. cerevisiae MCRl gene, encoding cytochrome b5 reductase
  • SEQ ID NO: 20 is the S. cerevisiae FASl gene, encoding FAS beta-subunit
  • SEQ ID NO: 21 is the S. cerevisiae FAS2 gene, encoding FAS alpha-subunit
  • SEQ ID NO: 22 is the S. cerevisiae LROl gene, encoding phospholipid :diacylglycerol acyltransferase
  • SEQ ID NO: 23 is the S. cerevisiae SP014 gene, encoding Phospholipase D
  • SEQ ID NO: 24 is the S. cerevisiae INO2 gene, encoding a transcription activator
  • SEQ ID NO: 25 is the S. cerevisiae INO4 gene, encoding a transcription activator
  • SEQ ID NO: 26 is the S. cerevisiae OPIl gene, encoding a transcriptional regulator
  • SEQ ID NO: 27 is the sequence of primer AD-fw
  • SEQ ID NO: 28 is the sequence of primer T2-2
  • SEQ ID NO: 29 is the sequence of primer Int3'
  • SEQ ID NO: 30 is the sequence of primer Int5'
  • SEQ ID NO: 31 is the sequence of primer AD-rv
  • SEQ ID NO: 32 is the sequence of primer T-DGA
  • SEQ ID NO: 33 is the sequence of primer TPIl-fw
  • SEQ ID NO: 34 is the sequence of primer HX7-fw
  • SEQ ID NO: 35 is the sequence of primer TPIl-rvP
  • SEQ ID NO: 36 is the sequence of primer HX7-rvP
  • SEQ ID NO: 37 is the sequence of primer TPIl-rv
  • SEQ ID NO: 38 is the sequence of primer HX7-rv
  • SEQ ID NO: 39 is the sequence of primer Ext 5'
  • SEQ ID NO: 40 is the sequence of primer Ext 3'
  • SEQ ID NO: 41 is the sequence of primer CYBU-fw
  • SEQ ID NO: 42 is the sequence of primer CYBU-rv
  • SEQ ID NO: 43 is the sequence of primer CYBD-fw
  • SEQ ID NO: 44 is the sequence of primer CYBD-rv
  • SEQ ID NO: 45 is the sequence of primer CYB5-C
  • SEQ ID NO: 46 is the sequence of primer TPI-C
  • SEQ ID NO: 47 is the sequence of primer MCRU-fw
  • SEQ ID NO: 48 is the sequence of primer MCRU-rv
  • SEQ ID NO: 49 is the sequence of primer MCRD-fw
  • SEQ ID NO: 50 is the sequence of primer MCRD-rv
  • SEQ ID NO: 51 is the sequence of primer MCRl-C
  • SEQ ID NO: 52 is the sequence of the yeast ADHl promoter
  • SEQ ID NO: 53 is the sequence of the yeast TDH3 promoter
  • SEQ ID NO: 54 is the sequence of Kluyveromyces lactis URA3
  • SEQ ID NO: 55 is the sequence of the yeast TPIl promoter
  • SEQ ID NO: 56 is the sequence of the yeast HXT7 promoter
  • SEQ ID NO: 57 is the sequence of the KLURA5 fragment
  • SEQ ID NO: 58 is the sequence of the KLURA3 fragment
  • SEQ ID NO: 59 is the sequence of primer GPPl-UP-fw
  • SEQ ID NO: 60 is the sequence of primer GPPl-UP-rv
  • SEQ ID NO: 61 is the sequence of primer GSK3-fw
  • SEQ ID NO: 62 is the sequence of primer SK33-rv-t2
  • SEQ ID NO: 63 is the sequence of primer GSK3-rv
  • SEQ ID NO: 64 is the sequence of primer GPPl-D-fw2
  • SEQ ID NO: 65 is the sequence of primer GPPl-D-rv
  • SEQ ID NO: 66 is the sequence of primer GPPl-UP-rvMF
  • SEQ ID NO: 67 is the sequence of primer FAD3-fwMF
  • SEQ ID NO: 68 is the sequence of primer FAD3-rv
  • SEQ ID NO: 69 is the sequence of primer F2D-fw
  • SEQ ID NO: 70 is the sequence of primer F2D-rv
  • SEQ ID NO: 71 is the sequence of primer F2U-fw
  • SEQ ID NO: 72 is the sequence of primer F2U-rv
  • SEQ ID NO: 73 is the sequence of primer ADF2-rv
  • SEQ ID NO: 74 is the sequence of primer FlD-fw
  • SEQ ID NO: 75 is the sequence of primer FlD-rv
  • SEQ ID NO: 76 is the sequence of primer FlU-fw
  • SEQ ID NO: 77 is the sequence of primer FlU-rv
  • SEQ ID NO: 78 is the sequence of primer ADFl-rv
  • SEQ ID NO: 79 is the sequence of primer ADHlted
  • SEQ ID NO: 80 is the sequence of primer FlC-rv
  • SEQ ID NO: 81 is the sequence of primer F2C-rv
  • SEQ ID NO: 82 is the sequence of primer INO2_up_fw
  • SEQ ID NO: 83 is the sequence of primer INO2_up_rv
  • SEQ ID NO: 84 is the sequence of primer INO2_S_fw
  • SEQ ID NO: 85 is the sequence of primer INO2_S_rv
  • SEQ ID NO: 86 is the sequence of primer INO2_C
  • SEQ ID NO: 87 is the sequence of primer INO4_up_fw
  • SEQ ID NO: 88 is the sequence of primer INO4_up_rv
  • SEQ ID NO: 89 is the sequence of primer INO4_S_fw
  • SEQ ID NO: 90 is the sequence of primer INO4_S_rv
  • SEQ ID NO: 91 is the sequence of primer INO4_C
  • SEQ ID NO: 92 is the sequence of primer OPIl_up_fw
  • SEQ ID NO: 93 is the sequence of primer OPIl_up_rv
  • SEQ ID NO: 94 is the sequence of primer OPIl_D_fw
  • SEQ ID NO: 95 is the sequence of primer OPI_D_rv
  • SEQ ID NO: 96 is the sequence of primer LR01_UP_fw
  • SEQ ID NO: 97 is the sequence of primer LR01_UP_rv
  • SEQ ID NO: 98 is the sequence of primer LROl_S_fw
  • SEQ ID NO: 99 is the sequence of primer LROl_S_rv
  • SEQ ID NO: 100 is the sequence of primer LROl-C
  • SEQ ID NO: 101 is the sequence of primer SPO14_up_fw
  • SEQ ID NO: 102 is the sequence of primer SPO14_up_rv
  • SEQ ID NO: 103 is the sequence of primer SPO14_S_fw
  • SEQ ID NO: 104 is the sequence of primer SPO14_S_rv
  • SEQ ID NO: 105 is the sequence of primer SPO14_C
  • SEQ ID NO: 106 is a nucleotide sequence from Aspergillus oryzae encoding a FAS beta-subunit
  • SEQ ID NO: 107 is the amino acid sequence encoded by SEQ ID NO: 106
  • SEQ ID NO: 108 is a nucleotide sequence from Aspergillus oryzae encoding a FAS alpha-subunit
  • SEQ ID NO: 109 is the amino acid sequence encoded by SEQ ID NO: 108
  • SEQ ID NO: 110 is a nucleotide sequence of from Aspergillus oryzae encoding a NADH-cytochrome b-5 reductase
  • SEQ ID NO: 111 is the amino acid sequence encoded by SEQ ID NO: 110
  • SEQ ID NO: 112 is a nucleotide sequence of from Aspergillus oryzae encoding a cytochrome b5
  • SEQ ID NO: 113 is the amino acid sequence encoded by SEQ ID NO: 112
  • SEQ ID NO: 114 is the sequence of primer Eloa-fw-MF
  • SEQ ID NO: 115 is the sequence of primer Elo-T-rv
  • SEQ ID NO: 116 is the sequence of primer TRPl-5end-fw
  • SEQ ID NO: 117 is the sequence of primer TRPl-5end-rv
  • SEQ ID NO: 118 is the sequence of primer TRPl-3end-fw
  • SEQ ID NO: 119 is the sequence of primer TRPl-3end-rv
  • SEQ ID NO: 120 is the sequence of primer PYKl-fw
  • SEQ ID NO: 121 is the sequence of primer PYKl-rv
  • SEQ ID NO: 122 is the sequence of primer D6E_2212
  • SEQ ID NO: 123 is the sequence of primer TRPl-C-rv
  • SEQ ID NO: 124 is the nucleotide sequence of DCIl from S. cerevisiae
  • SEQ ID NO: 125 is the amino acid sequence encoded by SEQ ID NO: 124
  • SEQ ID NO: 126 is the sequence of primer DCIl-UP-fw
  • SEQ ID NO: 127 is the sequence of primer DCIl-UP-rv
  • SEQ ID NO: 128 is the sequence of primer DCIl-D-fw
  • SEQ ID NO: 129 is the sequence of primer DCIl-D-rv
  • SEQ ID NO: 130 is a codon-optimized nucleotide sequence from Ostreococcus tauri encoding a delta-6 desaturase
  • SEQ ID NO: 131 is the amino acid sequence encoded by SEQ ID NO: 130
  • SEQ ID NO: 132 is the sequence of primer D6D-OTi-fw
  • SEQ ID NO: 133 is the sequence of primer D6D-OTi-rv
  • SEQ ID NO: 134 is the nucleotide sequence of FOX2 from S. cerevisiae
  • SEQ ID NO: 135 is the amino acid sequence encoded by SEQ ID NO: 134
  • SEQ ID NO: 136 is the sequence of primer FOX2-UP-fw
  • SEQ ID NO: 137 is the sequence of primer FOX2-UP-rv
  • SEQ ID NO: 138 is the sequence of primer FOX2-D-fw
  • SEQ ID NO: 139 is the sequence of primer FOX2-D-rv
  • SEQ ID NO: 140 is the sequence of primer D12Di-fw
  • SEQ ID NO: 141 is the sequence of primer D12Di-rv
  • SEQ ID NO: 142 is the nucleotide sequence of POTl from S. cerevisiae
  • SEQ ID NO: 143 is the amino acid sequence encoded by SEQ ID NO: 142
  • SEQ ID NO: 144 is the sequence of primer POTl-UP-fw
  • SEQ ID NO: 145 is the sequence of primer POTl-UP-rv
  • SEQ ID NO: 146 is the sequence of primer POTl-D-fw
  • SEQ ID NO: 147 is the sequence of primer POTl-D-rv
  • SEQ ID NO: 148 is the sequence of primer D5Di-fw
  • SEQ ID NO: 149 is the sequence of primer D5Di-rv
  • SEQ ID NO: 150 is a nucleotide sequence from Ostreococcus tauri encoding a delta 6- desaturase
  • SEQ ID NO: 151 is the sequence of primer OtD6D-fw
  • SEQ ID NO: 152 is the sequence of primer OtD6D-rv
  • SEQ ID NO: 153 is the sequence of primer OtlT3_fwl
  • SEQ ID NO: 154 is the sequence of primer OtlT3_fw2
  • SEQ ID NO: 155 is the sequence of primer OtlT3_rvl
  • SEQ ID NO: 156 is the sequence of primer OtlT3_rv2
  • SEQ ID NO: 157 is the sequence of the S. cerevisiae PYKl promoter
  • SEQ ID NO: 158 is the sequence of primer OPIl_up_fw
  • SEQ ID NO: 159 is the sequence of primer OPIl_up_rv
  • SEQ ID NO: 160 is the sequence of primer OPIl_D_fw
  • SEQ ID NO: 161 is the sequence of primer OPI_D_rv
  • SEQ ID NO: 162 is the sequence of primer 7001
  • SEQ ID NO: 163 is the sequence of primer 7002
  • SEQ ID NO: 164 is the sequence of primer 7003
  • SEQ ID NO: 165 is the sequence of primer 7004
  • SEQ ID NO: 166 is the sequence of S. cerevisiae INO2 on plasmid pSF003
  • SEQ ID NO: 167 is the coding sequence of a putative cytochrome b5 from Y.lipolytica
  • SEQ ID NO: 168 is the coding sequence of a putative cytochrome b5 reductase from Y.lipolytica
  • SEQ ID NO: 169 is the coding sequence of a putative phospholipid-DAG acyltransferase from Y.lipolytica
  • SEQ ID NO: 170 is the coding sequence of a putative fatty acid synthase alpha subunit from Y.lipolytica
  • SEQ ID NO: 171 is the coding sequence of a putative Ino4-like transcriptional regulator from Y.lipolytica EXAMPLES
  • E. coli DH5 ⁇ cells were made competent by the Inoue method as described in Sambrook et al., supra. E. coli cells were typically grown at 37°C in Luria Bertani (LB) medium, supplied with 50 mg/l ampicillin where necessary.
  • LB Luria Bertani
  • Yeast cells were typically grown at 30 0 C in YPD medium or synthetic complete drop-out medium, and were made competent by a LiAc-based method (Sambrook et al., supra).
  • Genomic modifications were performed by means of homologous recombination using PCR-generated targeting substrates and the K.lactis URA3 gene as a selectable marker, essentially as described in Erdeniz, N., Mortensen, L). H., Rothstein, R. (1997) Genome Res. 7: 1174-83.
  • Information on primer design for fusion PCR can be found in the same publication. Generally, fusion of DNA fragments was made possible by using primers with appropriately designed 5' overhangs for amplification of the original DNA fragments. In all cases, PCR- generated fragments were excised from a 1% agarose gel and purified before proceeding with fusion PCR.
