EP2344656A1 - Micro-organisme permettant de produire de l'acide succinique - Google Patents

Micro-organisme permettant de produire de l'acide succinique

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Publication number
EP2344656A1
EP2344656A1 EP09748218A EP09748218A EP2344656A1 EP 2344656 A1 EP2344656 A1 EP 2344656A1 EP 09748218 A EP09748218 A EP 09748218A EP 09748218 A EP09748218 A EP 09748218A EP 2344656 A1 EP2344656 A1 EP 2344656A1
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genes
microorganism
gene
promoter
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Christine Lang
Andreas Raab
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Novozymes Berlin GmbH
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OrganoBalance GmbH
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • 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
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01037—Malate dehydrogenase (1.1.1.37)
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01042—Isocitrate dehydrogenase (NADP+) (1.1.1.42)
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    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/05—Oxidoreductases acting on the CH-CH group of donors (1.3) with a quinone or related compound as acceptor (1.3.5)
    • C12Y103/05001—Succinate dehydrogenase (ubiquinone) (1.3.5.1)
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
    • C12Y203/03009—Malate synthase (2.3.3.9)
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    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/03—Oxo-acid-lyases (4.1.3)
    • C12Y401/03001—Isocitrate lyase (4.1.3.1)
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    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01001—Acetate-CoA ligase (6.2.1.1)

Definitions

  • the invention relates to a microorganism which is genetically modified with respect to the wild type and is suitable for the production of organic acids, in particular succinic acid, uses of such a microorganism and process for its preparation.
  • Dicarboxylic acids have great economic potential because they can be used as precursors for many chemicals.
  • succinic acid serves as a precursor for the production of plastics based on 1,4-butanediol, tetrahydrofuran and gamma-butyrolactone.
  • Succinic acid is today produced chemically by catalytic hydrogenation of maleic anhydride to form succinic anhydride and subsequent addition of water, or by direct catalytic hydrogenation of maleic acid.
  • Succinic acid is also produced by many microorganisms starting from sugars or amino acids under physiological environmental conditions. Under anaerobic conditions, in addition to succinic acid, further fermentation end products such as ethanol, lactic acid, acetic acid and formic acid are generally formed. The biosynthesis of succinic acid with its high oxygen content requires a reductive CO 2 fixation.
  • Succinic acid is a metabolite that is normally enriched by anaerobic fermentation processes. While the yield and enrichment of the product under anaerobic conditions is often better than under aerobic conditions, the disadvantage lies in an exclusively anaerobic process in a technical limitation of biomass production and a low productivity of the microbial producer. Thus, a relatively low biomass / product Efficiency the result. In addition, it is difficult to handle strictly anaerobic microorganisms technically.
  • Reference US 7,063,968 describes a microbial isolate from bovine rumen, Mannheimia sp. 55E which is capable of synthesizing organic acids under both aerobic and anaerobic conditions. However, this is not a specific enrichment of succinic acid, but a mixture of various organic acids, such as formic acid, acetic acid, lactic acid and succinic acid. Disadvantage of this producer is that an economic use of the strain is difficult, if not impossible, since consuming enrichment and purification processes would have to be used to recover succinic acid.
  • the invention is based on the technical problem of specifying a microorganism with which an improved yield of organic acids, in particular succinic acid, can be achieved in microbiological production processes.
  • the invention teaches an isolated genetically modified microorganism, wherein the wild type a) the genes idhl and idpl are deleted or inactivated, and / or b) the genes sdh2 and sdhl are deleted or inactivated, and / or c) the gene PDC2 is deleted or inactivated or under the
  • the foreign gene which replaces or supplements the ICL1 gene may have at least 75% homology to Sequence No. 1.
  • the foreign gene that replaces or supplements the ACS1 gene may have at least 75% homology to Sequence # 2.
  • the Foreign gene which replaces or supplements the MLS1 gene may have at least 75% homology to Sequence No. 3.
  • the foreign gene that replaces or supplements the gene MDH3 may have at least 75% homology to sequence # 4.
  • the homology is greater than 80%, more preferably greater than 90%, most preferably greater than 95%. Of course, it can also be identical herewith.
  • the invention provides an optimized process for the production of succinic acid and other organic acids of respiratory central metabolism by means of a yeast strain, in particular a Saccharomyces cerevisiae yeast strain.
  • This method allows more efficient production of organic carboxylic acids, especially dicarboxylic acids and hydroxy fatty acids, of the respiratory central metabolism of yeast, such as succinic acid, in terms of production time and yields.
  • it allows a two-step production process by separating growth and production phases without the use of an antibiotic-dependent promoter system.
  • the invention makes possible a process in which biomass is first enriched in a first growth phase and succinic acid is efficiently produced in a second phase and is to be released into the culture medium.
  • the separation of growth and production phase contributes significantly to the efficiency of the entire production process of organic acids in yeast, since growth of the cells and production of the desired metabolite are otherwise always competing factors.
  • the formation of biomass is undesirable because it consumes carbon or substrate used, which ultimately results in a reduction in the yield of the metabolite to be produced, for example succinic acid.
