Classifications

• • 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)
  • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
• • 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)
  • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
• • 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
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/44—Polycarboxylic acids

Definitions

• the invention relates to a process for the production of dicarboxylic acids by fermentation using a mutant strain of Yarrowia lipolytica yeast from a bioconversion substrate.
• the dicarboxylic acids also called “diacids”
• dicarboxylic acids are used as raw materials for example in the synthesis of polyamides and polyesters, lubricating oils, plasticizers or perfumes.
• DGA ⁇ acyl-CoA: diacylglycerol acyltransferase
• TGL ⁇ triacylglycerol lipase
• G3P glycerol-3-phosphate dehydrogenase
• SCP2 putative sterol transporter
• LR0 ⁇ ⁇ LR0 lecithin cholesterol acyltransferase
• IFP 621 unknown function
• IPF 905 unknown function
• IPF 2569 NADH-ubiquinone reductase subunit
• Yarrowia lipolytica is very different from that of Candida tropicalis. Unlike Candida tropicalis, which is a diploid yeast, Yarrowia lipolytica is indeed a haploid species. In this last microorganism, the gene deletion operations are therefore much more efficient and safer, because of the presence of a single set of chromosomes.
• a promoter of the POX2 gene encoding Pacyl-CoA oxidase is used to overexpress genes such as, for example, those encoding cytochrome P450 monooxygenase and for NADPH-cytochrome reductase.
• the pPOX2 promoter has the property of being a strong promoter inducible by the bioconversion substrates.
• the overexpression of a gene of interest is achieved by the addition of a single gene copy under the control of the pPOX2 promoter, making it possible to obtain Yarrowia lipolytica mutants which are efficient, stable and non-reverting, unlike the Candida tropicalis mutants obtained by a multicopy amplification system.
• Another advantage of Yarrowia lipolytica on Candida tropicalis for the production of diacids from esters of fatty acids or natural oils, for example vegetable oils, is the following: the transformation of natural oils into diacids by Candida tropicalis requires hydrolysis at least partial chemical substrate before the implementation of fermentation (see US Patent 5,962,285).
• This hydrolysis is carried out by a saponification conducted in the presence of calcium hydroxide or magnesium. It produces the corresponding salts of fatty acids (soaps).
• Yarrowia lipolytica has the ability to assimilate triglycerides as a carbon source.
• the first step of this catabolism involves the hydrolysis of triglycerides to free fatty acids and glycerol by the lipolytic enzymes (lipases), identified by Peters and Nelson in 1951. An extracellular lipase activity and two membrane lipases of 39 and 44 kDa (Barth et al.
• the strains used in the process of the invention are derived from the wild strain Yarrowia lipolytica W29 (ATCC 20460, listed under CLIB89 in the Biotechnology Yeast-CLIB Collection).
• new mutant strains derived from the strain Yarrowia lipolytica ATCC 20460 can be used via the strain PoId [auxotrophic strain for leucine (leu-) and uracil (ura-)], described in the review by G. Barth et al. . It is listed under CLIB139 in the CLIB.
• the mutant strain chosen is cultured in a medium consisting essentially of an energetic substrate which comprises at least one carbon source and one nitrogen source until the end of the growth.
• the bioconversion substrate alkane or mixture of alkanes, fatty acid or mixture of fatty acids, fatty acid ester or mixture of fatty acid esters or natural oil or mixture of these different substrates
• the culture medium may comprise a supply of secondary energy substrate generally consisting of at least one polyhydroxy compound, such as, for example, glycerol or a sugar.
• POX genes of the wild-type strain whose sequences are different from those of Candida tropicalis are cloned.
• Disruption cassettes of the genes coding for the isozymes of acyl-CoA oxidase are then constructed.
• the genes of acyl-CoA oxidase are disrupted using the selectable marker URA3.
• Promoter and terminator regions are amplified by a first PCR, using specific oligonucleotide pairs, eliminating the full frame of the open reading frame
• a second PCR is then performed with the external primers and PCR products of the promoters and terminators, which fuse via a common 20 bp extension with a site for the restriction enzyme I-SceI.
• the PCR product is cloned to give a series of plasmids (designated pPOX-PT) containing the promoter-terminator module
• a URA3 gene is introduced into the I-SceI site of the POX-PT cassette.
• a series of pPOX-PUT plasmids containing the promoter-URA3 ⁇ terminator module is constructed (disruption cassette 1). These constructs are called pPOX1-P ⁇ JT, pPOX2-POT, pPOX3-PUT, pPOX4-PDT and pPOX5-P ⁇ JT for the plasmids containing the disruption cassette 1, on the one hand, and pPOX1-PT, pPOX2-PT , pPOX3-P1, pPOX4-PT, and pPOX & PT for the plasmids containing the disruption cassette 2, on the other hand.