  • Transformants were generally selected on -LIRA plates, and pop-out of the K.lactis URA3 marker gene was selected for by plating on 5-FOA medium (5-fluoroorotic acid, 750 mg/l).
  • 5-FOA medium 5-fluoroorotic acid, 750 mg/l.
  • Correct integration of promoters and heterologous genes was verified by PCR, always using one primer annealing to a sequence outside of the target sequence for integration and one primer annealing inside the sequence to be integrated.
  • Gene deletions were also verified by PCR, using primers on both sides of the deleted gene.
  • PCR- verification of genomic modifications was performed by means of colony-PCR. For colony-PCR, a small amount of cells was dispersed in 10 ⁇ l H2O and was placed at -80 0 C for approximately 30 min, followed by 15 min. incubation at 37°C. The cell suspension was then used as template for PCR.
  • the asci were dissected on a YPD plate using a Singer MSM microscope and micromanipulator dissection microscope.
  • the mating types of the resulting tetrads were scored by replica-plating to a lawn of cells with either a or alpha mating type, incubating at 30 0 C to allow mating, replica-plating to sporulation medium, and visualizing sporulation by illuminating plates under a 302 nm UV-light source.
  • Auxotrophic markers were scored by replica plating to dropout plates. Genetic modifications that could not be scored by phenotype were scored by colony-PCR. In general, the same primer sets that were used for verification of genomic integrations or knockouts were also used for colony-PCR scoring of tetrads (see above).
  • yeast genes are written in capital letters, while deleted or mutated native yeast genes are written in small letters.
  • Yeast promoters are indicated by a small p, for example pADHl, pTDH3 for the ADHl and TDH3 promoters.
  • Overexpressions of native yeast genes by the promoter-replacement method are indicated by the promoter name followed by the gene name, for example pADHl- FASl, pTDH3-DGAl for overexpression of FASl with the ADHl promoter and overexpression of DGAl with the TDH3 promoter.
  • Disruption of native yeast genes are indicated by a double colon, for example poxl : :pTDH3-M. alpina olel, which means that the POXl gene has been disrupted and that the TDH3 promoter and the M. alpina olel gene has been integrated in its place. Plasmids are written in brackets.
  • Over-expression of native yeasts genes with constitutive yeast promoters is carried out by means of a promoter-replacement method based on a linear, PCR- generated gene-targeting substrate and using K. lactis URA3 as a recyclable marker.
  • An intermediate strain is first generated, where the marker gene is integrated in combination with two copies of the strong constitutive promoter sequence as a direct repeat on each side of the marker gene.
  • the marker gene is then looped out through recombination mediated by the direct repeat, an event which is selected for by plating the intermediate strain on medium containing 5- fluoroorotic acid (5-FOA), which is toxic to cells expressing the URA3 gene.
  • 5-FOA 5- fluoroorotic acid
  • the above described gene-targeting substrate can be constructed by means of multiple rounds of fusion-PCR. However, to avoid introduction of PCR-generated mutations, it is beneficial to use a bi-partite (figure 6) or even a quadruple gene- targeting substrate (figure 7).
  • Bi-partite gene targeting substrate For overexpression with the strong ADHl and TDH3 yeast promoters, these promoters have been introduced into intermediate working vectors on either side of K.lactis URA3, resulting in the vectors pWADl, pWAD2, pW-TDl and pW-TD2 (PCT/DK2005/000372, and figures 8, 9).
  • fragments can be amplified that contain (in the 5' to 3' direction) 1) the ADHl or TDH3 promoter coupled to two thirds of K.lactis URA3 towards the 5' end, using the primers AD-fw or T2-2 and Int3', and 2) two thirds of K.lactis URA3 towards the 3' end coupled to the ADHl or TDH3 promoter, using the primers Int5' and AD-rv or T-DGA.
  • Target sequences corresponding to a 300-500 bp sequence upstream of the gene to be overexpressed and a 300-500 bp starting with ATG of the gene to be overexpressed are amplified from genomic yeast DNA using suitable primers.
  • the reverse primer used for amplification of the upstream target sequence contains a 5' overhang that allows fusion to fragment 1 described above.
  • the forward primer used for amplification of the target sequence starting with ATG contains a 5' overhang that allows fusion with fragment 2 described above.
  • a target sequence upstream of the gene to be overexpressed and a target sequence starting with ATG of the gene to be overexpressed are amplified by PCR using genomic yeast DNA as template.
  • the wanted promoter sequence here the TPIl promoter or the HXT7 promoter
  • Two different primer sets are used in the amplification of the promoter sequence.
  • the forward primer TPIl- fw or HX7-fw contains a 5' overhang that allows fusion with the upstream target sequence and the reverse primer TPIl-rvP or HX7-rvP contains a 5' overhang that allows fusion with the 5' end of the marker gene K.lactis URA3.
  • the forward primer TPIl-fw or HX7- fw contains a 5' overhang that allows fusion with the 3' end of K.lactis URA3 and the reverse primer TPIl-rv or HX7-rv contains a 5' overhang that allows fusion with the target sequence starting with ATG.
  • Promoter fragment 1 and 2 are identical, except for the end-sequences introduced by the 5' overhangs of the primers used in amplification.
  • Promoter fragment 1 is fused to the upstream target sequence by PCR and promoter fragment 2 is fused to the target sequence starting with ATG by PCR.
  • These two target sequences are then used for transformation together with the fragments KLURA5, constituting two thirds of K. lactis URA3 towards the 5' end and KLURA3, constituting two thirds of K.lactis URA3 towards the 3' end.
  • KLURA5 and KLURA3 are amplified by PCR using a plasmid containing K.lactis URA3 as template and the primers Ext 5' and Int 3' for KLURA5 or Int 5' and Ext 3' for KLURA3.
  • K. lactis URA3 is used as a recyclable marker in the integration, and looping out of the marker gene is selected for by plating the intermediate strain on 5-FOA containing medium (figure 10). Targeting of the integration substrate to the desired location in the genome is performed, as described above, by means of PCR-generated target sequences.
  • an upstream and a downstream target sequence are amplified by PCR using yeast genomic DNA as template.
  • the primers that are used in the amplification contain suitable 5' overhang which later allows fusion of the upstream target sequence to the 5 ' end of the promoter sequence, and fusion of the downstream target sequence to the 3 ' end of the heterologous gene to be integrated.
  • the heterologous gene is amplified from a suitable source, e.g. a cDNA preparation, by PCR using primers with suitable 5' overhangs.
  • overhangs allow fusion of the heterologous gene with the downstream target sequence at the 5' end of the heterologous gene and fusion with the chosen promoter sequence (for example the TPIl promoter or the HXT7 promoter) at the 3' end of the heterologous gene.
  • the chosen promoter sequence is amplified by PCR using yeast genomic DNA as template.
  • the primers used for this amplification contain suitable 5' overhangs that later allows fusion of the promoter fragment to 1) the upstream target sequence at the 5' end of the promoter sequence and to the KLURA5 fragment at the 3' end of the promoter sequence and 2) the KLURA3 fragment at the 5' end of the promoter sequence and the downstream target sequence at the 3' end of the promoter sequence.
  • the upstream target sequence is fused to the promoter fragment by PCR.
  • the promoter fragment is fused to the heterologous gene.
  • the promoter-heterologous gene fusion product is further fused to the downstream target sequence by PCR.
  • a suitable ura3 yeast strain is transformed with 1) the upstream target fragment fused to the promoter fragment, 2) the KLURA5 fragment, 3) the KLURA3 fragment and 4) the promoter-heterologous gene-downstream target fusion product.
  • Transformants are selected on medium lacking uracil, and contain, inserted between the upstream and downstream target sequences, two copies of the chosen promoter as a direct repeat on each side of the marker gene K. lactis URA3, followed by the heterologous gene.
  • the resulting ura3 strain contains, inserted between the upstream and downstream target sequences, the chosen promoter followed by the heterologous gene.
  • the plasmid pESC-URA-elo-delta-5 (figure 11) contains a gene encoding delta-6 elongase from Mortierella alpina under the control of the GALl promoter and a gene encoding delta-5 desaturase from M. alpina under the control of the GALlO promoter. Furthermore it contains the yeast URA3 gene for selection in uracil- deficient medium.
  • the plasmid pESC-TRP-delta-12 delta-6 (figure 12) contains a gene encoding delta-12 desaturase from M. alpina under the control of the GALl promoter and a gene encoding delta-6 desaturase from M. alpina under the control of the GALlO promoter. Furthermore it contains the yeast TRPl gene for selection in tryptophane-deficient medium.
  • the plasmid pESC-LEU-SK33 contains a gene encoding omega-3 desaturase from Saccharomyces kluyveri under the control of the GALl promoter. It also contains the yeast LEU2 gene for selection in leucine-deficient medium.
  • Foaming was avoided by the addition of 100 ⁇ l Antifoam 204 (Sigma-Aldrich, St Louis, Missouri) per liter medium. Aerobic conditions were obtained by sparging the fermentor with sterile air at a flow rate of 1.5-2.5 l/min to ensure that the dissolved oxygen concentration was above 60%. The stirring speed was kept at 800 rpm and the carbon dioxide content of outflowing gas was measured with a Br ⁇ el and Kjaer acoustic gas analyzer (Br ⁇ el & Kjaer, Denmark). Following depletion of the carbon source, level controlled continuous fermentation mode at a dilution rate of 0.05 h-1 was applied.
  • the growth medium used in chemostat fermentations contained : 2.5 g/l glucose, 10 g/l galactose, 5 g/l (NH4)2SO4, 3 g/l KH2PO4, 0.5 g/l MgSO4 *7H2O, lml/l vitamin solution and 1 ml/1 trace metal solution.
  • the vitamin solution contained : 50 mg/L biotin, 1 g/L calcium panthotenate, 1 g/L nicotinic acid, 1 g/L thiamine HCI, 1 g/L pyridoxal HCI and 0.2 g/L para-aminobenzoic acid, while the trace metal solution contained: 15 g/L EDTA, 4.5 g/L ZnSO4-7H2O, 1 g/L MnCI2-2H2O, 0.3 g/L CoCI2-6H2O, 0.4 g/L Na2MoO4-2H2O, 4.5 g/L CaCI2-2H2O, 3 g/L FeSO4-7H2O, 1 g/L H3BO3 and 0.1 g/L KI.
  • Glucose, galactose, ethanol, glycerol, acetate, succinate, and pyruvate concentrations in the culture broth were determined by column liquid chromatography (CLC) using a Dionex Summit CLC system (Dionex, Sunnyvale, CA) after removing the cells from the culture broth via centrifugation.
  • An Aminex HPX-87H column (BioRad, Hercules, CA) was used at 60 0 C with a Waters 410 Differential refractive index detector (Millipore, Milford, MA) and a Waters 486 Tuneable Absorbance Detector (L)V detector) set at 210 nm. The two detectors were connected in series. As mobile phase 5 mM H2SO4 was used at a flow rate of 0.6 ml/min.
  • the cell dry weight was determined by filtering a known volume of the culture broth through a pre-dried, pre-weighed 0.45 ⁇ m Supor membrane (Pall Corporation, Ann Arbor, MI) filter. After washing with 1 volume of distilled water and drying in microwave oven for 15 minutes at 150 W, the filter was weighed again.
  • Supor membrane Pall Corporation, Ann Arbor, MI
  • the biomass was separated through centrifugation for 5 minutes at 5000 rpm.
  • the biomass was re-dissolved in 10 ml distilled water and the resulting cell suspension was broken using the glass bead method to generate cell extract.
  • the cell extract was prepared by addition of 1 ml glass beads with a particle size of 250-500 ⁇ m (Sigma-Aldrich, St Louis, Missouri) to 1 ml cell suspension in a micro tube with screw cap (Sarstedt, Germany). For each cell suspension 6 tubes were processed. The tubes were shaken at level 4 for 20 seconds in a FastPrep FP120 instrument (Qbiogene, France). This was done in total 6 rounds for each tube with a 5 minutes intervening cooling of the tubes on ice after 3 rounds. The cell extracts were combined in 2 ml eppendorf tubes by transferring 600 ⁇ l cell extract to generate 3 eppendorf tubes each containing 1.2 ml glass bead free cell extract.
  • Dry lipid was generated as for the determination of total lipid (Example 9) and was dissolved in 1 ml toluene and 2 ml 1 % sulphuric acid in methanol was added. The tube was closed after mixing and sparging with nitrogen and left at 50 0 C over night for transesterification of the lipids. The sample was then washed with 5 ml 5% NaCI solution. Methyl esters were subsequently extracted twice by adding 5 ml hexane, vortexing the sample and collecting the organic upper phase. The organic phase was washed with 4 ml 2% sodium carbonate and the organic phase was collected again.
  • the GC/MS system was a Hewlett Packard HP G1723A with a gas chromatograph-quadruple mass selective detector (EI) operated at 70 eV.
  • the column used was Supelco SPTM-2380.
  • the MS was operated in SCAN Mode.
  • the oven temperature was initially 170 0 C and in the following risen to 220 0 C at 4°C/min. The final temperature was held for 15 min.
  • the flow through the column was 0.6 ml
  • the native yeast gene CYB5 was overexpressed with the TPIl promoter using a quadruple gene targeting substrate according to the following procedure:
  • a target sequence upstream of CYB5 was amplified from genomic DNA by PCR using the primers CYBU-fw and CYBU-rv and was fused to the TPIl promoter fragment 1 by PCR. Furthermore a target sequence corresponding to the 5' end of CYB5 was amplified from genomic DNA using the primers CYBD-fw and CYBD-rv. The downstream target sequence was fused to the TPIl promoter fragment 2 by PCR.
