WO2020208842A1 - 遺伝子組換え微生物及びこれを用いた目的物質の生産方法 - Google Patents

遺伝子組換え微生物及びこれを用いた目的物質の生産方法 Download PDF

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WO2020208842A1
WO2020208842A1 PCT/JP2019/035286 JP2019035286W WO2020208842A1 WO 2020208842 A1 WO2020208842 A1 WO 2020208842A1 JP 2019035286 W JP2019035286 W JP 2019035286W WO 2020208842 A1 WO2020208842 A1 WO 2020208842A1
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amino acid
microorganism
wild
genetically modified
activity
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French (fr)
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徹 中屋敷
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Green Earth Institute Co Ltd
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Green Earth Institute Co Ltd
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Priority to JP2021513154A priority Critical patent/JP7360741B2/ja
Priority to CN201980069550.XA priority patent/CN112888776B/zh
Priority to CN202310343756.5A priority patent/CN116445387A/zh
Priority to KR1020217036600A priority patent/KR20210151897A/ko
Priority to EP19924352.8A priority patent/EP3954756A4/en
Priority to US17/602,998 priority patent/US12600991B2/en
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Definitions

  • the present invention is a modified phosphoenols that reduce or inactivate certain enzymatic activities such as lactate dehydrogenase enzyme activity, succinate dehydrogenase enzyme activity or fumaric acid reductase activity and are resistant to feedback inhibition by aspartic acid.
  • the present invention relates to a recombinant microorganism having pyruvate carboxylase activity and a method for producing a substance using the same. This application is based on the Japanese Patent Application No. submitted to the Japan Patent Office on April 12, 2019. Priority is claimed based on JP2019-76629 (Japanese Patent Application No. 2019-76629), and the contents of the above-mentioned Japanese patent application are incorporated herein by reference for all purposes.
  • amino acids made from petroleum-derived raw materials there are also amino acids made from petroleum-derived raw materials.
  • aspartame which is widely used in medical products, food additives, artificial sweeteners, etc.
  • aspartic acid which is widely used as a raw material for polyaspartic acid, which is a biodegradable resin
  • Fumaric acid synthesized in is used as a raw material. More specifically, a method of synthesizing aspartic acid by adding ammonia to fumaric acid synthesized from petroleum and allowing an aspartate enzyme produced by a microorganism to act on the fumaric acid is adopted.
  • Patent Document 1 describes a mutant aspartic acid dehirogenase in which the enzyme activity is enhanced by introducing a mutation into aspartic acid dehirogenase derived from Bacillus subtilis, and a method for producing L-aspartic acid using the same. ing.
  • the method for producing L-aspartic acid described in Patent Document 1 actually purifies the above-mentioned mutant aspartic acid dehydrogenase expressed in Escherichia coli, and produces aspartic acid in an in vitro enzyme reaction system using the purified mutant enzyme. The method of making it is adopted. Therefore, strictly speaking, the method described in Patent Document 1 cannot be said to be a fermentation production technique for aspartic acid.
  • Patent Document 2 aspartic acid derived from Pseudomonas erginoza PA01 strain and Ralstonia eutropha JM134 strain, which exhibit high catalytic activity even at room temperature. A dehirogenase has been found, and a method for producing L-aspartic acid using these aspartic acid dehydrogenases is disclosed. More specifically, in Patent Document 2, an enzyme mixture was prepared by treating Escherichia coli expressing the above-mentioned predetermined aspartate dehydrogenase with toluene, and aspartic acid was produced by an in vitro enzyme method using the enzyme mixture.
  • Patent Document 4 describes an Enterobacteriaceae bacterium that produces L-aspartic acid or a metabolite derived from it by introducing aspartate dehydrogenase, and L-aspartic acid using the bacterium or derived from it. A method for producing a metabolite is disclosed.
  • the bacteria disclosed in Patent Document 4 are specifically obtained by introducing various heterologous aspartate dehydrogenase genes derived from Thermotoga maritima, Corynebacterium glutamicum, etc. into Escherichia coli (Escherichia coli) or the like. It is a recombinant.
  • Patent Document 4 shows that L-aspartic acid and its downstream metabolites can be increased to a certain extent according to Escherichia coli imparted with the enzyme activity by introducing such a heterologous aspartate dehydrogenase gene. ..
  • aspartic acid is widely used as a raw material for medical products, food additives, aspartame used as an artificial sweetener, and polyaspartic acid which is a biodegradable resin.
  • aspartic acid has an important role in microorganisms as an intermediate for additional metabolites such as ⁇ -alanine and asparagine, and strengthening the metabolic system to aspartic acid is aspartic acid. It can be said that it is also important in the development of fermentation production technology for the above metabolites derived from.
  • the subject of the present invention is to enable the production of aspartic acid or a metabolite derived from it from a sugar source by direct fermentation by a microorganism instead of the above-mentioned conventional method using aspartate dehydrogenase, and can be applied to industrial production.
  • the purpose is to provide technology that can achieve a certain level of production efficiency.
  • Patent Document 4 describes a method for producing L-aspartic acid or a metabolite downstream thereof using Escherichia coli or the like modified to have various heterogeneous aspartate dehydrogenases, and additionally.
  • Some metabolizing enzymes such as ⁇ -ketoglutarate dehydrogenase and further strengthening some metabolizing enzymes such as phosphoenolpyruvate carboxylase.
  • Patent Document 4 since the technique disclosed in Patent Document 4 is a technique that depends on the expression of various heterologous aspartate dehydrogenases in Escherichia coli or the like, a sufficient expression level of the heterologous enzyme protein in the cells. It can be said that it is difficult to realize. Therefore, Patent Document 4 does not suggest any knowledge regarding the fermentative production of aspartic acid and related metabolites that can be applied to industrial production.
  • Patent Document 5 discloses a method for producing an amino acid using a coryneform bacterium inactivated by lactate dehydrogenase. However, in the first place, Patent Document 5 is a technique focusing on the production of total amino acids, and the patent document does not describe or suggest findings on the fermentative production of aspartic acid and related metabolites that can be applied to industrial production. ..
  • Patent Document 6 Escherichia coli and corynebacterium into which a mutant phosphoenolpyruvate carboxylase having a mutation that releases feedback inhibition of phosphoenolpyruvate carboxylase by aspartic acid in phosphoenolpyruvate carboxylase derived from Escherichia coli was introduced. -It is disclosed that glutamicum was produced and that various amino acids were produced using these. However, Patent Document 6 only describes that glutamic acid and lysine were actually produced, and neither describes nor suggests the findings regarding the fermentative production of aspartic acid and related metabolites that can be applied to industrial production.
  • Patent Document 7 introduces a modified phosphoenolpyruvate carboxylase gene derived from alfalfa, which does not require acetyl-CoA as a coenzyme and has a property desensitized to feedback inhibition by aspartic acid.
  • Recombinant bacteria such as Corinebacterium glutamicum have been disclosed, and formally, a method for producing an amino acid using the recombinant bacterium is also described.
  • Patent Document 7 only describes that lysine was actually produced, and does not describe or suggest any knowledge regarding the fermentative production of aspartic acid or related metabolites that can be applied to industrial production.
  • Non-Patent Document 1 predetermined metabolic enzymes such as succinate dehydrogenase (SDH) and pyruvate: quinone oxide reductase (PQO) were inactivated and a predetermined single amino acid substitution was introduced in corinebacterium glutamicum. Overexpression of mutant pyruvate carboxylase and wild phosphoenolpyruvate carboxylase has been shown to improve succinate production. Since Non-Patent Document 7 discloses a technique focusing on the production of succinic acid, neither description nor suggestion is made regarding the fermentative production of aspartic acid and related metabolites that can be applied to industrial production.
  • SDH succinate dehydrogenase
  • PQO quinone oxide reductase
  • Non-Patent Document 2 when a predetermined one amino acid substitution such as N917G is applied to phosphoenolpyruvate carboxylase derived from corinebacterium glutamicum, the enzyme activity is maintained and compared with the wild type. Therefore, it has been shown that feedback inhibition by aspartic acid and the like is reduced, and that the amount of lysine produced is improved in corynebacterium glutamicum into which the mutant ppc having the above-mentioned one amino acid mutation is introduced. Since Non-Patent Document 2 only shows the production of ricin using the above-mentioned mutant ppc, the findings on the fermentative production of aspartic acid and related metabolites that can be applied to industrial production are described and suggested. Absent.
  • Non-Patent Document 3 when a predetermined one amino acid substitution such as D299N is applied to phosphoenolpyruvate carboxylase derived from corinebacterium glutamic acid, the enzyme activity is maintained and compared with the wild type. , Feedback inhibition by aspartic acid, ⁇ -ketoglutaric acid, etc. can be reduced, and the production of glutamic acid and aspartic acid can be improved in corinebacterium glutamicum introduced with the above-mentioned monoamino acid-substituted mutant ppc. It is shown.
  • a predetermined one amino acid substitution such as D299N
  • Non-Patent Document 3 is not a document that describes findings on fermentative production of aspartic acid and related metabolites that can be applied to industrial production.
  • Non-Patent Document 4 states that, with respect to phosphoenolpyruvate carboxylase derived from Escherichia coli, the mutant enzyme in which the 620th lysine is replaced with serine has the property that the feedback inhibition by aspartic acid and malic acid is reduced. It is stated to show.
  • Non-Patent Document 4 is a mere academic study in which the effect of amino acid mutation on the above feedback inhibition on phosphoenolpyruvate carboxylase derived from Escherichia coli was investigated by an in vitro assay using a purified recombinant enzyme from the viewpoint of enzyme kinetics. This is a paper. Therefore, Non-Patent Document 4 is not a document that describes findings on fermentative production of aspartic acid and related metabolites that can be applied to industrial production.
  • the present inventor inactivates predetermined enzyme activities such as succinate dehydrogenase activity or fumaric acid reductase activity and lactate dehydrogenase activity in microorganisms, and inhibits feedback by aspartic acid. It was found that the production efficiency of aspartic acid and its related metabolites was improved by imparting modified phosphoenolpyruvate carboxylase activity showing resistance to aspartic acid. The present invention has been completed based on such findings.
  • Condition (IV) The pyruvate: quinone oxidoreductase activity is reduced or inactivated as compared with the wild-type microorganism described above.
  • at least two of the conditions (I), (II) and (IV) may be satisfied, and in certain embodiments, the conditions (I) and (II). ), Both of the conditions (I) and (IV), or both of the conditions (II) and (IV) may be satisfied.
  • a nucleic acid encoding a mutant phosphoenolpyruvate carboxylase derived from a bacterium has been introduced in a form capable of expressing the mutant phosphoenolpyruvate carboxylase, and the mutant phosphoenolpyruvate carboxylase is the above gene.
  • nucleic acid encoding mutant phosphoenolpyruvate carboxylase derived from bacteria is referred to as "nucleic acid encoding exogenous phosphoenolpyruvate carboxylase derived from microorganisms, plants, prokaryotes or bacteria”.
  • An embodiment can also be adopted by reading ".
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase is selected from the group consisting of the amino acid substitutions shown in the following (a) to (f) based on the amino acid sequence shown in SEQ ID NO: 2.
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase is selected from the group consisting of the amino acid substitutions shown in (g) to (l) below with reference to the amino acid sequence shown in SEQ ID NO: 2.
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase includes the amino acid substitution shown in (g) above and at least one of the amino acid substitutions shown in (h) to (l) above. , [8].
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase comprises the amino acid substitution shown in (g) above and the amino acid substitution shown in (i) or (l) above, [8] or The transgenic microorganism according to [9].
  • any one of [4] to [10], wherein the mutant phosphoenolpyruvate carboxylase has an amino acid sequence shown in any one of (A), (B) and (C) below.
  • the amino acid sequence of wild-type phosphoenolpyruvate carboxylase possessed by a microorganism belonging to a coryneform bacterium has an amino acid mutation capable of reducing feedback inhibition by aspartic acid in the wild-type phosphoenolpyruvate carboxylase activity.
