WO2010101053A1

WO2010101053A1 - Method for producing l-amino acid

Info

Publication number: WO2010101053A1
Application number: PCT/JP2010/052845
Authority: WO
Inventors: 佑介萩原; 由利長井
Original assignee: 味の素株式会社
Priority date: 2009-03-06
Filing date: 2010-02-24
Publication date: 2010-09-10
Also published as: JP2012100537A

Abstract

Disclosed is a method for producing L-amino acid, wherein a bacterium, which belongs to the family Enterobacteriaceae and has an L-amino acid-producing ability, while being modified so as to have a reduced ribonuclease G activity, is cultured in a culture medium that contains ethanol as a carbon source, so that L-amino acid is produced and accumulated in a culture, and then the thus-produced L-amino acid is collected from the culture.

Description

Method for producing L-amino acid

The present invention relates to a method for producing an L-amino acid using a microorganism. L-amino acids are used in various fields such as seasonings, food additives, feed additives, chemical products, and pharmaceuticals.

L-amino acids are industrially produced by fermentation using microorganisms belonging to the genera Brevibacterium, Corynebacterium, Escherichia and the like. In these production methods, strains isolated from nature, artificial mutants of the strains, and microorganisms modified so as to increase the activity of basic L-amino acid biosynthetic enzymes by recombinant DNA technology are used. It is used. (Patent Documents 1 to 9)

Generally, when amino acids are produced using microorganisms, a saccharide is used as a carbon source as a main component, but ethanol can also be used as a carbon source in the same manner as a saccharide (Patent Document 10). ).

Ribonuclease G was found as a ribonuclease involved in the maturation of the 5 'end of 16S rRNA (Non-patent Documents 1 and 2). Ribonuclease G is said to cleave the AU-rich region of single-stranded RNA, but details of the cleavage sequence and the like have not been elucidated (Non-Patent Documents 3 to 5).

Although little is known about the physiological role of ribonuclease G, ribonuclease G is involved in the degradation of eno mRNA and adhE mRNA encoding alcohol dehydrogenase, and the results of microarray analysis indicate that some glycolytic enzymes It has been reported that it is involved in specific degradation of mRNAs of a plurality of genes including genes to be encoded (Non-Patent Documents 6 to 8). In addition, when comparing the degradation activity for 16S rRNA and adhE mRNA, it is reported that rng: cat does not degrade both, whereas rng430 (G341S) only degrades adhE mRNA (Non-patent Document 9). ).

In addition, it has been reported that in strains lacking both the rng gene and the cra gene, pyruvic acid accumulates when cultured using glucose as a carbon source (Non-patent Document 10).

However, it has never been known that changing the activity of ribonuclease G has an effect on L-amino acid production from ethanol.

European Patent Publication EP0643135B European Patent Publication EP0733712B European Patent Publication EP1477565A European Patent Publication EP0796912A European Patent Publication EP0837134A International Publication WO01 / 53459 European Patent Publication EP1170376A International Publication WO2005 / 010175 International Publication WO96 / 17930 WO2008 / 010565

An object of the present invention is to provide a method for producing L-amino acid by fermentation using a substrate containing ethanol, which has been further improved over the prior art.

As a result of intensive studies to solve the above problems, the present inventors have found that the ability of intestinal bacteria to produce L-amino acids from ethanol is greatly improved by reducing the activity of ribonuclease G. The present invention has been completed.

That is, the present invention is as follows.
(1) Bacteria belonging to the family Enterobacteriaceae and having L-amino acid-producing ability are cultured in a medium containing ethanol as a carbon source, and L-amino acid is produced and accumulated in the culture. A method for producing an L-amino acid, wherein the bacterium is a bacterium modified so that the activity of ribonuclease G is reduced.
(2) The method as described above, wherein the rng gene encoding ribonuclease G is inactivated, whereby the activity of ribonuclease G is reduced.
(3) The said method that the said rng gene is DNA which codes the amino acid sequence of sequence number 2, or its variant.
(4) The method as described above, wherein the bacterium is modified so as to assimilate ethanol aerobically.
(5) The method as described above, wherein the bacterium retains the adh gene modified so as to be expressed under the control of a non-natural promoter functioning under aerobic conditions, and thereby can assimilate ethanol aerobically.
(6) The method as described above, wherein the bacterium is modified so as to retain a mutant adhE gene and thereby can assimilate ethanol aerobically.
(7) The method as described above, wherein the mutant adhE gene encodes a protein having the amino acid sequence of SEQ ID NO: 4 or a conservative variant thereof except that the glutamic acid residue at position 568 is substituted with another amino acid residue.
(8) The L-amino acid is L-lysine, L-glutamic acid, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L- The method as described above, which is one or more L-amino acids selected from the group consisting of tryptophan, L-proline, and L-cysteine.
(9) The L-amino acid is L-lysine, and the bacterium is dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semi The activity of one or more enzymes selected from the group consisting of aldehyde dehydrogenase, tetrahydrodipicolinate succinylase, and succinyldiaminopimelate deacylase is enhanced and / or the activity of lysine decarboxylase is weakened Said method being.
(10) The L-amino acid is L-threonine, and the bacterium is selected from the group consisting of aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase, or Said method wherein the activity of two or more enzymes is enhanced.
(11) The method as described above, wherein the bacteria belonging to the family Enterobacteriaceae are Escherichia bacteria, Enterobacter bacteria or Pantoea bacteria.
(12) The method as described above, wherein the bacterium is Escherichia coli.
(13) The method as described above, wherein ethanol is contained in the medium in an amount of 0.001 w / v% or more.

<1> Bacteria belonging to the family Enterobacteriaceae used in the present invention The bacteria used in the present invention belong to the family Enterobacteriaceae, have the ability to produce L-amino acids, and have a reduced activity of ribonuclease G. Bacteria that have been modified to The bacterium of the present invention belongs to the family Enterobacteriaceae and can be obtained by modifying a bacterium having an L-amino acid-producing ability so that the activity of ribonuclease G is reduced. Examples of the bacterium used as a parent strain of the bacterium of the present invention, which is modified so that the activity of ribonuclease G is reduced, and a method for imparting or enhancing L-amino acid-producing ability are shown below. In addition, the bacterium of the present invention has been modified so that L-amino acid-producing ability is imparted to a bacterium belonging to the family Enterobacteriaceae modified so that the activity of ribonuclease G is reduced or the activity of ribonuclease G is decreased. It can also be obtained by enhancing the L-amino acid producing ability of bacteria belonging to the family Enterobacteriaceae.