  • the separation of growth and production phase is by means of a genetically modified or mutated by genetic mutation microorganism and an associated fermentation process in which initially during a first phase, an optimal biomass increase of the microbial producer is guaranteed and Subsequently, in a second phase (production phase), the enrichment of carboxylic acids, such as succinic acid, from primary carbon sources (eg glucose) and CO 2 (anaplerotische Auf spallretress, CO 2 fixation) is connected, feasible.
  • primary carbon sources eg glucose
  • CO 2 anaplerotische Auf spallretress, CO 2 fixation
  • the invention provides a further, optimized possibility of separating growth and production phase, which makes possible the use of Antibiotics are not required, as well as an optimized process for the production of succinic acid or other organic acids in yeast.
  • Feature (a) is the following.
  • yeast Saccharomyces cerevisiae despite the deletions of the sdh2 and idhl genes, which result in an interruption of the citrate cycle, a growth rate comparable to unmodified wild-type yeast can be measured.
  • a growth of a yeast strain with an idhl deletion is possible only because this deletion does not lead to a complete disappearance of isocitrate dehydrogenase activity.
  • the reason for this are three other isoenzymes of isocitrate dehydrogenase, which can compensate for the elimination of the main dimeric enzyme encoded by the IDH1 and IDH2 genes with respect to the formation of ⁇ -ketoglutarate.
  • ⁇ -ketoglutarate is essential for growth of the yeast cell on minimal media, since this intermediate forms the amino acid glutamate, without which no growth is possible.
  • the effective separation of growth and production phase is thus only possible by the complete suppression of isocitrate dehydrogenase activity in the production strain, because it ensures a glutamate auxotrophy, with the result that the yeast has no growth in medium without supplemented glutamate.
  • This can be used to control the fermentation process via the supplementation of glutamate to the culture medium.
  • the amount of glutamate added to the culture medium can effectively control the duration of the growth phase as well as the desired cell density in this phase. As the amount increases, so does duration and cell density.
  • Another advantage of the complete suppression of isocitrate dehydrogenase activity is that all of the carbon in the respiratory system of the yeast is diverted into the glyoxylate cycle in the direction of succinic acid and can not flow to ⁇ -ketoglutarate, which would lead to yield losses.
  • succinic acid should be enriched as end product and not further metabolized by the yeast cell. This can not be achieved by the unique deletion of the sdh2 gene.
  • a further subunit of the heterotetrameric enzyme succinate dehydrogenase, encoded by the sdhl gene is deleted. (Kubo et al., 2000) detected a residual activity of succinate dehydrogenase in a yeast strain with an sdh2 deletion that was suppressed by additional deletion of the sdhl gene.
  • Yield losses can also result from the further metabolism of the succinate formed via the enzyme succinate-semialdehyde dehydrogenase.
  • This enzyme is part of the glutamate degradation pathway and catalyzes the conversion of succinate to succinate semialdehyde.
  • This intermediate is then metabolized via gamma-amino butyric acid to glutamate. In this way not only yield losses can be incurred, but also ⁇ -ketoglutarate and glutamate can be synthesized, which makes it impossible to control the fermentation process via glutamate supplementation.
  • the additional deletion of the gene uga2, which codes for the succinate semialdehyde dehydrogenase, in an optimized production process for the production of succinic acid advantageous, glyoxylate, which is necessary for the circulation of Glyoxylatzyklus, not only from that by the isocitrate lyase catalyzed reaction whereby isocitrate is cleaved into succinate and glyoxylate, but also by the reaction of alanine glyoxylate aminotransferase.
  • This enzyme catalyzes the formation of glyoxylate and alanine from pyruvate and glycine.
  • the circulation of the glyoxylate cycle may also be affected by the reaction of the Alanine-glyoxylate aminotransferase reaction which provides glyoxylate.
  • the isocitrate lyase activity which produces the desired product succinate, is not necessary for the circulation of the glyoxylate cycle, with the result that the yeast partially reverses the alanine glyoxylate aminotransferase-catalyzed alternative reaction to the glyoxylate Synthesis uses.
  • succinate which leads to yield losses. Therefore, the additional deletion of the gene agx ] , which codes for the alanine glyoxylate aminotransferase, is advantageous in the context of an optimized production process for the production of succinic acid.
  • the released energy of this controlled oxyhydrogen reaction is used to transport protons against a gradient into the intermembrane space of the mitochondria, which then drive back into the mitochondria through the complex V of the respiratory chain, the so-called "proton pump", generating energy in the form of ATP .
  • the yeast produces mainly ethanol in addition to glycerine and generates only 2 energy equivalents in the form of ATP per molecule of glucose, compared to respiration, in which 38 ATP can be generated per molecule of glucose.
  • NADH is reoxidized to NAD by the synthesis of ethanol or glycerol and not by oxygen, as it is by respiration, because oxygen is not available as an ultimate electron acceptor under anaerobic conditions.