  • the yeast strain FS01440 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2) was transformed with the two linear fusion substrates described above and additionally the overlapping marker fragments KLURA5 and KLURA3. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. Transformants were then transferred to plates containing 5- FOA. Pop-out recombinants were streak-purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2 pTPIl-CYB5 and was named FS01455. Correct integration of the TPIl promoter was verified by PCR using the primers CYB5-C and TPI-C.
  • the yeast strain FS01455 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl PADH1-FAS2 pTPIl-CYB5) was then transformed with the plasmids pESC-URA- elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain FS01467 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2 pTPIl-CYB5 [pESC-URA-elo-delta-5] [pESC-TRP-delta-12 delta-6]).
  • the native yeast gene MCRl was overexpressed with the TPIl promoter using a quadruple gene targeting substrate according to the following procedure:
  • a target sequence upstream of MCRl was amplified from genomic DNA by PCR using the primers MCRU-fw and MCRU-rv and was fused to the TPIl promoter fragment 1 by PCR. Furthermore a target sequence corresponding to the beginning of MCRl was amplified from genomic DNA using the primers MCRD-fw and MCRD-rv. The downstream target sequence was fused to the TPIl promoter fragment 2 by PCR.
  • the yeast strain FS01440 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2) was transformed with the two linear fusion substrates described above and additionally the overlapping marker fragments KLURA5 and KLURA3. Transformants were selected on medium lacking uracil and were streak-purified on the same medium.
  • the resulting strain was named FS01456 and had the genotype MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2 pTPIl-K.lactis URA3-PTPI1-MCR1.
  • TPIl promoter Correct integration of the TPIl promoter was verified by PCR using the primers MCRl-C and TPI-C.
  • FS01456 was then transferred to plates containing 5-FOA. Pop- out recombinants were streak-purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl PADH1-FAS2 pTPIl-MCRl and was named FS01457.
  • the yeast strain FS01457 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2 pTPIl-MCRl) was then transformed with the plasmids pESC-URA- elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain FS01468 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2 pTPIl-MCRl [pESC-URA-elo-delta-5] [pESC-TRP-delta-12 delta-6]).
  • Strain FS01456 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2 pTPIl-K.lactis URA3-pTPIl-MCRl) was crossed with strain FS01408 (MAT ⁇ ura3- 52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl). The resulting diploid was allowed to sporulate and the ascospores were dissected.
  • a strain with the genotype MATa ura3-52 trpl-289 poxl : :pTDH3- M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-K.lactis URA3-pTPIl-MCRl was identified and was named FS01488.
  • a strain with the genotype MATalpha ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl- FAS2 pTPIl-K.lactis ura3-pTPIl-MCRl was identified and was named FS01492.
  • FS01488 was transferred to 5-FOA plates and the resulting pop-out recombinants were streak purified on the same medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl and was named FS01494.
  • FS01494 was transformed with the plasmids pESC-URA-elo- delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain FS01490 (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl [pESC-URA-elo-delta-5] [pESC-TRP-delta-12 delta-6]).
  • S. kluyveri FAD3 To integrate S. kluyveri FAD3 into the genome of S. cerevisiae, a fragment containing the GALl promoter fused to S. kluyveri FAD3 was amplified from plasmid pESC-LEU-SK33, using the primers GSK3-fw and SK33-rv-t2.
  • an upstream targeting sequence was amplified by PCR using genomic yeast DNA as template and using the primers GPPl-UP-fw and GPPl-UP-rv
  • a downstream targeting sequence was amplified by PCR using genomic yeast DNA as template and using the primers GPPl-DOWN-fw-2 and GPPl-DOWN-rv.
  • GALl promoter fragment was amplified by PCR, using plasmid pESC-LEU-SK33 as template and using primers GSK3-fw and GSK3-rv.
  • the upstream target sequence was then fused to the GALl promoter fragment using the primers GPPl-UP-fw and GSK3-rv, while the fragment containing the GALl promoter fused to S. kluyveri FAD3 was fused by PCR to the downstream target sequence, using primers GSK3-fw and GPPl-DOWN-rv.
  • the yeast strain FS01458 (MATa ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M.
  • alpha OLEl pADHl-TSC13 pADHl-FASl was transformed with the two resulting fusion products and additionally the overlapping marker fragments KLURA5 and KLURA3.
  • Transformants were selected on medium lacking uracil, and popout of the marker gene was selected for by plating on 5-FOA containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 leu2-3 poxl: :pTDH3-M.alpina OLEl pADHl-TSC13 pADHl-FASl gppl : :pGALl-S. kluyveri FAD3 and was named FS01480.
  • strain FS01480 was crossed to strain FS01492 (MATalpha ura3-52 trpl-289 poxl : :pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl-K.lactis ura3- pTPIl-MCRl).
  • the resulting diploid was allowed to sporulate and the ascospores were dissected.
  • alpha OLEl pADHl-FASl PADH1-FAS2 pTPIl-K.lactis URA3-pTPIl-MCRl gppl : :pGALl-S. kluyveri FAD3 was selected and named FS01504. Following looping out of the selection marker in FS01504 on medium containing 5-FOA, strain FS01505 was obtained (MATa ura3- 52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl gppl : :pGALl-S.kluyveri FAD3).
  • promoter replacement with a quadruple gene targeting substrate was carried out according to the procedure described in Example 2.
  • Target sequences upstream and downstream of the GALl promoter, respectively, were amplified by PCR using genomic DNA from FS01505 as template and the primer pairs GPPl- UP-fw and GPPl-UP-rvMF for the upstream target and FAD3-fwMF and FAD3-rv for the downstream target.
  • the upstream target was fused to the HXT7 promoter fragment 1, while the downstream target was fused to the HXT7 promoter fragment 2 by PCR.
  • the yeast strain FS01505 was then transformed with the two fusion products, in addition to the two overlapping marker fragments KLURA5 and KLURA3. Transformants were selected on medium lacking uracil and the resulting strain was named FS01510 (MATa ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3- M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl gppl : :pHXT7-Klactis URA3-pHXT7-S.kluyveri FAD3).
  • Popout of the selection marker in FS01510 was selected for by plating the strain on medium containing 5-FOA, and the resulting strain was named FS01511 (MATa ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3- M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl gppl : :pHXT7-S.kluyveri FAD3).
  • FS01511 was transformed with the plasmids pESC-URA-elo-delta-5 and pESC- TRP-delta-12 delta-6, resulting in the yeast strain FS01524 (MATa ura3-52 trpl- 289 Ieu2-3_112 poxl : :pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl- MCRl gppl : :pHXT7-S.kluyveri FAD3 [pESC-URA-elo-delta-5] [pESC-TRP-delta-12 delta-6])
  • FS01511 is crossed to a sutaible strain, e.g. FS01408 (MATalpha ura3-52 trpl-289 poxl : :pTDH3-M. alpha OLEl).
  • the resulting diploid strain is allowed to sporulate and the ascospores are dissected.
  • strains with desired genotypes are selected, for example a strain with the same genotype as FS01511 but without the Ieu2-3_112 mutation, and a strain with the same genotype as FS01511 but without the Ieu2-3_112 mutation and without MCRl over-expression.
  • strains are transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, and the influence of MCRl over-expression on EPA production is investigated.
  • strains with MATalpha mating type and containing the gppl : :pHXT7-S.kluyveri FAD3 modification are selected and are further used in the construction of new strains.
  • the yeast strain FS01455 (MATa ura3-52 trpl-289 pADHl-TSC13 pADHl-FASl pADHl-FAS2 pTPIl-CYB5) is crossed to a suitable yeast strain with MATalpha mating type and containing the gppl : :pHXT7-S.kluyveri FAD3 modification.
  • the resulting diploid strain is allowed to sporulate and the ascospores are dissected.
  • a strain containing both CYB5 over- expression and the gppl : :pHXT7-S.isseyveri FAD3 modification is selected.
  • the strain is transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP- delta-12 delta-6, and the influence of CYB5 over-expression on EPA production is investigated.
  • yeast strain FS01468 carries MCRl overexpression with the TPIl promoter in the mentioned genetic background, and in addition expresses the pathway to ARA from multi-copy plasmids.
  • Lipid content (weight % of dw) 0.2 11.8 +/- 1. 1
  • CYB5 results in increased fatty acid desaturation and increased gamma-linolenic acid production
  • CYB5 was overexpressed with the TPIl promoter in a genetic background carrying overexpressions of the native yeast genes FASl, FAS2 and TSC13.
  • the strain FS01467 overexpresses CYB5 in the mentioned genetic background and also expresses the pathway to ARA from high-copy plasmids.
  • cDNA is first prepared from M. alpina.
  • M. alpina CBS 608.70 is cultivated in 100 ml GY medium (20 g/L glucose, 10 g/L yeast extract pH 6.0) at room temperature for 3 days. Biomass is collected by filtration and total RNA is isolated using Trizol reagent (Gibco BRL). Approximately 5 ⁇ g of RNA is used for reverse transcription (Superscript II RT, Invitrogen) using Oligo(dT)12-18 as primer. After first strand cDNA sythesis, complementary RNA is removed by RNAse digestion. The cDNA is then used as template for PCR (Phusion enzyme, Finnzymes), using primers designed to target the cytochrome b5 encoding gene (SEQ ID NO 1) and containing suitable 5' overhangs.
  • the strategy described in example 3 is used.
  • the gene targeting fragments are transformed into a suitable yeast strain, for example FS01396 (MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpina OLEl pADHl-FASl pADHl-FAS2).
  • FS01396 MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpina OLEl pADHl-FASl pADHl-FAS2
  • the resulting strain carries the M. alpina gene encoding cytochrome b5 integrated in the genome under the control of a strong yeast promoter and is ura3.
  • the strain is then transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, and the influence of the heterologous cytochrome b5 on ARA production is evaluated.
  • cDNA is first prepared from M. alpina. M. alpina CBS 608.70 is cultivated in 100 ml GY medium (20 g/L glucose, 10 g/L yeast extract pH 6.0) at room temperature for 3 days. Biomass is collected by filtration and total RNA is isolated using Trizol reagent (Gibco BRL). Approximately 5 ⁇ g of RNA is used for reverse transcription (Superscript II RT, Invitrogen) using Oligo(dT)12-18 as primer. After first strand cDNA sythesis, complementary RNA is removed by RNAse digestion. The cDNA is then used as template for PCR (Phusion enzyme, Finnzymes), using primers designed to target the cytochrome b5 reductase encoding gene (SEQ ID NO 3) and containing suitable 5' overhangs.
  • PCR Phusion enzyme, Finnzymes
  • the strategy described in example 3 is used.
  • the gene targeting fragments are transformed into a suitable yeast strain, for example FS01396 (MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpina OLEl pADHl-FASl pADHl-FAS2).
  • FS01396 MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpina OLEl pADHl-FASl pADHl-FAS2
  • the resulting strain carries the M.
  • the strain is then transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, and the influence of the heterologous cytochrome b5 reductase on ARA production is evaluated.
  • FAS2 was over-expressed by replacing its native promoter with the ADHl promoter, using a bi-partite gene targeting substrate as described in Example 2.
  • a target sequence upstream of FAS2 was amplified from genomic DNA by PCR using the primers F2U-fw and F2U-rv and a target sequence corresponding to the beginning of FAS2, starting with ATG, was amplified from genomic DNA using the primers F2D-fw and F2D-rv.
  • the ADHl promoter/K.lactis URA3 fragment 1 was amplified by PCR using the plasmid pWAD2 as template and the primers AD-fw and Int3 ' , and was fused to the upstream target sequence by PCR using primers F2U-fw and Int3 ' .
  • lactis URA3/ADH1 promoter fragment 2 was amplified by PCR using the plasmid pWADl as template and the primers Int5 ' and ADF2-rv, and was fused to the downstream target by PCR using the primers Int5 ' and F2D- rv.
  • the yeast strain FS01202 (MATa ura3-52) was transformed with the two linear fusion substrates described above. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. Correct integration of the ADHl promoter was verified by PCR using the primers ADH-ted and F2C-rv. To select for looping out of the marker gene, the transformant was transferred to plates containing 5-FOA. Pop-out recombinants were streak-purified on 5-FOA- containing medium. The resulting strain had the genotype MATa ura3-52 pADHl- FAS2 and was named FS01352. Correct integration of the ADHl promoter and absence of PCR-generated mutations was verified by sequencing of the modified region.
  • FS01352 was crossed to FS01269 (MATalpha trpl-289) and the resulting diploid strain was allowed to sporulate and the ascospores dissected.
  • the strain FS01518 MATa ura3-52 trpl-289 pADHl-FAS2 was selected since it allows transformation of the URA3- and TRPl-based plasmids overexpressing the genes in the ARA pathway.
  • FS01518 was transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain FS01520 (MATa ura3-52 trpl-289 pADHl-FAS2 [pESC-URA-elo-delta- 5] [pESC-TRP-delta-12 delta-6]).
  • FASl was over-expressed by replacing its native promoter with the ADHl promoter, using a bi-partite gene targeting substrate as described in Example 2.
  • a target sequence upstream of FASl was amplified from genomic DNA by PCR using the primers FlU-fw and FlU-rv and a target sequence corresponding to the beginning of FASl, starting with ATG, was amplified from genomic DNA using the primers FlD-fw and FlD-rv.