  • the amino acid mutation is based on the amino acid sequence shown in SEQ ID NO: 2.
  • II Substitution of the amino acid corresponding to the 813th lysine to a predetermined amino acid (however, the amino acid after the substitution is not lysine); or (l) the amino acid corresponding to the 917th asparagine.
  • Amino acid substitution to a given amino acid (however, the amino acid after substitution shall not be asparagin), Including at least To the amino acid sequence of the wild-type phosphoenolpyruvate carboxylase, resistance to feedback inhibition by aspartic acid is higher than that of a protein having only the amino acid substitutions specified in (g), (i) or (l). Is high, Mutant phosphoenolpyruvate carboxylase.
  • the amino acid corresponding to the 813th lysine is preferably replaced with alanine, asparagine, glycine or serine.
  • the amino acid corresponding to the 917th asparagine is preferably replaced with alanine, phenylalanine, glycine or serine, and more preferably replaced with phenylalanine or glycine.
  • [16] The mutant phosphoenolpyruvate carboxylase according to [14] or [15], which has the amino acid sequence shown in any one of (J), (K) and (L) below:
  • (J) An amino acid sequence obtained by introducing the above amino acid substitution into the amino acid sequence shown in any one of SEQ ID NOs: 2 to 13 (preferably SEQ ID NOs: 2 to 12, more preferably SEQ ID NO: 2 to 11);
  • (K) An amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence specified in (J) above (provided that the amino acid substitution is maintained);
  • (L) An amino acid sequence having at least 60% sequence identity with respect to the amino acid sequence specified in (J) above (provided that the amino acid substitution is maintained).
  • [19] A nucleic acid encoding the mutant phosphoenolpyruvate carboxylase according to any one of [14] to [18]. [20] The nucleic acid according to [19], which is a DNA fragment.
  • Condition (IV) satisfies that pyruvic acid: quinone oxidase reductase is reduced or inactivated as compared with the wild-type microorganism corresponding to the above-mentioned recombinant microorganism, [1] to [ 13] and the genetically modified microorganism according to any one of [21] to [24].
  • [26] The genetically modified microorganism according to any one of [1] to [13] and [21] to [25], which is a genetically modified microorganism belonging to a bacterium.
  • the genetically modified microorganism according to [28] which is a genetically modified microorganism belonging to the genus Corynebacterium.
  • the genetically modified microorganism according to [31] which is a genetically modified microorganism belonging to the genus Escherichia.
  • the genetically modified microorganism according to [32] which is a genetically modified microorganism of Escherichia coli.
  • any one of [31] to [33] satisfies that the pyruvate formic acid lyase activity is reduced or inactivated as compared with the wild-type microorganism.
  • condition (I) and / or condition (II) and / or condition (IV) and / or condition (V) in the above-mentioned genetically modified microorganism is satisfied on the chromosomal DNA of the above-mentioned genetically modified microorganism.
  • the coding region of the acid dehydrogenase gene or the fumaric acid reductase gene and / or the lactate dehydrogenase gene coding region and / or the pyruvate: quinone oxide reductase gene coding region and / or the pyruvate formate lyase gene coding region is completely or The genetically modified microorganism according to any one of [1] to [13] and [21] to [34], which is realized by being partially destroyed.
  • condition (I) and / or condition (II) and / or condition (IV) and / or condition (V) in the genetically modified microorganism is a succinate dehydrogenase on the chromosomal DNA of the recombinant microorganism.
  • Gene or fumarate reductase gene coding region and / or lactate dehydrogenase gene coding region and / or pyruvate quinone oxide reductase gene coding region and / or pyruvate formate lyase gene coding region upstream of each gene
  • the genetically modified microorganism according to any one of [1] to [13] and [21] to [35], which is realized by completely or partially disrupting the expression control region.
  • a target substance is produced using the cells of the recombinant microorganism according to any one of [1] to [13] and [21] to [36] or a processed product thereof. That; and (q) recovering the above-mentioned target substance, A method of producing a target substance, including.
  • step (p) the cells of the recombinant microorganism or a processed product thereof are reacted in the reaction medium (X) under reducing conditions in which the recombinant microorganism does not substantially grow.
  • the redox potential of the reaction medium (X) is a predetermined value in the range of ⁇ 200 millivolts to ⁇ 500 millivolts.
  • the reaction medium (X) contains a saccharide.
  • step (P') Culturing and growing the recombinant microorganism in a predetermined medium (Y) under aerobic conditions.
  • Y a predetermined medium
  • the cells of the genetically modified microorganism or the processed product thereof, which are further contained and propagated in the step (p') are subjected to the test in the step (p). The method described.
  • the present invention it is possible to improve the production efficiency of aspartic acid and metabolites produced by metabolic pathways derived from the aspartic acid, and as a result, the yield of the target substance can be improved.
  • the efficiency of conversion of a starting substrate such as a saccharide to a target substance can be improved, and as a result, energy saving of a bioprocess, cost reduction, and efficient substance production can be realized.
  • Genetically modified microorganisms that satisfy at least one of the following conditions (I), (II) and (IV) and satisfy the following condition (III):
  • Condition (I) The succinate dehydrogenase activity or the fumaric acid reductase activity is reduced or inactivated as compared with the wild-type microorganism corresponding to the above-mentioned recombinant microorganism;
  • Condition (II) Lactate dehydrogenase activity is reduced or inactivated as compared with the above wild-type microorganisms;
  • the following genetically modified microorganisms are also provided. Genetically modified microorganisms that satisfy all of the following conditions (I) to (III): Condition (I) The succinate dehydrogenase activity or the fumaric acid reductase activity is reduced or inactivated as compared with the wild-type microorganism corresponding to the above-mentioned recombinant microorganism; Condition (II) Lactate dehydrogenase activity is reduced or inactivated as compared with the above wild-type microorganisms; Condition (III) More than modified phosphoenolpyruvate carboxylase activity showing resistance to feedback inhibition by aspartic acid in wild phosphoenolpyruvate carboxylase activity, or wild phosphoenolpyruvate carboxylase activity shown by the above wild microorganisms.
  • Condition (I) The succinate dehydrogenase activity or the fumaric acid reductase activity is reduced or inactivated as compared with the wild-type microorganism
  • the genetically modified microorganism according to the first aspect of the present invention and the genetically modified microorganism according to the second aspect of the invention are collectively referred to as “the genetically modified microorganism of the present invention” or “the present invention. Such genetically modified microorganisms "may be referred to.
  • microorganism may be interpreted literally, and more specifically, in the present invention, "microorganism” or “genetically modified microorganism” is a prokaryote of a bacterium, an archaea, a cyanobacterial bacterium, or the like. It can be a living thing.
  • the "microorganism” or “genetically modified microorganism” is preferably a fungus or a bacterium, and more preferably a bacterium.
  • Saccharomyces yeast e.g., Saccharomyces cerevisiae
  • Schizosaccharomyces Romi KES genus yeasts e.g., Schizosaccharomyces pombe
  • Pichia yeast e.g., Pichia pastoris
  • click Louis web b Kluyveromyces yeast Kluyveromyces lactis
  • Hansenula polymorpha Yarrowia yeasts (e.g., Yarrowia lipolytica)
  • Cryptococcus fungi e.g., Cryptococcus sp.
  • Saccharomyces cerevisiae Schizosaccharomyces pombe , Pichia pastoris and the like can be conveniently used in the present invention because gene recombination technology and a heterologous protein expression system have been established.
  • the bacteria e.g., Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Lactobacillus spp (e.g., Lactobacillus acidophilus), clostridium bacteria (e.g., Clostridium thermocellum, Clostridium acetobutylicum) Rod Examples include the genus Pseudomonas (eg, Rhodopseudomonas pulustris ), the genus Rhodopseudom ( Rhodobacter capsaturus ), and the bacteria belonging to the corine-type bacteria described in detail below.
  • Escherichia e.g., Escherichia coli
  • Bacillus e.g., Bacillus subtilis
  • Lactobacillus spp e.g., Lactobacillus acidophilus
  • clostridium bacteria e.g., Clostridium thermocellum, Clo
  • microorganism or “genetically modified microorganism” in the present invention has already established a gene recombination technique and a protein expression system, and enables substance production under reducing conditions in which cells do not substantially proliferate. It is preferably a bacterium or a coryneform bacterium, more preferably an Escherichia coli or a coryneform bacterium, and most preferably a Corynebacterium spp.
  • the recombinant microorganism according to the present invention is a microorganism belonging to a Gram-positive bacterium (for example, actinomycete).
  • the recombinant microorganism according to the present invention may be a microorganism belonging to a Gram-negative bacterium.
  • Gram-negative bacteria are specifically microorganisms belonging to the phylum Proteobacteria, and more specifically, microorganisms belonging to the Alpha-, Beta-, Gamma-, Delta-, Epsilon- or Zeta-Proteobacteria, and oligos. Includes microorganisms belonging to the Proteobacteria.
  • Examples of Gram-negative bacteria that can be preferably used in the present invention include microorganisms belonging to the family Enterobacteriaceae, Vibrio or Pseudomonadaceae.
  • coryneform bacterium refers to a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology (Vol. 8, p. 599, 1974). More specifically, as coryneform bacteria, Corynebacterium spp., Brevibacterium spp., Arthrobacter spp., Mycobacterium spp., Micrococcus ( Examples thereof include Micrococcus spp., Microbacterium spp.
  • Corynebacterium spp. include the following species and strains.
  • Corynebacterium glutamicum eg, FERM P-18976 strain, ATCC13032 strain, ATCC31831 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 strain, ATCC13287 strain, ATCC1375 strain, ATCC1373 strain , ATCC13761 strain, ATCC14020 strain
  • Corynebacterium acetoglutamicum eg, ATCC15806 strain
  • Corynebacterium acetoacidophilum eg, ATCC13870 strain
  • Corynebacterium melassecola eg, ATCC17965 strain
  • Corynebacterium efficiens eg, YS-314 strain, YS-314 T strain (NBRC100395 T strain)
  • Corynebacterium alkanoliticum eg, ATCC21511 strain
  • Corynebacterium marinum eg, DSM44953 strain
  • Corynebacterium humireducens eg, NBRC106098 strain
  • Corynebacterium halotolerans eg, YIM70093 strain
  • Brevibacterium divaricatum (Brevibacterium divaricatum) (for example ATCC14020 shares); Brevibacterium flavum [for example, MJ-233 (FERM BP-1497) strain, MJ-233AB-41 (FERM BP-1498) strain, ATCC13826 strain, ATCC14067 strain, ATCC13826 strain]; Brevibacterium imariophyllum (eg, ATCC14068 strain); Brevibacterium lactofermentum (Corynebacterium glutamicum) (Brevibacterium lactofermentum (Corynebacterium glutamicum) ) ( for example ATCC13869 shares); Brevibacterium roseum (eg, ATCC13825 strain); Brevibacterium saccharoliticum (eg, ATCC14066 strain); Brevibacterium thiogenitalis (eg, ATCC19240 strain); Brevibacterium album (eg, ATCC15111 strain); Brevibacterium cer
  • genus Earthrobacter include the following species and strains.
  • examples thereof include Arthrobacter globiformis (ATCC8010 strain, ATCC4336 strain, ATCC21056 strain, ATCC31250 strain, ATCC31738 strain, ATCC35698 strain, NBRC3062 strain, NBRC12137T strain) and the like.
  • Micrococcus spp. are Micrococcus freudenreichi [for example, No. 239 (FERM P-13221) strain]; Micrococcus luteus [eg, NCTC2665 strain, No. 240 (FERM P-13222) strain]; Micrococcus ureae (for example, IAM1010 strain); Micrococcus roseus (for example, IFO3764 strain) and the like.
  • Microbacterium spp Are Microbacteriaium ammoniaphilum (eg, ATCC15354 strain).
  • coryneform bacterial strain is, for example, an ATCC strain, it is sold from the American Type Culture Collection (PO Box 1549 Manassas, VA 20108 USA) that provides the strain. Can receive. Other strains can also be sold from each microbial preservation institution that provides these strains.