  • the yeast Saccharomyces cerevisiae is "Crabtree" positive. This means that the yeast on primary carbon sources, such as for example, glucose, even under aerobic conditions, and does not breathe. This fermentation activity can already be observed from a glucose concentration of about 100 mg / l glucose in the culture medium, since from this concentration the limit of the respiratory capacity of the yeast cell has been reached. The reason for this is that, in the presence of glucose, a large number of the respiratory central metabolism genes, ie the citrate and glyoxylate cycles (see FIGS. 1 a.) And b.)) And the respiratory chain, are strongly repressed by transcription (Gancedo 1998). This phenomenon is also referred to as glucose or catabolite repression.
  • Fermentation is another aspect which may be detrimental to the biotechnological production of succinic acid because it leads to the formation of undesirable by-products.
  • especially the formation of glycerol, acetate and ethanol is problematic because it leads to serious yield losses.
  • All organisms which have the crabtree effect such as the yeast Saccharomyces cerevisiae, ferment even under aerobic conditions.
  • the formation of fermentation end products, especially of ethanol is not undesirable and should be avoided. Under aerobic conditions, this can be achieved in part by making a continuous addition of a small amount of glucose to the culture medium, which prevents or reduces the glucose repression and thus the fermentation under aerobic conditions.
  • ethanol Under anaerobic conditions, oxygen is not available as a final electron acceptor, so the yeast must ferment in any case to reoxidize NADH and thus remain metabolically active.
  • the pyruvate decarboxylase activity can be switched off, which is catalysed in the yeast Saccharomyces cerevisiae by 3 pyruvate decarboxylase isoenzymes, encoded by the genes PDC1, PDC5 and PDC6.
  • PDC6 is only weakly expressed in both glucose and ethanol growth. (Velmurugan et al., 1997).
  • the gene PDC2 encodes a transcriptional inducer, which is mainly responsible for the expression of the genes PDC1 and PDC5.
  • the PDC2 gene thus offers the possibility with only a single deletion of the major part of the pyruvate decarboxylase activity, which of 3 Genes is encoded to abolish in the cell.
  • one or more of the genes PDC1, PDC5 and / or PDC6 may be deleted. This has the great advantage that thus the problematic ethanol formation can be avoided with only a single modification in the metabolism of the yeast. In order to minimize or reduce the yield losses in the biotechnical production of succinic acid in yeast, byproduct formation, especially of ethanol should be avoided or greatly reduced.
  • the PDC2 gene may be preceded by a repressible promoter or an inducible promoter.
  • the repressible promoter ensures sufficient transcription of the downstream gene in the growth phase and stops transcription in the production phase by adding an additive to the culture medium.
  • the inducible promoter is induced during the growth phase by adding an inducer to the culture medium, whereby the downstream gene PDC2 is sufficiently transcribed and growth of the yeast culture becomes possible. Once the inductor is exhausted no further growth is possible and the production phase is initiated. Without pyruvate decarboxylase activity, no growth of the yeast cell is possible because insufficient acetyl-CoA, formed via acetaldehyde and acetate, is available for fatty acid biosynthesis.
  • This enzyme which is essential for the efficient production of organic acids, undergoes inactivation by phosphorylation and in the presence of glucose enhanced proteolytic degradation (Lopez-Boado et al 1988, Ordiz et al., 1996).
  • glucose enhanced proteolytic degradation Lipez-Boado et al 1988, Ordiz et al., 1996.
  • glucose-induced negative regulatory effects at the protein level can also be assumed. This mainly concerns acetyl-CoA synthetase (Acslp), malate synthase (MIs Ip) and malate dehydrogenase (Mdh3p).
  • the glucose-induced protetolytic degradation or inactivation of these enzymes can be prevented by expressing heterologous isoenzymes in the yeast Saccharomyces cerevisiae.
  • These enzymes are derived from a microorganism which naturally has a glyoxylate cycle and "crabtree" is negative, since enzymes of the glyoxylate cycle from such an organism are not subject to glucose-induced negative regulation or proteolytic degradation at the protein level.
  • An active glycolysis cycle is essential for the efficient production of organic acids with high yields on primary carbon sources.
  • suitable "crabtree" donor organisms are Escherichia coli, Anaerobiospirillum, Actinobacillus, Mannheimia or Corynebacterium.
  • deletion of a gene refers to the complete removal of the gene from the genome of the microorganism and / or the removal of the thereby encoded active enzyme from the microorganism.
  • Inactivation of a gene refers to the reduction or complete elimination of the activity of the enzyme or protein encoded by the gene. This can be checked by measuring the relevant enzymatic activity with standard tests or by determining the relevant enzyme or protein by means of, for example, immunological detection reactions. Inactivation can be carried out, for example, by reducing or inhibiting gene expression (transcription and / or translation).
  • the introduction of antisense nucleic acids by addition or incorporation of nucleic acid sequences into the genome which are transcribable to the antisense nucleic acid
  • the introduction of mutations into the endogen which reduce or completely eliminate the activity of the gene product
  • the introduction of gene specific DNA binding factors, such as zinc finger transcription factors, that reduce gene expression cause the replacement of the Endogens by a foreign gene which encodes u r f a corresponding, but inactivated or reduced active enzyme or protein.