<1-1> Bacteria used as parent strain of the present invention The bacterium of the present invention belongs to the family Enterobacteriaceae and has the ability to produce L-amino acids.
The Enterobacteriaceae family includes bacteria belonging to genera such as Escherichia, Enterobacter, Erbinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, and Yersinia. In particular, enterobacteria are identified by the taxonomy used in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347). Bacteria classified in the family are preferred.

The bacterium belonging to the genus Escherichia is not particularly limited, but means that the bacterium is classified into the genus Escherichia according to the classification known to experts in microbiology. Examples of bacteria belonging to the genus Escherichia used in the present invention include, but are not limited to, Escherichia coli (E. coli).

The bacteria belonging to the genus Escherichia that can be used in the present invention are not particularly limited. For example, Neidhardt et al. (Neidhardt, F. C. Ed. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology / Second Edition pp 2477-2483. Table 1 1. American Society for Microbiology Press, Washington, DC). Specific examples include Escherichia coli W3110 (ATCC 32525) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild type K12 strain.

These strains can be sold, for example, from the American Type Culture Collection (address P.O. Box 1549 Manassas, VA 20108, United States of America). That is, the registration number corresponding to each strain is given, and it can receive distribution using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.

The bacterium belonging to the genus Pantoea means that the bacterium is classified into the genus Pantoea according to the classification known to microbiologists. Certain types of Enterobacter agglomerans were recently reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii and others (Int. J. Syst. Bacteriol., 43, 162-173 (1993)). In the present invention, the bacteria belonging to the genus Pantoea include bacteria that have been reclassified to the genus Pantoea in this way.

The bacterium used in the present invention is a bacterium having an ethanol-assimilating ability, and originally has an ethanol-assimilating bacterium, an ethanol-assimilating recombinant strain, or an ethanol-assimilating ability. It may be a mutant strain.
Regarding Escherichia coli, the presence of adhaldehyde having acetaldehyde dehydrogenase activity and alcohol dehydrogenase activity that reversibly catalyze the following reaction is known as an enzyme that produces ethanol under anaerobic conditions. The sequence of the adhE gene encoding AdhE of Escherichia coli is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
Acetyl-CoA + NADH + H ⁺ → Acetaldehyde + NAD ⁺ + CoA
Acetaldehyde + NADH + H ⁺ → Ethanol + NAD ⁺

In the present invention, it is preferable to use bacteria that can assimilate ethanol aerobically. Although Escherichia coli cannot assimilate ethanol under aerobic conditions, a strain modified so as to assimilate ethanol aerobically may be used. In order to modify bacteria that are not aerobically assimilating ethanol aerobically so that they can assimilate ethanol, for example, expression under the control of a non-native promoter that functions under aerobic conditions. Examples include retaining a modified adh gene, or retaining a mutant adhE gene having a mutation in the coding region that enables aerobic assimilation of ethanol. Furthermore, this mutant adhE gene may be expressed under the control of a non-natural promoter that functions under aerobic conditions.

In Escherichia coli, alcohol dehydrogenase can be expressed under aerobic conditions and ethanol can be assimilated aerobically by replacing the promoter upstream of the gene encoding alcohol dehydrogenase with a promoter that functions aerobically. (WO2008 / 010565 pamphlet). As the non-natural promoter that functions under aerobic conditions, any promoter that can express the adhE gene beyond a certain level under aerobic conditions can be used. Aerobic conditions can be those normally used for culturing bacteria that are supplied with oxygen by methods such as shaking, aeration and agitation. Specifically, any promoter known to express a gene under aerobic conditions can be used. For example, promoters of genes involved in glycolysis, pentose phosphate pathway, TCA cycle, amino acid biosynthesis pathway, etc. can be used. Furthermore, P _tac promoter λ phage, lac promoter, trp promoter, all trc promoter, P _R promoter, or P _L promoters are known to be strong promoters which function under aerobic conditions, the use of these Is preferred.

As described above, Escherichia coli cannot assimilate ethanol under aerobic conditions, but it is known that ethanol can be assimilated aerobically even by mutation of AdhE (Clark （D. P ., And Cronan, J. E. Jr. 1980. J. Bacteriol. 144: 179-184; Membrillo-Hernandez, J. et al. 2000. J. Biol. Chem. 275: 33869-33875). Specifically, AdhE mutants having such mutations include mutants in which the glutamic acid residue at position 568 of AdhE of Escherichia coli is substituted with an amino acid residue other than glutamic acid and aspartic acid, such as lysine (Glu568Lys, E568K (International publication pamphlet WO2008 / 010565).

Furthermore, the AdhE mutant may contain the following additional mutations.
A) Substitution of a glutamic acid residue at position 560 to another amino acid residue, such as a lysine residue B) Substitution of a phenylalanine residue at position 566 to another amino acid residue, such as a valine residue,
C) Glutamic acid residue at position 22, methionine residue at position 236, tyrosine residue at position 461, isoleucine residue at position 554, and other amino acid residues at position 786, such as a glycine residue, respectively. Substitution to valine residue, cysteine residue, serine residue, and valine residue, or D) Combination of the above mutations.

“Aerobically assimilate ethanol” means that it can grow in aerobic conditions in a minimal liquid medium or solid medium using ethanol as a single carbon source. “Aerobic conditions” can be those commonly used for culturing bacteria to which oxygen is supplied by methods such as shaking, aeration and agitation, as described above. “Aerobic assimilation of ethanol” means a cell-free extract measured by the method of Clark and Cronan (J. Bacteriol., 141, 177-183 (1980)) with respect to the level of AdhE protein. The alcohol dehydrogenase activity in is meant to be 1.5 units or more, preferably 5 units or more, and more preferably 10 units or more per mg of protein.

The bacterium of the present invention may be a strain modified so that the activity of pyruvate synthase or pyruvate: NADP ⁺ oxidoreductase is increased. In order to modify pyruvate synthase or pyruvate: NADP ⁺ oxidoreductase activity to increase, pyruvate synthase or pyruvate: NADP ⁺ oxidoreductase activity is affected by the parent strain such as a wild strain or an unmodified strain. It is preferable to modify so as to increase. In addition, when the microorganism originally does not have pyruvate synthase activity, the microorganism modified to have the enzyme activity has pyruvate synthase or pyruvate: NADP ⁺ oxidoreductase activity in an unmodified strain. It is increasing compared to this.