  • the ADHl promoter/K.lactis URA3 fragment 1 was amplified by PCR using the plasmid pWAD2 as template and the primers AD-fw and Int3 ' , and was fused to the upstream target sequence by PCR using primers FlU-fw and Int3 ' .
  • the K. lactis URA3/ADH1 promoter fragment 2 was amplified by PCR using the plasmid pWADl as template and the primers Int5 ' and ADFl-rv, and was fused to the downstream target by PCR using the primers Int5 ' and FlD- rv.
  • the yeast strain FS01202 (MATa ura3-52) was transformed with the two linear fusion substrates described above. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. Correct integration of the ADHl promoter was verified by PCR using the primers ADH-ted and FlC-rv. To select for looping out of the marker gene, the transformant was transferred to plates containing 5-FOA. Pop-out recombinants were streak-purified on 5-FOA- containing medium. The resulting strain had the genotype MATa ura3-52 pADHl- FASl and was named FS01351. Correct integration of the ADHl promoter and absence of PCR-generated mutations was verified by sequencing of the modified region.
  • FS01351 was crossed to FS01269 (MATalpha trpl-289) and the resulting diploid strain was allowed to sporulate and the ascospores dissected.
  • the strain FS01517 MATa ura3-52 trpl-289 pADHl-FASl was selected since it allows transformation of the URA3- and TRPl-based plasmids overexpressing the genes in the ARA pathway.
  • FS01517 was transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain FS01519 (MATa ura3-52 trpl-289 pADHl-FASl [pESC-URA-elo-delta- 5] [pESC-TRP-delta-12 delta-6]).
  • yeast strain FS01519 MATa ura3-52 trpl-289 pADHl-FASl [pESC-URA-elo-delta- 5] [pESC-TRP-delta-12 delta-6]).
  • the FASl overexpressing strain FS01351 (MATa ura3-52 pADHl-FASl) was first crossed to the strain FS01269 (MATalpha trpl-289). Diploids were allowed to sporulate, and the asci were dissected into spore tetrads. From the set of haploid strains derived from the cross, a strain with the genotype MATalpha trpl-289 pADHl-FASl was selected and named FS01342.
  • FS01342 (MATalpha trpl-289 pADHl-FASl) was then crossed to FS01352 (MATa ura3-52 pADHl-FAS2). Following transfer of the diploids to sporulation medium, asci were dissected into ascospore tetrads. From the set of haploid strains derived from the cross, a strain with the genotype MATa ura3 trpl pADHl-FASl pADHl- FAS2 was selected and was named FS01372.
  • FS01372 was transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in strain FS01373 (MATa ura3-52 trpl-289 pADHl-FASl pADHl-FAS2 [pESC-URA-elo- delta-5] [pESC-TRP-delta-12 delta-6].
  • the strain FS01520 overexpressing FAS2 and also expressing the pathway to ARA from high-copy plasmids was analysed in chemostat cultivation.
  • the strains FS01324 no overexpression of FASl and FAS2 genes, MATa ura3-52 trpl-289 [pESC-URA-elo- delta-5] [pESC-TRP-delta-12 delta-6]
  • FS01373 overexpression of both FASl and FAS2 genes, MATa ura3-52 trpl-289 pADHl-FASl pADHl-FAS2 [pESC-URA- elo-delta-5] [pESC-TRP-delta-12 delta-6]
  • FS01324 no overexpression of FASl and FAS2 genes, MATa ura3-52 trpl-289 [pADHl-FASl pADHl-FAS2 [pESC-URA- elo-delta-5] [p
  • the native yeast gene LROl (SEQ ID NO: 22) was overexpressed with the TPIl promoter using a quadruple gene targeting substrate according to the following procedure:
  • a target sequence upstream of LROl was amplified from genomic DNA by PCR using the primers LR01_UP_fw and LR01_UP_rv and was fused to the TPIl promoter fragment 1 by PCR. Furthermore a target sequence corresponding to the 5' end of LROl was amplified from genomic DNA using the primers LROl_S_fw and LR01_S_rv. The downstream target sequence was fused to the TPIl promoter fragment 2 by PCR.
  • the yeast strain FS01494 (MATa ura3-52 trpl-289 poxl : pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl) was transformed with the two linear fusion substrates described above and additionally the overlapping marker fragments KLURA5 end and KLURA3. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. Transformants were then transferred to plates containing 5-FOA. Pop-out recombinants were streak- purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl- MCRl pTPIl-LROl. Correct integration of the TPIl promoter was verified by PCR using the primers LROl-C and TPI-C.
  • the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl- FASl pADHl-FAS2 pTPIl-MCRl pTPIl-LROl) was then transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in a yeast strain with the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl pTPIl-LROl [pESC-URA-el ⁇ -delta-5] [pESC-TRP-delta-12 delta-6]).
  • the native yeast gene SP014 (SEQ ID NO: 23) was overexpressed with the TPIl promoter using a quadruple gene targeting substrate according to the following procedure:
  • a target sequence upstream of SP014 was amplified from genomic DNA by PCR using the primers SPO14_UP_fw and SPO14_UP_rv and was fused to the TPIl promoter fragment 1 by PCR. Furthermore a target sequence corresponding to the 5' end of SP014 was amplified from genomic DNA using the primers SPO14_S_fw and SPO14_S_rv. The downstream target sequence was fused to the TPIl promoter fragment 2 by PCR.
  • the yeast strain FS01494 (MATa ura3-52 trpl-289 poxl : pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl) was transformed with the two linear fusion substrates described above and additionally the overlapping marker fragments KLURA5 end and KLURA3. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. Transformants were then transferred to plates containing 5-FOA. Pop-out recombinants were streak- purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl- MCRl pTPIl-SPO14. Correct integration of the TPIl promoter was verified by PCR using the primers SPO14-C and TPI-C.
  • the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl- FASl pADHl-FAS2 pTPIl-MCRl pTPIl-SPO14) was then transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl PADH1-FAS2 pTPIl-MCRl pTPIl-SP014 [pESC-URA-el ⁇ -delta-5] [pESC-TRP-delta- 12 delta-6]).
  • the native yeast gene INO2 (SEQ ID NO: 24) was overexpressed with the PYKl promoter using a quadruple gene targeting substrate according to the following procedure:
  • a target sequence upstream of INO2 was amplified from genomic DNA by PCR using the primers INO2_UP_fw and INO2_UP_rv and was fused to the PYKl promoter fragment 1 by PCR. Furthermore a target sequence corresponding to the 5' end of INO2 was amplified from genomic DNA using the primers INO2_S_fw and INO2_S_rv. The downstream target sequence was fused to the PYKl promoter fragment 2 by PCR.
  • the yeast strain FS01494 (MATa ura3-52 trpl-289 poxl : pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl) was transformed with the two linear fusion substrates described above and additionally the overlapping marker fragments KLURA5 end and KLURA3. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. Transformants were then transferred to plates containing 5-FOA. Pop-out recombinants were streak- purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-molel pADHl-FASl pADHl-FAS2 pTPIl-MCRl pPYKl-INO2. Correct integration of the PYKl promoter was verified by PCR using the primers INO2-C and PYK-C.
  • the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl- FASl pADHl-FAS2 pTPIl-MCRl pPYKl-INO2) was then transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl PADH1-FAS2 pTPIl-MCRl pPYKl-INO2 [pESC-URA-el ⁇ -delta-5] [pESC-TRP-delta- 12 delta-6]).
  • the native yeast gene INO4 was overexpressed with the HXT7 promoter using a quadruple gene targeting substrate according to the following procedure:
  • a target sequence upstream of INO4 (SEQ ID NO: 25) was amplified from genomic DNA by PCR using the primers INO4_UP_fw and INO4_UP_rv and was fused to the HXT7 promoter fragment 1 by PCR. Furthermore a target sequence corresponding to the 5' end of INO4 was amplified from genomic DNA using the primers INO4_S_fw and INO4_S_rv. The downstream target sequence was fused to the HXT7 promoter fragment 2 by PCR.
  • the yeast strain FS01494 (MATa ura3-52 trpl-289 poxl : pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl) was transformed with the two linear fusion substrates described above and additionally the overlapping marker fragments KLURA5 end and KLURA3. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. Transformants were then transferred to plates containing 5-FOA. Pop-out recombinants were streak- purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl- MCRl pHXT7-INO4. Correct integration of the HXT7 promoter was verified by PCR using the primers INO4-C and HXT-C.
  • the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl- FASl pADHl-FAS2 pTPIl-MCRl pHXT7-INO4) was then transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl PADH1-FAS2 pTPIl-MCRl pHXT7-INO4 [pESC-URA-el ⁇ -delta-5] [pESC-TRP-delta- 12 delta-6]).
  • the native yeast gene OPIl (SEQ ID NO: 26) is deleted using a quadruple gene targeting substrate according to the following procedure:
  • a target sequence upstream of OPIl is amplified from genomic DNA by PCR using the primers OPIl_up_fw and OPIl_up_rv and is fused to the two thirds of the K. lactis URA3 gene to the 5' end by PCR. Furthermore a target sequence corresponding to the downstream region of OPIl is amplified from genomic DNA using the primers OPIl-D-fw and OPIl_d_rv. The downstream target sequence is fused to the two thirds of the K. lactis URA3 gene to the 3' end by PCR.
  • the yeast strain FS01494 (MATa ura3-52 trpl-289 poxl : pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl) is transformed with the two linear fusion substrates described above containing the upstream target region and the downstream target region of the gene to be deleted fused to either two thirds of the K. lactis URA3 gene.
  • Transformants are selected on medium lacking uracil and are streak-purified on the same medium. Transformants are transferred to plates containing 5-FOA. Pop-out recombinants are streak-purified on 5-FOA-containing medium.
  • the resulting strain has the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl opil ⁇ . Correct deletion of the OPIl gene is verified by PCR using the primers OPIl_up_fw and OPIl D rv.
  • the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl- FASl pADHl-FAS2 pTPIl-MCRl opil ⁇ ) is transformed with the plasmids pESC- URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in the yeast strain (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl opil ⁇ [pESC-URA-elo-delta-5] [pESC-TRP-delta-12 delta-6]).
  • the FAS2 gene product of S. kluyveri contains three domains- ⁇ -ketoacyl synthase, ⁇ -ketoacyl reductase and acyl carrier protein.
  • S. kluyveri FAS2 in S. cerevisiae, the gene is amplified by PCR using genomic DNA from S. kluyveri as template.
  • the defined primers used for the amplification are designed to match the published sequence encoding FAS2 from S. kluyveri.
  • the amplified gene is then put under the control of a constitutive promoter through integration into the genome of a suitable S. cerevisiae strain, e.g.
  • FS01516 (MATa ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl), using the strategy described in Example 3.
  • the resulting strain is transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in an arachidonic acid producing strain.
  • the strain is transformed with the plasmids pESC-URA-elo- delta-5, pESC-TRP-delta-12 delta-6 and pESC-LEU-SK33, resulting in an eicosapentaenoic acid producing strain.
  • A. nidulans produces more C18 than C16 and FAS catalyzes the synthesis of long- chain saturated fatty acids from acetyl-CoA, malonyl-CoA and NADPH.
  • the nucleotide sequences encoding the alpha and beta subunits of FAS in A. nidulans are amplified by PCR using cDNA from A. nidulans as template.
  • the defined primers used for the amplification are designed to match the published sequences encoding FAS in A. nidulans.
  • the resulting fragments of the expected sizes are excised from an agarose gel and purified using GFX-columns (Amersham).
  • the amplified genes are put under the control of constitutive promoters through integration into the genome of a suitable S. cerevisiae strain, e.g. FS01516 (MATa ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl), using using the strategy described in Example 3.
  • a suitable S. cerevisiae strain e.g. FS01516 (MATa ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl), using using
  • the resulting strain is transformed with the plasmids pESC-URA-elo-delta-5 and pESC-TRP-delta-12 delta-6, resulting in an arachidonic acid producing strain. Furthermore, in a separate experiment, the strain is transformed with the plasmids pESC-URA-elo-delta-5, pESC-TRP-delta-12 delta-6 and pESC-LEU-SK33, resulting in an eicosapentaenoic acid producing strain.
  • Deletion of native yeasts genes is carried out by means of a replacement method based on a linear, PCR-generated gene-targeting substrate and using K. lactis URA3 as a recyclable marker.
  • An intermediate strain is first generated, where the marker gene flanked with two direct repeat sequences is integrated at the locus of the gene to be deleted.
  • the marker gene is then looped out through recombination between the two direct repeat sequences flanking K. lactis URA3. This event is selected for by plating the intermediate strain on medium containing 5-fluoroorotic acid (5-FOA), which is toxic to cells expressing the URA3 gene.
  • 5-FOA 5-fluoroorotic acid
  • the result is a strain, in which the native gene has been replaced with one copy of the direct repeat sequence. Integration of the above described direct repeat sequences and marker gene is directed to the correct location in the genome by means of PCR-generated target sequences.
  • a target sequence upstream of the gene to be deleted and a target sequence downstream of the gene to be deleted are amplified from genomic yeast DNA. Furthermore, the vector pWJ1042 is used as template to obtain marker fragments for integration.
  • the vector contains the K.lactis URA3 gene flanked by two direct repeat sequences. By using the primers cKL3' and 5'int the two thirds of K.lactis URA3 towards the 5' end are amplified (fragment 1), and using the primers dKL5' and 3'int the two thirds of K.lactis URA3 towards the 3' end are amplified (fragment 2).
  • the reverse primer used for amplification of the upstream target sequence contains a 5' overhang that allows fusion to fragment 1 described above.
