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Definitions

• the present invention relates to mutant yeasts capable of producing an unusual fatty acid by bioconversion or neosynthesis and the use of these yeasts for the production of this unusual fatty acid.
• Industrial fatty acids come mainly from mineral oils or vegetable oils. These industrial fatty acids are intended for various applications, such as food, paints and varnishes, lubricants, waterproofing agents, plastics and polymers. They may be of very diverse structures, for example polyunsaturated fatty acids, with very short or very long chains, having hydroxyl or epoxy groups, or having one or more conjugated double or triple bonds.
• the cost of obtaining industrial fatty acids from mineral oils is expensive because of the increasing cost of the raw material and the chemical treatment costs of these mineral oils.
• Some plant species produce fatty acids with interesting properties in the industrial field but these species are generally wild, exotic and / or non-agronomic species.
• microorganisms especially yeast strains
• production of industrial fatty acids represents an alternative to the use of fossil and plant resources.
• an unusual fatty acid is a fatty acid that is not naturally synthesized by it.
• Ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid, C18: 1-OH) is an omega-9-hydroxylated fatty acid, which is unusual in yeasts.
• Ricinoleic Acid (RA) and its derivatives have several industrial applications, for example in food as additives, textiles as surfactants or pigment-wetting agents, paper as antifoam agents or waterproofing additives. , plastics for the manufacture of Nylon-1 1, plasticizers, tubes or films, perfumes and cosmetics as emulsifiers or deodorants, electronics for the manufacture of capacitor fluids, polyurethane or resins of polyamide, pharmaceuticals, paints, inks, adhesives and lubricants.
• ricinoleic acid has been proposed as a component of biodiesel and as a lubricant additive to replace sulfur-based lubricant compounds in petroleum diesel.
• Ricinoleic acid accounts for about 90% of the total fatty acids of castor beans (Ricinus communia) (Yamamoto et al., 2008).
• the high ricinoleic acid content in castor oil combined with high oil content in castor beans and the multitude of ricinoleic acid applications, makes castor oil a high-value oilseed crop.
• a major disadvantage to extensive castor culture is the high content in its seeds of ricin, an extremely toxic protein (Knight, 1979). The use of ricin has long raised public health concerns.
• the genetic engineering strategy in these model species consists of a heterologous expression of castor oleate ( ⁇ 12) hydroxylase or Claviceps purpurea, encoded by the gene FAH12, Indeed, during the biosynthesis of ricinoleic acid, the oleic acid is hydroxylated by the Fahl2p enzyme in the sn-2 position of phosphatidylcholine mainly in the endoplasmic reticulum (Bafor et al., 1991). The hydroxylation takes place at the 12-position of the esterified oleic acid.
• Oleate desaturases and oleate hydroxylases belong to the same family of membrane proteins and have a similar peptide sequence and function. They share the same oleic substrate and their reactions are very competitive (Broun et al., 1998). Oleate ( ⁇ 12) desaturase (FAD2) preferentially forms linoleic acid and oleate hydroxyase preferably forms oleic acid.
• RcFAH12 and Ricinus communis acyl-CoA: diacylglycerol acyltransferase leads to an increase in the accumulation of hydroxylated fatty acids up to 30% of the total fatty acids in the seeds, but this represents an ever lower content than that found in castor seeds (Lu et al, 2006, Burgal et al, 2008). It is nevertheless necessary to overexpress at least one acyl-CoA: diacylglycerol acyltransferase (DGA) in these plants.
• DGA diacylglycerol acyltransferase
• pombe yeasts which do not contain genes encoding oleate ( ⁇ 12) desaturases - the heterologous expression of C. purpurea oleate ( ⁇ 12) hydroxyase CpFAH12) leads to an accumulation of ricinoleic acid at 8% and 53% of total fatty acids (Mavraganis et al., 2010, Holic et al, 2012).
• Pacyl-CoA diacylglycerol acyltransferase of C. purpurea (CpDGAT2)
• RcFAH12 the ricinoleic acid content increases slightly up to 10% of total fatty acids.
• Vernolic acid (12,13-epoxy-9-cis-octadecenoic acid, 3 ⁇ 4 ⁇ 32 0 3 ) is an epoxidized omega-9 fatty acid, which is also unusual in yeasts. Vernolic acid has several industrial applications, for example in glues, paints, plastics, inks, textiles and the pharmaceutical industry. Vernolic acid represents at least 60% of the total fatty acids of the seeds of Vernonia galamensis and Euphorbia lagascae.
• Some oleaginous microorganisms are capable of converting substrates, such as fats, sugars or glycerol, into lipids, especially triglycerides and fatty acids. These oleaginous microorganisms have the capacity to accumulate significant amounts of lipids, at least 20% of their dry matter.
• yeasts there are a few oleaginous species, so-called unconventional, among which we can mention those belonging to the genera Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon or Yarrowia (see for reviews Beopoulos et al, 2009, Papanikolaou et al. , 201 and 201 lbs).
• Yarrowia lipofytica is a hemiascomycete yeast. It is considered as a model of bioconversion for the production of proteins, enzymes and lipid derivatives (see for review Nicaud, 2012). It is naturally present in polluted environments of oil and in particular in the heavy fractions. Y. lipofytica is one of the most studied oleaginous yeasts because of its ability to accumulate lipids up to more than 50% of its dry matter according to a defined culture profile, or even more than 80% of its material.
• Y. lipofytica is also able to accumulate more than 90% of neutral lipids, in the form of triacylglycerols (TAG).
• TAG triacylglycerols
• lipofytica can be efficiently cultured on a wide variety of hydrophobic compounds (free fatty acids, triacylglycerols, n-alkanes, etc.) as the sole source of carbon and energy, through the expression of multigene families encoding enzymes. key involved in the decomposition of these compounds (eg, acyl-CoA oxidases, lipases) (Papanikolaou et al., 2001, Beopoulos et al, 2009a, Papanikolaou et al, 2010, 201 1a and 201 lb). The synthesis of lipids in Y.
• lipofytica is carried out either by the de novo biosynthesis of fatty acids via the production of fatty acid precursors such as acetyl-CoA and raalonyl-CoA and their integration in the pathway of lipid biosynthesis (Kennedy's pathway), either by the ex novo accumulation, via the incorporation of the pre-existing fatty acids in the fermentation medium or deriving from the hydrolysis of the oils, fats, triglycerides and methyl esters of the culture medium and their accumulation inside the cell.
• the main pathways of de novo lipid biosynthesis in Y. lipolytica and Saccharomyces cerevisiae (S. cerevisiae, so-called non-oleaginous yeast) are well preserved.
• the genes involved in the metabolism of fatty acids in yeasts, in particular in Y. lipolytica are described in Beopoulos et al (2009) and International Application WO 2010/004141.