  • the genetically modified microorganism according to the present invention can be produced by subjecting the microorganisms exemplified above to a predetermined genetic manipulation.
  • the activity of pyruvate: quinone oxidoreductase is reduced or inactivated as compared with the wild-type microorganism
  • the recombinant microorganism according to the present invention is prepared.
  • the pyruvate: quinone oxidoreductase activity is significantly reduced or completely inactivated compared to the wild-type microorganisms used as starting materials.
  • the TCA cycle (citric acid cycle) shown in FIG. 1 generally metabolizes clockwise under aerobic conditions.
  • the TCA cycle metabolizes counterclockwise. That is, when the embodiment satisfying the condition (I) is adopted in the present invention, the conversion of succinic acid to fumaric acid is suppressed under aerobic conditions, so that a larger amount of citric acid, cis-aconitic acid, is used.
  • Acids, D-isocitric acid, ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid or additional metabolites derived from it can be efficiently produced, while under reducing or anaerobic conditions, higher amounts of oxalo Acetic acid, L-citric acid, fumaric acid or additional metabolites derived from it can be efficiently produced.
  • the further metabolites derived from the above metabolites in the TCA cycle are biosynthesized in the genetically modified microorganism of the present invention satisfying the condition (I) via the metabolic system originally retained by the wild-type microorganisms. It may be one that is biosynthesized via a newly constructed metabolic system by introducing a predetermined gene.
  • the embodiment satisfying the condition (IV) when the embodiment satisfying the condition (IV) is adopted in the present invention, the conversion from pyruvic acid to acetic acid is suppressed, so that the pyruvic acid is used as in the embodiment satisfying the condition (II).
  • the metabolic pathway to oxaloacetate proceeds efficiently.
  • the production of oxaloacetate, L-malic acid, fumaric acid or further metabolites derived thereto can be efficiently carried out. Therefore, in the embodiment satisfying both the conditions (II) and (IV), the production efficiency of oxaloacetate, L-malic acid, fumaric acid or further metabolites derived thereto can be further improved.
  • Embodiments can be preferably adopted in the present invention.
  • the recombinant microorganism according to the present invention satisfies at least two of the conditions (I), (II) and (IV).
  • the recombinant microorganism according to the present invention preferably satisfies both the conditions (I) and (II), both the conditions (I) and (IV), or the conditions (II) and (IV). , It is more preferable that all of the conditions (I), (II) and (IV) are satisfied.
  • the metabolic pathway from pyruvate to the TCA cycle and metabolism in the TCA cycle proceed efficiently, with oxaloacetate, L-apple acid, fumaric acid or derived metabolites in the TCA cycle, or citric acid.
  • downstream metabolites such as acid, cis-aconitric acid, D-isocitric acid, ⁇ -ketoglutaric acid, succinyl CoA, and succinic acid can be efficiently produced, these metabolites and further metabolism from these metabolites This is because the production of the induced substance can be efficiently achieved.
  • a downstream metabolite such as succinic acid or a metabolite derived thereto (for example, a metabolite derived from glutamic acid or glutamic acid).
  • a downstream metabolite such as succinic acid or a metabolite derived thereto (for example, a metabolite derived from glutamic acid or glutamic acid).
  • Efficient production is possible, and under reducing conditions, it is possible to efficiently produce downstream metabolites such as fumaric acid or metabolites derived from them (for example, aspartic acid or metabolites derived from aspartic acid).
  • downstream metabolites such as fumaric acid or metabolites derived from them (for example, aspartic acid or metabolites derived from aspartic acid).
  • condition (V) The pyruvate formic acid lyase activity is reduced or inactivated as compared with the wild-type microorganism.
  • condition (V) "the activity of pyruvate lyase lyase is reduced or inactivated as compared with the wild-type microorganism” means that the recombinant microorganism according to the present invention is produced. It means that the pyruvate lyase activity is significantly reduced or completely inactivated compared to the wild-type microorganism used as a starting material.
  • the genetically modified microorganism according to the present invention is a microorganism belonging to a Gram-negative bacterium, it is preferable that the condition (V) is satisfied.
  • the reason is as follows. Wild-type microorganisms belonging to Gram-negative bacteria express pyruvate formic acid lyase activity that is not normally found in Gram-positive bacteria. As described below, this pyruvate formic acid lyase activity creates a secondary biosynthetic pathway for synthesizing organic acids such as formic acid and acetic acid from pyruvate, as described below.
  • the pyruvate succinate lyase activity is originally an enzyme activity that can be exhibited by a wild-type microorganism used as a starting material in producing the recombinant microorganism according to the present invention. More specifically, it can be described by a systematic classification according to the type of reaction between the substrate and the enzyme and an EC number recognized as an international enzyme classification based on the reaction type, and the enzyme activity under each condition is described below. It contains the enzyme activity shown in Table 1.
  • the satisfaction of the conditions (I) and / or (II) and / or (IV) and / or (V) may be realized by using various methods of genetic engineering and molecular biology.
  • a method by gene disruption or mutagenesis targeting these genes on the genome, a method by antisense inhibition at the mRNA expression level (antisense RNA), or the like can be adopted.
  • a genetically modified microorganism that has been genetically engineered to express a peptide or protein that inhibits the activity of each enzyme is also included in the present invention.
  • the enzyme protein that can impart each enzyme activity requires a process of activation by a predetermined endogenous activator, the endogenous activator. By inactivating the enzyme, the expression of each enzyme activity may be suppressed and each condition may be satisfied.
  • amino acid mutations means amino acid mutations that can cause reduction or inactivation of each enzyme activity.
  • the condition (I) is that the endogenous activator that activates the enzymatic activity of the enzyme protein that can impart each enzymatic activity is inactivated by the method according to the above embodiments (I) to (III). ) And / or (II) and / or (IV) and / or (V).
  • embodiments (I) to (IV) can be independently adopted in order to reduce or inactivate each enzyme activity specified in each condition.
  • at least two embodiments of embodiments (I) to (IV) may be adopted as long as there is no particular contradiction.
  • both the conditions (I) and (II) may be adopted, and more specifically, both the coding region and the gene expression regulation region of each gene are microbial. May be disrupted in the genome of.
  • both embodiments (I) and (II) may be adopted to satisfy condition (I)
  • embodiment (III) may be adopted to satisfy condition (II).
  • the disruption of the gene coding region and the gene expression regulation region (target region) in the genetically modified microorganism can be described by, for example, a homologous recombination method, a genome editing technology (CRISPR / CAS system), a transposon method, a mutation introduction method, or the like. Can be realized by. Among these methods, it is convenient to adopt the homologous recombination method in that the destruction of the target region can be achieved relatively inexpensively and efficiently.
  • the following is an example of a gene disruption method by homologous recombination.
  • the method for producing a genetically modified microorganism according to the present invention is not limited to the methods shown below, and any method can be adopted.
  • Corynebacterium glutamicum which is one of the microorganisms that can be preferably used in the present invention
  • the entire genome sequence has been determined in a large number of strains such as ATCC13032 strain, R strain, ATCC21831 strain, and ATCC14067 strain, and their nucleotide sequences have been determined. Etc. are known.
  • the entire genome sequence has been determined for Corynebacterium strains such as Corynebacterium maris DSM45190 strain; Corynebacterium doosanense CAU212 strain (DSM45436 strain), and their nucleotide sequences and the like are known.
  • nucleotide sequence of each enzyme gene that imparts the activity of each enzyme according to the conditions (I), (II), (IV) and (V) and the amino acid sequence of the enzyme.
  • NCBI National Center for Biotechnology Information Support Center
  • Tables 2 to 10 below show the conditions (I) and / or (II) and / or (IV) and / or the microorganisms used as starting materials and those microorganisms in producing the recombinant microorganisms according to the present invention. Information on genes and the like that can be destroyed in order to reduce or inactivate the enzyme activity according to (V) is illustrated. Needless to say, the information on the microorganisms that can be used in the present invention and the genes that can be targeted for reducing or inactivating the activity of each enzyme is limited to those shown in the table below. is not.
  • the proteins related to these enzymes include sdhCAB (sdhCABD depending on the bacterial species), ldhA, ld and lldD (genes encoding enzyme proteins exhibiting lactate dehydrogenase activity), pokeB (pqo), genes described as pflABCD, etc. It can be encoded in (see Tables 2-10).
  • succinate dehydrogenase (Sdh) is classified into the transmembrane protein (subunit C) encoded by the sdhC gene, the flavin protein subunit (subunit A) encoded by the sdhA gene, and the sdhB gene.
  • the microorganism that can be used as a starting material in producing the recombinant microorganism according to the present invention is not limited to such a microorganism whose genomic nucleotide sequence and the like are known, and the enzyme protein. Microorganisms for which the coding region and its surrounding regions are unknown are also available.
  • the coding region of each enzyme gene should be appropriately cloned by various genetic engineering techniques, and the nucleotide sequence should be determined as necessary. For example, the area to be destroyed can be identified and cloned. For example, when an alignment analysis is performed on the amino acid sequences of known homologous enzyme proteins (Tables 2 to 10), a plurality of constant amino acid storage regions are found.
  • a degenerate primer is designed in each of the amino acid storage regions found on the N-terminal side and the C-terminal side of the enzyme protein, and the genomic DNA of the microorganism to be cloned is used as a template and the degenerate primer is used as a pair.
  • the nucleotide sequence of this partial coding region is appropriately determined, and the cloned partial coding region is targeted for gene disruption based on the method for producing a gene-disrupted strain described later, and the conditions (I) and (II) , (IV) and (V) may be satisfied, and the recombinant microorganism according to the present invention may be prepared.
  • primers may be appropriately designed in the opposite direction, the coding region of the full length of the target enzyme gene and its peripheral region may be cloned using a method such as inverse PCR, and the nucleotide sequence of those regions may be determined. Good.
  • Such a PCR-based cloning method is convenient in that the genomic region to be disrupted can be easily cloned, but as another method, a gene library of the target microorganism is prepared and an appropriate probe is designed. Then, the enzyme genes to be disrupted and their peripheral regions may be cloned by various hybridization methods, and their nucleotide sequences may be determined. Furthermore, for microbial species for which the homolog gene sequence cannot be used, the protein purification technique and each enzyme activity measurement method are combined according to the conventional method, the target enzyme is identified, the peptide sequence is partially determined, and the like. The target enzyme gene may be cloned by the above-mentioned various genetic engineering techniques.
  • a drug resistance gene such as a canamycin resistance gene is inserted into the region to be disrupted.
  • a plasmid vector for gene disruption can be mentioned. According to such a plasmid vector for gene disruption, there are regions on both sides of the drug resistance gene that are homologous to the region to be disrupted on the genome of the microorganism.
  • homologous recombination occurs between the microbial genome and the gene disruption plasmid in the form in which the drug resistance gene is inserted into the region to be disrupted on the microbial genome, so that the target enzyme gene can be disrupted. Become.
  • a plasmid vector for gene disruption a fragment in which regions located on both sides of a portion of the microbial genome to be disrupted (that is, 5'upstream and 3'upstream of the region to be removed from the genome) are tandemly linked. It is also possible to utilize the plasmid vector containing it.
  • a disruption plasmid is, for example, a plasmid vector in which the 5'upstream region and the 3'downstream region of the enzyme gene targeted for destruction are amplified by the PCR method and the fragments are tandemly linked. It can be obtained by inserting it at a predetermined location such as a multi-cloning site.
  • the entire area from 5'upstream to 3'downstream of the enzyme gene to be disrupted is amplified by the PCR method, cloned using various plasmid vectors, and then a reverse primer is designed inside the cloned region, and inverse PCR is performed.
  • a plasmid vector for gene disruption into which a deletion mutation of the enzyme gene has been introduced may be prepared.