The “pyruvate synthase” in the present invention is an enzyme (EC 1.2) that catalyzes the following reaction for producing pyruvate from acetyl-CoA and CO _{2 in the} presence of an electron donor, for example, in the presence of ferredoxin or flavodoxin. .7.1). Pyruvate synthase is sometimes abbreviated as PS and is sometimes named pyruvate oxidoreductase, pyruvate ferredoxin oxidoreductase, pyruvate flavodoxin oxidoreductase, or pyruvate oxidoreductase. As the electron donor, ferredoxin or flavodoxin can be used.

Reduced ferredoxin + acetyl-CoA + CO ₂ → oxidized ferredoxin + pyruvate + CoA

Confirmation that the activity of pyruvate synthase is enhanced is achieved by preparing a crude enzyme solution from the microorganism before enhancement and the microorganism after enhancement and comparing the activity of pyruvate synthase. The activity of pyruvate synthase can be measured, for example, according to the method of Yoon et al. (Yoon, K. S. et al. 1997. Arch. Microbiol. 167: 275-279). For example, when pyruvic acid is added to a reaction solution containing oxidized methyl viologen as an electron acceptor, CoA, and a crude enzyme solution, the amount of reduced methyl viologen that increases due to decarboxylation of pyruvic acid is measured spectroscopically. It can be measured by measuring. One unit (U) of enzyme activity is expressed as a reduction amount of 1 μmol of methyl viologen per minute. When the parent strain has pyruvate synthase activity, the enzyme activity is preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more that of the parent strain. If the parent strain does not have pyruvate synthase activity, it is sufficient that pyruvate synthase is produced by introducing the pyruvate synthase gene, but the enzyme activity is enhanced to such an extent that it can be measured. Is preferably 0.001 U / mg (bacterial protein) or more, more preferably 0.005 U / mg or more, and still more preferably 0.01 U / mg or more. Pyruvate synthase is sensitive to oxygen and is generally difficult to express and measure (Buckel, W.and Golding, B. T. 2006. Ann. Rev. of Microbiol. 60: 27-49). Therefore, when measuring enzyme activity, it is preferable to carry out the enzyme reaction by reducing the oxygen concentration in the reaction vessel.

As a gene encoding pyruvate synthase, it is possible to use a pyruvate synthase gene of a bacterium having a reductive TCA cycle such as Chlorobium tepidum, Hydrogenobacter thermophilus, etc. . It is also possible to use a pyruvate synthase gene derived from bacteria belonging to the group of enterobacteria such as Escherichia coli. In addition, genes encoding pyruvate synthase are autotrophic methane producers such as Methanococcus maripaludis, Methanococcus janasti, Methanothermobacter thermautotrophicus, and other methanothermobacter thermautotrophicus (Autotrophic （methanogens) pyruvate synthase gene can be used.

Specifically, as the pyruvate synthase gene of Chlorobium tepidum, the base sequence shown in SEQ ID NO: 9 located at base numbers 1534432 to 1537989 of the genome sequence of Chlorobium tepidum (GenBank Accession No. NC_002932) The gene which has can be illustrated. SEQ ID NO: 10 shows the amino acid sequence encoded by the same gene (GenBank Accession No. AAC76906). Hydrogenobacter thermophilus pyruvate synthase is composed of δ subunit (GenBank Accession No. BAA95604), α subunit (GenBank Accession No. BAA95605), β subunit (GenBank Accession No. BAA95606), γ subunit. It is known to form a complex with four subunits of the unit (GenBank Accession No. BAA95607) (Ikeda, T. et al. 2006. Biochem. Biophys. Res. Commun. 340: 76-82 ). Furthermore, the pyruvate synthase gene comprising 4 genes of HP1108, HP1109, HP1110, and HP1111 located at base numbers 1170138 to 1173296 of the genome sequence of Helicobacter pylori (GenBank Accession No. Illustrates the pyruvate synthase gene encoded by the four genes SSO1208, SSO7412, SSO1207, and SSO1206 indicated by nucleotide numbers 1047593 to 1044711 in the genome sequence of Sulfolobus solfataricus (GenBank Accession No. NC 002754) be able to. Furthermore, the pyruvate synthase gene is based on the homology with the genes exemplified above, based on the genus Chlorobium, the genus Desulfobacter, the genus Aquifex, the genus Hydrogenobacter, It may be cloned from bacteria of the genus Thermoproteus, Pyrobaculum, or the like.

In Escherichia coli, the ydbK gene (b1378) having the nucleotide sequence shown in SEQ ID NO: 11 located at nucleotide numbers 1435284 to 1438808 in the genome sequence of K-12 strain (GenBank Accession No. U00096) is homologous in sequence. From the nature, it is expected to encode pyruvate flavodoxin oxidoreductase, ie, pyruvate synthase. SEQ ID NO: 12 shows the amino acid sequence encoded by the same gene (GenBank Accession No. AAC76906). Furthermore, the pyruvate synthase gene is highly homologous to the Escherichia coli pyruvate synthase gene (ydbK), and the genera Escherichia, Salmonella, Serratia, Enterobacter, Shigella It may be a pyruvate synthase gene belonging to the group of enterobacteria such as (Shigella) and Citrobacter.