• ⁇ -oxidation is a pathway of fatty acid degradation that occurs only in peroxisomes. This pathway allows the formation of acetyl-CoA from even-chain fatty acids and propionyl-CoA from odd-chain fatty acids.
• the ⁇ -oxidation comprises four successive reactions during which the carbon chain of the acyl-CoA is reduced by two carbon atoms. Once the reaction has been performed, the reduced acyl-CoA of two carbons can return to the spiral of ⁇ -oxidation (Lynen's helix) and undergo a further reduction of two carbons.
• acyl-CoA decarboxylation cycles may be interrupted depending on the nature of the acyl-CoA, the availability of substrate, the presence of coenzyme A, acetyl-CoA or the NAD + / NADH ratio.
• TAG triacylglycerol fatty acids
• the active form of acyl-CoA formed is oxidized by a molecule of Flavin Adenine Dinucleotide (F AD) to form a trans-A 2 -enoyl-CoA molecule by means of an acyl-CoA oxidase (AOX).
• F AD Flavin Adenine Dinucleotide
• ⁇ -oxidation in Y, lipolytica has been widely described (Wang et al, 1999b, Mlickova et al 2004).
• the trans-A 2 -enoyl-CoA is then hydrated with 2-enoyl-CoA hydratase.
• the formed 3-hydroxyacyl CoA molecule is oxidized by NAD + to form a 3-ketoacyl-CoA molecule.
• lipolytica in which the beta-oxidation of fatty acids is invalidated due to the deletion of the 6 endogenous POX genes have been described by Beopoulos et al (2008) and in International Application WO 2012/001144. strains mutants show an increase in lipid accumulation relative to the parent strains.
• Y. lipolytica is currently used for the industrial processing of ricinoleic acid into ⁇ -decalactone, an aromatic compound with fruity and oily notes that can be found naturally in fruit and fermented foods (Schrader et al. , 2004).
• the ⁇ -oxidation pathway responsible for the degradation of lipid reserves, was abolished by the deletion of the 6 POX genes coding for the 6 endogenous Acyl-CoA oxidases (AOX1 to AOX6).
• the accessibility of the oleic substrate in the form of phospholipids for an enzyme was facilitated by the invalidation of the 3 genes encoding endogenous triacylglycerol acyltransferases (DGA1, DGA2, LRO1), in order to prevent the storage of oleic acid in the form of TAG.
• DGA1, DGA2, LRO1 endogenous triacylglycerol acyltransferases
• lipolytica matrix containing the poxl-6 ⁇ , d ⁇ gA, dga2A, lrolAfad2 genetic modifications is named JMY2159. It is incapable of degrading oleic acid, storing it in the form of triglycerides and converting it by desaturation to linoleic acid.
• JMY2159 matrix strain From this JMY2159 matrix strain, the inventors then obtained genetically modified Y. lipolytica yeast mutant strains possessing a considerable capacity for lipid accumulation and capable of synthesizing ricinoleic acid up to more than 7% of their concentration. dry matter.
• This rate of ricinoleic acid production of strain JMY2556 can be further increased by increasing the copy number of the CpFAH12 and YlLRO1 genes, or by inhibiting the expression of the endogenous 2-methylcitrate dehydratase of the strain and / or overexpressing a gene.
• acylglycerol acyltransferase monoacylglycerol acyltransferase, patatin-like triacylglycerol lipase, at least one of the two subunits of PATP citrate lyase, diacylglycerol: choline-O phosphotransferase, ethanolamine phosphotransferase, phospholipase A 2 , an acyl-CoA: lysophosphatidylcholine acyltransferase, a cytochrome-b 5 reductase, inositol / phosphatidyl inositol phosphatase and elongase.
• genetically modified Y. lipolytica yeast mutant strains capable of synthesizing ricinoleic acid are also capable of secreting it.
• This property of these mutant strains has an advantage for the production of ricinoleic acid in large quantities (it is not necessary to lyse the cells to obtain ricinoleic acid).
• Yarrowia lipolytica can also be done in other oleaginous yeasts. These mutant oleaginous yeast strains can be used as alternatives to ricinoleic acid production by castor, as they are easy to grow, irrespective of season and climate.
• the subject of the present invention is therefore a process for obtaining a mutant strain of oleaginous yeast useful as a yeast matrix strain for obtaining other mutant strains of oleaginous yeast comprising:
• Steps (a) to (d) of the method according to the present invention can be implemented in any order, simultaneously or sequentially.
• step (a) above the inhibition of beta-oxidation of fatty acids defined in step (a) above is obtained by:
• Oleaginous yeast strains are well known to those skilled in the art. They have the ability to accumulate significant amounts of lipids, at least 20% of their dry matter (Ratledge, 1994). They generally belong to the genus Candida, Cryptococcus, Lipomyces, Rhodosporidium (e.g., Rhodosporidium toruloides), Rhodotorula (e.g., Rhodotura tinota), Trichosporon or Yorrowia.
• Lipomyces starkeyi see Joint Genome institute (JGI) and Rhodosporidium toruloides, see Kumar et al (2012) and Zhu et al. (2012).
• JGI Joint Genome institute
• Rhodosporidium toruloides see Kumar et al (2012) and Zhu et al. (2012).
• a more particularly preferred strain in the sense of the present invention is a Yarrowia yeast strain, more preferably Yarrowia lipolytica.
• yeast strain is meant a yeast strain from which other genetic modifications can be made in said strain.
• Inhibition of the expression or activity of an enzyme defined in the present invention may be total or partial. It can be obtained in various ways by methods known in themselves to those skilled in the art.
• this inhibition can be obtained by mutagenesis of the gene encoding said enzyme.
• Mutagenesis of the gene coding for said enzyme may occur at the level of the coding sequence or sequences for regulating the expression of this gene, in particular at the level of the promoter, leading to an inhibition of the transcription or translation of said enzyme.
• Mutagenesis of the gene encoding said enzyme can be performed by genetic engineering. For example, it is possible to delete all or part of said gene and / or to insert an exogenous sequence. Methods for deleting (deleting) or inserting a given genetic sequence into yeast, particularly Y. polytica, are well known to those skilled in the art (see for review Barth and Gaillardin, 1996, Madzak et al, 2004).
• POP IN / POP OUT which has been used in yeasts, particularly in Y, lipolytica, for the deletion of the LEU2, URA3 and XPR2 genes (Barth and Gaillardin, 1996). It is also possible to use the SEP method (Maftahi et al., 1996) which has been adapted from Y. lipolytica for the deletion of FOX genes (Wang et al., 1999b).
• SEP / Cre method developed by Fickers et al (2003) and described in International Application WO 2006/064131.
• An advantageous method according to the present invention consists in replacing the coding sequence of the gene coding for said enzyme with an expression cassette containing the sequence of a gene coding for a selection marker. It is also possible to introduce one or more point mutations in the gene coding for said enzyme, having the consequence of shifting the reading frame and / or of introducing a stop codon into the sequence and / or of inhibiting the transcription or translation of the gene encoding said enzyme.