• the transformation of Yarrowia lipolytica can be carried out by various methods.
• the presence of disruption is verified by PCR according to the technique of Gussow et al. : Direct Clone Characterization of Plates and Colonies by Polymerase Chain Reaction. Nucleic Acids Res. 17, 1989, 4000, then confirmed by Southern blot hybridization.
• Disruption of a gene and excision of the marker can still be done by a method involving recombination or recombinase.
• markers can be used with either a repeat sequence (allowing the recombination that is selected) or a lox sequence that is recognized by the Cre recombinase. Excision occurs when one expresses Recombinase Cre, Fickers et al., 2003 New Disruption Cassettes for Rapid Disease Gene and Marker Rescue in the Yeast Yarrowia lipolytica. J. Microbiol. Methods 55/3: 727-737.
• strain MTLY74 Leu + Ura- was constructed.
• the first step is the construction of the auxotrophic MTLY40 strain for uracil (Leu +, Ura-) by conversion of the URA3 marker by the ura3-41 marker by transforming the PCR fragment containing this marker and selecting the Ura- in the presence of 5FOA.
• Loss of the plasmid pRRQ2 is performed by cultivation on YPD rich medium and isolating a clone (Leu-, Ura-, Hy 9 -).
• 2b From mutant MTLY66, a mutant strain Yarrowia lipolytica MTLY74 Leu + Ura- was constructed which overexpresses NADPH-cytochrome reductase, expressing it under the control of the strong pPOX2 promoter, induced by bioconversion substrates, of the fatty acid type, fatty acid ester or natural oil.
• MTLY80 expressing NADPH-cytochrome reductase and cytochrome P450 monooxygenase ALK2 under the control of the pPOX2 promoter inducible by fatty acids, fatty acid esters or natural oils
• mutant Yarrowia lipolytica strain MTLY80 was constructed which overexpresses the genes encoding NADPH-cytochrome reductase [CPR] and cytochrome P450 monooxygenase (ALK2) under bioconversion conditions, under the control of the strong pPOX2 promoter. induced by bioconversion substrates of the fatty acid, fatty acid ester or natural oil type.
• CPR NADPH-cytochrome reductase
• ALK2 cytochrome P450 monooxygenase
• the ALK2 gene coding for cytochrome P450 monooxygenase was introduced into a vector containing the URA3 selection gene, for example JMP61, under the control of the pPOX2 promoter that is inducible by fatty acids, fatty acid esters or natural oils.
• the marker-promoter-gene cassette (URA3-pPOX2-> AKL2) is introduced by transformation.
• MTLY81 expressing NADPH-cytochrome reductase without the cytochrome P450 monooxygenase genes (ALK1 or ALK2) under the control of the pPOX2 promoter inducible by fatty acids, fatty acid esters or natural oils
• a mutant Yarrowia lipolytica strain MTLY81 was constructed which overexpressed the gene coding for NADPH-cytochrome reductase (CPR) under the control of the strong pPOX2 promoter induced by fatty acid type bioconversion substrates, ester of fatty acid or natural oil.
• CPR NADPH-cytochrome reductase
• Mutant MTLY74 prototroph was made by transformation with plasmid JMP61 carrying the marker URA3.
• the MTLY37 strain or the MTLY66 strain 1) constructing a PCR ("Polymerase Chain Reaction") disruption cassette or by cloning, using a counter-selectable marker, for example the URA3 marker (with which one can select for the Ura + phenotype or for the Ura-phenotype). ), or by using a marker with either a repeat sequence (allowing the recombination that is selected) or a lox sequence that is recognized by the Cre recombinase
• the strains are selected with the gene of interest deleted (transformation and selection of the transformants, advantageously Ura + if the marker is URA3) and the disruption of the gene is verified; 3) the strains are selected with the deleted marker (transformation and selection of the transformants); advantageously 5FOA R if the marker is URA3 or advantageously a plasmid expressing the recombinase if the marker has the lox sequence and the disruption of the gene is verified.
• strain FT120 Leu-Ura-, ⁇ pox1-6 was constructed.
• mutant strain MTLY95 ⁇ pox1-6 was constructed by insertion of deletion of the POX1 and POX6 genes and deletion of the marker according to the method described previously.
• mutant strain Yarrowia lipolytica FT101 Leu + Ura- which overexpresses the gene coding for NADPH-cytochrome reductase (CPR) under bioconversion conditions was constructed, expressing it under the control of the strong pPOX2 promoter, induced by bioconversion substrates, fatty acid type, fatty acid ester or natural oil.
• strain FT120 To obtain this strain, one can proceed as for the construction of strain FT120:
• the strains are selected with the deleted marker (transformation and selection of the transformants); advantageously 5FOA R if the marker is URA3 or advantageously a plasmid expressing the Cre recombinase if the marker has the lox sequence and the disruption of the gene is verified.