  • a promoter under the control of which endogenous is concerned, be deleted or mutated, so that a transcription is reduced or inhibited.
  • microorganisms such as yeast cells
  • transformation of microorganisms can be carried out in a customary manner, and reference is made to the references Schiestl RH, et al., Curr Genet. Dec, 16 (5-6): 339-346 (1989), or Manivasakam P., et al., Nucleic Acids Res. Sep 11, 21 (18): 4414-4415 (1993), or Morgan AJ, Experientia Suppl., 46: 155-166 (1983).
  • Vehicles suitable for transformation, in particular plasmids are known for example from the references Naumovski L., et al., J Bacteriol, 152 (1): 323-331 (1982), Broach JR, et al., Gene, 8 (1): 121-133 (1979), Sikorski RS, et al., Genetics, 122 (1) 19-27 (1989).
  • These vectors are Yep24, Yepl3, pRS vector series, as well as YCp 19 or pYEXBX.
  • suitable in the context of the invention expression cassettes is typically carried out by fusion of Promo sector with the coding for the gene nucleic acid sequence and optionally a terminator by conventional recombination and cloning techniques, such as in the references Maniatis T., et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory, ColD Spring Harbor, NY, USA 5 1989, or Sihlavy TJ, et al., Experiments with Gene Fusion, Co.D. Spring Harbor Laboratory, ColD Spring Harbor, NY, USA, 1984, or Ausubel FM, et al. 5 Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, 1987.
  • the invention further relates to the use of a microorganism according to the invention for producing an organic carboxylic acid of the glyoxylate and / or citrate cycle, in particular an organic dicarboxylic acid, preferably succinic acid, as well as its use in a process for the preparation of an organic carboxylic acid of the glyoxylate and / or Citrate cycle, especially of an organic dicarboxylic acid, preferably of succinic acid, with the following process steps: A) in a growth process step, the microorganism is cultured and propagated under preferably aerobic conditions, optionally with addition of an inducer substance for inducing the inducible promoter and / or glutamate, B) then the microorganism is cultured in a production phase under preferably anaerobic conditions, optionally with the addition of an inducer substance Repression of the repressible promoter, C) subsequent to step B) or during step B), the carboxylic acid is separated from the culture supernatant and optionally purified.
  • step A) is carried out up to a cell density of at least 100 g of dry biomass / l, preferably at least 120 g / l, most preferably at least 140 g / l.
  • Step b) may be carried out to a carboxylic acid concentration of at least 0.4 mol / l, preferably at least 0.8 mol / l, most preferably at least 1.0 mol / l.
  • a pH in the range of 4 to 9, preferably 6 to 8, and a
  • step B) a pH in the range of 4 to 9, preferably 6 to 8, and a salt concentration in the range of 0.01 to 0.5 mol / l, preferably from 0.05 to 0.2 mol / l, most preferably from 0.05 to 0.1 mol / l.
  • the step A) is preferably carried out at a temperature of 20 to 35 ° C, preferably 28 to 30 0 C, and for a period of 1 to 1000 h, preferably from 2 to 500 h, most preferably 2 to 200 h performed.
  • step B) is conducted at a temperature of 15 to 40 0 C, preferably from 20 to 35 0 C, most preferably 28 to 30 0 C, and for a duration of 1 to 1,000 hours, preferably from 2 to 500 h, most preferably 2 to 200 hours, preferably.
  • WMVIII medium Liang C, Looman AC, Appl Microbiol Biotechnol. 44 (1-2): 147-156 (1995)
  • the amount of tetracycline in the culture medium is preferably below 20 mg / l, preferably below 10 mg / l, most preferably below 1 mg / l, up to values below the detection limit and / or the CuSO 4 concentration in the Culture medium, if used, preferably above 1 ⁇ M, most preferably above 5 ⁇ M. Examples of suitable ranges are 1 to 3 ⁇ M or 3 to 15 ⁇ M.
  • WMVIU medium is suitable, and a conventional molasses medium can also be used.
  • the amount of tetracycline, if used, is preferably above 1 mg / l, most preferably above 3 mg / l. Examples of suitable ranges are 1 to 3 mg / l or 3 to 15 mg / l.
  • the CuSO 4 concentration used in the culture medium is preferably below 20 ⁇ M, preferably below 10 ⁇ M, most preferably below 1 ⁇ M, up to values which are below the detection limit
  • Stage C) can then be carried out on stage B). Then the culture supernatant is separated from the microorganisms, for example by filtration or centrifugation. However, stage C) can also be carried out during stage B), continuously or discontinuously. In the latter case, at least part of the culture supernatant is removed and replaced by new culture medium and this process may be repeated several times.
  • the succinic acid is obtained from the removed culture supernatant. Continuous separation may be via suitable membranes or by passing a stream of culture medium through a succinic acid separation device.