• Mutagenesis of the gene coding for said enzyme can also be carried out using physical agents (for example radiation) or chemical agents. This mutagenesis also makes it possible to introduce one or more point mutations in the gene coding for said enzyme.
• the mutated gene coding for said enzyme may be identified, for example, by PCR using primers specific for said gene.
• selection markers for the complementation of an auxotrophy also commonly referred to as auxotrophy markers, are well known to those skilled in the art.
• the selection marker URA3 is well known to those skilled in the art. More specifically, a yeast strain including the URA3 gene (sequence available in Genolevures databases (http://genolevures.org) under the name YALI0E26741g or UniProt under access number Q12724), encoding orotidine- S'-phosphate decarboxylase, is inactivated (eg by deletion), will not be able to grow on a medium not supplemented with uracil.
• the integration of the selection marker URA3 in this yeast strain will then restore the growth of this strain on a medium lacking uracil.
• the selection marker LEU2 described in particular in US Pat. No. 4,937,189 is also well known to those skilled in the art. More specifically, a yeast strain whose LEU2 gene (YALI0C00407g), encoding ⁇ -isopropylmalate dehydrogenase, is inactivated (for example by deletion), will not be able to grow on a medium not supplemented with leucine. As previously, the integration of the selection marker LEU2 in this yeast strain will then restore the growth of this strain on a medium not supplemented with leucine.
• the ADE2 selection marker is also well known to those skilled in the field of yeast transformation.
• said mutant yeast strain is auxotrophic for leucine (Leu " ) and optionally for Porotidine-5 '-phosphate decarboxylase (Ura " ).
• 3 POX1, POX2, POX3, POX4, POX5 and POX6 genes encode 6 isoforms of acyl-coenzymeA oxidases (AOX, EC 6.2.1.3) involved, at least partially, in the ⁇ -oxidation of fatty acids. Inhibition, partial or total, of the expression or the activity of these isoenzymes leads to the accumulation by yeasts of dodecanedioic acid, without consumption of the accumulated lipids. More particularly, the coding sequence of the POX1-6 genes and the peptide sequence of AOX1-6 of Y. lipolytica CLIB122 are available in Genolevures databases (http://genolevures.org/) or GenBank under access numbers.
• POX1 I AOX1 YALI0E32835g / YALI0E32835p
• POX2I AOX2 YAL10F10857g / YAL10F10857p
• POX3! AOX3 YALI0D24750g / YALI0D24750p
• POX4 / AOX4 YALI0E27654g / YALI0E27654p
• POX5 I AOX5 YALIOC23859g / YALI0C23859p
• lipolytica acyl-CoA oxidases have 45% identity or 50% similarity with those of other yeasts.
• the degree of identity between the acyl-CoA oxidases varies from 55 to 70% (or 65 to 76% similarity) (International Application WO 2006/064131).
• a method of inhibiting the expression of Endogenous AOX in a strain of Y. lipolytica has been described in International Applications WO 2006/064131, WO 2010/004141 and WO 2012/001144,
• the multifunctional enzyme has three domains: two domains with 3-hydroxyacyi-CoA dehydrogenase activity (EC 4.2.1.74, domains A and B) and a domain with enoyl-CoA hydratase activity (EC 4.2. 1.17, domain C).
• This enzyme is encoded by the gene MFE1 ("Multifunctional enzyme type 1"). More particularly, the coding sequence of the MFE1 gene and the peptide sequence of Y. lipolytica CLIB122 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase are available in Genolevures or GenBank databases under the access number or the name. next: YALIOE 15378g / YALI0E15378p. A method of inhibiting the expression of said endogenous multifunctional enzyme in a strain of Y, lipolytica has been described by Haddouche et al. (201 1).
• 3-oxoacyl-coenzyme A thiolase (E.C. 2.3.1.16) is encoded by the gene POT1 ("Peroxisomal Oxoacyl Thiolase 1"). More particularly, the coding sequence of the POT1 gene and the peptide sequence of the 3-oxoacyl-CoA thiolase of Y. lipolytica CLIB122 are available in the Genolevures or GenBank databases under the accession number or the following name: YALIO 18568g / YALIO 18568p. A method of inhibiting the expression of endogenous 3-oxoacyl-coenzyme A thiolase in a Y. lipolytica strain has been described by Berninger et al. (1993).
• acyl-CoA diacylglycerol acyltransferases
• DGA1 and DGA2 are encoded by two genes: DGA1 and DGA2 (Beopoulos et al, 2009 and 2012, International Application WO 2012/001144). More particularly, the coding sequence of the DGA1 gene and the peptide sequence of the acyl-CoA: diacylglycerol acyltransferases 1 of Y. lipolytica CLIB122 are available in the Genolevures or GenBank databases under the accession number or the following name: YALI0E32769g / YALI0E32769p. The coding sequence of the DGA2 gene and the peptide sequence of the acyl-CoA: diacylglyceryl acyltransferases 2 of Y.
• lipolytica CLIB122 are available in the Génolevures or GenBank databases under the accession number or the following name: YALI0D07986g / YALI0D07986p.
• Rhodoturula glutanis an acyl-CoA: diacylglycerol acyltransferase has been described by Rani et al. (2013).
• a method of inhibiting the expression of one or both endogenous DGATs (DGAT1 and / or DGAT2) in a Y. lipolytica strain has been described by Beopoulos et al (2012).
• yeasts naturally have a gene encoding an oleate ( ⁇ 12) desaturase (EC 1.14.19.6) which is encoded by the FAD2 gene.
• Y. lipolytica possesses this gene whereas S. this evisiae (which is not considered as an oleaginous yeast) does not possess it (Ratledge, 2004, Beopoulos et al, 2008).
• the coding sequence of the FAD2 gene and the peptide sequence of the oleate ( ⁇ 12) desaturase of Y. lipolytica CLIB122 are available in the Genolevures or GenBank databases under the access number or the following name: YALIOB10153g / YALI0B10153p . If the yeast strain does not possess a gene encoding an oleate ( ⁇ 12) desaturase, then step (c) of the method according to the present invention will not be implemented.
• phospholipid: diacylglycerol acyltransferase (PDAT, E.C. 2.3.1.158) is encoded by the LRO1 gene (Beopoulos et al, 2009 and 2012; International Application WO 2012/001144). More particularly, the coding sequence of the LRO1 gene and the peptide sequence of the phospholipid: diacylglycerol acyltransferase of Y. lipolytica CLIB122 are available in the Genolevures or GenBank databases under the accession number or the following name: YALI0E16797g / YALI0E16797p. A method of inhibiting endogenous PDAT in a Y. lipolytica strain has been described by Beopoulos et al (2012).