The pyruvate synthase of Methanococcus maripaludis is the base number of the genome sequence of Genococcus maripardis (GenBank Accession No. NC_005791) (Hendrickson, EL et al. 2004. J. Bacteriol. 186: 6956-6969). It is encoded by the porCDABEF operon located between 1462535 and 1466397 (Lin, WC et al. 2003. Arch. Microbiol. 179: 444-456). This pyruvate synthase contains four subunits, γ, α, β, and δ. In addition to these subunits, PorE and PorF are also known to be important for the activity of pyruvate synthase. (Lin, W. and Whitman, WB 2004. Arch. Microbiol. 181: 68-73). The γ subunit is encoded by the porA gene of base numbers 1465867 to 1466397 (complementary strand) of the genome sequence, the base sequence is shown in SEQ ID NO: 13, and the amino acid sequence encoded by the same gene is shown in SEQ ID NO: 14 ( GenBank Accession No. NP_988626). The δ subunit is encoded by the genome sequence base numbers 1465595 to 1465852 (complementary strand) porB gene, the base sequence is shown in SEQ ID NO: 15, and the amino acid sequence encoded by the same gene is shown in SEQ ID NO: 16 (GenBank). Accession No. NP_988627). The α subunit is encoded by the porC gene of nucleotide numbers 1464410 to 1455773 (complementary strand) of the genome sequence. SEQ ID NO: 17 shows the nucleotide sequence, and SEQ ID NO: 18 shows the amino acid sequence encoded by the same gene. (GenBank Accession No. NP_988625). The β subunit is encoded by the porD gene at base numbers 1463497 to 14439393 (complementary strand) of the genome sequence. SEQ ID NO: 19 shows the base sequence, and SEQ ID NO: 20 shows the amino acid sequence encoded by the same gene. (GenBank Accession No. NP_988624). PorE is encoded by the porE gene of nucleotide numbers 1462970 to 1463473 (complementary strand) of the genome sequence, SEQ ID NO: 21 shows the nucleotide sequence, and SEQ ID NO: 22 shows the amino acid sequence encoded by the same gene (GenBank Accession No. NP_988623). PorF is encoded by the porF gene of base numbers 1462535 to 1462951 (complementary strand) of the genome sequence, SEQ ID NO: 23 shows the base sequence, and SEQ ID NO: 24 shows the amino acid sequence encoded by the same gene (GenBank Accession No. NP_988622).
Autotrophic methanogenic archaea Methananocaldococcus jannaschii and Methanothermobacter thermautotrophicus are also known to have the same pyruvate synthase gene These can be used.

In the present invention, “pyruvate: NADP ⁺ oxidoreductase” means reversibly catalyzing the following reaction for producing pyruvic acid from acetyl-CoA and CO _{2 in the} presence of an electron donor, for example, in the presence of NADPH or NADH. Means enzyme (EC 1.2.1.15). Pyruvate: NADP ⁺ oxidoreductase is sometimes abbreviated as PNO and sometimes as pyruvate dehydrogenase. However, in the present invention, “pyruvate dehydrogenase activity” is an activity that catalyzes a reaction of oxidatively decarboxylating pyruvate to produce acetyl-CoA, as described later. Acid dehydrogenase (PDH) is a separate enzyme from pyruvate: NADP ⁺ oxidoreductase. Pyruvate: NADP ⁺ oxidoreductase can use NADPH or NADH as an electron donor.

NADPH + Acetyl-CoA + CO ₂ → NADP ⁺ + Pyruvate + CoA

Confirmation that the activity of pyruvate: NADP ⁺ oxidoreductase was enhanced was made by preparing a crude enzyme solution from the microorganism before enhancement and the microorganism after enhancement, and comparing the activity of pyruvate: NADP ⁺ oxidoreductase. Achieved. The activity of pyruvate: NADP ⁺ oxidoreductase can be measured, for example, according to the method of Inui et al. (Inui, H. et al. 1987. J. Biol. Chem. 262: 9130-9135). For example, when pyruvate is added to a reaction solution containing oxidized methyl viologen as an electron acceptor, CoA, and a crude enzyme solution, the amount of reduced methyl viologen that increases due to the decarboxylation of pyruvate is measured spectroscopically. It can be measured by measuring. One unit (U) of enzyme activity is expressed as a reduction amount of 1 μmol of methyl viologen per minute. When the parent strain has pyruvate: NADP ⁺ oxidoreductase activity, the enzyme activity is preferably increased 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more compared to the parent strain. Is desirable. If the parent strain does not have pyruvate: NADP ⁺ oxidoreductase activity, it is sufficient that pyruvate: NADP ⁺ oxidoreductase is generated by introducing the pyruvate synthase gene, but the enzyme activity is measured. It is preferably strengthened to the extent possible, preferably 0.001 U / mg (bacterial protein) or more, more preferably 0.005 U / mg or more, and still more preferably 0.01 U / mg or more. Pyruvate: NADP ⁺ oxidoreductase is sensitive to oxygen and is generally difficult to express and measure activity (Inui, H. et al. 1987. J. Biol. Chem. 262: 9130). -9135; Rotte, C. et al. 2001. Mol. Biol. Evol. 18: 710-720).

The gene encoding pyruvate: NADP ⁺ oxidoreductase is a photosynthetic eukaryotic microorganism and is also classified as a protozoan. The pyruvate: NADP ⁺ oxidoreductase gene of Euglena gracilis (Nakazawa, M. et al. 2000) FEBS Lett. 479: 155-156), the protist Cryptosporidium parvum pyruvate: NADP ⁺ oxidoreductase gene (Rotte, C. et al. 2001. Mol. Biol. Evol. 18: 710 -720) and homologous genes are known to exist in the diatom Tharassiosira pseudonana (Ctrnacta, V. et al. 2006. J. Eukaryot. Microbiol. 53: 225-231) .

Specifically, a gene having the base sequence shown in SEQ ID NO: 25 can be exemplified as a pyruvate: NADP ⁺ oxidoreductase gene of Euglena gracilis (GenBank Accession No. AB021127). SEQ ID NO: 26 shows the amino acid sequence encoded by the same gene (GenBank Accession No. BAB12024).

The microorganism of the present invention is modified by increasing the activity of recycling the oxidized form of the electron donor necessary for the activity of pyruvate synthase to the reduced form as compared with the parent strain, for example, a wild strain or an unmodified strain, The microorganism may be modified so that the activity of pyruvate synthase is increased. Examples of the activity of recycling the oxidized form of the electron donor to the reduced form include ferredoxin-NADP ⁺ reductase activity. In addition to enhancing the recycling activity of the electron donor, the microorganism may be modified so that the activity of pyruvate synthase is increased by modifying the activity to increase pyruvate synthase activity. The parent strain may have a gene that inherently encodes the electron donor recycling activity, or originally does not have the electron donor recycling activity. Activity may be imparted by introducing a gene to be encoded, and L-amino acid producing ability may be improved.

“Ferredoxin-NADP ⁺ reductase” refers to an enzyme (EC 1.18.1.2) that reversibly catalyzes the following reaction.