  • the invention further relates to a method for producing a microorganism according to the invention, wherein a) the genes idhl and idpl are deleted or inactivated, and / or b) the genes sdh2 and sdhl are deleted or inactivated, and / or c) the
  • Gene PDC2 is deleted or inactivated or under the control of a repressible or inducible by exposure of the microorganism to an inducer substance
  • Promoter is provided, and / or d) one or more genes from the group consisting of ICLl, MLSl, ACSl and MDH3 replaced or supplemented by a corresponding foreign gene or corresponding foreign genes from a crabtree negative organism.
  • Fig. 1 a representation of the citrate and Glyoxylatzyklus with the genes involved, metabolites and enzymes or proteins, and
  • Fig. 2 metabolism of succinate to glutamate in the wild type.
  • Example 1 Preparation of a microorganism having a glyoxylate cycle which is transcriptional and at the protein level not subject to glucose repression for the production of organic acids of the respiratory central metabolism, in particular of succinic acid
  • the glucose-induced proteolytic degradation or inactivation of the enzymes of the glyoxylate cycle can be prevented by expressing heterologous isoenzymes in the yeast Saccharomyces cerevisiae.
  • These enzymes are derived from a microorganism which naturally has a glyoxylate cycle and "crabtree" is negative, since enzymes of the glyoxylate cycle from such an organism do not undergo the glucose-induced negative regulation, or the proteolytic Subject to degradation at the protein level.
  • Spenderorganisrniis come here for example Escherichia coli, Anaerobiospirillum, Actinobacillus, Mannheimia and Corynebacterium in question.
  • the transcriptional deregulation of these enzymes is achieved by placing the genes under the control of a constitutive promoter.
  • the genes acs acetyl-CoA synthetase
  • aceA isocitrate lyase
  • aceB malate synthase A
  • mdh malate dehydrogenase
  • the constitutive ADHI promoter is used, which by modification of the natural sequence leads to a constitutive, glucose and ethanol independent expression over a very long period of time (Lang and Looman 1995).
  • the coding nucleic acid sequence for the expression cassette from ADHlprom - acs (aceA, aceB, mdh) - TRPlterm. was amplified by PCR using standard methods from the vector pFlatl-acs (aceA, aceB, mdh). The DNA fragment obtained was cloned after a Klenow treatment in the vector pUG6 in the EcoRV interface blunt-end and gave the vector pUG6 acs (aceA, aceB, MDH).
  • an extended fragment from the vector pUG6-acs (aceA, aceB, mdh) was amplified by PCR, so that the resulting fragment consists of the following components: loxP-kanMX-loxP-ADHlprom-acs (aceA, aceB, mdh) - tryptophan terminator.
  • primer oligonucleotide sequences were selected which contain on the 5 'and 3' overhangs each 5 'or 3' sequence of the acs (aceA, aceB, mdh) gene and in the annealing region the sequences 5 'of the loxP regions and 3 'of the tryptophan terminator.
  • the selection marker used is in each case the resistance to G418.
  • the respective yeast strain is transformed with the cre recombinase vector pSH47 (Guldener et al., 1996).
  • This vector expresses the cre recombinase in the yeast, with the result that the sequence region within the two loxP sequences recombines out.
  • only one of the two loxP sequences and the respective expression cassette remains in the original corresponding gene locus.
  • the yeast strain loses the G418 resistance again and thus is suitable for integrating or removing further genes into the yeast strain by means of this cre-lox system.
  • the vector pSH47 can then be removed by counterselection on YNB agar plates supplemented with uracil (20 mg / L) and FOA (5-fluoroorotic acid) (1 g / L).
  • uracil (20 mg / L)
  • FOA 5-fluoroorotic acid
  • the cells carrying this plasmid must first be cultured under non-selective conditions and then be grown on FOA-containing selective plates. Under these conditions, only cells can grow that are unable to synthesize uracil itself. In this case, these are cells that no longer contain a plasmid (pSH47).
  • Example 2 Preparation of a microorganism for the biotechnological production of succinic acid and other organic acids, which is a more efficient manufacturing process by reducing
  • succinic acid must be enriched as a final product and must not be further metabolized by the yeast cell.
  • Yield losses can also result from the further metabolism of the succinate formed via the enzyme succinate-semialdehyde dehydrogenase.
  • This enzyme is part of the glutamate degradation pathway and catalyzes the conversion of succinate to succinate semialdehyde.
  • This intermediate is then metabolized via gamma-amino butyric acid to glutamate.
  • not only yield losses can be incurred, but also ⁇ -ketoglutarate and glutamate can be synthesized, which makes it impossible to control the fermentation process via glutamate supplementation. Therefore, the additional deletion of the gene uga2, which codes for the succinate-semialdehyde dehydrogenase, in an optimized production process for the production of succinic acid is advantageous.
  • Glyoxylate which is necessary for the circulation of the Glyoxylatzyklus, can be formed not only from the reaction catalyzed by the isocitrate lyase, wherein isocitrate is cleaved in succinate and glyoxylate, but also by the reaction of alanine glyoxylate aminotransferase.
  • This enzyme catalyzes the formation of glyoxylate and alanine from pyruvate and glycine.