• the present invention also relates to a process for obtaining a mutant strain of oleaginous yeast capable of synthesizing an unusual omega-9 fatty acid, comprising the steps (a) to (c) defined above, optionally the step (d) defined above, and furthermore the expression in said strain of a heterologous enzyme having oleate hydroxylase activity (EC 1.14.99.33) or oleate epoxidase (EC 1.14.99.- Lee et al, 1998) .
• these mutant strains of oleaginous yeast capable of synthesizing an unusual omega-9 fatty acid are also capable of secreting said unusual omega-9 fatty acid.
• heterologous enzyme an enzyme that does not naturally possess oleaginous yeasts. It can be an enzyme from any prokaryotic or eukaryotic organism.
• Enzymes exhibiting oleate hydroxylase or oleate epoxidase activity are known to those skilled in the art.
• the use of a particular enzyme having a hydroxylase or epoxidase activity is dependent on the unusual omega-9 fatty acid that one of ordinary skill in the art wishes to produce in the mutant strain of oleaginous yeast.
• the unusual omega-9 fatty acid is ricinoleic acid and said method comprises the expression in said strain of a heterologous enzyme having an oleate ( ⁇ 12) hydroxylase activity, such as a oleate ( ⁇ 12) hydroxylase (FAH12) or a mutated oleate ( ⁇ 12) desaturase (FAD2) to exhibit oleate ( ⁇ 12) hydroxylase activity, preferably an oleate ( ⁇ 12) hydroxylase.
• a heterologous enzyme having an oleate ( ⁇ 12) hydroxylase activity such as a oleate ( ⁇ 12) hydroxylase (FAH12) or a mutated oleate ( ⁇ 12) desaturase (FAD2) to exhibit oleate ( ⁇ 12) hydroxylase activity, preferably an oleate ( ⁇ 12) hydroxylase.
• said oleate ( ⁇ 12) hydroxylase is the oleate ( ⁇ 12) hydroxylase of Ricinus communis (RcFAH12) or comes from a fungus of the division of ascomycetes, preferably from the family of Clavicipitaceae, more preferably of the genus Claviceps and most preferably of the species Claviceps purpurea (CpFAH12).
• the peptide sequence of the enzyme RcFAH12 is available in the GenBank database under accession number GI: 187940238.
• a nucleotide sequence encoding RcFAH12 optimized for its expression in yeast is represented by the sequence SEQ ID NO: 21.
• the peptide sequence of the CpFAH12 enzyme is available in the GenBank database under accession number GI: 194271137.
• a nucleotide sequence encoding CpFAH12 optimized for its expression in yeast is represented by the sequence SEQ ID NO: 22.
• the mutated oleate ( ⁇ 12) desaturase with oleate ( ⁇ 12) hydroxylase activity can be obtained by domain exchange of the oleate ( ⁇ 12) desaturase by one or more domains of an oleate ( ⁇ 12) hydroxylase conferring the hydroxylase activity ( H2 and / or H3 domains), and / or by mutagenesis (eg, substitution) of one or more amino acids of the oleate ( ⁇ 12) desaturase by one or more amino acids of an oleate ( ⁇ 12) desaturase conferring an activity hydroxylase.
• mutated oleate desaturases with oleate hydroxylase activity have been described by Broun et al (1998) and Broadwater et al. (2002).
• the mutated oleate ( ⁇ 12) desaturase comes from a fungus of the family Clavicipitaceae, more preferably from the genus Claviceps and more preferably from the species Claviceps purpurea (CpFAD2, Meesapyodsuk et al., 2007) or comes from a yeast of the family of hemiascomycetes, preferably of the genus Yarrowia and more preferably of the species Yarrowia lipolytica (Y1FAD2: YALI0B10153g / YALI0B101539).
• C. purpurea the amino acid sequence of oleate desaturase (CpFAD2) has 86% identity with the amino acid sequence of oleate hydroxylase (CpFAH12). The difference in function between the desaturase and the hydroxylase of C. purpurea is therefore contained in the amino acids corresponding to the 14% of divergent sequences between these two enzymes.
• Mutated CpFAD2 (chimeric proteins) with oleate activity ( ⁇ 12) hydroxylase are represented as SEQ ID NO: 49, 50 and 51, preferably SEQ ID NO: 51,
• the overexpression of an enzyme (endogenous, orthologous, heterologous) defined in the present invention in a mutant yeast strain according to the present invention can be obtained in various ways by methods known per se.
• Overexpression of an enzyme defined in the present invention may be effected by placing one or more (preferably two or three) copies of the open reading phase of the sequence encoding said enzyme under the control of appropriate regulatory sequences.
• Said regulatory sequences comprise promoter sequences placed upstream (5 ') of the open reading phase of the sequence encoding said enzyme, and terminator sequences placed downstream (3') of the open reading phase of the sequence encoding said enzyme.
• Promoter sequences that can be used in yeast are well known to those skilled in the art and can correspond in particular to inducible or constitutive promoters.
• promoters that can be used in the process according to the present invention, mention may be made in particular of the promoter of a Y. lipolytica gene which is strongly repressed by glucose and which is inducible by fatty acids or triglycerides.
• the promoter of the FBA1 gene coding for fructose-bisphosphate aldolase (Application US 2005/0130280), the promoter of the GPM gene coding for phosphoglycerate mutase (International Application WO 2006/0019297), the promoter of the YAT1 gene encoding the transporter of ammonium (US 2006/0094102), the GPAT gene promoter encoding glycerol-3-phosphate O-acyltransferase (US Application 2006/0057690), the TEF gene promoter (Muller et al., 1998; US Application 2001/6265185). ), the hybrid promoter hp4d (International Application WO 96/41889) or the hybrid promoters XPR2 described in Mazdak et al. (2000).
• Terminator sequences that can be used in yeast are also well known to those skilled in the art. Examples of terminator sequences that may be used in the process according to the invention include the terminator sequence of the PGK1 gene and the terminator sequence of the LIP2 gene described in International Application WO 01/83773.
• the nucleotide sequence of the coding sequences of the heterologous genes can be optimized for its expression in yeast by methods well known to those skilled in the art (see for review Hedfalk, 2012). Overexpression of an endogenous enzyme can be achieved by replacing sequences controlling the expression of said endogenous enzyme with regulatory sequences allowing for stronger expression, such as those described above. Those skilled in the art can thus replace the copy of the gene encoding an endogenous enzyme in the genome, as well as its own regulatory sequences, by transforming the mutant yeast strain with a linear polynucleotide comprising the open reading phase of the sequence encoding said endogenous enzyme under the control of regulatory sequences such as those described above.
• said polynucleotide is flanked by sequences that are homologous to sequences located on either side of the gene encoding said endogenous chromosomal enzyme.