  • the forward primer used for amplification of the downstream target sequence contains a 5' overhang that allows fusion with fragment 2 described above.
  • Example 32 Construction of a fungal vector for expression of the gene encoding delta-6 desaturase from Mortierella alpina in species belonging to the genus Aspergillus.
  • the plasmid that is used in the following examples is derived from pARpl that contains the AMAl initiating replication sequence from Aspergillus nidulans, which also sustains autonomous plasmid replication in A. niger and A. oryzae (Gems et al., 1991). Moreover, the plasmid is a shuttle vector, containing the replication sequence of Escherichia coli, and the inherent difficult transformations in A. niger and A. oryzae can therefore overcome by using E. coli as an intermediate host for the construction of recombinant plasmids. The plasmid contains one or more marker genes to allow the microorganism that harbour them to be selected from those which do not.
  • the selection system can be either based upon dominant markers e.g. resistance against hygromycin B, phleomycin and bleomycin, or heterologous markers e.g amino acids and the pyrG gene.
  • the plasmid contains promoter- and terminator sequences that allow the expression of the recombinant genes. Suitable promoters are taken from genes of Aspergillus nidulans e.g. alcA, glaA, amy, niaD, and gpdA.
  • the plasmid contains suitable unique restriction sites to facilitate the cloning of DNA fragments and subsequent identification of recombinants.
  • the plasmid used in the following examples contains the strong constitutive gpdA-promoter and auxotropic markers, all originating from A. nidulans; the plasmid containing the gene methG that is involved in methionine biosynthesis, is designated as pAMAl-MET; the plasmid containing the gene hisA that is involved in histidine biosynthesis, is designated as pAMAl-HIS.
  • the gene encoding delta-6 desaturase from M. alpina is reamplified by PCR from the plasmid pESC-TRP-delta-12 delta-6 (example 4), using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-HIS vector that contains the gpdA promoter from Aspergillus nidulans.
  • the resulting plasmid, pAMAl-HIS-delta-6 contains the gene delta-6 desaturase from M. alpina under the control of the gpdA promoter from Aspergillus nidulans.
  • delta-6 desaturase The sequence of the gene encoding delta-6 desaturase is verified by sequencing of clones of pAMAl-HIS-delta-6.
  • Aspergillus oryzae strains are transformed with the vector described in example 32.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989). Transformants are selected on minimal medium lacking histidine.
  • a strain of A. oryzae that is auxotrophic for histidine is transformed with the vector pAMAl-HIS-delta-6 (example 32), yielding the strain FSAN-delta-6.
  • Example 34
  • the gene encoding a delta-6 elongase from Mortierella alpina is reamplified by PCR from the plasmid pESC-URA-elo-delta-5 (example 4), using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from Aspergillus nidulans.
  • the resulting plasmid, pAMAl-MET-elo contains the delta-6 elongase from M. alpina under the control of the gpdA promoter from A.
  • the gene encoding a delta-5 desaturase from M. alpina is reamplified by PCR from the plasmid pESC-URA- elo-delta-5 (example 4) using forward- and reverse primers, with 5' overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-HIS vector to yield pAMAl-HIS- delta-5.
  • the gpdA promoter and the delta-5 desaturase from M is reamplified by PCR from the plasmid pESC-URA- elo-delta-5 (example 4) using forward- and reverse primers, with 5' overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-HIS vector to yield pAMAl-HI
  • alpina are reamplified as one fragment by PCR from the plasmid pAMAl-HIS-delta-5 using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmid pAMAl-MET-elo.
  • the resulting plasmid, pAMAl-MET-elo-delta-5 contains the delta-6 elongase and the delta-5 desaturase from M. alpina that are each under the control of an individual pgdA promoter from A. nidulans.
  • the sequence of the delta-6 elongase and the delta-5 desaturase is verified by sequencing of clones of pAMAl-MET- elo-delta-5.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 32 and 34.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989). Transformants are selected on minimal medium lacking methionine and histidine. A strain of A.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-HIS-delta-6 (example 32) and pAMAl-MET- elo-delta-5 (example 34), yielding the strain FSAN-delta-6 -elo -delta-5.
  • the gene encoding omega-3 desaturase from S. kluyveri is reamplified by PCR from the plasmid pESC-LEU-SK33 (example 4), using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A. nidulans.
  • the resulting plasmid, pAMAl-MET-SK33 contains the gene encoding omega-3 desaturase from S. kluyveri under the control of the gpdA promoter from A. nidulans.
  • the gpdA promoter and the gene encoding omega-3 desaturase from S. kluyveri are reamplified as one fragment by PCR from the plasmid pAMAl-MET-SK33 using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmid pAMAl-HIS-delta-6 (example 32).
  • the resulting plasmid, pAMAl-HIS-delta-6 - SK33 contains the genes encoding delta-6 desaturase from M. alpina and omega- 3 desaturase from S.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 34 and 36.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989).
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A is selected from minimal medium lacking methionine and histidine.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vector pAMAl-MET- elo-delta- 5 (example 34) and the vector pAMAl-HIS-delta-6 -SK33 (example 36), yielding the strain FSAN-elo -delta-5 -delta-6 -SK33.
  • cDNA is first prepared from M. alpina as described in example 18. The cDNA is then used as template for PCR, using primers designed to target the cytochrome b5 encoding gene (SEQ ID NO 1) and containing suitable 5' overhangs. The introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A. nidulans.
  • the resulting plasmid, pAMAl-MET-cytb5het contains the gene encoding cytochrome b5 from M. alpina under the control of the gpdA promoter from A. nidulans.
  • the gpdA promoter and the gene encoding cytochrome b5 from M. alpina are reamplified as one fragment by PCR from the plasmid pAMAl-MET-cytb5het using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmids pAMAl-HIS-delta-6 (example 32) or pAMAl-HIS-delta-6 -SK33 (example 36).
  • the resulting plasmid, pAMAl-HIS-delta-6 - cytb5het contain the genes encoding delta-6 desaturase from M. alpina and cytochrome b5 from M. alpina and the resulting plasmid, pAMAl-HIS-delta-6 - SK33-cytb5het, contain the genes encoding delta-6 desaturase from M.
  • alpina omega-3 desaturase from S. kluyveri 5 and cytochrome b5 from M. alpina. All the genes are under the control of an individual pgdA promoter from A. nidulans. The sequence of the gene encoding cytochrome b5 from M. alpina is verified by sequencing of clones of pAMAl-HIS- delta-6 - cytb5het and pAMAl-HIS-delta-6 - SK33-cytb5het.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989). Transformants are selected on minimal medium lacking methionine and histidine. A strain of A. oryzae that is auxotrophic for histidine and methionine is transformed with the vector pAMAl-HIS-delta-6 -
  • strain FSAN-elo -delta-5 -delta-6 -cytb5het the arachidonic acid producing strain FSAN-elo -delta-5 -delta-6 -cytb5het.
  • a strain of A. oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - SK33-cytb5het (example 38), yielding the eicosapentaenoic acid producing strain FSAN-elo -delta-5 -delta-6 -
  • S. kluyveri FAS2 (SEQ ID NO 6) in Aspergillus
  • the gene is amplified by PCR using genomic DNA from S. kluyveri as template.
  • the defined primers used for the amplification are designed to match the published sequence encoding FAS2 from S. kluyveri and contain suitable 5' overhangs.
  • the introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A. nidulans.
  • the resulting plasmid, pAMAl-MET-FAS2sk contains S.
  • the gpdA promoter and S. kluyveri FAS2 are reamplified as one fragment by PCR from the plasmid pAMAl-MET- FAS2sk using forward- and reverse primers, with 5' overhangs containing suitable restriction sites. The introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmids pAMAl-HIS-delta-6 (example 32) or pAMAl-HIS-delta-6 -SK33 (example 36).
  • the resulting plasmid, pAMAl-HIS- delta-6 - FAS2sk contain the genes encoding delta-6 desaturase from M. alpina and S. kluyveri FAS2 and the resulting plasmid, pAMAl-HIS-delta-6 - SK33- FAS2sk, contain the genes encoding delta-6 desaturase from M. alpina, omega-3 desaturase from S. kluyveri and S. kluyveri FAS2. All the genes are under the control of an individual pgdA promoter from A. nidulans. The sequence of the S. kluyveri FAS2 is verified by sequencing of clones of pAMAl-HIS-delta-6 - FAS2sk and pAMAl-HIS-delta-6 - SK33- FAS2sk.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 34 and 38.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989).
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A is selected from minimal medium lacking methionine and histidine.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - FAS2sk (example 40), yielding the arachidonic acid producing strain FSAN-elo -delta-5 -delta-6 - FAS2sk. Furthermore, in a separate experiment, a strain of A.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - SK33- FAS2sk (example 40), yielding the eicosapentaenoic acid producing strain FSAN-elo -delta-5 -delta-6 - SK33-FAS2sk.
  • cDNA is first prepared from M. alpina as described in example 19. The cDNA is then used as template for PCR, using primers designed to target the cytochrome b5 reductase (SEQ ID NO 3) and containing suitable 5' overhangs. The introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A. nidulans.
  • the resulting plasmid, pAMAl-MET-cytb5r contains the gene encoding a cytochrome b5 reductase from M. alpina under the control of the gpdA promoter from A. nidulans.
  • the gpdA promoter and the gene encoding a cytochrome b5 reductase from M. alpina are reamplified as one fragment by PCR from the plasmid pAMAl-MET- cytb5r using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmids pAMAl-HIS-delta-6 (example 32) or pAMAl-HIS-delta-6 -SK33 (example 36).
  • the resulting plasmid, pAMAl-HIS-delta-6 - cytb5r contain the genes encoding delta-6 desaturase from M. alpina and the gene encoding a cytochrome b5 reductase from M. alpina and the resulting plasmid, pAMAl-HIS- delta-6 - SK33- cytb5r, contain the genes encoding delta-6 desaturase from M. alpina, omega-3 desaturase from S. kluyveri and the gene encoding a cytochrome b5 reductase from M. alpina. All the genes are under the control of an individual pgdA promoter from A.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 34 and 42.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989).
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A is selected from minimal medium lacking methionine and histidine.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - cytb5r (example 42), yielding the arachidonic acid producing strain FSAN-elo -delta-5 -delta-6 - cytb5r. Furthermore, in a separate experiment, a strain of A.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - SK33- cytb5r (example 42), yielding the eicosapentaenoic acid producing strain FSAN-elo -delta-5 -delta-6 - SK33- cytb5r.
  • a fungal vector for expression of the gene encoding delta-6 desaturase from M. alpina and Aspergillus oryzae FASl in species belonging to the genus Aspergillus To isolate FASl from Aspergillus oryzae (SEQ ID NO 106), cDNA is first prepared from Aspergillus oryzae as described for Aspergillus nidulans in example 30. The cDNA is then used as template for PCR, using primers designed to target the A. oryzae FASl (SEQ ID NO 106) and containing suitable 5' overhangs.
  • restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A. nidulans.
  • the resulting plasmid, pAMAl-MET- FASlory contains the A. oryzae FASl under the control of the gpdA promoter from A. nidulans.
  • the gpdA promoter and the A. oryzae FASl are reamplified as one fragment by PCR from the plasmid pAMAl-MET-FASlory r using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmids pAMAl-HIS-delta-6 (example 32) or pAMAl-HIS-delta-6 -SK33 (example 36).
  • the resulting plasmid, pAMAl- HIS-delta-6 -FASlory contain the genes encoding delta-6 desaturase from M. alpina and the A. oryzae FASl and the resulting plasmid, pAMAl-HIS-delta-6 - SK33- FASlory, contain the genes encoding delta-6 desaturase from M. alpina, omega-3 desaturase from S.
  • A. oryzae FASl All the genes are under the control of an individual pgdA promoter from A. nidulans.
  • the sequence of the A. oryzae FASl is verified by sequencing of clones of pAMAl-HIS-delta-6 - FASlory and pAMAl-HIS-delta-6 - SK33- FASlory.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 34 and 44.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989).
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A is selected from minimal medium lacking methionine and histidine.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - FASlory (example 44), yielding the arachidonic acid producing strain FSAN-elo -delta-5 -delta-6 - FASlory. Furthermore, in a separate experiment, a strain of A.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - SK33- FASlory (example 44), yielding the eicosapentaenoic acid producing strain FSAN-elo -delta-5 -delta-6 - SK33- FASlory.
  • cDNA is first prepared from Aspergillus oryzae as described for Aspergillus nidulans in example 30.
  • the cDNA is then used as template for PCR, using primers designed to target the A. oryzae FAS2 (SEQ ID NO 108) and containing suitable 5' overhangs.
  • the introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A. nidulans.
  • FAS2ory contains the A. oryzae FAS2 under the control of the gpdA promoter from
  • the gpdA promoter and the A. oryzae FAS2 are reamplified as one fragment by PCR from the plasmid pAMAl-MET-FAS2ory r using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmids pAMAl-HIS-delta-6 (example 32) or pAMAl-HIS-delta-6 -SK33 (example 36).
  • the resulting plasmid, pAMAl- HIS-delta-6 -FAS2ory contain the genes encoding delta-6 desaturase from M.
  • alpina and the A. oryzae FAS2 and the resulting plasmid, pAMAl-HIS-delta-6 - SK33- FAS2ory contain the genes encoding delta-6 desaturase from M. alpina, omega-3 desaturase from S. kluyveri and the A. oryzae FAS2. All the genes are under the control of an individual pgdA promoter from A. nidulans. The sequence of the A. oryzae FAS2 is verified by sequencing of clones of pAMAl-HIS-delta-6 - FAS2ory and pAMAl-HIS-delta-6 - SK33- FAS2ory.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 34 and 46.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989).