  • glyoxylate need not necessarily be provided from the isocitrate-lyase reaction
  • circulation of the glyoxylate cycle can also be ensured by the reaction of the alanine-glyoxylate-aminotransferase reaction which provides glyoxylate.
  • the isocitrate-lyase activity by which the desired product succinate is formed is not necessary for the circulation of the glyoxylate cycle, with the result that the yeast partially reverses the alanine-glyoxylate aminotransferase-catalyzed alternative reaction to the glyoxylate Synthesis uses. There is no succinate, which leads to yield losses.
  • the additional deletion of the gene agxl, which codes for the alanine glyoxylate aminotransferase, in an optimized production process for the production of succinic acid is advantageous.
  • the idhl deletion does not lead to a complete disappearance of isocitrate dehydrogenase activity.
  • the reason for this are 3 other isoenzymes of isocitrate dehydrogenase, which can compensate for the elimination of the main dimeric enzyme encoded by the IDH1 and IDH2 genes with respect to the formation of ⁇ -ketoglutarate.
  • the gene idhl which codes for an isoenzyme of isocitrate dehydrogenase, must be deleted in order to completely suppress the isocitrate dehydrogenase activity on glucose.
  • the complete suppression of isocitrate dehydrogenase activity has the advantage that all the carbon in the respiratory system of the yeast is diverted into the glyoxylate cycle in the direction of succinic acid and can not flow to ⁇ -ketoglutarate, which would lead to yield losses.
  • yield losses in the biotechnical production of succinic acid in yeast can be minimized or reduced by deleting the genes sdhl, agxl, uga2 and idpl in addition to the genes sdh2 and idh.
  • the coding nucleic acid sequence for the deletion cassette loxP-kanMX-loxP was amplified by PCR using standard methods from the vector pUG6 (Guldener et al., 1996), so that the resulting fragment consists of the following components: loxP-kanMX-loxP.
  • primer oligonucleotide sequences were selected which contain at the 5 'and 3' overhangs each 5 'or 3' sequence at the beginning and at the end of the native locus of the genes to be deleted ⁇ sdhl, agxl, uga2, idpl) and in annealing region sequences 5 'of the loxP region and 3' of the second loxP region.
  • the selection marker is resistance to G418 (encoded by kanMX).
  • the resulting yeast strain is cre Recombinase vector pSH47 (Guldener et al., 1996). This vector expresses the cre recombinase in the yeast, with the result that the sequence region recombines out within the two loxP sequences. As a result, only one of the two loxP sequences remains at the deleted gene locus (sdhl, agxl, uga2, idpl).
  • the consequence is that the yeast strain loses the G418 resistance again and thus is suitable for integrating or removing further genes into the yeast strain by means of this cre-lox system.
  • the vector pSH47 can then be removed by counterselection on YNB agar plates supplemented with uracil (20 mg / L) and FOA (5-fluoroorotic acid) (1 g / L).
  • the cells carrying this plasmid must first be cultivated under non-selective conditions and then be grown on FOA-containing selective plates. Under these conditions, only cells can grow that are unable to synthesize uracil itself. In this case, these are cells that no longer contain a plasmid (pSH47).
  • all genes to be deleted (sdhl, agxl, uga2, idpl) were iteratively deleted.
  • Table 1 shows by way of example the yield increases by deletions described above after culturing the strains indicated in the table for 72 hours in WM8 medium (Lang and Looman, 1995) with 3.52 g / l ammonium sulfate and 0.05 M Na 2 HPO 4 and 0.05 M NaH 2 PO 4 as a buffer, and with histidine 100 mg / l, leucine 400 mg / l, uracil 100 mg / l.
  • the C source was 5% glucose. It was inoculated l% from a 48h preculture. Cultivation was carried out in 100 ml shake flasks on a shaking incubator at 30 ° C. and 150 rpm.
  • strain 5 and 6 did not grow in medium without glutamate, biomass was first generated with these strains in 75 ml of standard WM8 medium with NaGlutamat and glucose 5% in 250 ml baffled flasks. The cells were washed and resuspended in the above-mentioned medium for further cultivation.
  • Table 1 Succinate titer after 72 hours cultivation of the indicated strains in WM8 medium under the conditions described above
  • Example 3 Preparation of a microorganism for the biotechnological production of succinic acid and other organic acids, which allows a more efficient production process by reducing by-product formation, in particular of ethanol and acetate.
  • Fermentation is another important aspect, which is detrimental in the biotechnological production of succinic acid, since it leads to the formation of unwanted by-products. In this context, especially the formation of acetate and ethanol is problematic because it leads to serious losses in yield.
  • One way to prevent alcoholic fermentation, ie the formation of ethanol, is to turn off ethanol biosynthesis starting from pyruvate. This also prevents acetate formation via acetaldehyde.
  • the pyruvate decarboxylase activity which is in the yeast Saccharomyces cerevisiae by 3 pyruvate decarboxylase isoenzymes encoded by the genes PdCl 1 PDC5 and PDC6, catalyzed.
  • the gene PDC2 encodes a transcriptional inducer, which is mainly responsible for the expression of the genes PDC1 and PDC5.