• Selection markers may be inserted between the sequences ensuring the recombination in order to allow, after transformation, to isolate the cells where the integration of the fragment has occurred by highlighting the corresponding markers.
• the promoter and terminator sequences used belong to genes different from that encoding the endogenous enzyme to be overexpressed, so as to minimize the risks of unwanted recombination in the genome of the yeast strain.
• the overexpression of an endogenous enzyme can also be obtained by introducing into the yeast strain according to the invention supernumerary copies of the gene coding for said endogenous enzyme under the control of regulatory sequences such as those described above.
• Said additional copies encoding said endogenous enzyme may be carried by an episomal vector, i.e. capable of replicating in yeast.
• these additional copies are carried by an integrative vector, i.e. integrating at a given location in the yeast genome (Mazdak et al, 2004).
• the polynucleotide comprising the gene encoding said endogenous enzyme under the control of regulatory regions is integrated by targeted integration.
• Targeted integration of a gene into the yeast genome is a molecular biology technique well known to those skilled in the art; a DNA fragment is cloned into an integrative vector, introduced into the cell to be transformed, which DNA fragment then integrates by homologous recombination into a targeted region of the recipient genome (Orr-Weaver et al, 1981).
• Yeast transformation methods are also well known to those skilled in the art and are described, in particular, by Ito et al (1983), Klebe et al. (1983) and Gysler et al (1990). Selection markers may also be inserted between the sequences ensuring the recombination in order to allow, after transformation, to isolate the cells where the integration of the fragment has occurred by highlighting the corresponding markers. Said additional copies may also be carried by PCR fragments whose ends are homologous to a given yeast locus, thus allowing the integration of said copies into the yeast genome by homologous recombination.
• Said additional copies may also be carried by autocloning vectors or PCR fragments whose ends have a zeta region absent from the yeast genome, thus allowing integration of said copies into the yeast genome by random insertion as described. in US Application 2012/0034652.
• Any known gene transfer method of the prior art can be used to introduce into a yeast strain a disabling cassette of a gene or introduce a gene encoding an enzyme.
• the process for obtaining a mutant strain of oleaginous yeast capable of synthesizing ricinoleic acid according to the present invention further comprises the overexpression in said yeast strain of an enzyme capable of catalyze the formation of triacylglycerol (TAG) from the 1, 2-j * R-diacylglycerol.
• TAG triacylglycerol
• This enzyme capable of catalyzing the formation of triacylglycerol (TAG) from 1,2-iH-diacylglycerol can be an acyl-CoA: diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) or a phospholipid: diacylglycerol acyltransferase (PDAT, EC 2.3 .1.158).
• This enzyme may be endogenous to said yeast strain.
• this enzyme is a PDAT, more preferably the endogenous PDAT of said yeast strain.
• PDAT allows the transfer of ricinoleic acid from phospholipid to diacylglycerol.
• a monoacylglycerol acyltransferase advantageously a yeast monoacylglycerol acyltransferase, advantageously also endogenous monoacylglycerol acyltransferase of said strain (this process for obtaining a mutant strain of oleaginous yeast and the strain obtained by this method being particularly advantageous) and / or
• a patatin-like triacylglycerol lipase advantageously a patatin-like triacylglycerol lipase of yeast, advantageously still the endogenous patatin-like triacylglycerol lipase of said strain and / or the inhibition of the expression or the activity of the endogenous 2-methylcitrate dehydratase of said strain (this process for obtaining a mutant strain of oleaginous yeast and the strain obtained by this method being particularly advantageous) and or
• a diacylglycerol choline-0 phosphotransferase
• a diacylglycerol yeast choline-O phosphotransferase
• diacylglycerol endogenous choline-O phosphotransferase of said strain
• an ethanolamine phosphotransferase advantageously a yeast ethanoamine phosphotransferase, advantageously also endogenous ethanolamine phosphotransferase of said strain and / or
• a phospholipase A 2 advantageously a yeast phospholipase A 2 , advantageously also endogenous phospholipase A 2 of said strain and / or
• an acyl-CoA lysophosphatidylcholine acyltransferase
• a yeast acyl-CoA lysophosphatidylcholine acyltransferase
• an endogenous acyl-CoA lysophosphatidylcholine acyltransferase of said strain and / or
• cytochrome-bs reductase advantageously of an endogenous cytochrome-b 5 reductase of said strain (this process for obtaining a mutant strain of oleaginous yeast and the strain obtained by this method being particularly advantageous) and / or the overexpression of an inositol / phosphatidyl inositol phosphatase, advantageously endogenous inositol / phosphatidyl inositol phosphatase of said strain and / or
• the enzymes overexpressed in said yeast strain may be from any prokaryotic or eukaryotic organism.
• the coding sequence of the genes encoding these enzymes can be optimized for its expression in yeast by methods well known to those skilled in the art (see for review Hedfalk, 2012).
• at least one of the overexpressed enzymes is endogenous to said strain, preferably all the overexpressed enzymes are endogenous to said strain.
• monoacylglycerol acyltransferase In yeasts, monoacylglycerol acyltransferase (MAGT; 1-acyl-sn-glycerol-3-phosphate acyltransferase, EC 2.3.1.51) is encoded by the SLC1 gene (Beopoulos et al, 2009 and 2012; International Application WO 2012/001 144 ). More particularly, the coding sequence of the SLC1 gene and the peptide sequence of the Y. lipolytica CLIB122 monoacylglycerol acyltransferase are available in the Genolevures or GenBank databases under the accession number or the following name: YALI0E18964g / YALI0E18964p.
• patatin-like triacylglycerol lipase (TGL5, triacylglycerol lipase, E.C. 3.1.1.3) is encoded by the TGL5 gene (Beopoulos et al, 2009 and 2012). More particularly, the coding sequence of the TGL5 gene and the peptide sequence of the patatin-like triacylglycerol lipase of Y. lipolytica CLIB122 are available in Génolevures or GenBank databases under the accession number or the following name: YALI0D16379g / YALI0D16379p.
• 2-methyl citrate dehydratase (EC 4.2.1.79) is a mitochondrial protein that catalyzes the conversion of 2-methylcitrate to 2-methyl-cis aconitate in the 2-methyl citrate cycle of propionate metabolism (Uchiyama et al. al, 1982, Tabuchi et al, 1981). It is encoded by the PHD1 gene. More particularly, the coding sequence of the PHD1 gene and the peptide sequence of Y. lipolytica CLIB122 methylhydrate dehydratase are available in the Génolevures or GenBank databases under the accession number or the following name: YALI0F02497g / YALI0F02497p.
• citrate lyase In yeasts, citrate lyase (E.C. 2.3.3.8) consists of two subunits encoded by two genes (ACL1 and ACL2, respectively) (Beopoulos et al, 2009). ATP citrate lyase from certain oleaginous yeasts has been characterized by Boulton et al (1981). More particularly, the coding sequence of the ACL1 and ACL2 genes and the peptide sequence of Y. lipolytica CLIB122 citrate lyase subunits A and B are available in Genolevures or GenBank databases under access numbers or names. following: ACL1! sub-unit A: YALI0E34793g / YALI0E347939p, and ACL2! subunit B: YALI0D2443 lg / YALI0D2443 lp.