  • the sequence length of the region homologous to the microbial genome sequence to be gene disrupted is not limited as long as homologous recombination can occur, but is generally about 500 bp or more, preferably about about 500 bp or more. It is better to have about 1000 bp. Further, it is convenient to use a plasmid having a replication origin of Escherichia coli because the construction work becomes simple if the plasmid for gene disruption can be constructed using Escherichia coli for cloning. Further, it is preferable that the plasmid for gene disruption does not have an origin of replication capable of autonomous replication in the microorganism targeted for gene disruption.
  • gene disruption plasmid has an origin of replication of the microorganism, it is recommended to remove the origin of replication by restriction enzyme treatment or the like before introducing it into a coryneform bacterium.
  • gene disruption plasmids are lethal, allowing for positive selection of drug resistance genes that allow drug selection and SacB genes that can produce toxins that inhibit the growth of Gram-negative bacteria in the presence of sucrose.
  • a combination with a sex gene may be used.
  • a gene disruption plasmid is used, a strain that has undergone homologous recombination by selection with a drug is isolated, and then selection is performed by culturing in a medium containing sucrose, and the vector is subjected to the second homologous recombination. Since it is possible to isolate a gene-disrupted strain in which a portion has been eliminated, it is possible to efficiently obtain a gene-disrupted strain.
  • the introduction of the plasmid vector for gene disruption into a microorganism is not particularly limited, but a transformation method established for various microorganisms may be used.
  • the coryneform bacterium preferably adopted in the present invention can be carried out by using an electric pulse method (for example, the method described in Van der Rest et al. Appl. Microbiol Biotechnol 52, pp541-545, 1999). It is convenient. This is because the electric pulse method enables efficient introduction of nucleic acid into coryneform bacterial cells. The destruction of the target region on the genome of the recombinant microorganism can be confirmed based on the PCR method, the Southern hybridization method, various enzyme activity measurement methods, and the like.
  • the recombinant microorganism according to the present invention further comprises, as condition (III), "modified phosphoenolpyruvate carboxylase activity exhibiting resistance to feedback inhibition by aspartic acid in wild phosphoenolpyruvate carboxylase activity, or the above-mentioned wild type phosphoenolpyruvate carboxylase activity. Having an exogenous phosphoenolpyruvate carboxylase activity that is more resistant to feedback inhibition by aspartic acid than the wild phosphoenolpyruvate carboxylase activity exhibited by type microorganisms ”can be satisfied.
  • phosphoenolpyruvate carboxylase activity specifically refers to an enzymatic activity that catalyzes a reaction defined in EC 4.11.31, and is widely possessed by a wide variety of plants and microorganisms.
  • Phosphoenolpyruvate carboxylase (PEPC) exerts enzymatic activity. The metabolic reactions catalyzed by PEPC are shown below.
  • Non-Patent Documents 2-4 that is, the "modified phosphoenolpyruvate carboxylase activity" in the present invention is different from that of the corresponding wild-type microorganism or the wild-type phosphoenolpyruvate carboxylase possessed by the microorganism. It is defined by an enzymatic property that exhibits phosphoenolpyruvate carboxylase activity but has significantly reduced feedback inhibition by aspartic acid in the enzymatic activity.
  • the meaning of the term "extrinsic phosphoenolpyruvate carboxylase activity, which is more resistant to feedback inhibition by aspartic acid than the wild-type phosphoenolpyruvate carboxylase activity exhibited by wild-type microorganisms,” is as follows. That is, the above term is possessed by a wild-type microorganism corresponding to the species to which the genetically modified microorganism according to the present invention belongs, or a wild-type microorganism used as a starting material in producing the genetically modified microorganism according to the present invention.
  • exogenous phosphoenolpyruvate carboxylase activity that is more resistant to feedback inhibition by aspartic acid as compared with "resistance to feedback inhibition by aspartic acid” shown by wild-type phosphoenolpyruvate carboxylase.
  • exogenous phosphoenolpyruvate carboxylase activity is specifically imparted by a heterologous phosphoenolpyruvate carboxylase possessed by a strain line or species different from the above-mentioned "corresponding wild-type host microorganism”. obtain.
  • the "species different from the wild-type host microorganism” includes various species such as microorganisms (for example, fungi, prokaryotes such as individual bacteria and bacteria), plants, and animals such as mammals. Furthermore, the addition of “foreign phosphoenolpyruvate carboxylase activity" in the recombinant microorganism according to the present invention is more specifically PEPC isolated from "a strain line or species different from the wild-type host microorganism". This can be achieved by introducing a nucleic acid encoding a gene.
  • modified phosphoenolpyruvate carboxylase (activity) shows resistance to feedback inhibition by aspartic acid in wild-type phosphoenolpyruvate carboxylase activity” and “foreign phosphoenolpyruvate carboxylase (activity)”.
  • the resistance to feedback inhibition by aspartic acid is higher than that of wild-type phosphoenolpyruvate carboxylase activity exhibited by wild-type microorganisms, for example, the measurement methods described in Non-Patent Documents 2 to 4, Yoshinaga, T. .. Izui, Kand Katsuki, HJ. It can be confirmed by using the measuring method or the like described in Biochem, 68, 747-750 (1970).
  • phosphoenolpyruvate carboxylase may be referred to as "PEPC” or "ppc".
  • condition (III) is not particularly limited, but can be realized in the following embodiments. That is, by introducing an amino acid mutation into the protein sequence of wild-type phosphoenolpyruvate carboxylase held by various microorganisms by a genetic engineering method, "phosphoenolpyruvate carboxylase activity" is maintained while “wild". A gene encoding a mutant enzyme that has acquired "resistance to feedback inhibition by aspartic acid in type phosphoenolpyruvate carboxylase activity" may be artificially prepared.
  • a base substitution technique such as a random mutagenesis method based on error-prone PCR or a PCR-based site-specific mutagenesis method using a mutagen primer may be used.
  • a molecular evolution method such as DNA shuffling to a plurality of types of wild-type PEPC-encoding DNA, a mutant PEPC having higher superiority may be prepared.
  • the nucleic acid encoding the mutant PEPC obtained as described above can be introduced into various microorganisms to prepare a genetically modified microorganism satisfying (III). More specifically, a nucleic acid encoding the mutant PEPC may be introduced into various microorganisms in a form capable of expressing the mutant PEPC.
  • a gene expression system suitable for each microbial species has already been established in many microbial species including coryneform bacteria.
  • the known technology may be used for introducing the above-mentioned mutant PEPC into the microorganism.
  • gene recombination technology and gene expression system technology may be independently developed and used for introducing mutant PEPC into microorganisms.
  • the mutant PEPC satisfying (III) is not particularly limited, but is a mutant obtained by introducing a predetermined mutation into a wild-type PEPC derived from a bacterium. It is preferably an enzyme. Further, such a mutant PEPC is a mutant enzyme obtained by introducing a predetermined mutation into a wild-type PEPC derived from a Corynebacterium, more preferably a Corynebacterium genus.
  • Table 11 lists examples of bacterially derived PEPCs preferably used in the present invention.
  • the following embodiments (i) and (ii) are assumed as specific configurations of the mutant PEPC satisfying the condition (III), for example.
  • (I) A mutant PEPC in which one or more amino acids are deleted, substituted or added to the amino acid sequence of wild-type PEPC.
  • the range of "one or more” is, for example, 1 to 100, 1 to 50, 1 to 30, preferably at least 2 or more, 2 to 20, more preferably 2 to 10, and even more. It is preferably 2 to 5, particularly preferably 2 to 4, 2 to 3, for example 2.
  • a chimeric PEPC composed by combining a part of the amino acid sequences of two or more wild-type PEPCs.
  • a nucleic acid encoding a mutant phosphoenolpyruvate carboxylase derived from a bacterium has been introduced in a form capable of expressing the mutant phosphoenolpyruvate carboxylase.
  • Mutant phosphoenolpyruvate carboxylase has at least one amino acid mutation that satisfies condition (III) for the recombinant microorganism.
  • the above-mentioned mutant phosphoenolpyruvate carboxylase is preferably a mutant PEPC derived from a coryneform bacterium or a Corynebacterium spp. Or an Escherichia spp., And more preferably a mutant PEPC derived from a Corynebacterium spp. , Particularly preferably a mutant PEPC derived from Corynebacterium glutamicum.
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase is based on the amino acid substitutions shown in (a) to (f) below with reference to the amino acid sequence shown in SEQ ID NO: 2.
  • amino acids shown in (a) to (f) above are intended to specify the amino acid substitution site in the PEPC amino acid sequence to be introduced into the mutation based on the amino acid contained in the amino acid sequence shown in SEQ ID NO: 2.
  • the "corresponding amino acids" in the above (a) to (f) are more specifically referred to as Clustal W and Clustal X (Bioinformatics, Vol. 23, Issue 21, November 2008, pp. 2947-2948; Bioinformatics, Volume 23, Issue 21, 1 Noveller 2007, pp2497-2948), etc., 1: 1 based on the identity of the PEPC amino acid sequence to be introduced into the amino acid sequence shown in SEQ ID NO: 2.
  • 3A to 3B show the wild-type PEPC proteins of 11 other Corynebacterium spp. As shown in Table 11 with respect to the amino acid sequence (SEQ ID NO: 2) of wild-type PEPC derived from the Corynebacterium glutamicum ATCC13032 strain. An example is shown in which multiple alignment analysis is performed on the sequences (SEQ ID NOs: 3 to 13) by BluestalW and the amino acids substituted in the above (a) to (f) are identified.
  • the "amino acid corresponding to the 299th aspartic acid" in the above (a) is aspartic acid (D) in all nine wild PEPCs belonging to the genus Corynebacterium, and coryne. It is threonine (T) in the wild-type PEPC of the Arthrobacter globiformis NBRC12137 strain, which is a type of bacterium, and glutamic acid (E) in the wild-type PEPC of the Escherichia coli K-12 strain. Further, as shown in FIG.
  • the “amino acid corresponding to the 653rd lysine” in the above (b) is used for the nine wild-type PEPCs belonging to the genus Corynebacterium . In ammoniagenes, it is arginine (R), and C.I. In doosanense, it is histidine (H), but in all other bacterial species, it is identical to lysine (K) in the reference sequence. Further, as shown in FIG. 3C, the "amino acid corresponding to the 813th lysine" in the above (c) is the same for the lysine (K) in the reference sequence in all bacterial species. Further, as shown in FIG.
  • the "amino acid corresponding to the 869th serine” in the above (d) is the same for the serine (S) in the reference sequence in all bacterial species.
  • the "amino acid corresponding to the 873rd arginine” in the above (e) is the same for arginine (R) in the reference sequence in all bacterial species.
  • the "amino acid corresponding to the 917th asparagine” in the above (f) is C.I. In ammoniagenes, it is "threonine", and C.I.
  • valine (V) in doosanense is identical to asparagine (N) in the reference sequence in all other strains.
  • the 299th aspartic acid is expressed as “D299” by using the one-letter notation of the amino acid
  • amino acid substitution of the 299th aspartic acid with asparagine is expressed as "D299N”.
  • Other amino acids and amino acid substitutions may be expressed in a similar manner.
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase is a group consisting of the amino acid substitutions shown in the following (g) to (l) with reference to the amino acid sequence shown in SEQ ID NO: 2. Includes at least one selected from.
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase is one of the amino acid substitutions shown in (g) above and the amino acid substitutions shown in (h) to (l) above. Includes at least one.
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase is the amino acid substitution shown in (g) above and the amino acid substitution shown in (i) to (l) above. Includes at least one of them.
  • the at least one amino acid mutation in the mutant phosphoenolpyruvate carboxylase comprises the amino acid substitution shown in (g) above and the amino acid substitution shown in (i) or (l) above. including.
  • the mutant phosphoenolpyruvate carboxylase may be a mutant PEPC having the amino acid sequence shown in any one of (A) to (C) below.
  • A In the amino acid sequence shown in any one of SEQ ID NOs: 2 to 13 (preferably SEQ ID NOs: 2 to 12, more preferably SEQ ID NO: 2 to 11), from the amino acid substitutions shown in (a) to (l) above. Amino acid sequence obtained by introducing at least one selected from the group (provided that the amino acid before substitution and the amino acid after substitution are different);
  • B In the amino acid sequence defined in (A) above, an amino acid sequence in which one or more amino acids are deleted, substituted and / or added (provided that at least one amino acid substitution is maintained. );
  • C An amino acid sequence having at least 60% sequence identity to the amino acid sequence specified in (A) above (provided that at least one amino acid substitution is maintained).