Reduced ferredoxin + NADP ⁺ → oxidized ferredoxin + NADPH + H ⁺

This reaction is a reversible reaction, and reduced ferredoxin can be produced in the presence of NADPH and oxidized ferredoxin. Ferredoxin can be substituted for flavodoxin, and what is named flavodoxin-NADP ⁺ reductase also has an equivalent function. Ferredoxin-NADP ⁺ reductase has been confirmed to exist widely from microorganisms to higher organisms (Carrillo, N. and Ceccarelli, EA 2003. Eur. J. Biochem. 270: 1900-1915; Ceccarelli, EA et al. 2004. Biochim Biophys. Acta. 1698: 155-165), some have been named ferredoxin-NADP ⁺ oxidoreductase, NADPH-ferredoxin oxidoreductase.

Confirmation that the activity of ferredoxin-NADP ⁺ reductase is enhanced is achieved by preparing a crude enzyme solution from the microorganism before modification and the microorganism after modification, and comparing the activity of ferredoxin-NADP ⁺ reductase. The activity of ferredoxin-NADP ⁺ reductase can be measured, for example, according to the method of Blaschkowski et al. (Blaschkowski, H. P. et al. 1982. Eur. J. Biochem. 123: 563-569). For example, it can be measured by spectroscopically measuring the decreasing amount of NADPH using ferredoxin as a substrate. One unit (U) of enzyme activity is expressed as an oxidation amount of 1 μmol NADPH per minute. When the parent strain has ferredoxin-NADP ⁺ reductase activity, it is not necessary to enhance if the activity of the parent strain is sufficiently high. Preferably, the enzyme activity is increased 3 times or more.

A gene encoding ferredoxin-NADP ⁺ reductase has been found in many biological species and can be used as long as it has activity in the target L-amino acid producing strain. In Escherichia coli, the fpr gene has been identified as flavodoxin-NADP ⁺ reductase (Bianchi, V. et al. 1993. J. Bacteriol. 175: 1590-1595). It is also known that Pseedomonas putida has NADPH-Putidaredoxin reductase gene and Putidaredoxin gene as operons (Koga, H. et al. 1989). J. Biochem. (Tokyo) 106: 831-836).

As the Escherichia coli flavodoxin-NADP ⁺ reductase, the nucleotide sequence shown in SEQ ID NO: 27, which is located in the base number 4111749-4112495 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096) The fpr gene having SEQ ID NO: 28 shows the amino acid sequence of Fpr (GenBank Accession No. AAC76906). Further, a ferredoxin-NADP ⁺ reductase gene has been found at the base numbers 25526234 to 2527211 of the genome sequence of Corynebacterium glutamicum (GenBank Accession No. BA00036) (GenBank Accession No. BAB99777).

The activity of pyruvate synthase requires that ferredoxin or flavodoxin be present as an electron donor. Therefore, the microorganism may be modified so that the activity of pyruvate synthase is increased by modifying the ferredoxin or flavodoxin so as to improve the production ability.
Further, in addition to modification so that pyruvate synthase activity, or flavodoxin-NADP ⁺ reductase and pyruvate synthase activities are enhanced, modification may be made so that ferredoxin or flavodoxin production ability is improved.

The “ferredoxin” in the present invention is a protein that contains a non-heme iron atom (Fe) and a sulfur atom and binds an iron-sulfur cluster called a 4Fe-4S, 3Fe-4S, or 2Fe-2S cluster. The one that functions as a transmitter. “Flavodoxin” refers to a protein that functions as a one- or two-electron transmitter containing FMN (Flavin-mononucleotide) as a prosthetic genus. Ferredoxin and flavodoxin are described in McLean et al. (McLean, K. J. et al. 2005. Biochem. Soc. Trans. 33: 796-801).

The parent strain used for the modification may have a gene that inherently encodes ferredoxin or flavodoxin, or originally has no ferredoxin or flavodoxin gene, but introduces a ferredoxin or flavodoxin gene. By doing so, activity may be imparted and L-amino acid producing ability may be improved.

Confirmation that ferredoxin or flavodoxin production ability is improved compared to the parent strain, for example, wild strain or non-modified strain, can be confirmed by comparing the amount of ferredoxin or flavodoxin mRNA with the wild-type or non-modified strain. . Examples of the expression level confirmation method include Northern hybridization and RT-PCR (Sambrook, J. et al. 1989. Molecular CloningA Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). The expression level may be any as long as it is increased compared to the wild strain or the unmodified strain, for example, 1.5 times or more, more preferably 2 times or more, more preferably compared to the wild strain or the non-modified strain. It is desirable that it rises 3 times or more.

Confirmation that ferredoxin or flavodoxin production is improved compared to the parent strain, for example, wild strain or unmodified strain, should be detected by SDS-PAGE, two-dimensional electrophoresis, or Western blot using an antibody. (Sambrook, J. et al. 1989. Molecular Cloning A Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). The production amount may be any as long as it is improved as compared to the wild strain or the unmodified strain, but for example, 1.5 times or more, more preferably 2 times or more, more preferably compared to the wild strain or the non-modified strain. It is desirable that it rises 3 times or more.

The activity of ferredoxin and flavodoxin can be measured by adding to an appropriate redox reaction system. For example, Boyer et al. Discloses a method of reducing the produced ferredoxin with ferredoxin-NADP ⁺ reductase and quantifying the reduction of cytochrome C by the resulting reduced ferredoxin (Boyer, ME et al. 2006. Biotechnol. Bioeng. 94: 128-138). The activity of flavodoxin can be measured by the same method using flavodoxin-NADP ⁺ reductase.

The gene encoding ferredoxin or flavodoxin is widely distributed, and any encoded ferredoxin or flavodoxin can be used as long as pyruvate synthase and an electron donor regeneration system are available. . For example, in Escherichia coli, the fdx gene exists as a gene encoding ferredoxin having a 2Fe-2S cluster (Ta, D. T. and Vickery, L. E. 1992. J. Biol. Chem. 267: 11120 -11125), the yfhL gene is predicted as a ferredoxin gene having a 4Fe-4S cluster. The flavodoxin gene includes fldA gene (Osborne, C. et al. 1991. J. Bacteriol. 173: 1729-1737) and fldB gene (Gaudu, P. and Weiss, B. 2000. J. Bacteriol. 182: 1788-1793) is known. In the genome sequence of Corynebacterium glutamicum (GenBank Accession No. BA00036), multiple ferredoxin genes fdx (GenBank Accession No. BAB97942) at base numbers 562643 to 562963 and fer (GenBank Accession No at base numbers 1148953 to 1149270) (BAB98495) has been found. In Chlorobium tepidum, there are many ferredoxin genes, but ferredoxin I and ferredoxin II have been identified as 4Fe-4S type ferredoxin genes that serve as electron acceptors for pyruvate synthase (Yoon, K. S Et al. 2001. J. Biol. Chem. 276: 44027-44036). Ferredoxin genes or flavodoxin genes derived from bacteria having a reductive TCA cycle such as Hydrogenobacter thermophilus can also be used.