• diacylglycerol choline-O phosphotransferase (EC 2.7.8.2) is encoded by the CPT1 gene. More particularly, the coding sequence of the CPT1 gene and the peptide sequence of diacylglycerol: choline-O phosphotransferase of Y. lipolytica CLIB122 are available in Genolevures or GenBank databases under the access number or the following name: YALI0E26565g / YALI0E26565p.
• EPT1 ethanolamine phosphotransferase
• EPT1 E.C. 2.7.8.1
• the coding sequence of the EPT1 gene and the peptide sequence of Y. lipolytica CL11B122 ethanolamine phosphotransferase are available in the Genolevures or GenBank databases under the accession number or the following name: YALIOC 10989g / YALIOC 10989p.
• phospholipase A 2 (PLA2, EC 3.1.1.3, 3.1.1.13, 3.1.1.4 and 2.3.1.51) is encoded by the LPA1 gene. More particularly, the coding sequence of the LPA1 gene and the peptide sequence of Y. lipolytica CLIB122 phospholipase A 2 are available in the Génolevures or GenBank databases under the accession number or the following name: YALIOF1001Og / YALIOF1001Op.
• acyl-CoA lysophosphatidyl choline acyltransferases (LPCAT) (EC 2.3.1.51, 2.3.1.23, 2.3.1.-) are coded by 3 genes, LCA1, LCA2 and LCA3, respectively. coding of the LCA1, LCA2 and LCA3 genes and the peptide sequence of Y.
• LPCAT lysophosphatidyl choline acyltransferases
• lipolytica CLIB122 acyl-CoA lysophosphatidylcholine acyltransferases 1, 2 and 3 are available in the Genolevures or GenBank databases under the following access numbers or names: LCA1: YALI0F19514g / YALI0F19514p; LCA2: YALI0C20625g / YAL1OC20625p and LCA3: YALIOC14036g / YAL1OC14036p.
• cytochrome-bs reductases In yeasts, cytochrome-bs reductases (E.C. 1.6.2.2) are encoded by two genes, MCR1 and CBRI respectively (Sickmann et al., 2003, Dujon et al., 2004). More particularly, the coding sequence of the MCR1 and CBRI genes and the peptide sequence of Y. lipolytica CLIB122 cytochrome-b5 reductases are available in the Genolevures or GenBank databases under the following accession numbers or names: MCR1: YALI0D11330g / YALI0D1 1330p and CBRI: YALI0D04983g / YALIOD04983p.
• inositol / phosphatidylinositol phosphatase (E.C. 3.1.3.-) is encoded by the SAC1 gene (Whitters et al, 1993). More particularly, the coding sequence of the SAC1 gene and the peptide sequence of the inositol / phosphatidyl inositol phosphatase of Y. lipolytica CLIB122 are available in the Genolevures or GenBank databases under the accession number or the following name: YALI0D05995g / YALI0D05995p .
• elongases In yeasts, elongases (EC 2.3.1.199) are encoded by two genes, ELO1 and EL02, respectively. More particularly, the coding sequence of the ELO1 and EL02 genes and the peptide sequence of the elongases A and B respectively of Y. lipolytica.
• CLIB 122 are available in Genolevures or GenBank databases under the following access numbers or names: ELOl; YALIOF06754g, and EL02: YAL1B20196g.
• the unusual omega-9 fatty acid is vernolic acid and said The method comprises expressing in said strain a heterologous enzyme having oleate ( ⁇ 12) epoxidase activity.
• ⁇ oleate ( ⁇ 12) epoxidase is that of Crepis a ⁇ pina, Crupis Palaestina or Vernonia ga ⁇ amensis (Lee et al, 1998).
• the present invention also relates to:
• mutant strain of oleaginous yeast useful as a yeast matrix strain for obtaining other oleaginous yeast mutant strains obtainable by a method according to the present invention defined above and
• a mutant strain of oleaginous yeast capable of synthesizing an unusual omega-9 fatty acid, preferably capable of producing ricinoleic acid or vernolic acid, more preferably ricinoleic acid, obtainable by a method according to the present invention defined above.
• mutant strains of oleaginous yeast useful as strains of yeast matrices for obtaining other mutant strains of oleaginous yeast, those of genotype:
• mutant strains of oleaginous yeast capable of synthesizing ricinoleic acid include those of genotype:
• these mutant strains of oleaginous yeast capable of synthesizing an unusual omega-9 fatty acid, such as ricinoleic acid or vernolic acid, are also capable of secreting said unusual omega-9 fatty acid.
• the present invention also relates to the use of a mutant strain of oleaginous yeast capable of synthesizing an unusual omega-9 fatty acid according to the present invention as defined above for the production of an omega-9 fatty acid.
• Said omega-9 fatty acid is preferably ricinoleic acid or vernolic acid, more preferably ricinoleic acid.
• the present invention also relates to a method for producing omega-9 fatty acid, comprising a step of culturing on a suitable medium of a mutant strain of oleaginous yeast capable of synthesizing an unusual omega-9 fatty acid according to the present invention.
• Said omega-9 fatty acid is preferably ricinoleic acid or vernolic acid, more preferably ricinoleic acid.
• Said process for producing omega-9 fatty acid comprises a first step of culturing said mutant oleaginous yeast strain according to the present invention in a suitable medium and a second step of harvesting the omega-9 fatty acids produced by the omega-9 fatty acid culture. 'Step 1.
• Said appropriate medium may comprise different carbonaceous sources for growth such as for example glucose, sucrose or glycerol. Complex sources of supply of this carbon substrate can also be used such as molasses. It may also include vegetable oils or oleic acid as a bioconversion substrate.
• Said appropriate medium may be a rich medium based on yeast extract, tryptone or peptone (for example yeast extract 5g / L-glucose 50g / L), or a conventional minimum medium as described by Mlickova et al. (2004) or optimized for said yeast (International Application WO 2007/144445, Emond et al, 2010) comprising trace elements, iron and vitamins as well as orthophosphoric acid and ammonia or any other source of nitrogen known to those skilled in the art.
• Oleaginous yeast culture can be carried out as a fermentor.
• Figure 1 Table describing plasmids and E. coli strains coli and
• Y. lipolytica used to obtain mutant strains of Y. lipolytica according to the present invention.
• Figure 2 Schematic representation of the construction of mutant strains of Y. lipolytica according to the present invention and their genotype.
• Figure 3 Primer pairs used for cloning genes of interest.