  • the mutant phosphoenolpyruvate carboxylase may be a mutant PEPC having the amino acid sequence shown in any one of (D) to (F) below.
  • D In the amino acid sequence shown in any one of SEQ ID NOs: 2 to 13 (preferably SEQ ID NOs: 2 to 12, more preferably SEQ ID NO: 2 to 11), the amino acid substitution shown in (g) above and the above (h) )-Amino acid sequence obtained by introducing at least one of the amino acid substitutions shown in (l) (however, the amino acid before the substitution and the amino acid after the substitution are different);
  • E An amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence specified in (D) above (provided that each of the above amino acid substitutions is maintained);
  • F An amino acid sequence having at least 60% sequence identity to the amino acid sequence specified in (D) above (provided that each of the above amino acid substitutions is maintained).
  • the mutant phosphoenolpyruvate carboxylase may be a mutant PEPC having the amino acid sequence shown in any one of (G) to (I) below.
  • G In the amino acid sequence shown in any one of SEQ ID NOs: 2 to 13 (preferably SEQ ID NOs: 2 to 12, more preferably SEQ ID NO: 2 to 11), the amino acid substitution shown in (g) above and the above (i).
  • the mutant phosphoenolpyruvate carboxylase may be a mutant PEPC having the amino acid sequence shown in any one of (J) to (L) below.
  • (J) In the amino acid sequence shown in any one of SEQ ID NOs: 2 to 13 (preferably SEQ ID NOs: 2 to 12, more preferably SEQ ID NO: 2 to 11), the amino acid substitution shown in (g) above and the above (i).
  • the range of "1 or more” is, for example, 1 to 100, 1 to 50, 1 to 30, preferably 1 to 1. 20, 1 to 15, 1 to 10, more preferably 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, One or two.
  • "at least 60%” is preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, 90. It can be read as%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • any one of “SEQ ID NOs: 2 to 13 preferably SEQ ID NOs: 2 to 12, more preferably SEQ ID NO: 2 to 11)).
  • the embodiment in which "the amino acid sequence shown in one" is read as "the amino acid sequence shown in SEQ ID NO: 2" is particularly preferable.
  • the mutant PEPC having the specified amino acid sequence in any of the above (A) to (L) retains the phosphoenolpyruvate carboxylase activity and satisfies the condition (III). There is no change in the purpose.
  • aspartate dehydrogenase (AspDH, EC 1.4.1.21), aspartate amino group transfer enzyme (AspC, EC2.6) .1.1) and the aspartate ammonia-lyase (Aspartate EC4.3.1.1,) (see FIG. 1)
  • aspartate dehydrogenase (AspDH, EC 1.4.1.21)
  • aspartate amino group transfer enzyme (AspC, EC2.6) .1.1)
  • aspartate ammonia-lyase (Aspartate EC4.3.1.1,) (see FIG. 1)
  • Genes encoding these enzymes may be additionally introduced for the enhancement of these enzymes.
  • Examples of the enzyme gene introduced into the recombinant coryneform bacterium of the present invention include enzyme genes disclosed in Patent Documents 2 and 2, and other Japanese Patent Application Laid-Open No. 2016-516435. The disclosure contents of these prior art documents are also incorporated in the present specification.
  • a starting substrate such as a saccharide can be used more efficiently for the production of a substance, and aspartic acid or a metabolite derived from the aspartic acid can be used. It can be expected that the production efficiency of target substances such as these will be significantly improved.
  • a method for producing the following target substance is provided.
  • (P) Producing a target substance using a bacterial cell of a genetically modified microorganism according to the present invention or a processed product thereof; and (q) Recovering the target substance.
  • a method of producing a target substance including.
  • the transgenic microorganism according to the present invention may be cultured under aerobic conditions in which it can grow substantially to produce the target substance.
  • aerobic conditions in coryneform bacteria, the TCA cycle shown in FIG. 1 proceeds in a clockwise direction. Therefore, taking this into consideration, it is aerobic according to the type of target substance to be produced as a target. If the embodiment relating to substance production under the conditions is suitable, the embodiment may be selected.
  • E.I. Microorganisms such as Escherichia spp. And coryneform bacteria such as cory do not grow substantially in the medium or reaction solution under the reducing conditions, and the unique metabolic system under the reducing conditions functions. Therefore, when the coryneform bacterium according to the present invention and its cell-treated product are reacted in a medium or reaction solution under reducing conditions in this way, it becomes possible to eliminate waste of nutrient sources due to proliferation and division of bacterial cells. It is possible to improve the efficiency of conversion of nutrient sources into target substances.
  • the recombinant microorganism according to the present invention has reduced or inactivated one or more enzyme activities under the conditions (I), (II) and (IV), and has resistance to feedback inhibition by metabolites. Since it retains the phosphoenolpyruvate carboxylase activity shown, it is expected that the efficiency of conversion of nutrient sources into target substances will be significantly improved. Furthermore, according to an embodiment in which the reaction proceeds under reducing conditions in which such microorganisms do not substantially proliferate, the generation of heat of fermentation is generated as compared with the bioprocess under aerobic conditions accompanied by cell division / proliferation. Since it can be prevented and it is not necessary to secure sufficient aeration during culturing, it is possible to simplify the equipment required for the bioprocess and reduce the energy, which is friendly to the global environment and leads to cost reduction.
  • the object is to react the cells of the recombinant microorganism or the processed product thereof in the reaction medium (X) under the reducing condition in which the recombinant microorganism does not substantially grow. It is preferable to produce a substance.
  • Step (p') In a more preferred embodiment, the method according to the invention is performed prior to Step (p). (P') Further comprising pre-culturing and growing the recombinant microorganism in a predetermined medium (Y) under aerobic conditions. The cells of the genetically modified microorganism grown in the step (p') or a processed product thereof are tested in the step (p).
  • the embodiment in which the step (p') is included in the method of the present invention is also assumed in the embodiment relating to the production of substances under aerobic conditions, and in particular, the recombinant microorganism is substantially substantially. It is preferably applied when the substance is produced in the reaction medium (X) under reducing conditions that do not proliferate.
  • the genetically modified microorganism is previously grown to a certain degree under aerobic conditions, and then, in the step (p), a sufficient amount of the grown genetically modified microorganism is subjected to the genetic recombination.
  • the genetically modified microorganism can be used as if it were a chemical catalyst to efficiently produce the substance.
  • the recombinant microorganism may be recovered from the reaction medium (X) after production of the substance in the reaction medium (X) and reused for the reaction in the step (p) after two cycles. It is possible.
  • specific configurations and elements that can be adopted in these steps will be described in detail in the order of step (p'), step (p), and step (q).
  • the medium (Y) is not particularly limited, and an appropriate medium (Y) may be selected and used according to the type of the recombinant microorganism used in the method.
  • an appropriate medium (Y) may be selected and used according to the type of the recombinant microorganism used in the method.
  • a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts and other nutritional substances can be used as the medium (Y).
  • the components contained in the medium are, for example, as follows.
  • Examples of the carbon source include carbohydrates, more specifically carbon-containing substances such as polysaccharides and saccharides containing monosaccharides, and various materials containing these, and examples thereof include the following components.
  • Monosaccharides such as glucose, fructose, mannose, xylose, arabinose, galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, trehalose; polysaccharides such as cellulose, starch, glycogen, agarose, pectin, alginic acid; sugar honey Etc .; non-edible agricultural waste such as rice straw, forest residue, bagas, corn stover, etc.
  • non-edible biomass sources made from non-edible herbaceous plants and woody plants
  • switch glass napier glass
  • a saccharified solution containing multiple sugars such as glucose and xylose obtained by saccharifying energy crops such as misscansus with saccharifying enzymes; sugar alcohols such as mannitol, sorbitol, xylitol and glycerin; , Organic acids such as gluconic acid; alcohols such as ethanol, propanol and butanol; hydrocarbons such as normal paraffin.
  • the carbon source may be used alone or in combination of two or more.
  • inorganic or organic ammonium compounds such as ammonium carbonate ((NH 4 ) 2 CO 3 ), ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate, urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like can be used.
  • corn steep liquor, meat extract, protein hydrolyzate (casamino acid, tryptone, peptone, NZ-amine, etc.), nitrogen-containing organic compounds such as amino acids, and the like can also be used.
  • one type can be used alone or two or more types can be used in combination.
  • the concentration of the nitrogen source in the medium may be appropriately adjusted according to conditions such as the type and properties of the genetically modified microorganism to be adopted, the type of nitrogen compound, and the like, and is not particularly limited, but is not particularly limited, for example, about 0.1. It can be up to 10 w / v%.
  • Inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate (hydrate), sodium chloride, iron (II) sulfate heptahydrate, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, Calcium carbonate and the like can be mentioned.
  • the inorganic salt may be used alone or in combination of two or more.
  • the concentration of the inorganic salt in the medium may be appropriately adjusted according to conditions such as the type and properties of the genetically modified microorganism to be adopted, the type of the inorganic salt, and the like, and is not particularly limited, but is, for example, about 0.01. It may be set to ⁇ 1 (w / v%).
  • other nutritional substances include meat extract, peptone, polypeptone, yeast extract, dried yeast, corn steep liquor, skim milk powder, skim soybean hydrochloric acid hydrolyzate, animal and plant or microbial cell extracts and their decomposition products. Be done.
  • concentration of other nutritional substances in the medium may be appropriately adjusted according to conditions such as the type and properties of the genetically modified microorganism to be adopted, the type of nutritional substance, and the like, and is not particularly limited. For example, about 0. It may be 1 to 10 (w / v%).
  • vitamins can be added as needed.
  • vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol and the like.
  • a defoaming agent such as a silicone-based defoaming agent or a polyether-based defoaming agent may be added, and various defoaming agents for bacterial media are commercially available. You may use it.
  • the pH of the medium (Y) is not particularly limited as long as the genetically modified microorganism to be adopted can grow, but is preferably about 6 to 8.
  • the medium (Y) is medium A [Inui, M. et al. Et al. , Metabolic analysis of Corynebacterium glutamicum lutating lactitate and succinate products under oxygen development connections. J. Mol. Microbiol. Biotechnol. 7: 182-196 (2004)], BT medium [Omumasaba, C.I. A. Et al. , Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8: 91-103 (2004)], the NA medium and the like described in Examples of the present specification can be preferably used.
  • the bacterial cells obtained by culturing and growing the recombinant microorganism of the present invention in the medium (Y) as described above or the bacterial cell-treated product thereof may be subjected to the step (p).
  • the culture conditions of the genetically modified microorganism are appropriately set so that the genetically modified microorganism can sufficiently grow and a sufficient amount of bacterial cells or a processed product thereof can be obtained.
  • the culture can be carried out under aerobic conditions at a culture temperature of about 25 ° C. to 38 ° C. and a culture time of about 12 hours to 48 hours.
  • the specific form of the "bacteria or a processed product thereof" is not particularly limited as long as it can produce the target substance.
  • the recombinant microorganism in step (p'), is cultivated and propagated in the medium (Y), and then the recombinant microorganism is recovered or separated from the medium (Y).
  • the medium (Y) containing the genetically modified microorganism may be used as it is in the step (p), and the target substance may be produced using the cells of the genetically modified microorganism.
  • a carbon source that can be a component of the reaction medium (X) described later is added to the medium (Y) containing the recombinant microorganism obtained in the step (p'), if necessary.
  • Nitrogen source, inorganic salts, vitamins, reducing agent, etc. may be added and used in the reaction for producing the target substance in step (p).
  • the cell itself obtained by separating and recovering the recombinant microorganism cultured and propagated in the medium (Y) in the step (p') from the medium (Y), or the bacterium thereof.