Specifically, as the ferredoxin gene of Escherichia coli, the fdx shown in SEQ ID NO: 29 located at nucleotide numbers 2654770 to 2655105 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096) Examples thereof include the gene and the yfhL gene shown in SEQ ID NO: 31 located at base numbers 2676885 to 2697945. SEQ ID NO: 30 and SEQ ID NO: 32 show the amino acid sequences of Fdx and YfhL (GenBank Accession No. AAC75578 and AAC75615, respectively). Examples of the flavodoxin gene of Escherichia coli include the fldA gene shown in SEQ ID NO: 33 located at base numbers 710688 to 710158 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096), and bases An example of the fldB gene shown in SEQ ID NO: 35 located at numbers 3037877 to 3038398 is shown. SEQ ID NO: 34 and SEQ ID NO: 36 show the amino acid sequences encoded by the fldA gene and the fldB gene (GenBank Accession No. AAC73778 and AAC75933, respectively).

As the ferredoxin gene of Chlorobium tepidum, the ferredoxin I gene shown in SEQ ID NO: 37 located at base numbers 1184078 to 1184266 of the genomic sequence of Chlorobium tepidum (GenBank Accession NC_002932), and base numbers 1184476 to A ferredoxin II gene represented by SEQ ID NO: 39 located at 1184664 can be exemplified. SEQ ID NO: 38 and SEQ ID NO: 40 show the amino acid sequences encoded by ferredoxin I and ferredoxin II (GenBank Accession No. AAM72491 and AAM72490, respectively). In addition, the Ferrobacter thermophilus ferredoxin gene (GenBank Accession No. BAE02673) and the Sulfolobus solfataricus base sequence 2345414 to 2345728 are shown. An example is the ferricoxin gene of Taricus. Furthermore, based on the homology with the genes exemplified above, the genera Chlorobium, Desulfobacter, Aquifex, Hydrogenobacter, Thermoproteus, Thermoproteus May be cloned from bacteria belonging to the genus Pyrobaculum, and also γ-proteobacteria such as Enterobacter, Klebsiella, Serratia, Erbinia, Yersinia, Corynebacterium glutamicum, etc. It may be cloned from coryneform bacteria such as Brevibacterium lactofermentum, Pseudomonas bacteria such as Pseudomonas aeruginosa, and Mycobacterium bacteria such as Mycobacterium tuberculosis.

The modification for enhancing the expression of the gene of the present invention as described above can be performed in the same manner as the method for enhancing the expression of the target gene described for imparting L-amino acid-producing ability. The gene of the present invention can be obtained by a PCR method using a chromosomal DNA of a microorganism holding them as a template.

For example, the pyruvate synthase gene of Chlorobium tepidum is prepared by PCR using primers prepared based on the nucleotide sequence of SEQ ID NO: 9, for example, primers shown in SEQ ID NOs: 41 and 42, and chromosomal DNA of Chlorobium tepidum as a template. It can be obtained by the method (polymerase chain reaction) (see White, TJ et al. 1989. Trends Genet. 5: 185-189).
Escherichia coli pyruvate synthase gene is a primer prepared based on the nucleotide sequence of SEQ ID NO: 11, for example, using primers shown in SEQ ID NOs: 43 and 44, by PCR using Escherichia coli chromosomal DNA as a template, Can be acquired.

Euglena gracilis pyruvate: NADP ⁺ oxidoreductase gene is a PCR method using a primer prepared based on SEQ ID NO: 13, for example, the primers shown in SEQ ID NOs: 45 and 46, using Euglena gracilis chromosomal DNA as a template Can be obtained.

Escherichia coli flavodoxin-NADP ⁺ reductase gene is a PCR prepared using a primer prepared based on the nucleotide sequence of SEQ ID NO: 27, for example, the primers shown in SEQ ID NOs: 47 and 48, using Escherichia coli chromosomal DNA as a template. It can be obtained by law.

Escherichia coli ferredoxin gene fdx is a primer prepared based on the nucleotide sequence of SEQ ID NO: 29, for example, using primers shown in SEQ ID NO: 49, 50, by PCR method using Escherichia coli chromosomal DNA as a template, Can be acquired.

The Escherichia coli flavodoxin gene fldA was prepared using a primer prepared based on the nucleotide sequence of SEQ ID NO: 33, and the flavodoxin gene fldB was prepared using a primer prepared based on the nucleotide sequence of SEQ ID NO: 35. Each can be obtained by PCR using chromosomal DNA as a template.

The ferredoxin I gene of Chlorobium tepidum was prepared using a primer prepared based on the nucleotide sequence of SEQ ID NO: 37, and the ferredoxin II gene was prepared using a primer prepared based on the nucleotide sequence of SEQ ID NO: 39. Each can be obtained by PCR using tepidum chromosomal DNA as a template.

The gene of the present invention derived from other microorganisms is also a PCR method using primers prepared with oligonucleotides prepared based on the sequence information of each of the above genes or gene or protein sequences known in the microorganism, or It can be obtained from a chromosomal DNA or a chromosomal DNA library of a microorganism by a hybridization method using an oligonucleotide prepared based on the sequence information as a probe. In addition, chromosomal DNA is obtained from microorganisms as DNA donors, for example, by Saito and Miura's method (Saito, H. and Miura, K. I. 1963. Biochem.phyBiophys. Acta, 72, 619-629; Book, edited by Japanese Society for Biotechnology, pages 97-98, Bafukan, 1992).