  • the PDC2 gene offers the possibility of eliminating in the cell the major part of the pyruvate decarboxylase activity, which is encoded by 3 genes, with only a single deletion. This has the great advantage that thus the problematic ethanol production can be avoided with only a single modification in the metabolism of the yeast.
  • the PDC2 gene can be preceded by an inducible promoter.
  • the inducible promoter is added during the growth phase by adding a
  • the CUP1 promoter was chosen and integrated chromosomally in front of the "open reading frame" of the PDC2 gene so that it is under the control of the copper-inducible CUP1 promoter.
  • the coding nucleic acid sequence for the CUPI promoter cassette was amplified by PCR using standard methods, so that the resulting fragment consists of the following components: loxP-kanMX-loxP-CUPlpr.
  • primer oligonucleotide sequences were selected which contain on the 5 'and 3' overhangs each 5 'or 3' sequence of the native promoter of the PDC2 gene and in the annealenden the sequences 5 'of the loxP region and 3' of CUPlprom.
  • the selection marker is resistance to G418.
  • the resulting strain contains a copy of the PDC2 gene under the control of the copper-regulated CUPI
  • the resulting yeast strain is transformed with the cre recombinase vector pSH47 (Guldener et al., 1996).
  • This vector expresses the cre recombinase in the yeast, with the result that the sequence region recombines out within the two loxP sequences.
  • only one of the two loxP sequences and the CUPI promoter cassette remains in front of the coding sequence of the PDC2 gene.
  • the consequence is that the yeast strain loses G418 resistance again and is thus able to integrate or remove further genes into the yeast strain by means of this cre-lox system.
  • the vector pSH47 can then be removed by counterselection on YNB agar plates supplemented with uracil (20 mg / L) and FOA (5-fluoroorotic acid) (1 g / L).
  • uracil (20 mg / L)
  • FOA 5-fluoroorotic acid
  • the cells carrying this plasmid must first be cultivated under non-selective conditions and then be grown on FOA-containing selective plates. Under these conditions, only cells can grow that are unable to synthesize uracil itself. In this case, these are cells that no longer contain a plasmid (pSH47).
  • Example 4 Preparation of a microorganism for the biotechnological production of succinic acid and other organic acids, which involves a separation of growth and production phase via glutamate
  • Another advantage of the complete suppression of isocitrate dehydrogenase activity is that all the carbon in the respiratory system of the yeast is diverted into the glyoxylate cycle in the direction of succinic acid and can not flow off to ⁇ -ketoglutarate, which would lead to yield losses (see FIG e.)).
  • the coding nucleic acid sequence for the deletion cassette loxP-kanMX-loxP was amplified by PCR using standard methods from the vector pUG6 (Guldener et al., 1996), so that the resulting fragment consists of the following components: loxP-kanMX-loxP.
  • primer oligonucleotide sequences were selected which contain at the 5 'and 3' overhangs each 5 'or 3' sequence at the beginning and at the end of the native locus of the gene to be deleted idpl and in the annealenden the sequences 5 'of loxP Region and 3 'of the second loxP region.
  • the selection marker is resistance to G418 (encoded by kanMX).
  • the resulting yeast strain is transformed with the cre recombinase vector pSH47 (Guldener et al., 1996).
  • This vector expresses the cre recombinase in the yeast, with the result that the sequence region recombines out within the two loxP sequences.
  • only one of the two loxP sequences remains at the deleted gene locus idpl.
  • the consequence is that the yeast strain loses the G418 resistance again and thus is suitable for integrating or removing further genes into the yeast strain by means of this cre-lox system.
  • the vector pSH47 can then be removed by counterselection on YNB agar plates supplemented with uracil (20 mg / L) and FOA (5-fluoroorotic acid) (1 g / L).
  • uracil (20 mg / L)
  • FOA 5-fluoroorotic acid
  • the cells carrying this plasmid must first be cultured under non-selective conditions and then be grown on FOA-containing selective plates. Under these conditions, only cells can grow that are unable to synthesize uracil itself. In this case, these are cells that no longer contain a plasmid (pSH47).
  • the production strain AH22ura3AsdhJAsdh2AidhlAidpl was evaluated on its growth characteristics with and without supplemented glutamate.
  • the reference strain was AH22um3Asdhl Asdh2Aidhl, ie the strain without additional deletion of idpl.
  • the two strains were dissolved in 20 ml of WM8 medium in 100 ml flasks once with 3.52 g / l ammonium sulfate (as nitrogen source) and 0.05 M K2HPO4, and 0.05 M KH2PO4 as buffer and in standard WM8 medium (Lang and Looman 1995) containing 10 g of NaGlutamate as a nitrogen source. After 64 h, the optical density of the 4 cultures was determined. The results are shown in Table 2.
  • Table 2 Optical density of the indicated strains after 64 hours cultivation in WM8 medium with and without glutamate. In the medium without glutamate, NH3SO4 was used as nitrogen source.