• FIG. 4 Fatty acid composition (percentages with respect to total fatty acids) of mutant yeast (A) strains expressing R. communis oleate hydroxylase (RcFAH12) or C. purpurea (CpFAH12) in different genetic backgrounds and ( B) co-expressing RcFAH12 or CpHAH12 and R. communis oleate desaturase (RcDGAT2), C. purpurea (CpDGAT2) or Y. lipolytica (Y1LRO1) in different genetic backgrounds.
• RcFAH12 R. communis oleate hydroxylase
• CpFAH12 C. purpurea
• Y1LRO1 Y. lipolytica
• Figure 5 Evolution of the amount (in g / L of culture) of ricinoleic acid produced during fermentation by strain JMY3030.
• the extracellular quantity of ricinoleic acid is shown in black.
• the intracellular amount of ricinoleic acid is represented in gray.
• the label above gives the extracellular ricinoleic acid%.
• Figure 6 Sequence of primers used for cloning genes involved in the lipid metabolism of Y. lipolytica.
• the sense primers (for) contain a BamHI site
• the anti-sense primers (rev) contain an April site. Introduced sites are underlined.
• BamH1 internal sites in the ACL2 gene were removed by base modification (in bold) at the BamHI site.
• BamHI, BamHI, and VllII sites are underlined.
• Figure 7 Diagram of the strategy adopted for the amplification of the ACL2 gene of Y. Upolytica while eliminating the two BamHI restriction sites.
• Figure 8 Schematic representation of the construction of mutant strains of Y. Upolytica from strain JMY2853 (A) and JMY3431 (B) according to the present invention and their genotype.
• Figure 9 (A) Y. Upolytica yeast mutant strains obtained from strain JMY2853 (Ura-, Leu-) by overexpression or deletion of a target gene. The results of the genetic modifications on the production of ricinoleic acid are represented in percentage compared to the strain JMY2556. The strain JMY2556 (Ura +, Leu-) was used as a control because it has the same auxotrophies as the strains constructed. (B) Y. Upolytica yeast mutant strains obtained from strain JMY3431 (Ura-, Leu-) by overexpression of a target gene. The results of the genetic modifications on ricinoleic acid production are represented as a percentage with respect to strain JMY3030. * +: overexpression; -: deletion.
• Figure 10 Comparison of the amount (in ⁇ g / ml of culture) of ricinoleic acid produced in the neosynthesis by the strains derived from the strain (A) JMY2556 and by the strains derived from the strain (B) JMY3431 (JMY3030).
• Figure 11 (A) Schematic representation of different chimeric proteins between a desaturase (in gray) and a hydroxylase (in black). (B) Lipid composition of different strains after 96h of vial culture containing 5% glucose (YED 5 ) for the mutant strains of yeast QPF-CpFAH12, QPF-H2_hyd, QPF-H2 / H3_hyd and QPF-H2 / H3 Jiyd Cterm .
• the mutant strains of Y. Upolytica are derived from the auxotrophic strain of
• Upolytica Pold (Leu " Ura " CLIB 139, genotype MatA Ura3 ⁇ 302, Leu2-270, xpr2-322), itself derived from the wild strain of Y.
• Upolytica W29 (genotype MatA, ATCC 20460) by genetic modification. The Pold and W29 strains have been described by Barth and Gaillardin (1996). The strains used to obtain the strains according to the present The invention is shown in the table of Figure 1. Their construction is shown in Figure 2 and described in detail below.
• the medium and culture conditions of lipofytica were described by Barth and Gaillardin (1996). Rich medium (YPD), minium + glucose medium (YNB) and minimum medium + casamino acids (YNBcasa) or uracil (YNBura) were prepared as described by Mlickova et al. (2004).
• the minimum medium (YNB) contains 0.17% (w / v) yeast nitrogen base (without amino acid and ammonium sulfate, YNBww, Difco, Paris, France), 0.5% (w / v) of NH 4 Ci, 0.1% (w / v) yeast extract (Bacto-DB) and 50 mM phosphate buffer (pH 6.8).
• the glucose medium for the neosynthesis of ricinoleic acid (YED 5 ) contains 1% (w / v) yeast extract (Bacto-DB) and 5% (w / v) glucose.
• Escherichia coli strain Machl-T1 (Invitrogen) was used for transformation and amplification of recombinant plasmid DNA.
• Cells were cultured on LB medium (Sambrook et al., 1989). Kanamycin (40 ⁇ g / mL) was used for plasmid selection.
• the lipofytica by transformation by the lithium acetate method.
• the Ura + and Leu + transformants were selected on YNBcasa and YNBura media, respectively.
• the corresponding ylA12-ver1 and ylA12-ver2 primers were used to verify gene disruption by PCR amplification of genomic loci. Excision of the markers was performed using the Cre-lox recombinase system by transformation with the pUB4-CreI replicative plasmid (JME547) as described by Fickers et al. (2003). The strains were then cured from the plasmid by successive replications on rich medium.
• the genes of interest were placed under the control of the constituent constitutive promoter TEF.
• Yarrowia llpolytica (Muller et al, 1998).
• the codons of the heterologous genes encoding hydroxylase and acyltransferase were optimized for yeast expression and synthesized by Genscript (New Jersey, USA).
• the coding genes were then inserted between the BamHI-AvrII restriction sites of the JMP62-derived expression vector containing the pTEF promoter and the URA3ex selection marker (JME1046) described by Nicaud et al. (2002).
• the JMP62 vectors containing the LEU2ex selection marker were obtained by replacing the marker using the Iscel restriction site upstream / downstream of URA3ex in the JME802 vector (Fickers et al, 2003, Nicaud et al, 2002). . Plasmids were digested with NotI prior to transformation. Transformants were selected by auxotrophy on the appropriate minimal medium.
• Lipids of the equivalent of 10 OD units of cells in lyophilized culture were extracted by the procedure described by Folch et al (1957) for TLC analysis (thin layer chromato graphy) or were directly converted to their methyl esters. for GC analysis, as described by Browse et al. (1986).
• GC analysis of the fatty acid methyl esters (FAMEs) was performed on a gas chromatograph (Varian 3900) equipped with a flame ionization detector and a Varian FactorFour vf-23ms column, with a specification washing at 260 ° C of 3pA (30m, 0.25mm, 0.25 ⁇ m).
• the fatty acids (FA) were identified by comparison with the commercial standard fatty acid methyl esters (FAME32, Supelco, methyl ricinoleate, Sigma) and quantified by the internal standard method with the addition of 50 g of Cl 7: 0 commercial (Sigma).
• the analysis of the culture supernatants was carried out as follows: a volume of culture is mixed with one volume of isopropanol, centrifuged (1 minute 13,000 rpm) and filtered through 0.2 ⁇ . Twenty microliters of the mixture are injected in HPLC with 254 nm UV detection on a Cl 8 reverse phase column (Luna® 5 ⁇ Cl 8 (2) 100 ⁇ , LC Column 151 ⁇ 4.6 mm, Ea) at 40 ° C. with as quenched a methanol / water / trifluoroacetic acid mixture 90/10 / 0.3 at 1 ml / min.