In addition to the enhancement of pyruvate synthase or pyruvate: NADP ⁺ oxidoreductase activity, the microorganism of the present invention preferably has reduced activity of malic enzyme. When the microorganism of the present invention is a bacterium belonging to the genus Escherichia, Enterobacter, Pantoea, Klebsiella, or Serratia, it is particularly preferable to reduce the activity of malic enzyme.

In the present invention, the activity of malic enzyme means an activity of reversibly catalyzing a reaction in which malic acid is oxidatively decarboxylated to produce pyruvic acid. The above reaction is NADP-type malic enzyme (also expressed as malate dehydrogenase (oxaloacetate-decarboxylating) (NADP ⁺ )) using NADP as an electron acceptor (EC: 1.1.1.40 b2463 gene (also referred to as maeB gene) SEQ ID NO: 51) or NAD type malic enzyme (also referred to as malate dehydrogenase (oxaloacetate-decarboxylating) (NAD ⁺ )) using NAD as an electron acceptor (EC: 1.1.1.38 sfcA gene (also referred to as maeA gene) sequence Catalyzed by two enzymes of number 53). The confirmation of malic enzyme activity can be measured according to the method of Bologna et al. (Bologna, F. P. et al. 2007. J. Bacteriol. 2007 189: 5937-5946).

NADP-dependent malic enzyme: NADP ⁺ + malate → NADPH + CO ₂ + pyruvate
NAD-dependent malic enzyme: NAD ⁺ + malate → NADH + CO ₂ + pyruvate

The decrease in enzyme activity can be carried out in the same manner as the decrease in ribonuclease G activity described later.

In the present invention, it is more preferable to reduce the activities of both NADP-type and NAD-type malic enzymes. In particular, the microorganism of the present invention belongs to the genus Escherichia, Enterobacter, Pantoea, Klebsiella, or Serratia. When the bacterium belongs, it is preferable to reduce the activity of both types of malic enzyme.

In addition to the enhanced activity of pyruvate synthase or pyruvate: NADP ⁺ oxidoreductase, the microorganism of the present invention preferably has reduced pyruvate dehydrogenase activity.

In the present invention, pyruvate dehydrogenase (hereinafter sometimes referred to as “PDH”) activity means an activity that catalyzes a reaction of oxidatively decarboxylating pyruvate to produce acetyl-CoA (acetyl-CoA). To do. The above reactions were carried out by PDH (E1p: pyruvate dehydrogenase, EC: 1.2.4.1 aceE gene SEQ ID NO: 55), dihydrolipoyltransacetylase (E2p: dihydrolipoyltransacetylase, EC: 2.3.1.12 aceF gene SEQ ID NO: 57), dihydrolipoamide dehydrogenase It is catalyzed by three enzymes (E3: dihydrolipoamide hydrogendehydrogenase; EC: 1.8.1.4 lpdA gene SEQ ID NO: 59). That is, these three types of subunits each catalyze the following reaction, and the activity of catalyzing the combined reaction of these three reactions is called PDH activity. Confirmation of PDH activity can be measured according to the method of Visser and Strating (Visser, J. and Strating, M. 1982. Methods Enzymol. 89: 391-399).

E1p: pyruvate + [dihydrolipoyllysine-residue succinyltransferase] lipoyllysine → [dihydrolipoyllysine-residue acetyltransferase] S-acetyldihydrolipoyllysine + CO ₂
E2p: CoA + enzyme N6- (S-acetyldihydrolipoyl) lysine → acetyl-CoA + enzyme N6- (dihydrolipoyl) lysine
E3: protein N6- (dihydrolipoyl) lysine + NAD ⁺ → protein N6- (lipoyl) lysine + NADH + H ⁺

The bacterium of the present invention is modified so that malate synthase, isocitrate triase, isocitrate dehydrogenase kinase / phosphatase operon (ace operon) is constitutively expressed, or expression of the operon is enhanced. Strains may be used. Constitutive expression of malate synthase, isocitrate triase, isocitrate dehydrogenase kinase / phosphatase operon (ace operon) means that the ace operon promoter is not repressed by the repressor protein iclR. It means that it has been released.

The fact that the ace operon is constitutively expressed and the expression of the operon is enhanced is that the proteins encoded by the ace operon are malate synthase (aceB), isocitrate triase (aceA), isocyto This can be confirmed by the fact that the enzyme activity of rate dehydrogenase kinase / phosphatase (aceK) is increased compared to the unmodified strain or the wild strain.

Enzyme activity was determined by measuring glyoxylic acid-dependent degradation of the thioester bond of acetyl-CoA by reducing A232 for malate synthase (Dixon, GH, Kornberg, HL, 1960, Biochem. J, 1; 41: p217-233), for isocitrate lyase, a method for measuring glyoxylic acid generated from isocitrate as a 2,4-dinitrophenylhydrazone derivative (Roche, TE. Williams JO, 1970, Biochim. Biophys. Acta, 22; 206 (1 ): p193-195), with respect to isocitrate dehydrogenase kinase, a method of measuring desorption of phosphate to isocitrate dehydrogenase using ³² P (Wang, JYJ and Koshland, DE, Jr., 1982, Arch Biochem. Biophys., 218, p59-67).

In order to release the suppression, for example, the binding site of the repressor (iclR) on the ace operon may be modified so that iclR cannot bind. In addition, the suppression can be released by replacing the promoter of the operon with a strong promoter (such as the lac promoter) that is not subject to expression suppression by iclR.

Also, the expression of the ace operon can be made constitutive by modifying the bacterium so that the expression of the iclR gene is reduced or deleted. Specifically, the expression control sequence of the gene encoding iclR is modified so that the gene does not express, or the coding region is modified so that the function of the repressor is lost, thereby suppressing the expression of the ace operon. Can be released.

The preferred form of the bacterium used in the present invention is the above-mentioned i) the property of aerobically assimilating ethanol, ii) the increased activity of pyruvate synthase or pyruvate: NADP ⁺ oxidoreductase, iii) the ace operon It has either constitutive expression or enhanced expression, iv) reduced pyruvate dehydrogenase activity, but preferably has the properties i) and ii), more preferably has these four properties preferable.