• strain FT130 Leu-Ura-, ⁇ pox1-6, ⁇ dgai was constructed.
• the MTLY66, MTLY81, FT120 and FT 130 strains were deposited in the National Collection of Microorganism Cultures under the respective registration numbers CNCM 1-3319, CNCM I-3320, CNCM I-3527 and CNCM I-3528.
• the mutant strains FT120 and FT130 were tested under identical conditions.
• the deletion of the POX1 and POX 6 genes makes it possible to reduce the degradation of the diacids for FT120.
• the deletion of an additional DGA1 gene coding for acyl-CoA diacylglycerol acyltransferase results in a decrease in the accumulation of the bioconversion substrate in the form of lipid bodies within the Yarrowia lipolytica cell.
• most of the diacids obtained in these examples are composed of diacids containing 18 carbon atoms, like the bioconversion substrate used, which is predominantly composed of 18-carbon fatty acids.
• Example 1 Process for producing dicarboxylic acids from oleic sunflower oil with the MTLY37 mutant
• a preculture of mutant MTLY37, preserved on agar medium of composition: yeast extract 10 gl '1 ; peptone 10 gl '1 ; glucose 10 gl '1 ; Agar 20 gl '1 , is carried out by seeding which provides an initial absorbance of the preculture medium close to 0.30.
• the preculture is carried out with orbital shaking (200 rpm) for 24 h at 30 ° C. in a 500 ml finned flask containing 25 ml of medium (10 ⁇ l -1 yeast extract, 10 ⁇ l -1 peptone, 20 ⁇ l -1 glucose).
• the medium used for the culture is deionized water, yeast extract at 10 gl -1 , tryptone at 20 gl -1 , glucose at 40 gl -1 , and oleic sunflower oil at 30 gl. "1 .
• Seeding of the fermenter is performed with the entire preculture vial.
• the culture is conducted at 30 ° C. in a 4 L fermentor with 2 l medium at a ventilation rate of 0.5 vvm and a stirring speed of 800 rpm provided by a double-acting centripetal turbine.
• the cell biomass is removed by centrifugation.
• the supernatant is then acidified to pH 2.5 by the addition of 6M HCl and the insoluble dicarboxylic acids are recovered by centrifugation of the acidified wort and dried.
• the dicarboxylic acid composition of the mixture is determined by gas chromatography on a DB1 column after conversion of the dicarboxylic acids to diesters according to the method described by Uchio et al: Microbial Production of Long-chain Dicarboxylic Acids from / 7-Alkanes. Part II. Production by Candida cloacae Mutant Unable to Assimilate Dicarboxylic Acid. Agr Biol. Chem. 36, No. 3, 1972, 426-433. The oven temperature of the chromatograph is programmed from 150 ° C. to 28 ° C. at a rate of 8 ° C. per min. The results show a maximum production of diacids in the supernatants of 5.9 gl -1 after 130 h.
• Example 1 is repeated replacing the tryptone with peptone at the same concentration in the culture medium. After 130 hours of culture, 9.9 g -1 of dicarboxylic acids are obtained, giving a production increase of about 68% compared to Example 1.
• EXAMPLE 3 Production of Dicarboxylic Acids by Mutant MTLY37 with Continuous Feeding of Oleic Sunflower Oil
• Example 2 is repeated. removing oleic sunflower oil from the culture medium and replacing it with a continuous injection of the same oil at a sub-limiting flow of 1 ml into the reactor.
• Example 4 Production of dicarboxylic acids from oleic sunflower oil with the mutant MTLY79 overexpressed for CPR and ALK1
• Example 3 is repeated replacing the MTLY37 mutant with the MTLY79 mutant overexpressing the CPR and ALK1 genes. After 130 hours of culture, 16 gl -1 of dicarboxylic acids are obtained.
• Example 5 Production of dicarboxylic acids from oleic sunflower oil with the MTLY80 mutant overexpressed for CPR and ALK2
• Example 3 is repeated replacing the MTLY37 mutant with the MTLY80 mutant overexpressing the CPR and ALK2 genes. After 130 hours of culture, 16 gl -1 of dicarboxylic acids are obtained.
• Example 3 is repeated replacing the MTLY37 mutant with the mutant MTLY81 overexpressing only the CPR gene. After 130 hours of culture, 16 g are obtained. I- 1 dicarboxylic acids.
• EXAMPLE 7 Production of Dicarboxylic Acids from Oleic Sunflower Oil with the FT120 Mutant Deleted for the Six POX ( ⁇ poxi-6) Genes and Overexpressing the CPR Gene
• Example 3 is repeated replacing the mutant MTLY37 with the mutant FT120 deleted the six POX genes and overexpressing the CPR gene. After 130 hours of culture, 18 gl -1 of dicarboxylic acids are obtained
• Example 8 Production of dicarboxylic acids from oleic sunflower oil with the mutant FT130 deleted for the six POX genes ( ⁇ pox1-6) and deleted for the DGA ⁇ gene ( ⁇ dgai) and overexpressing the CPR gene Example 3 is repeated, replacing the mutant MTLY37 with the mutant FT130 deleted from the six POX genes and the DGA ⁇ gene and overexpressing the CPR gene. After 130 hours of culture, 23 gl -1 of dicarboxylic acids are obtained