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A is selected from minimal medium lacking methionine and histidine.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - FAS2ory (example 46), yielding the arachidonic acid producing strain FSAN-elo -delta-5 -delta-6 - FAS2ory. Furthermore, in a separate experiment, a strain of A.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - SK33- FAS2ory (example 46), yielding the eicosapentaenoic acid producing strain FSAN-elo -delta-5 -delta-6 - SK33- FAS2ory.
  • the plasmid, pAMAl-MET-FAS2ory contains the A. oryzae FAS2 under the control of the gpdA promoter from A. nidulans.
  • the gpdA promoter and the A. oryzae FAS2 are reamplified as one fragment by PCR from the plasmid pAMAl-MET-FAS2ory r using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmid pAMAl-MET- elo-delta-5 (example 34).
  • the resulting plasmid, pAMAl-MET- elo-delta-5 -FAS2ory contains the delta-6 elongase and the delta-5 desaturase from M. alpina and the A. oryzae FAS2. All the genes are under the control of an individual pgdA promoter from A. nidulans. The sequence of the A. oryzae FAS2 is verified by sequencing of clones of pAMAl-MET- elo- delta-5 -FAS2ory.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 44 and 48.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989).
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A is selected from minimal medium lacking methionine and histidine.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-HIS-delta-6 - FASlory (example 44) and pAMAl-MET- elo-delta-5 -FAS2ory (example 48), yielding the arachidonic acid producing strain FSAN-elo -delta-5 - FAS2ory-delta- 6 - FASlory. Furthermore, in a separate experiment, a strain of A.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-HIS-delta- 6 - SK33- FASlory (example 44) and pAMAl-MET- elo-delta-5 -FAS2ory (example 48), yielding the eicosapentaenoic acid producing strain FSAN-elo - delta-5 - FAS2ory-delta-6 - SK33- FASlory.
  • cDNA is first prepared from Aspergillus oryzae as described for Aspergillus nidulans in example 30. The cDNA is then used as template for PCR, using primers designed to target the the gene encoding NADH- cytochrome b-5 reductase from Aspergillus oryzae (SEQ ID NO 110) and containing suitable 5' overhangs. The introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A.
  • the resulting plasmid, pAMAl-MET-cyt5rory contains the gene encoding NADH-cytochrome b-5 reductase from A. oryzae under the control of the gpdA promoter from A. nidulans.
  • the gpdA promoter and the gene encoding NADH-cytochrome b-5 reductase from A. oryzae are reamplified as one fragment by PCR from the plasmid pAMAl-MET- cyt5rory using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmids pAMAl-HIS-delta-6 (example 32) or pAMAl-HIS-delta-6 -SK33 (example 36).
  • the resulting plasmid, pAMAl-HIS-delta-6 - cyt5rory contains the genes encoding delta-6 desaturase from M. alpina and the gene encoding NADH- cytochrome b-5 reductase from A. oryzae and the resulting plasmid, pAMAl-HIS- delta-6 - SK33- cyt5rory, contains the genes encoding delta-6 desaturase from M. alpina, omega-3 desaturase from S. kluyveri and the gene encoding NADH- cytochrome b-5 reductase from A. oryzae. All the genes are under the control of an individual pgdA promoter from A.
  • NADH-cytochrome b-5 reductase from A. oryzae is verified by sequencing of clones of pAMAl-HIS-delta-6 - cyt5rory and pAMAl-HIS-delta-6 - SK33- cyt5rory.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 34 and 50.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see, e.g., Sambrook et al., 1989).
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A is selected from minimal medium lacking methionine and histidine.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - cyt5rory (example 50), yielding the arachidonic acid producing strain FSAN-elo -delta-5 -delta-6 - cyt5rory. Furthermore, in a separate experiment, a strain of A.
  • oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - SK33- cyt5rory (example 50), yielding the eicosapentaenoic acid producing strain FSAN-elo -delta-5 -delta-6 - SK33- cyt5rory.
  • cDNA is first prepared from Aspergillus oryzae as described for Aspergillus nidulans in example 30.
  • the cDNA is then used as template for PCR, using primers designed to target the the gene encoding a cytochrome b5 from Aspergillus oryzae (SEQ ID NO 112) and containing suitable 5' overhangs.
  • the introduction of said restriction sites at the 5' and 3' ends of the gene allows ligation of the restricted PCR product into a digested pAMAl-MET vector that contains the gpdA promoter from A. nidulans.
  • the resulting plasmid, pAMAl-MET- cytb5 contains the the gene encoding a cytochrome b5 from A. oryzae under the control of the gpdA promoter from A. nidulans.
  • the gpdA promoter and the gene encoding a cytochrome b5 from A. oryzae are reamplified as one fragment by PCR from the plasmid pAMAl-MET- cytb5 using forward- and reverse primers, with 5' overhangs containing suitable restriction sites.
  • the introduction of said restriction sites at the 5' and 3' ends of the DNA fragment allows ligation of the restricted PCR product into the digested plasmids pAMAl-HIS-delta-6 (example 32) or pAMAl-HIS-delta-6 -SK33 (example 36).
  • the resulting plasmid, pAMAl-HIS-delta-6 - cytb5 contains the genes encoding delta-6 desaturase from M. alpina and the gene encoding a cytochrome b5 from A. oryzae and the resulting plasmid, pAMAl-HIS-delta-6 - SK33- cytb5, contains the genes encoding delta-6 desaturase from M. alpina, omega-3 desaturase from S. kluyveri and the gene encoding a cytochrome b5 from A. oryzae. All the genes are under the control of an individual pgdA promoter from A. nidulans.
  • the sequence of the gene encoding a cytochrome b5 from A. oryzae is verified by 5 sequencing of clones of pAMAl-HIS-delta-6 - cytb5 and pAMAl-HIS-delta-6 - SK33- cytb5.
  • Aspergillus oryzae strains are transformed with the vectors described in examples 34 and 52.
  • the transformation of the fungal cell is conducted in accordance with methods known in the art, for instance, by electroporation or by conjugation (see,
  • Transformants are selected on minimal medium lacking methionine and histidine.
  • a strain of A. oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - cytb5 (example 52), yielding the arachidonic acid producing strain FSAN-elo -delta-5 -delta-6 - cytb5.
  • a strain of A. oryzae that is auxotrophic for histidine and methionine is co-transformed with the vectors pAMAl-MET- elo- delta-5 (example 34) and pAMAl-HIS-delta-6 - SK33- cytb5 (example 52), yielding the eicosapentaenoic acid producing strain FSAN-elo -delta-5 -delta-6 - SK33- cytb5.
  • the recombinant strains described in examples 33, 35, 37, 39, 41, 43, 45, 47, 49, 30 51 and 53 can be grown in fermentors operated as batch, fed-batch or chemostat cultures. Batch and Fed-batch cultivations
  • the biomass Prior to lipid extraction, the biomass was separated through centrifugation for 5 minutes at 5000 rpm. The biomass was re-dissolved in 10-15 ml distilled water and the resulting cell suspension was broken using the glass bead method to generate cell extract.
  • the cell extract was prepared by addition of 1 ml glass beads with a particle size of 250-500 ⁇ m (Sigma-Aldrich, St Louis, Missouri) to 1 ml cell suspension in a micro tube with screw cap (Sarstedt, Germany). For each cell suspension 2 tubes were processed. The tubes were shaken at level 4 for 20 sec. in a FastPrep FP120 instrument (Qbiogene, France).
  • the cell extracts were combined in 2 ml eppendorf tubes by transferring 600 ⁇ l cell extract from each of the tubes to a eppendorf tube there by containing 1.2 ml glass bead free cell extract.
  • Fatty acid methyl esters were produced by acidic transmethylation.
  • FAME were analysed on a gas chromatograph (GC) (GC-2010, Shimadzu) coupled to a mass-selective-detector (MS) (GCMS-QP2010, Shimadzu)) and a flame- ionisation-detector (FID).
  • GC-MS-FID was operated with a split/splitless auto- injector (AOS-20i, Shimadzu) and GCMSsolution software, Lab solution (version 2.50, Shimadzu).
  • Sample injection volumes were 1 - 5 ⁇ l (2-6mg/mL) and the split ratio 10: 1 - 50: 1 operated at an injector temperature of 250°C. Number of rinses with sample prior to injection was 1 and after injection number of rinses with solvent was 5. Samples were split and components separated in parallel on two identical capillary columns (50mx0.25mmID, 0.25 ⁇ m film thickness) (CP-Select CB for FAME, Varian). One column was fitted to a mass spectrometer (MS-quadropole) and one to a flame-ionization-detector (FID) for identification/structural clarification and quantification, respectively. Helium was used as carrier gas and operated at a linear velocity of 36 cm/sec (18 cm/sec pr column). Purge flow was set at 5 3ml_/min.
  • MS-quadropole mass spectrometer
  • FID flame-ionization-detector
  • the MS was operated in the SCAN mode (46m/z-500m/z) using electronic 15 ionisation (EI) at 7OeV, with a scan speed of 1000 amu/sec and scan events every
  • Ionsource temperature was set at 105°C and interface temperature at
  • FA yield (mg FA/g DW) was determined by calculation based on the ISTD and divided by the dry weight (DW) of the biomass in 1 ml of the initial cell suspension.
  • Ostreococcus tauri delta 6-desaturase sequence (SEQ ID NO 150) was obtained from a sequence database (Pubmed), based on its accession number AY746357, found in literature. Using a backtranslation tool, the amino acid sequence (SEQ ID NO 131) was translated into a codon-optimized DNA sequence (SEQ ID NO: 130), with the options: standard genetic code, optimization according to S. cerevisiae codon usage, and discarding codons below 50% of theoretical ratio. The reverse complementing strand of the optimized gene sequence was obtained using a "reverse complement" tool (ex. from Saccharomyces genome database). The sequences of both strands were divided into oligomers 40 bp in size. The oligomers overlapped between the strands, and the outermost oligomers were smaller than 40 bp.
  • the oligomers were mixed together, and the assembly was done by PCR.
  • These primers introduced Notl and BgIII cloning sites upstream and downstream of the start and stop codons, respectively.
  • the desaturase sequence was cloned into the plasmid pESC-TRP (figure 18), using the same restriction sites, yielding pESC-TRP-OtD6 (figure 19).
  • the plasmid pESC-TRP-OtD6 was sequenced, and the two point mutations found in the desaturase gene sequence were corrected by site directed mutagenesis, using a multi site-directed mutagenesis kit (QuickChange® Multi Site-Directed Mutagenesis Kit, Stratagene).
  • Two forward (OtlT3_fwl and OtlT3_fw2, SEQ ID NO 153 and SEQ ID NO 154, respectively), and two reverse (OtlT3_rvl and OtlT3_rv2, SEQ ID NO 155 and SEQ ID NO 156, respectively) primers with approximately 45 bp were designed in a way that the point mutation should be close to the middle of the primer, with 10-15 bases of sequence on both sides. Both forward and both reverse primers anneal to the same strand of the template plasmid.
  • the mutagenesis reaction was carried out according to specified instructions.
  • the repaired delta 6 desaturase sequence was restricted from pESC-TRP-OtD6 with Notl and BgIII restriction enzymes and cloned into the vector pESC-TRP- delta-12 delta-6 (figure 12). Correct integration of the gene was verified by restriction reaction using Xmal.
  • a target sequence upstream of DCIl was amplified from genomic DNA by PCR using the primers DCIl-UP-fw (SEQ ID NO: 126) and DCIl-UP-rv (SEQ ID NO: 127) and a target sequence downstream of DCIl was amplified from genomic DNA by PCR using the primers DCIl-D-fw (SEQ ID NO: 128) and DCIl-D-rv (SEQ ID NO: 129).
  • the ADHl promoter/K.lactis URA3 fragment 1 was amplified by PCR using the plasmid pWAD2 as template and the primers AD-fw and Int3 ' , and was fused to the upstream target sequence by PCR using primers DCIl-UP-fw and Int3 ' .
  • the K. lactis URA3/ADH1 promoter fragment 2 was amplified by PCR using the plasmid pWADl as template and the primers Int5 ' and AD-rv.
  • SEQ ID NO: 130 was amplified from plasmid pESC-TRP-delta-12 delta-60t (Example 59) by PCR using the primers D6D-OTi-fw (SEQ ID NO: 132) and D6D-OTi-rv (SEQ ID NO: 133). This DNA fragment was fused to the target sequence downstream of DCIl by PCR using the primers D6D-OTi-fw and DCIl-D- rv.
  • the yeast strain FS01494 (MATa ura3-52 trpl-289 poxl : pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl) was transformed with the two linear fusion substrates described above and the K. lactis URA3/ADH1 promoter fragment 2. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. To select for looping out of the marker gene, the transformant was transferred to plates containing 5-FOA. Pop-out recombinants were streak-purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl dcil : :pADHl-OtD6D and was named FS01559.
  • the native yeast gene FOX2 (SEQ ID NO: 134) was deleted using a bi-partite gene targeting substrate (Example 3) according to the following procedure: A target sequence upstream of FOX2 was amplified from genomic DNA by PCR using the primers FOX2-UP-fw (SEQ ID NO: 136) and FOX2-UP-rv (SEQ ID NO: 137) and a target sequence downstream of FOX2 was amplified from genomic DNA by PCR using the primers FOX2-D-fw (SEQ ID NO: 138) and FOX2-D-rv (SEQ ID NO: 139).