  • the cell-treated product obtained by subjecting the body to a predetermined physical or chemical treatment may be used in the step (p). Examples of the method for separating and recovering the recombinant microorganism from the medium (Y) include centrifugation, separation by various filters, decantation and the like.
  • the "treated cell product" is not particularly limited as long as the reaction for producing the target substance in step (p) can be realized, but more specifically, for example, with respect to the recovered cell cells. Examples thereof include those subjected to various chemical treatments, those immobilized on carriers such as acrylamide, carrageenan, and other suitable polymers.
  • composition of reaction medium (X) realizes a reaction medium (X) under reducing conditions in which the recombinant microorganism does not substantially proliferate and the production reaction of the target substance by the recombinant microorganism proceeds.
  • the reaction medium (X) may contain, for example, a carbon source, a nitrogen source, an inorganic salt, or the like, and may be a natural one derived from an organism or the like, or an artificially synthesized one.
  • the components contained in the reaction medium (X) are, for example, as follows.
  • Examples of the carbon source include carbohydrates, more specifically polysaccharides, saccharides containing monosaccharides, and various materials containing these, and examples thereof include the following components.
  • Monosaccharides such as glucose, fructose, mannose, xylose, arabinose, galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, trehalose; polysaccharides such as cellulose, starch, glycogen, agarose, pectin, alginic acid; Sugar honey, etc .;
  • Non-edible agricultural waste such as rice straw, forest residue, bagas, corn stover, etc.
  • Non-edible biomass sources made from non-edible herbaceous plants and woody plants
  • Switch glass Napier glass
  • a saccharified solution containing multiple sugars such as glucose and xylose obtained by saccharifying energy crops such as miscanthus with saccharifying enzymes; sugar alcohols such as mannitol, sorbitol, xylitol and glycerin; Organic acids such as acid and gluconic acid; alcohols such as ethanol, propanol and butanol; hydrocarbons such as normal paraffin.
  • the carbon source one type may be used alone or two or more types may be used in combination.
  • the concentration of the carbon source in the reaction medium (X) is preferably about 1 to 20 (w / v%), more preferably about 2 to 10 (w / v%), and about 2 to 5 (w / v%). v%) is even more preferable.
  • the concentration of saccharides in the reaction medium (X) is, for example, about 1-20 (w / v%), more preferably about 2-10 (w / v%), even more preferably about 2-5 (w). / V%).
  • inorganic or organic ammonium compounds such as ammonium carbonate ((NH 4 ) 2 CO 3 ), ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate, urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like can be used.
  • nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolyzate, and amino acid can also be used.
  • one type can be used alone or two or more types can be used in combination.
  • the concentration of the nitrogen source in the reaction solution may be appropriately adjusted according to the type of the recombinant microorganism to be used, the type and properties of the desired target substance, the reaction conditions, the type of the nitrogen compound, etc., and is particularly limited. Although it is not a compound, it can be adjusted to, for example, about 0.1 to 10 (w / v%).
  • Inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate (hydrate), sodium chloride, iron (II) sulfate heptahydrate, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, Calcium carbonate and the like can be mentioned.
  • Inorganic salts may be used alone or in combination of two or more.
  • the concentration of the inorganic salt in the reaction solution may be appropriately adjusted according to the type of the recombinant microorganism to be used, the type and properties of the desired target substance, the reaction conditions, the type of the inorganic salt, etc., and is particularly limited. Although it is not a substance, it may be, for example, about 0.01 to 1 (w / v%).
  • vitamins can be added to the reaction medium (X) as needed.
  • vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol and the like.
  • the pH of the reaction medium (X) is not particularly limited as long as the reaction for producing the desired target substance proceeds, but is generally preferably about 6.0 to 8.0. More preferably, it is 6.5 to 8.0, for example, around 7.5.
  • specific preferred basic compositions of the reaction medium (X) include the above-mentioned BT medium and the like, and based on the composition of these media, as described above, the concentration of the carbon source (sugar) and nicotinic acid.
  • the reaction medium (X) can be prepared by appropriately adjusting the concentration (xn), biotin concentration (xb), and the like of at least one of the above and its derivatives.
  • the reduction condition in which the coryneform bacterium does not substantially grow means that the reaction medium is in a reduced state to the extent that the recombinant microorganism does not actually grow, but more specifically, the reaction. It is defined by the redox potential of the medium.
  • the redox potential of the reaction medium (X) is preferably about ⁇ 200 mV to ⁇ 500 mV, more preferably about ⁇ 250 mV to ⁇ 500 mV.
  • the redox potential of the reaction medium (X) can be measured using an oxidation-reduction potential meter. Since there are commercially available redox potential meters, those commercially available products may be used for measuring the redox potential of the reaction medium (X) in the present invention.
  • the reduced state of the reaction medium can be estimated with a resazurin indicator (decolorization from blue to colorless in the reduced state) as a simple method, but if more accurate control is desired, a redox potentiometer (for example, BROADLEY) It may be measured using ORP Electrodes) manufactured by JAMES.
  • the method for adjusting the reaction medium (X) under the reducing conditions is not particularly limited, and various methods can be used.
  • the following known method for preparing an aqueous solution for a reaction solution can be used. it can. That is, as the solvent of the reaction medium, an aqueous solution for a reaction solution may be used instead of distilled water or the like, and the method for adjusting the aqueous solution for the reaction solution is, for example, a method for preparing a culture solution for an absolutely anaerobic microorganism such as a sulfate-reducing microorganism. (Pfennig, N.
  • an aqueous solution for a reaction solution under reducing conditions can be obtained by heat-treating or depressurizing distilled water or the like to remove the dissolved gas.
  • it is dissolved by treating distilled water or the like for about 1 to 60 minutes, preferably about 5 to 40 minutes under reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less.
  • the gas, especially the dissolved oxygen, can be removed to prepare an aqueous solution for the reaction solution under reducing conditions (anaerobic state).
  • an appropriate reducing agent for example, thioglycolic acid, ascorbic acid, cystine hydrochloride, mercaptoacetic acid, thiol acetic acid, glutathione, sodium sulfide, etc.
  • an appropriate reducing agent for example, thioglycolic acid, ascorbic acid, cystine hydrochloride, mercaptoacetic acid, thiol acetic acid, glutathione, sodium sulfide, etc.
  • An appropriate combination of these methods is also a method for preparing an aqueous solution for a reaction solution under effective reducing conditions.
  • the reaction system is made of nitrogen gas or the like. Examples thereof include a method of encapsulating with an inert gas or carbon dioxide gas.
  • the pH maintenance adjusting solution of the reaction system is added or various nutrient dissolving solutions are added. In some cases, it may be necessary to add oxygen as appropriate. In such a case, it is effective to remove oxygen from the added solution in advance.
  • the method of the present invention includes a step (p')
  • the above-mentioned predetermined operation is performed on the medium (Y) in which the gene-recombinant microorganism of the present invention has grown in the step (p').
  • the medium (Y) adjusted so as to satisfy the reduction conditions in which the transgenic microorganism does not substantially proliferate by adding a reducing agent or the like is used as the reaction medium (X) in the step (p). You may.
  • the reaction temperature in the step (p) may be any range as long as it produces a desired target substance, and may be appropriately set according to the properties of the genetically modified microorganism to be adopted, and is not particularly limited. Typically, it is about 20 to 50 ° C., preferably about 25 to 47 ° C., more preferably about 27 to 37 ° C., and the target substance can be efficiently produced within such a temperature range.
  • the reaction time may be appropriately adjusted so as to obtain the desired target substance, and is not particularly limited, but is, for example, about 1 hour to about 7 days, and from the viewpoint of a more efficient target substance, about 1 hour to. It is preferably about 3 days, for example, about 1 hour to 48 hours.
  • the reaction may be a batch type, a fed-batch type, or a continuous type. Of these, the batch type is preferable.
  • step (p) After the reaction in step (p) is completed, the recombinant microorganism and its cell-treated product are recovered from the reaction medium (X) by an appropriate operation such as centrifugation, and the recovered genetically modified microorganism is reused in the step. (P) may be repeated a plurality of times. Such a configuration in which the step (p) is repeated a plurality of times by reusing the genetically modified microorganism leads to a reduction in production cost and can realize efficient production of the target substance, and thus is a preferred embodiment in the present invention. is there.
  • step (q) After producing the target substance in step (p), the target substance is recovered as step (q).
  • the term "recovering the target substance (that)” is used by collecting a genetically modified microorganism containing the target substance and / or a culture solution or a reaction medium. It is a concept that includes the recovery of substances.
  • the target substance may be recovered by collecting the transgenic microorganism containing the target substance and / or the culture solution or the reaction medium, but the target substance is contained.
  • the target substance may be recovered by separating and / or purifying the target substance from the culture solution or reaction medium (X), the recombinant microbial cell, or the processed product thereof.
  • the separation and / or purification process of the target substance In the embodiment in which the separation and / or purification process of the target substance is adopted, the purity and the like required for the separation and purification process of the target substance in consideration of the type of the target substance and the use of the target substance are obtained. Appropriate separation / purification techniques may be adopted accordingly. Although not particularly limited, for example, various filtration techniques such as various crystallization methods and ultrafiltration methods, various chromatography techniques such as ion exchange chromatography, affinity chromatography, hydrophobic chromatography, and reverse phase chromatography. , Concentration method, dialysis, activated charcoal adsorption method, etc. can be appropriately combined to recover the target substance. Since various techniques for separating and purifying these substances are known, they may be appropriately used. Further, the method of the present invention may further include steps such as optionally washing, drying, crushing, powdering or granulating, and / or packing the target substance.
  • ⁇ Type of target substance> various target substances can be produced in high yield by utilizing the genetically modified microorganism according to the present invention.
  • the type of target substance varies depending on the type of recombinant microorganism to be adopted, but specifically, nucleic acid-related compounds (for example, adenine, guanine, shikimic acid, timine, uracil, 5'-guanylate, adenosine, etc.
  • ATP ATP, CDP-choline
  • various physiologically active substances such as hormone substances; carbohydrates and sugars; vitamin-related substances and coenzymes (for example, vitamin C, vitamin B2, B12, sorbose, NAD, FAD, coenzyme A); proteins, Peptides, amino acids; amino acid derivatives such as L-3,4-dihydroxyphenylalanine (L-DOAP), 5-hydroxytryptophane, pyrrolidone carboxylic acid; alcohols such as ethanol, butanol, isopropanol; phenol, catechol, 4-hydroxybenzoic acid , 4-Aminobenzoic acid, anthranilic acid, gallic acid, succinic acid, fumaric acid, malic acid, shikimic acid, 3-dehydroshikimic acid, 3-dehydroquinic acid, protocatechuic acid, corrismic acid and other various organic compounds. ..
  • the target substance is at least one selected from the group consisting of amino acids, alcohols, aromatic compounds, and organic acids.
  • the target substance is preferably an L-amino acid or a derivative thereof.
  • the amino acids are valine, leucine, isoleucine, glutamine, aspartic acid, glutamic acid, arginine, alanine, proline, cysteine, lysine (lysine), threonine, asparagine, phenylalanine, serine, methionine, glycine, tyrosine, histidine, Contains tryptophan, cystine and theanin.
  • the L-amino acid derivative is specifically a metabolite derived from the L-amino acid in the metabolic system of a recombinant microorganism.
  • the target substance is preferably L-aspartic acid or a metabolite derived from it.
  • Metabolites derived from L-aspartic acid include amino acids and amino acid derivatives such as L-threonine, L-lysine, L-arginine and L-homoserin.
  • the substance of interest is citric acid, cis-aconitic acid, D-isocitric acid, ⁇ -ketoglutaric acid, succinyl-CoA, succinic acid or additional metabolites derived from it.
  • these metabolites can be efficiently produced by culturing or reacting the recombinant microorganism according to the present invention under aerobic conditions (Fig. 1).
  • the target substance is oxaloacetate, L-malic acid, fumaric acid or a metabolite derived from it. These metabolites can be efficiently produced by reacting the genetically modified microorganism according to the present invention under reducing conditions in which the recombinant microorganism does not substantially proliferate (FIG. 1).