• Pre-coated TLC plates (G60 silica, 20 x 20 cm, 0.25 mm thick) from Merck (Germany) were used.
• the lipid classes were separated with hexane / ethyl ether / acetic acid solvent, 80/20/1 (v / v / v).
• the plates were sprayed with 1% sulfuric vanillin in ethanol and incubated at 105 ° C for 10 min.
• the different lipid classes were identified using commercial standards (Nu-shek, USA).
• RcFAH12 and CpFAH12 were optimized for their expression in yeast (SEQ ID NO: 21 and 22 respectively).
• the Y. lipolytica gene encoding endogenous ⁇ 12 desaturase was deleted before the heterologous expression of oleate hydroxylases. This deletion was carried out both in the Pold strain and the strain JMY1233 (poxl-6). The resulting mutants, designated JMY1366 and JMY1762 respectively, were unable to synthesize linoleic acid.
• the JMY2159 strain containing deletions invalidating ⁇ -oxidation (poxl-6A), the synthesis of TAGs (dgalA dgalA Irol) and ⁇ 12 desaturation (fad2A) is named PQF for simplification.
• the deleted mutant strain of TAG acyltransferases expressing RcFAH12 contains 7% ricinoleic acid, which suggests that the absence of enzymatic activity in the above constructs is due largely to the unavailability of the substrate (oleic acid esterified in TAG).
• the homologous construct expressing CpFAH12 contained up to 29% ricinoleic acid ( Figure 4A).
• strain PQF-CpFAH12 JMY2324) and 1.5: 1 for strain PQF-CpFAH12x2 (JMY2511).
• the fraction of unsaturated C18 fatty acids oleic acid, linoleic acid and ricinoleic acid
• fad2A ⁇ RcF ⁇ H12 strain JMY1760
• poxl-6Afad2A-RcFAH12 strain JMY1763
• All strains derived from PQF accumulated lipids at about 5% dry weight.
• the level of ricinoleic acid synthesis is related to the accumulation capacity of the yeast strains, it has been independently expressed, under the control of the pTEF promoter, the ricinoleic acid-specific acyltransferase DGAT2.
• R. communis R. communis
• C. pur pur ae CpDGAT2
• the coding sequences of RcDGAT2 and CpDGAT2 were optimized for their expression in yeast (SEQ ID NO: 23 and 24 respectively).
• the strain CpFAH12 overexpressing the native LRO1 acyltransferase of Y, lipolytica (JMY2556 [Ura +, Leu-] or JMY2853 [Ura-, Leu-], the number of the strain varying according auxotrophies) has accumulated ricinoleic acid up to 42% of its total lipids, with a fraction representing 20% of the total ricinoleic acid, esterified to TAG. This corresponds to a 1, 4-fold increase in ricinoleic acid accumulation relative to the parental strain (JMY2511). The accumulation of lipids reached 13% of the dry weight of the cells, corresponding to an increase of 2.5 times compared to the strains not expressing LROl.
• the amount of ricinoleic acid produced reached 700 ⁇ / ⁇ . (or 63mg / g dry weight).
• the oleate hydroxylase is capable of continuing the hydroxylation reaction following the formation of linoleic acid (22%), but with a hydroxylation ratio on the desaturation of raising to 2: 1.
• strain JMY2853 served as a matrix yeast strain for multicopy overexpression of the genes coding for the CpFAH12 and Y1L 01 enzymes.
• a strain comprising 3 copies of CpFAH12 and 2 copies of the YlLRO1 acyltransferase was obtained (strain JMY3030). It produces up to 53% of ricinoleic acid in neosynthesis on a glucose medium.
• Strain JMY3030 (containing 3 copies of CpFAH12 and 2 copies of YlLRO1) was used as a 10 L fermenter (4 L liquid volume).
• the culture conditions are perfectly controlled (regulation of pH, temperature and aeration).
• the quantity of biomass and thus of producing cells is then improved, which makes it possible to increase the production of ricinoleic acid.
• the fermentation was conducted in fed-batch mode, with glucose as a carbon source for growth and oleic acid as a bioconversion substrate.
• the culture was carried out on the minimum medium optimized for Yarrowia lipo ⁇ ytica (synthetic), with addition of trace elements, iron and vitamins, as described in International Application WO 2007/144445, with 160 g / L of glucose and 24 mg of glucose. g / L of oleic acid in total and a pH adjusted to 6 with orthophosphoric acid and ammonia.
• the culture was inoculated to a biomass concentration of 0.48 g / L and the average growth rate was 0.19 h "1.
• the cell concentration reached 90 g C dw / L.
• Four controlled additions of oleic acid were made, firstly by adding 20% (v / v) oleic acid emulsion and then non-emulsified oleic acid (80% pure).
• the final concentration of ricinoleic acid produced is 12 g / L, with a purity of 60% on the total lipids.
• the insertion of the genes of interest into the JMP62-Ura3ex-pTEF vector is made by amplifying the genes with the primers described in FIG. 6 in which the BamHI and Avril restriction enzyme sequences at the 5 'ends have been introduced. and 3 'respectively. Plasmids and PC products are digested with enzymes and ligated to obtain the expression vectors.
• the expression vectors were verified by sequencing.
• Phospholipase A2 (YALIOF1001Og), TGL5 (YALIOD 16379g), LCAT3 (YALI0C14036g) and LR02 (YALI0E08206g) were made from synthetic genes directly synthesized and cloned (Euro fins).
• the genes capable of increasing the neosynthesis of ricinoleic acid identified have been the subject of new strains constructs from the JMY3431 matrix strain.
• the insertion of the genes of interest into the JMP62-Ura3ex-pTEF vector is made by amplifying the genes with the primers described in FIG. 6 in which the BamHI and Avril restriction enzyme sequences at the 5 'ends have been introduced. and 3 'respectively. Plasmids and PCR products are digested with enzymes and ligated to obtain the expression vectors.
• EXAMPLE V EVOLUTION OF OLEATATASE DEATURASE OF F. LIPOLYTICA IN OLATEATE H YDROX YL A SE
• hydroxylases and desaturases are homologous enzymes that belong to the same protein family and share a strong similarity, both in their sequence and in their function: both modify oleic acid (either by creating a desaturation or creating a hydroxylation).
• C. purpurea P hydroxylase and Y. lipolytica desaturase from two different organisms share only 47% identical amino acids. It was therefore chosen to work initially with C. purpurea desaturase which has 86% amino acids identical to those of its hydroxylase. Thus, the difference in function between the desaturase and the hydroxylase of this fungus is contained in the 14% of divergent sequences between these two enzymes.

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