  • the TDH3 promoter/K.lactis URA3 fragment 1 was amplified by PCR using the plasmid pW-TD2 as template and the primers T2-2 and Int3 ' , and was fused to the upstream target sequence by PCR using primers FOX2-UP-fw and Int3 ' .
  • the K. lactis URA3/TDH3 promoter fragment 2 was amplified by PCR using the plasmid pW-TDl as template and the primers Int5 ' and T-DGA.
  • alpina was amplified from the plasmid pESC-TRP-delta-12 delta-6 (Example 4) by PCR using the primers D12Di-fw (SEQ ID NO: 140) and D12Di-rv (SEQ ID NO: 141). This DNA fragment was fused to the target sequence downstream of FOX2 by PCR using the primers D12Di-fw and FOX2-D-rv.
  • the yeast strain FS01494 (MATa ura3-52 trpl-289 poxl : pTDH3-M. alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl) was transformed with the two linear fusion substrates described above and the K. lactis URA3/TDH3 promoter fragment 2. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. To select for looping out of the marker gene, the transformant was transferred to plates containing 5-FOA. Pop-out recombinants were streak-purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl fox2: :pTDH3-MaD12D and was named FS01557.
  • a target sequence upstream of POTl was amplified from genomic DNA by PCR using the primers POTl-UP-fw (SEQ ID NO: 144) and POTl-UP-rv (SEQ ID NO: 145) and a target sequence downstream of POTl was amplified from genomic DNA by PCR using the primers POTl-D-fw (SEQ ID NO: 146) and POTl-D-rv (SEQ ID NO: 147).
  • the ADHl promoter/K.lactis URA3 fragment 1 was amplified by PCR using the plasmid pWAD2 as template and the primers AD-fw and Int3 ' , and was fused to the upstream target sequence by PCR using primers POTl-UP-fw and Int3 ' .
  • the K. lactis URA3/ADH1 promoter fragment 2 was amplified by PCR using the plasmid pWADl as template and the primers Int5 ' and AD-rv.
  • alpina was amplified from the plasmid pESC-URA-elo-delta-5 (Example 4) by PCR using the primers D5Di-fw (SEQ ID NO: 148) and D5Di-rv (SEQ ID NO: 149). This DNA fragment was fused to the target sequence downstream of POTl by PCR using the primers D5Di-fw and POTl-D-rv.
  • the yeast strain FS01509 (MATalpha ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3- M. alpina OLEl pADHl-FASl pTPIl-MCRl) was transformed with the two linear fusion substrates described above and the K. lactis URA3/ADH1 promoter fragment 2. Transformants were selected on medium lacking uracil and were streak-purified on the same medium. To select for looping out of the marker gene, the transformant was transferred to plates containing 5-FOA. Pop-out recombinants were streak-purified on 5-FOA-containing medium.
  • the resulting strain had the genotype MATalpha ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3- M. alpina OLEl pADHl-FASl pTPIl-MCRl potl: :pADHl-MaD5D and was named FS01577.
  • alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl dcil : :pADHl-0tD6D was crossed to the strain FS01577 (MATalpha ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M.
  • alpina OLEl pADHl-FASl pTPIl- MCRl potl : :pADHl-MaD5D) Example 62. Diploids were allowed to sporulate, and the asci were dissected into spore tetrads.
  • alpina OLEl pADHl-FASl pTPIl-MCRl potl : :pADHl- MaD5D dcil : :pADHl-OtD6D was selected and named FS01578.
  • the strain FS01557 (MATa ura3-52 trpl- 289 poxl : :pTDH3-M. alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl fox2: :pTDH3-MaD12D) (Example 61) was crossed to the strain FS01577 (MATalpha ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M.
  • alpina OLEl pTPIl-MCRl pADHl-FASl pADHl-FAS2 fox2: :pTDH3-MaD12D potl : :pADHl-MaD5D was selected and named FS01580.
  • the strain FS01578 (MATa ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3- M.alpina OLEl pADHl-FASl pTPIl-MCRl potl : :pADHl-MaD5D dcil : :pADHl- OtD6D) (Example 63) was crossed to the strain FS01580 (MATalpha ura3-52 trpl- 289 poxl : :pTDH3-M.alpina OLEl pTPIl-MCRl pADHl-FASl pADHl-FAS2 fox2: :pTDH3-MaD12D pot
  • Diploids were allowed to sporulate, and the asci were dissected into spore tetrads. From the set of haploid strains derived from the cross, a strain with the genotype MATa ura3-52 trpl-289 poxl: :pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl fox2: :pTDH3-MaD12D potl : :pADHl-MaD5D dcil : :ADHlp-OtD6D was selected and named FS01591.
  • alpha OLEl pADHl-FASl pTPIl-MCRl potl : :pADHl-MaD5D dcil : :pADHl-OtD6D fox2: :pTDH3-MaD12D was selected from the same cross and was named FS01610.
  • K. lactis URA3 is used as a recyclable marker in the integration, and looping out of the marker gene is selected for by plating the intermediate strain on 5-FOA containing medium (figure 10). Targeting of the integration substrate to the desired location in the genome is performed, as described above, by means of PCR-generated target sequences.
  • Target sequences corresponding to a 300-500 bp sequence upstream of the native gene to be deleted and target sequences corresponding to a 300-500 bp sequence downstream of the native gene to be deleted are amplified from genomic yeast DNA using suitable primers.
  • the heterologous gene is amplified from a suitable source, e.g. a cDNA preparation, by PCR using primers with suitable 5' overhangs.
  • overhangs allow fusion of the heterologous gene with the downstream target sequence at the 5' end of the heterologous gene and fusion with the chosen promoter sequence (the ADHl promoter or the TDH3 promoter) at the 3' end of the heterologous gene.
  • the reverse primer used for amplification of the upstream target sequence contains a 5' overhang that allows fusion to fragment 1 described above.
  • the forward primer used for amplification of the downstream target sequence contains a 5' overhang that allows fusion with the heterologous gene.
  • a suitable ura3 yeast strain is transformed with 1) the upstream target fragment fused to fragment 1 described above, 2) fragment 2 described above, 3) the heterologous gene-downstream target fusion product.
  • Transformants are selected on medium lacking uracil, and contain, inserted between the upstream and downstream target sequences, two copies of the chosen promoter as a direct repeat on each side of the marker gene K. lactis URA3, followed by the heterologous gene.
  • the resulting ura3 strain contains, inserted between the upstream and downstream target sequences, the chosen promoter followed by the heterologous gene.
  • the medium for shake flask cultivations contained 20 g/L glucose, 15 g/L (NH4)2SO4, 1 g/L MgSO4-7H2O, 14.4 g/L KH2PO4, lmL/L vitamin solution and 1 mL/L trace metal solution.
  • the vitamin solution contained : 50 mg/L biotin, 1 g/L calcium panthotenate, 1 g/L nicotinic acid, 25 g/L myo-inositol, 1 g/L thiamine HCI, 1 g/L pyridoxal HCI and 0.2 g/L para-aminobenzoic acid, while the trace metal solution contained : 15 g/L EDTA, 4.5 g/L ZnSO4-7H2O, 1 g/L MnCI2-2H2O, 0.3 g/L CoCI2-6H2O, 0.4 g/L Na2MoO4-2H2O, 4.5 g/L CaCI2-2H2O, 3 g/L 5 FeSO4-7H2O, 1 g/L H3BO3 and 0.1 g/L KI.
  • the carbon source was autoclaved separately from the other medium components, and the vitamin and trace metal solutions were added following autoclavation by sterile filtration.
  • the yeast was inoculated at an initial cell density of approximately 0.1 (OD600) into 100 ml medium in 500 ml Erlenmeyer shake flasks with 2 baffles and was incubated at 30°C shaking at 150 rpm.
  • the medium for shake flask cultivations with galactose as carbon source was as described in Example 67, but with 20 g/L galactose as carbon source instead of glucose.
  • the carbon source was autoclaved separately from the other medium 20 components, and the vitamin and trace metal solutions were added following autoclavation by sterile filtration.
  • the pH of the medium was set to pH 6.5 prior to autoclavation.
  • Example 69 Cells were pre-grown in 500 ml Erlenmeyer shake flasks with 2 baffles in 100 mL 25 medium containing 5 g/L glucose, 20 g/L galactose and remaining medium components as specified in Example 67. Following depletion of glucose and while in the exponential growth phase on galactose, 1 ml of the pre-culture was transferred to 100 mL medium containing galactose as the sole carbon source in 500 ml Erlenmeyer shake flasks with 2 baffles. The shake flasks were incubated at 30 30°C with 150 rpm shaking.
  • Example 69 Example 69
  • a minimal growth medium with a molar C/N ratio of 15 was used.
  • the medium contained: 100 g/L glucose, 14.8 g/L (NH4)2SO4, 4.6 g/l KH2PO4, 2.8 g/l MgSO4 *7H2O, 2.4 ml/L vitamin solution and 2 ml/L trace metal solution.
  • the vitamin solution contained: 50 mg/L biotin, 1 g/L calcium panthotenate, 1 g/L nicotinic acid, 25 g/L myo-inositol, 1 g/L thiamine HCI, 1 g/L pyridoxal HCI and 0.2 g/L para- aminobenzoic acid, while the trace metal solution contained: 15 g/L EDTA, 4.5 g/L ZnSO4 7H2O, 1 g/L MnCI2-2H2O, 0.3 g/L CoCI2-6H2O, 0.4 g/L Na2MoO4-2H2O, 4.5 g/L CaCI2-2H2O, 3 g/L FeSO4-7H2O, 1 g/L H3BO3 and 0.1 g/L KI.
  • Example 70 Integration of M. alpina delta-6 elongase gene in the trp-289 locus of S. cerevisiae
  • MaD6E was amplified by PCR using the primers Eloa-fw-MF (SEQ ID NO: 114) and
  • Elo-T-rv (SEQ ID NO: 115) and using the plasmid pESC-URA-elo-delta-5 as template.
  • the primers contained 5' overhangs to allow fusion to the promoter (pPYKl) and target sequences (TRPl fragment 1).
  • the marker gene TRPl was amplified as to separate fragments by PCR using genomic DNA from a tryptophane-prototrophic strain of S.cerevisae as template.
  • TRPl fragment 1 consisted of 396 bp of the TRPl upstream region and the first 353 bp of the TRPl coding sequence, and was amplified using the primers TRPl-5end-fw (SEQ ID NO: 116) and TRPl-5end-rv (SEQ ID NO: 117), while TRPl fragment 2 consisted of pos 354 to 914 of the TRPl coding sequence and was amplified using primers TRPl-3end-fw (SEQ ID NO: 118) and TRPl-3end-rv (SEQ ID NO: 119).
  • TRPl- 3end-rv contained a 5' overhang allowing fusion to a PYKl promoter fragment.
  • the PYKl promoter fragment was amplified by PCR using genomic S.cerevisae DNA as template and using the primers PYKl-fw (SEQ ID NO: 120) and PYKl-rv (SEQ ID NO: 121). MaD6E was then fused to TRPl fragment 1 by PCR using primers Eloa-fw-MF and TRPl-5end-rv, resulting in Fusion fragment B (figure 15), while the PYKl promoter fragment was fused to TRPl fragment 2 by PCR using primers TRPl-3end-fw and PYKl-rv, resulting in fusion fragment A (figure 15).
  • the yeast strain FS01591 (MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpha OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl fox2: :pTDH3-MaD12D potl : :pADHl- MaD5D dcil : :ADHlp-OtD6D) was transformed with fusion fragments A and B, and transformants were selected on medium lacking tryptophane.
  • the genotype of the resulting strain FS01613 was MATa ura3-52 trpl-289-pPYKl-MaD6E-TRPl poxl: :pTDH3- M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl fox2: :pTDH3-MaD12D potl : :pADHl-MaD5D dcil : :pADHl-OtD6D.
  • FS01625 (MATalpha ura3-52 trpl-289 pTPIl-MCRl poxl : :pTDH3-M. alpha OLEl pTPIl-ACCl fox2: :pTDH3-MaD12D dcil : :pADHl-OtD6D potl : :pADHl-MaD5D gppl: :pHXT7-K.lactis URA3-pHXT7-S.kluyveri FAD3) was crossed to FS01613 (MATa ura3-52 trpl-289-pPYKl-MaD6E-TRPl poxl : :pTDH3-M.
  • FS01622 (MATa ura3-52 trpl-289-pPYKl-MaD6E-TRPl pTPIl-MCRl poxl : :pTDH3-M.alpina OLEl fox2: :pTDH3-MaD12D potl : :pADHl-MaD5D dcil : :pADHl-OtD6D gppl: :pHXT7-K.lactis URA3-pHXT7-S.kluyveri FAD3 pADHl-FASl pADHl-FAS2) was crossed to strain FS01620 (MATalpha ura3-52 trpl-289 Ieu2-3_112 poxl : :pTDH3-M.alpina OLEl pADHl-FASl fox2: :pTDH
  • FS01620 had previously been generated from a cross between FS01396 and FS01610. Following sporulation and dissection of the cross FS01622xFS01620, a strain was selected with the genotype MATa ura3-52 trpl- 289-pPYKl-MaD6E-TRPl poxl: :pTDH3-M.alpina OLEl fox2: :pTDH3-MaD12D potl : :pADHl-MaD5D dcil : :pADHl-OtD6D gppl: :pHXT7-K.lactis URA3-pHXT7- S.kluyveri FAD3 pADHl-FASl pADHl-FAS2 and was named FS01647.