  • the substance of interest is oxaloacetate, malic acid or a metabolite via these compounds on the biosynthetic pathway.
  • the target substance is aspartic acid or a metabolite derived from it.
  • the target substance is aspartic acid, beta-alanine, or asparagine.
  • the target substance may be one produced by a genetically modified microorganism by a metabolic system inherited from the wild type under the conditions prescribed in the present invention, or further artificially produced by genetic manipulation, mutation treatment, or the like. It may be produced in a metabolic system, or it may be produced by a combination of these two metabolic systems. Furthermore, in the method for producing the target substance according to the present invention, the final target substance is synthesized from the substance produced by the recombinant microorganism by a chemical synthesis process or a bioprocess by a cell-free enzyme metabolism. Embodiments that further include steps are also envisioned.
  • target substance produced in the present invention is not limited in any way, and examples thereof include pharmaceutical use, food use, industrial use, fuel use, and cosmetic use.
  • target substance produced in the present invention may be a substance actually used for various purposes, or may be an intermediate raw material for use in producing a final product.
  • the following mutant phosphoenolpyruvate carboxylase has an amino acid mutation in the amino acid sequence of wild-type phosphoenolpyruvate carboxylase possessed by a microorganism belonging to a coryneform bacterium, which can reduce feedback inhibition by aspartic acid in the wild-type phosphoenolpyruvate carboxylase activity.
  • the amino acid mutation is based on the amino acid sequence shown in SEQ ID NO: 2.
  • the mutant phosphoenolpyruvate carboxylase is a mutant PEPC having the amino acid sequence shown in any one of (J) to (L) below.
  • (J) In the amino acid sequence shown in any one of SEQ ID NOs: 2 to 13 (preferably SEQ ID NOs: 2 to 12, more preferably SEQ ID NO: 2 to 11), the amino acid substitution shown in (g) above and the above (i).
  • the range of "1 or more” is, for example, 1 to 100, 1 to 50, 1 to 30, preferably 1 to 20, 1 to 15, 1 to 10. More preferably, it is 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2.
  • "at least 60%” is preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, 90%, 91%, 92%, 93%, 94. It can be read as%, 95%, 96%, 97%, 98%, 99%.
  • amino acid sequence shown in any one of SEQ ID NOs: 2 to 13 is “SEQ ID NO: 2".
  • the embodiment to be read as “the amino acid sequence shown in” is particularly preferable.
  • nucleic acid encoding mutant PEPC ⁇ Nucleic acid encoding mutant PEPC> According to the fifth aspect of the present invention, the nucleic acid encoding the mutant phosphoenolpyruvate carboxylase according to the fourth aspect is provided.
  • the "nucleic acid” may be provided in any form of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Furthermore, the nucleic acid according to the present invention may be in the form of a single strand or a double strand.
  • the nucleic acid is specifically an isolated nucleic acid, cDNA or cRNA. Considering that DNA is chemically more stable than RNA, the nucleic acid of the present invention is preferably provided in the form of DNA. In addition, in the present invention, the nucleic acid may be chemically modified such as methylation.
  • the nucleic acid according to the present invention may be provided in the form of a plasmid, which includes an origin of replication and the like so as to enable autonomous replication in cells of a specific microorganism, although not necessarily essential.
  • a promoter sequence, Shine-Dalgarno sequence, etc. are used so that the mutant PEPC according to the present invention can be expressed in the cells of the microorganism. It may contain a gene regulatory sequence. May include the mutant PEPC coding region.
  • the genetically modified microorganism into which the nucleic acid according to the present invention has been introduced is included in the present invention as a part of the genetically modified microorganism according to the first or second aspect of the present invention.
  • the method for producing the target substance using the genetically modified microorganism into which the nucleic acid according to the present invention has been introduced is also a part of the method for producing the target substance according to the third aspect of the present invention. Is included in the present invention.
  • the amplified DNA fragment and plasmid pHSG299 (Takara Bio Inc.) were treated with restriction enzymes BamHI and HindIII, and then DNA ligation Kit Ver. 2 (Takara Bio Inc.) was used for ligation to obtain plasmid pGE015. Furthermore, by PCR reaction, the region of about 1000 bp upstream of the ldh gene coding region and the region of about 1000 bp downstream of the gene coding region were amplified by using the genomic DNA of the Corynebacterium glutamicum ATCC13032 strain as a template. In these PCR reactions, a pair of primers F2 and R2 was used for the upstream region, and primers F3 and R3 were used for the downstream region (Table 13). Note that FIG. 2A schematically shows the positional relationship between each primer and the gene coding region.
  • thermal cycler T100TM (BIO-RAD) was used for each PCR reaction, and PrimeStar MAX (Takara Bio Inc.) was used as the PCR enzyme reagent. The same applies to the PCR reaction in the procedure described below unless otherwise specified.
  • the DNA fragments in the upstream and downstream regions of the ldh gene amplified as described above were ligated to a pGE15 vector linearized by restriction enzyme treatment with EcoRI using an In-Fusion cloning kit (Takara Bio Co., Ltd.). It was cloned.
  • the plasmid thus obtained was named pGE033.
  • pGE33 In the plasmid pGE33, the upstream fragment of the ldh gene and the downstream fragment of the gene are in the form of being tandemly linked and inserted into the multicloning site, but the coding region of the ldh gene is deleted.
  • pGE33 is a plasmid that can replicate in E. coli but not in the cells of coryneform bacteria.
  • the plasmid pGE33 was introduced into the Corynebacterium glutamicum ATCC13032 strain by the electrical pulse method (2500V, 25 ⁇ F, 200 ⁇ ; Van der Rest et al. Appl. Microbiol Biotechnol 52, pp541-545, 1999).
  • the sample after the electric pulse method was A agar medium containing 25 ⁇ g / ml of casamino acid (composition in 1 L of medium: urea: 2 g, (NH 4 ) 2 SO 4 : 7 g, KH 2 PO 4 : 0.5 g, K 2 HPO.
  • pGE33 has a kanamycin resistance gene as a drug resistance marker, as described above, in the growth strain grown on the A agar medium containing kanamycin, pGE33 has one point with the wild-type ldh gene on the chromosome. It is a strain that has undergone homologous recombination and has been integrated into genomic DNA together with the plasmid.
  • the growth strain thus obtained was applied to an LB agar medium (composition in 1 L of medium: bactopeptone: 10 g, yeast extract: 5 g, sodium chloride: 10 g, agar: 16 g) to which 10% sucrose was added. It was cultured by the method.
  • the transformant carrying the SacB gene derived from pGE33 cannot survive on the medium supplemented with sucrose because a toxic substance is produced.
  • the transformant in which the plasmid-derived region containing the SacB gene was lost due to the homologous recombination again can survive on the medium to which sucrose was added, so that the plasmid-derived region was lost and the ldh gene was deleted.
  • the transformant is obtained as a growing strain.
  • the homologous recombination is performed again, the complete pGE33 form in which the entire plasmid region is removed returns to the trait of the wild-type ATCC13032 strain that carries the ldh gene in intact.
  • primers F2 and R3 are primers designed at the 5'end of the region about 1000 bp upstream of the ldh gene and the 3'end of the region about 1000 bp downstream of the gene, respectively, they may be strains lacking the ldh gene. For example, a DNA fragment of about 2 kb should be obtained. Using this as an index, agarose gel electrophoresis (Molecular Cloning, Sambrook et al., 1989 Cold Spring Harbor Laboratory Press) was performed on the product obtained by the above colony PCR, and colony cells in which a deletion of the ldh gene was confirmed were obtained. It was obtained as an ldh gene-deficient strain (GES168).
  • the sdhCAB gene was further deleted from the ldh gene-deficient strain (ATCC13032 ⁇ ldh strain) (GES168) according to the method for obtaining the ldh gene-deficient strain. That is, using the genomic DNA of the Corynebacterium glutamicum ATCC13032 strain as a template, a region of about 1000 bp upstream of the sdhCAB gene coding region and a region of about 1000 bp downstream of the gene coding region were amplified. In these PCR reactions, a pair of primers F4 and R4 was used for the upstream region, and primers F5 and R5 were used for the downstream region (Table 14).
  • sdhCAB constitutes an operon on the genome by the sdhC coding region, the sdhA coding region, and the sdhB coding region.
  • the positional relationship between each primer and each coding region is shown in FIG. 2B. Has been done.
  • the upstream and downstream DNA fragments of the SdhCAB gene amplified as described above were ligated to a pGE15 vector linearized by restriction enzyme treatment with EcoRI using an In-Fusion cloning kit (Takara Bio Co., Ltd.). , Cloned.
  • the plasmid thus obtained was named pGE020.
  • the plasmid pGE020 was introduced into the ATCC13032 ⁇ ldh strain (GES168) according to the above-mentioned electric pulse method in the same manner as when the ATCC13032 ⁇ ldh strain (GES168) was obtained, and ATCC13032 ⁇ ldh ⁇ sdh was screened by colony PCR and selection using a kanamycin-containing medium and a sucrose-containing medium. Acquired the stock (GES439)
  • the poxB gene was further deleted from the ATCC13032 ⁇ ldh ⁇ sdh strain (GES439). That is, using the genomic DNA of the Corynebacterium glutamicum ATCC13032 strain as a template, a region of about 1000 bp upstream of the pokeB gene coding region and a region of about 1000 bp downstream of the gene coding region were amplified. In these PCR reactions, a pair of primers F6 and R6 was used for the upstream region, and primers F7 and R7 were used for the downstream region (Table 15). Note that FIG. 2C schematically shows the positional relationship between each primer and the gene coding region.
  • the upstream and downstream DNA fragments of the pokeB gene amplified as described above were ligated to a pGE15 vector linearized by restriction enzyme treatment with EcoRI using an In-Fusion cloning kit (Takara Bio Co., Ltd.). , Cloned.
  • the plasmid thus obtained was named pGE191.
  • the plasmid pGE191 was introduced into the ATCC13032 ⁇ ldh ⁇ sdh strain (GES439) according to the above-mentioned electric pulse method in the same manner as when the ATCC13032 ⁇ ldh strain (GES168) was obtained, and ATCC13032 ⁇ ldh ⁇ sdh ⁇ pox by selection with a kanamycin-containing medium and a sucrose-containing medium and screening by colony PCR. Acquired the stock (GES524)
  • the DNA fragment related to the origin of replication of Escherichia coli was amplified by the PCR method using pMW119 (Takara Bio Inc.) as a template and the pair of primers F9 and R9 shown in Table 16. Further, the kanamycin resistance gene DNA fragment was amplified by the PCR method using pHSG299 (Takara Bio Inc.) as a template and the pair of primers F10 and R10 shown in Table 16.
  • plasmid pGEK004 was named a shuttle vector for Corynebacterium glutamicum ATCC13032.
  • the region of about 900 bp corresponding to the N-terminal side of PEPC was amplified by the PCR method using the ATCC13032 strain genomic DNA as a template and the pair of primers F12 and R12-1 shown in Table 18.
  • the region of about 3500 bp including the C-terminal region of PEPC was amplified by using the pair of primers F13-1 and R13 shown in Table 18 using the ATCC13032 strain genomic DNA as a template.
  • the primer R12-1 (reverse primer) used for amplifying the N-terminal fragment of PEPC and the primer F13-1 (forward primer) used for amplifying the C-terminal fragment of PEPC are the coding regions of PEPC. It overlaps in and contains the mutant codons associated with D299N (“GTT” / “AAC”, respectively, underlined in Table 18).
  • the corresponding wild-type codon is "GAC" (sense strand).
  • the three DNA fragments obtained as described above are cyclized by ligating them in tandem using an In-Fusion cloning kit (Takara Bio Inc.) to a vector fragment linearized by treating pGEK004 with a restriction enzyme with BamHI. , The plasmid pGE320 was obtained.
  • the C-terminal region of PEPC can be obtained.
  • the region containing about 530 bp was amplified.