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Abstract

L'invention concerne un micro-organisme isolé, génétiquement modifié, comparé au type sauvage présente les caractéristiques suivantes: a) les gènes idh1 et idp1 ont été supprimés ou inactivés, et/ou b) les gènes sdh2 et sdh1 ont été supprimés ou inactivés, et/ou c) le gène PDC2 a été supprimé ou inactivé ou est sous le contrôle d'un promoteur qui peut être supprimé ou induit par exposition du micro-organisme à une substance inductrice, et/ou d) un ou plusieurs gènes du groupe constitué de ICL1, MLS1, ACS1 und MDH3 ont été remplacés ou complétés par un ou plusieurs gènes étrangers correspondants provenant d'un organisme à Crabtree négatif. L'invention concerne également l'utilisation desdits micro-organismes.
EP09748218A 2008-10-17 2009-10-07 Micro-organisme permettant de produire de l'acide succinique Withdrawn EP2344656A1 (fr)

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US9605285B2 (en) 2011-01-25 2017-03-28 Cargill, Incorporated Compositions and methods for succinate production
TWI555800B (zh) 2011-04-04 2016-11-01 拜耳材料科學股份有限公司 聚胺基甲酸酯脲分散體
BR112015001601A2 (pt) 2012-07-25 2017-11-07 Bioamber Sas células de levedura tendo curso de tca redutor de piruvato em succinato e superexpressão de uma enzima transidrogenase nad(p)+ exógena
CA2884266C (fr) 2012-09-14 2022-05-24 Myriant Corporation Production d'acides organiques par fermentation a bas ph
CN105431529A (zh) * 2013-05-03 2016-03-23 德克萨斯大学系统董事会 用于真菌脂质生产的组合物和方法
EP3064519B1 (fr) 2015-03-06 2018-12-19 Covestro Deutschland AG Dispersion aqueuse d'urée de polyuréthane, contenant des groupes acides libres
ES2708202T3 (es) 2015-06-25 2019-04-09 Covestro Deutschland Ag Dispersiones de poliuretano desprovistas de urea
WO2017022583A1 (fr) * 2015-08-06 2017-02-09 花王株式会社 Nouveau promoteur
CN105838632B (zh) * 2016-05-19 2019-05-10 江南大学 一种产琥珀酸的酿酒酵母基因工程菌及其应用
WO2018131898A2 (fr) 2017-01-10 2018-07-19 경희대학교 산학협력단 Nouvelle utilisation de souche de méthylomonas sp. dh-1
KR101954530B1 (ko) * 2017-09-13 2019-05-23 경희대학교 산학협력단 메탄을 이용하여 숙신산을 생성하는 재조합 미생물 및 이의 용도
KR20200023451A (ko) 2017-06-30 2020-03-04 피티티 글로벌 케미칼 피씨엘 숙신산 생성이 증가된 유전자 변형 효모
EP3636321A1 (fr) 2018-10-10 2020-04-15 Covestro Deutschland AG Dispersions d'urée de polyuréthane provenant au moins partiellement de sources renouvelables et leur production et leurs utilisations
EP3888627A1 (fr) 2020-03-31 2021-10-06 Covestro Deutschland AG Dispersions de polyuréthane biobasées pour applications cosmétiques décoratives
EP3889196A1 (fr) 2020-03-31 2021-10-06 Covestro Deutschland AG Dispersions de polyuréthane biobasées pour applications de protection solaire
FR3137832A1 (fr) 2022-07-13 2024-01-19 L'oreal Emulsion huile-dans-eau comprenant un polyuréthanne et une charge spécifiques
FR3142671A1 (fr) 2022-12-06 2024-06-07 L'oreal Composition présentant une stabilité accrue

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5143834A (en) 1986-06-11 1992-09-01 Glassner David A Process for the production and purification of succinic acid
US5869301A (en) 1995-11-02 1999-02-09 Lockhead Martin Energy Research Corporation Method for the production of dicarboxylic acids
US6190914B1 (en) 1996-12-12 2001-02-20 Universiteit Van Amsterdam Methods for modulating metabolic pathways of micro-organisms and micro-organisms obtainable by said methods
JP3942718B2 (ja) * 1997-07-17 2007-07-11 宝ホールディングス株式会社 酒類、食品の製造方法
KR100372218B1 (ko) 2000-06-29 2003-02-14 바이오인포메틱스 주식회사 유기산을 생산하는 균주 및 이를 이용한 유기산의 생산방법
WO2006020663A2 (fr) * 2004-08-09 2006-02-23 Rice University Production de succinate aerobie dans des bacteries
DE102007019184A1 (de) 2007-04-20 2008-10-23 Organo Balance Gmbh Mikroorganismus zur Herstellung von Bernsteinsäure

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; 5 June 1993 (1993-06-05), HASELBECK R J ET AL: "Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases.", Database accession no. NLM8099357 *
HASELBECK R J ET AL: "Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 5 JUN 1993 LNKD- PUBMED:8099357, vol. 268, no. 16, 5 June 1993 (1993-06-05), pages 12116 - 12122, ISSN: 0021-9258 *
See also references of WO2010043197A1 *

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