  • the strain was transferred to 5-FOA containing medium to select for looping out of the K.lactis URA3 marker.
  • the resulting strain had the genotype MATa ura3-52 trpl- 289-pPYKl-MaD6E-TRPl poxl: :pTDH3-M.alpina OLEl fox2: :pTDH3-MaD12D potl : :pADHl-MaD5D dcil : :pADHl-OtD6D gppl: :pHXT7- S.kluyveri FAD3 pADHl- FASl pADHl-FAS2 and was named FS01648.
  • FS01648 was transformed with an empty pESC-URA vector in order to achieve prototrophy, and the transformed strain was named FS01658.
  • FS01639 has identical genetic background to FS01658 but additionally overexpresses MCRl from the TPIl promoter.
  • the strains were cultivated in triplicate in shake flasks with glucose as carbon source as described in Example 67, and were harvested for fatty acid analysis following depletion of the carbon source. Fatty acids were extracted, analyzed and quantified as described in examples 55-58.
  • results of the analysis show that MCRl overexpression results in an increased percentage of fatty acids with 2 or more double bonds in total fatty acid (Table 7). Specifically, the percentage of linoleic acid is increased from an average of 6.0% of total fatty acid to an average of 10.6 % of total fatty acid.
  • the EPA-producing strain FS01658 was cultivated in controlled chemostat cultivation using two different fermentation set-ups; one fermentor set-up (A) utilized two standard rushton turbine impellers (0 5.2 cm) supplied by the fermentor manufacturer (Biostat B, Sartorius), and in the other fermentor set-up (B) the lower rushton turbine was replaced by a custom-made rushton turbine with a larger diameter (0 7.2 cm).
  • the fermentors were run in chemostat mode as described in example 5, but at 30°C in stead of 17°C. Sterile air was supplied at a flow rate of 2.3 L/min and the stirring rate was set at 1000 rpm.
  • the medium was a mineral medium with 100 g/l glucose as described in example 69.
  • the dissolved oxygen concentration was around 42% in fermentor A and around 61% in fermentor B, indicating that the oxygen transfer in the cultivation broth was more efficient in the fermentor utilizing a custom-made large impeller.
  • Biomass samples for lipid analysis were taken at steady state and fatty acids were extracted, analyzed and quantified as described in Examples 55-58.
  • LROl was overexpressed with the PYKl promoter in a genetic background carrying deletion of POXl, overexpression of M.alpina OLEl and overexpressions of the native yeast genes FASl, FAS2 and MCRl.
  • the strain FS01539 overexpresses LROl in the mentioned genetic background and also expresses the pathway to ARA from high-copy plasmids.
  • the strain FS01490 (same genetic background but without CYB5 overexpression) was also analyzed under the same conditions. The strains were grown with galactose as carbon in chemostat cultivation as described in Example 5 and using the growth medium described in Example 6. Biomass samples were taken at steady state and lipids were extracted and analyzed as described in Examples 9 and 10.
  • Example 67 except that the concentration of glucose was 40g/L instead of 20g/L.
  • Cells were harvested in exponential phase for lipid extraction, and fatty acids were extracted, analyzed and quantified as described in examples 55-58.
  • the strain FS01639 (Mata ura3-52 trpl-289-pPYKl-MaD6E-TRPl pTPI-MCRl pox: :pTDH3 M.alpina OLEl fox2: :pTDH3-MaD12D potl : :pADHl-MaD5D dcil : :pADHl-0tD6D
  • the strain FS01640 over-expresses FAS2 alone (Mata ura3-52 trpl-289-pPYKl-MaD6E-TRPl pTPI-MCRl pox::pTDH3 M.alpina OLEl fox2::pTDH3-MaD12D potl: :pADHl-MaD5D dcil: :pADHl-OtD6D gppl::pHXT7-KLURA-pHXT7-S.
  • Table 10 Fatty acid composition (% of total fatty acid) of strains with genes encoding the EPA-pathway integrated in the genome of S. cerevisiae strains and 5 either over-expressing FASl, FAS2 or the FAS1/FAS2 complex.
  • the FAS1/FAS2 complex is over-expressed in strain FS01639, FAS2 in FS01640 and FASl in FS01641.
  • FS01542 (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl opil : :R [pESC-TRP-dl2d-d6d] [pESC-URA-d6elo-d5d]) was analyzed using FS01490 (MATa ura3-52 trpl-289 poxl : :pTDH3-M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl [pESCTRP-dl2d-d6d] [pESCURA-d6e-d5d]) as reference.
  • the deletion of OPIl increased the lipid yield to an average of 11.46% of dry weight (absolute SD 1.02%) compared with an average of 7.99% of dry weight
  • Plasmid pSF002 expressing INO4 from the GALl promoter in vector pESC-TRP Construction of plasmid pSF002 expressing INO4 from the GALl promoter in vector pESC-TRP
  • the coding sequence of INO4 was isolated by PCR from genomic DNA from yeast strain CEN-PK using gene specific primers 7003 (SEQ ID NO: 164) and 7004 (SEQ ID NO: 165).
  • Forward primer 7003 contains a BamHl cloning site upstream of the ATG.
  • Reverse primer 7004 contains a Xhol cloning site downstream of the stop codon.
  • the PCR reaction was purified with a PCR clean-up kit (Macherey-Nagel).
  • Vector p-ESC-TRP (Stratagene) and the PCR reaction were digested with restriction enzymes BamHl and Xhol (New England Biolabs) in reaction buffer 3, purified from an agarose gel using a gel-extraction clean-up kit (Macherey-Nagel) and ligated together.
  • the coding sequence of INO2 was isolated by PCR from genomic DNA from yeast strain CEN-PK using gene specific primers 7001 (SEQ ID NO: 162) and 7002 (SEQ ID NO: 163) described below.
  • Forward primer 7001 contains a BamHl cloning site upstream of the ATG.
  • Reverse primer 7002 contains a Xhol cloning site downstream of the stop codon.
  • the PCR reaction contained: 35 microliter water, 10 microliter 5xHF buffer, 1 microliter primer 7001 20 micromolar stock, 1 microliter primer 7002 20 micromolar stock, 2 microliter genomic DNA template at ca 50 nanogram/microliter, 1 microliter Phusion polymerase (Finnzymes) at 2 units/microliter.
  • the PCR program was: 98C 5 minutes, 1 cycle; 98C 30 seconds, 68C 30 seconds, 72C 30 seconds, 10 cycles; 95C 30 seconds, 6OC 30 seconds, 72C 30 seconds, 20 cycles; 72C 5 minutes. 1 cycle.
  • the PCR reaction was purified with a PCR clean-up kit (Macherey-Nagel).
  • Vector p-ESC-URA (Stratagene) and the PCR reaction were digested with restriction enzymes BamHl and Xhol (New England Biolabs) in reaction buffer 3, purified from an agarose gel using a gel-extraction clean-up kit (Macherey-Nagel) and ligated together.
  • Plasmids pSF002 and pSF003 were transformed together into yeast strains FS01494 (MATa ura3-52 trpl-289 poxl : :pTDH3-M. alpha OLEl pADHl-FASl PADH1-FAS2 pTPIl-MCRl), FS01541 (MATa ura3-52 trpl-289 poxl : :pTDH3- M.alpina OLEl pADHl-FASl pADHl-FAS2 pTPIl-MCRl opil : :R ) and FS01567 (MATalpha ura3-52 trpl-289 poxl : :pTDH3-M.alpina olel pADHl-FASl pTPIl- MCRl pTPIl-ACCl) selecting for growth on SC media lacking tryptophan and uracil. The same strains were also
  • INO2 and INO4 gene products positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INOl promoter. J Biol Chem 269: 15344-15349

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Abstract

L'invention concerne la production d'acides gras et, plus particulièrement, la production d'acide arachidonique (ARA) et d'acide eicosapentanoïque (EPA) d'acides gras polyinsaturés (PUFA) dans des cellules fongiques génétiquement modifiées, notamment, des cellules de Saccharomyces cerevisiae métaboliquement modifiées avec une teneur élevée en ARA et en EPA. L'invention se rapporte particulièrement à l'accroissement de la teneur en PUFA dans l'organisme hôte au moyen, par exemple, de diverses sur-expressions du cytochrome b5 et de la réductase du cytochrome b5 impliquées dans la désaturation d'acide gras, et de l'expression hétérologue du cytochrome b5 et de la réductase de cytochrome b5 ainsi que de l'expression de synthases d'acides gras hétérologues.
PCT/DK2007/050079 2006-06-29 2007-06-29 Cellules fongiques métaboliquement modifiées à teneur élevée en acides gras polyinsaturés WO2008000277A2 (fr)

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US60/817,643 2006-06-29

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WO2008000277A2 true WO2008000277A2 (fr) 2008-01-03
WO2008000277A3 WO2008000277A3 (fr) 2008-03-20
WO2008000277A8 WO2008000277A8 (fr) 2009-07-30

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009080630A2 (fr) * 2007-12-21 2009-07-02 Fluxome Sciences A/S Aliments et boissons à teneur accrue en acides gras polyinsaturés
EP2108702A1 (fr) * 2008-04-07 2009-10-14 Organo Balance GmbH Synthèse d'acides gras polyinsaturés dans la levure
FR2940315A1 (fr) * 2008-12-19 2010-06-25 Airbus France Nouveau procede de culture de levures du genre yarrowia
EP3075848A1 (fr) * 2015-04-01 2016-10-05 Johann Wolfgang Goethe-Universität Frankfurt am Main Production microbiologique d'acides gras courts et leurs utilisations
US11186616B2 (en) 2017-05-02 2021-11-30 Korea Research Institute Of Chemical Technology Increased production of ginsenosides through yeast cell organelle improvement
CN114478727A (zh) * 2022-01-17 2022-05-13 华中科技大学 高山被孢霉油脂合成相关转录因子ino4及其编码基因与应用
CN114555777A (zh) * 2019-07-26 2022-05-27 诺维信公司 经修饰的丝状真菌宿主细胞
WO2022238404A1 (fr) * 2021-05-10 2022-11-17 Biophero Aps Procédés et cellules améliorés pour augmenter l'activité enzymatique et la production de phéromones d'insectes

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EP0978563A1 (fr) * 1997-02-27 2000-02-09 Suntory Limited Gene de delta 9-desaturase
WO2005118814A2 (fr) * 2004-06-04 2005-12-15 Fluxome Sciences A/S Cellules metaboliquement transformees pour la production d'acides gras polyinsaturates

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EP0978563A1 (fr) * 1997-02-27 2000-02-09 Suntory Limited Gene de delta 9-desaturase
WO2005118814A2 (fr) * 2004-06-04 2005-12-15 Fluxome Sciences A/S Cellules metaboliquement transformees pour la production d'acides gras polyinsaturates

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BEAUDOIN FREDERIC ET AL: "Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 97, no. 12, 6 June 2000 (2000-06-06), pages 6421-6426, XP002200201 ISSN: 0027-8424 *
VEEN M ET AL: "Production of lipid compounds in the yeast Saccharomyces cerevisiae." APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 63, no. 6, February 2004 (2004-02), pages 635-646, XP002405982 ISSN: 0175-7598 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009080630A2 (fr) * 2007-12-21 2009-07-02 Fluxome Sciences A/S Aliments et boissons à teneur accrue en acides gras polyinsaturés
WO2009080630A3 (fr) * 2007-12-21 2009-12-10 Fluxome Sciences A/S Aliments et boissons à teneur accrue en acides gras polyinsaturés
EP2108702A1 (fr) * 2008-04-07 2009-10-14 Organo Balance GmbH Synthèse d'acides gras polyinsaturés dans la levure
WO2009124694A2 (fr) * 2008-04-07 2009-10-15 Organobalance Gmbh Synthèse d'acides gras polyinsaturés dans une levure
WO2009124694A3 (fr) * 2008-04-07 2010-01-14 Organobalance Gmbh Synthèse d'acides gras polyinsaturés dans une levure
WO2010076432A1 (fr) * 2008-12-19 2010-07-08 Airbus Operations Nouveau procédé de culture de levures du genre yarrowia
FR2940315A1 (fr) * 2008-12-19 2010-06-25 Airbus France Nouveau procede de culture de levures du genre yarrowia
EP3075848A1 (fr) * 2015-04-01 2016-10-05 Johann Wolfgang Goethe-Universität Frankfurt am Main Production microbiologique d'acides gras courts et leurs utilisations
WO2016156548A1 (fr) * 2015-04-01 2016-10-06 Johann Wolfgang Goethe-Universität Frankfurt am Main Production microbiologique d'acides gras à chaine courte et utilisations associées
US11078468B2 (en) 2015-04-01 2021-08-03 Johann Wolfgang Goethe-Universität Frankfurt am Main Microbiological production of short fatty acids and uses thereof
US11186616B2 (en) 2017-05-02 2021-11-30 Korea Research Institute Of Chemical Technology Increased production of ginsenosides through yeast cell organelle improvement
CN114555777A (zh) * 2019-07-26 2022-05-27 诺维信公司 经修饰的丝状真菌宿主细胞
WO2022238404A1 (fr) * 2021-05-10 2022-11-17 Biophero Aps Procédés et cellules améliorés pour augmenter l'activité enzymatique et la production de phéromones d'insectes
CN114478727A (zh) * 2022-01-17 2022-05-13 华中科技大学 高山被孢霉油脂合成相关转录因子ino4及其编码基因与应用

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WO2008000277A8 (fr) 2009-07-30

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