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (10)

Applications Claiming Priority (2)

Publications (2)

Family

ID=34954795

Family Applications (1)

Country Status (13)

Cited By (17)

Families Citing this family (12)

Citations (4)

Family Cites Families (1)

• 2004
  • 2004-12-15 FR FR0413468A patent/FR2879215B1/fr not_active Expired - Fee Related
• 2005
  • 2005-12-13 WO PCT/FR2005/003140 patent/WO2006064131A1/fr not_active Ceased
  • 2005-12-13 DK DK05826569.5T patent/DK1828392T3/da active
  • 2005-12-13 CN CN2005800483222A patent/CN101228282B/zh not_active Expired - Fee Related
  • 2005-12-13 US US11/721,726 patent/US20100041115A1/en not_active Abandoned
  • 2005-12-13 PL PL05826569T patent/PL1828392T3/pl unknown
  • 2005-12-13 JP JP2007546115A patent/JP2008523794A/ja active Pending
  • 2005-12-13 EP EP05826569A patent/EP1828392B1/fr not_active Expired - Lifetime
  • 2005-12-13 CA CA2590795A patent/CA2590795C/fr not_active Expired - Fee Related
  • 2005-12-13 AT AT05826569T patent/ATE471384T1/de active
  • 2005-12-13 DE DE602005021913T patent/DE602005021913D1/de not_active Expired - Lifetime
  • 2005-12-13 BR BRPI0519928-0A patent/BRPI0519928A2/pt not_active IP Right Cessation
  • 2005-12-13 ES ES05826569T patent/ES2346773T3/es not_active Expired - Lifetime
• 2012
  • 2012-03-06 JP JP2012049326A patent/JP5615859B2/ja not_active Expired - Fee Related
  • 2012-03-06 JP JP2012049322A patent/JP5462303B2/ja not_active Expired - Fee Related

Patent Citations (5)

Non-Patent Citations (9)

Also Published As

Similar Documents

Legal Events