  • the primer R12-2 reverse primer used for amplifying the N-terminal fragment of PEPC and the primer F13-2 (forward primer) used for amplifying the C-terminal fragment of PEPC are the coding regions of PEPC. It overlaps in and contains the mutant codons associated with K813S (“CGA” / “TCG”, respectively, underlined in Table 19). The corresponding wild-type codon is "AAG" (sense strand).
  • pGE321 (ppcN917G gene)
  • a DNA fragment containing the gapA gene promoter region was amplified by a PCR reaction in the same manner as in the preparation of pGE320.
  • the PEPC coding region first, by using the PCR method, using the ATCC13032 strain genomic DNA as a template and using the pair of the primer F12 shown in Table 18 and the primer R12-3 shown in Table 20, the N-terminal side of PEPC was used.
  • the C-terminal region of PEPC can be obtained.
  • the region containing about 530 bp was amplified.
  • the primer R12-3 reverse primer used for amplifying the N-terminal fragment of PEPC and the primer F13-3 (forward primer) used for amplifying the C-terminal fragment of PEPC are the coding regions of PEPC. It overlaps in and contains mutant codons relating to N917G (“GCC” / “GGC”, respectively, underlined in Table 20). The corresponding wild-type codon is "AAC" (sense strand).
  • the two DNA fragments obtained as described above are cyclized by ligating them in tandem using an In-Fusion cloning kit (Takara Bio Inc.) to a vector fragment linearized by treating pGEK004 with a restriction enzyme with BamHI. , The plasmid pGE333 was obtained.
  • the two DNA fragments obtained as described above are cyclized by ligating them in tandem using an In-Fusion cloning kit (Takara Bio Inc.) to a vector fragment linearized by treating pGEK004 with a restriction enzyme with BamHI. , The plasmid pGE322 was obtained.
  • each recombinant coryneform bacterial strain was pre-cultured (test tube) in 5 mL of A medium, and 2 mL of each obtained pre-culture solution was placed in a 500 mL flask in NA medium (urea: 2 g, (NH 4 )).
  • the results of the aspartic acid production test are shown in FIG. In the graph of FIG. 4, the vertical axis represents the aspartic acid concentration in the sample after the reaction. Further, Table 21 below shows the genotypes of the obtained recombinant coryneform bacteria and the aspartic acid production efficiency (%) calculated by the aspartic acid production test. The value of aspartic acid production efficiency (%) is the ratio of the produced aspartic acid to 0.5 mol of glucose taken up by the cells.
  • Test Example 2 (Test procedure) For each of the corynebacterium-glutamicum gene-deficient strains GES168 ( ⁇ ldh), GES439 ( ⁇ ldh ⁇ sdhCAB), and GES524 ( ⁇ ldh ⁇ sdhCAB ⁇ poxB) produced in the above-mentioned item (1) by the above-mentioned electric pulse method, the above (2-2) The plasmid pGE333 (ppcD299N / K813S) constructed in the above section was transformed to obtain a recombinant coryneform bacterium. Using each of the obtained recombinant coryneform bacteria, a production test of aspartic acid was carried out by the same method as in Test Example 1.
  • the results of the aspartic acid production test are shown in FIG. In the graph of FIG. 5, the vertical axis represents the aspartic acid concentration in the sample after the reaction. Furthermore, Table 22 below shows the genotypes of the obtained recombinant coryneform bacteria and the aspartic acid production efficiency (%) calculated by the aspartic acid production test. The value of aspartic acid production efficiency (%) is the ratio of aspartic acid actually produced to 0.5 mol of glucose taken up by the cells. This value is based on the fact that aspartic acid can theoretically produce 1 to 2 moles of glucose.
  • the aspartic acid production efficiency is significant as shown in FIGS. 5 and 22. No significant improvement was seen.
  • the ES439 / pGE333 strain which also has a mutant ppc gene having an amino acid substitution by the combination of D299N / K813S and lacks two genes, ldh gene and sdhCAB. As for, a remarkable improvement in aspartic acid production efficiency was observed.
  • Test Example 3 In Test Examples 1 and 2, recombinant coryneform bacteria were previously grown under aerobic culture conditions using A medium and NA medium, the culture medium was removed by centrifugation, and the isolated somatic cells were placed. The aspartic acid production reaction was carried out by suspending the cells in a fixed amount of BT solution. On the other hand, in this test example, after the recombinant coryneform bacterium was grown under aerobic culture conditions using A medium and NA medium, the culture solution was prepared without forming somatic cells by centrifugation or the like. Aspartic acid production reaction was carried out using it as it was. The procedure is shown below.
  • each sample culture solution was directly transferred to a 100 mL medium bottle containing a stir bar, and 5 mL of 50% glucose and 5 mL of 2M (NH 4 ) 2 CO 3 were further added.
  • This medium bottle was placed in a constant temperature bath at 33 ° C. and allowed to stand, and the reaction was carried out while stirring with the above stirrer. After 24 hours, 0.5 mL of the reaction solution was collected, and the supernatant was collected by centrifugation, and the amount of glucose consumed and the amount of amino acids produced were measured.
  • the amino acid analysis system Prominence was used for the identification and measurement of amino acids.
  • the results of the aspartic acid production test in this test example are shown in FIG. In the graph of FIG. 6, the vertical axis represents the aspartic acid concentration in the sample after the reaction. Further, Table 23 below shows the genotypes of the obtained recombinant coryneform bacteria and the aspartic acid production efficiency (%) calculated by the aspartic acid production test. The value of the aspartic acid production efficiency (%) is, as described above, the ratio of the aspartic acid actually produced to 0.5 mol of glucose taken up by the cells.
  • Test Examples 1 to 3 when the recombinant coryneform bacterium prescribed in the present invention is used, it is possible to improve the conversion efficiency of a starting substrate such as a saccharide into a target substance.
  • a predetermined gene was disrupted in Escherichia coli according to the following procedure.
  • the strain was pre-transformed with a bacteriophage recombinant expression vector pKD46 (Life Science Market).
  • the obtained transformant was inoculated into 100 mL of LB medium (containing arabinose at a final concentration of 10 mM) and cultured at 30 ° C. until the medium turbidity OD600 was around 0.6.
  • Competent cells were prepared by washing the obtained cells of recombinant Escherichia coli with 10% Glucerol three times and finally suspending them in 1 mL of 10% Glucerol.
  • primers F14 and R14 shown in Table 24 below, a DNA fragment containing the kanamycin resistance gene coding region was amplified by PCR reaction using pKD13 as a template.
  • primers F14 and R14 each contain nucleotide sequences homologous to the upstream and downstream regions of the pflB gene coding region in E. coli chromosomal DNA.
  • the obtained PCR product was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio Inc.).
  • the obtained transformant contained 50 ⁇ g / mL of kanamycin.
  • the growth strain was selected by growing on the LB agar medium contained in. Deletion of the pflB gene was confirmed by culturing the selected growth strain in a predetermined medium and performing organic acid analysis on the supernatant of the culture solution in which the bacterial cells were separated by centrifugation. That is, a strain in which formic acid production was not observed in the organic acid analysis was obtained as a pflB gene-deficient strain.
  • the organic acid analysis was performed by HPLC analysis using a TSKgel OApak column (Tosoh).
  • pCP20 (Life Science Market) was transformed into the BW251 13 ⁇ pflB :: Km strain obtained as described above at 30 ° C., and the obtained transformant was streaked onto an antibiotic-free LB medium plate at 42 ° C. Was cultured in.
  • pCP20 is a plasmid vector designed to express Flp recombinase, which sheds a cassette of the kanamycin resistance gene under high temperature culture temperature conditions.
  • a colony sensitive to kanamycin was selected and named BW25113 ⁇ pflB.
  • the purified PCR product was gene-introduced into the above-mentioned competent cells to prepare an ldhA gene-deficient strain.
  • the deletion of the ldhA gene was confirmed by culturing the transformant in a predetermined medium and analyzing the supernatant of the culture solution separated by centrifugation by organic acid analysis by the same method as described above. That is, a strain in which lactic acid production was not observed in the organic acid analysis was obtained as an ldhA gene-deficient strain.
  • kanamycin resistance gene was also carried out by a method using pCP20 (Life Science Market) as described above, and colonies showing sensitivity to kanamycin were selected and named BW25113 ⁇ pflB ⁇ ldhA strain.
  • the purified PCR product was gene-introduced into the above competent cells to prepare a frdA gene-deficient strain.
  • the deletion of the frdA gene was confirmed by culturing the transformant in a predetermined medium and analyzing the supernatant of the culture solution separated by centrifugation by organic acid analysis by the same method as described above. That is, in the organic acid analysis, a strain in which production of succinic acid was not observed was obtained as a frdA gene-deficient strain.
  • kanamycin resistance gene was also carried out by a method using pCP20 (Life Science Market) as described above, and colonies showing sensitivity to kanamycin were selected and named as BW25113 ⁇ pflB ⁇ ldhA ⁇ frdA strain.
  • Test Example 4 By transforming the BW25113 ⁇ pflB ⁇ ldhA ⁇ frdA strain obtained as described above with pGEK004, pGE333 (ppcD229N / K813S) and pGE322 (ppcD229N / N917G) constructed in Test Example 1, respectively, the present invention can be recombined. Obtained a strain of Kori.
  • Each of the above recombinant strains was pre-cultured in 5 mL of LB medium in vitro.
  • the resulting preculture Quat medium in 500mL flask (Composition of medium 1L: Bacto tryptone 12g, yeast extract 24 g, glycerol 4mL, KH 2 PO 4 2.31g, K 2 HPO 4 12.54g) in 100mL
  • the cells were inoculated and further cultured at 37 ° C. for 20 hours at 200 rpm with shaking. After culturing, the cells were centrifuged to remove the culture solution, and the obtained cells were suspended in 40 mL of BT solution, and the OD value of the suspension was adjusted to be around 15 to 20.
  • the suspension was transferred to a 100 mL medium bottle containing a stir bar and an additional 5 mL of 50% glucose and 5 mL of 2M (NH 4 ) 2 CO 3 were added.
  • This medium bottle was placed in a constant temperature bath at 33 ° C. and allowed to stand, and the reaction was carried out with stirring. After 20 hours, 0.5 mL of the reaction solution was collected from the medium bottle and centrifuged to obtain a supernatant, and the amount of glucose consumed and the amount of amino acids produced were measured.
  • An amino acid analysis system (Shimadzu Corporation) was used for the identification and measurement of amino acids.
  • the chromatograph obtained by amino acid analysis is shown in FIG. In the chromatograph shown in FIG. 7, the peak observed around 7 minutes is the peak of aspartic acid. Further, Table 27 below shows the genotype of the obtained recombinant Escherichia coli strain, the aspartic acid production efficiency (%) calculated by the aspartic acid production test, and the like. The value of aspartic acid production efficiency (%) is the ratio of aspartic acid actually produced to 0.5 mol of glucose taken up by the cells. This value is based on the fact that aspartic acid can theoretically produce 1 to 2 moles of glucose.
  • aspartic acid as a target substance and a metabolite produced by a metabolic pathway derived from the aspartic acid are improved in production efficiency, and as a result, the yield of the target substance is improved. It was shown that it would be possible to improve the rate. That is, according to the present invention, it has been clarified that the conversion efficiency of a starting substrate such as a saccharide into a target substance can be improved, and as a result, energy saving of a bioprocess, cost reduction, and efficient substance production can be realized.
  • cloning of various gene coding regions, promoter regions, etc. includes a step using an In-Fusion cloning kit (Takara Bio Inc.) as described above, but the primer pair used for PCR amplification is described. It is supplemented that the appropriate adapter sequence is added to each of the 5'ends of the forward / reverse primer according to the instructions of the cloning kit.
  • the present invention has high industrial applicability in the fields of biotechnology, substance production, and the like.

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WO2023068295A1 (ja) * 2021-10-21 2023-04-27 伊藤忠商事株式会社 バイオプロセス、微生物を培養する方法及び標的物質を製造する方法並びにバイオプロセス装置
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