In the present invention, the bacterium having L-amino acid-producing ability refers to a bacterium having the ability to produce L-amino acid and secrete it into the medium when cultured in the medium. Preferably, it refers to a bacterium capable of accumulating the target L-amino acid in the medium in an amount of preferably 0.5 g / L or more, more preferably 1.0 g / L or more. L-amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L- Includes lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. Among these, L-lysine, L-glutamic acid, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, and L-cysteine is preferred, and L-threonine, L-lysine and L-glutamic acid are particularly preferred.
In the present invention, L-amino acids include not only free L-amino acids but also salts including sulfates, hydrochlorides, carbonates, ammonium salts, sodium salts, and potassium salts.

Hereinafter, a method for imparting L-amino acid-producing ability to the bacterium as described above or a method for enhancing the bacterium L-amino acid-producing ability as described above will be described.

In order to confer L-amino acid-producing ability, acquisition of auxotrophic mutants, L-amino acid analog resistant strains or metabolic control mutants, and recombinant strains with enhanced expression of L-amino acid biosynthetic enzymes Can be applied to the breeding of amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (Amino Acid Fermentation, Academic Publishing Center, Inc., May 30, 1986, first edition) Issue, see pages 77-100). Here, in the breeding of L-amino acid-producing bacteria, the auxotrophy, analog resistance, metabolic control mutation and other properties imparted may be singly or may be two or more. Further, the L-amino acid biosynthetic enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.

In order to obtain an auxotrophic mutant, an analog resistant strain, or a metabolically controlled mutant having L-amino acid-producing ability, the parent strain or wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl. -Treated with a treatment with a mutant such as -N'-nitro-N-nitrosoguanidine, among the obtained mutant strains, shows auxotrophy, analog resistance, or metabolic control mutation, and has an ability to produce L-amino acid It can be obtained by selecting what it has.

Also, the imparting or enhancing of the ability to produce L-amino acid can be performed by enhancing the enzyme activity by gene recombination. The enzyme activity can be enhanced by, for example, a method of modifying a bacterium so that expression of a gene encoding an enzyme involved in L-amino acid biosynthesis is enhanced. As a method for enhancing the expression of a gene, introducing an amplified plasmid in which a DNA fragment containing the gene is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication replication function of the plasmid in a microorganism, Alternatively, it can be achieved by making multiple copies of these genes on the chromosome by joining, transferring, etc., and introducing mutations into the promoter regions of these genes (see International Publication WO95 / 34672).

When the target genes are introduced onto the amplification plasmid or chromosome, the promoter for expressing these genes may be any promoter that functions in coryneform bacteria, and the promoter of the gene itself used. Or may be modified. The expression level of the gene can also be regulated by appropriately selecting a promoter that functions strongly in coryneform bacteria, or by bringing the −35 and −10 regions of the promoter closer to the consensus sequence. The method for enhancing the expression of the enzyme gene as described above is described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like.

Hereinafter, specific methods for imparting L-amino acid-producing ability to bacteria and bacteria imparted with L-amino acid-producing ability will be exemplified.

L-threonine producing bacteria Preferred microorganisms having L-threonine producing ability include bacteria having enhanced activity of one or more L-threonine biosynthetic enzymes. L-threonine biosynthetic enzymes include aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA), homoserine kinase (thrB), threonine synthase (thrC), aspartate amino Examples include transferase (aspartate transaminase) (aspC). The parentheses are abbreviations for the genes (the same applies to the following description). Of these enzymes, aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase are particularly preferred. The L-threonine biosynthesis gene may be introduced into a bacterium belonging to the genus Escherichia in which threonine degradation is suppressed. Examples of the Escherichia bacterium in which threonine degradation is suppressed include, for example, the TDH6 strain lacking threonine dehydrogenase activity (Japanese Patent Laid-Open No. 2001-346578).

The enzyme activity of the L-threonine biosynthetic enzyme is suppressed by the final product, L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is desirable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine. The thrA, thrB, and thrC genes constitute the threonine operon, but the threonine operon forms an attenuator structure, and the expression of the threonine operon inhibits isoleucine and threonine in the culture medium. The expression is suppressed by attenuation. This modification can be achieved by removing the leader sequence or attenuator in the attenuation region (Lynn, S. P., Burton, W. S., Donohue, T. J., Gould, R. M ., Gumport, R. I., and Gardner, J. F. J. Mol. Biol. 194: 59-69 (1987); International Publication No. 02/26993; International Publication No. 2005/049808) .

A unique promoter exists upstream of the threonine operon, but it may be replaced with a non-natural promoter (see WO98 / 04715 pamphlet), and the expression of a gene involved in threonine biosynthesis is expressed by a lambda phage repressor and A threonine operon as governed by a promoter may be constructed. (See European Patent No. 0593792) In order to modify bacteria so that it is not subject to feedback inhibition by L-threonine, a strain resistant to α-amino-β-hydroxyvaleric acid (AHV) may be selected. Is possible.

Thus, the threonine operon modified so as not to be subjected to feedback inhibition by L-threonine has an increased copy number in the host or is linked to a strong promoter to improve the expression level. Is preferred. The increase in copy number can be achieved by transferring the threonine operon on the genome by transposon, Mu-fuzzy, etc., in addition to amplification by plasmid.

In addition to the L-threonine biosynthetic enzyme, it is also preferable to enhance the glycolytic system, TCA cycle, genes related to the respiratory chain, genes controlling gene expression, and sugar uptake genes. These genes effective for L-threonine production include transhydronase (pntAB) gene (European Patent 733712), phosphoenolpyruvate carboxylase gene (pepC) (International Publication No. 95/06114 pamphlet), phospho Examples include the enol pyruvate synthase gene (pps) (European Patent No. 877090), the pyruvate carboxylase gene of Coryneform bacteria or Bacillus bacteria (International Publication No. 99/18228, European Application Publication No. 1092776).

It is also preferable to enhance the expression of a gene conferring resistance to L-threonine and a gene conferring resistance to L-homoserine, or confer L-threonine resistance and L-homoserine resistance to the host. Examples of genes that confer resistance include rhtA gene (Res. Microbiol. 154: 123-135 (2003)), rhtB gene (European Patent Application Publication No. 0994190), rhtC gene (European Patent Application Publication No. 1013765) ), YfiK, yeaS gene (European Patent Application Publication No. 1016710). For methods for conferring L-threonine resistance to the host, the methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

Examples of L-threonine-producing bacteria or parent strains for inducing them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Patent No. 5,175,107, US Patent No. 5,705,371), E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E.coli NRRL-21593 (U.S. Pat.No. 5,939,307), E.coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E.coli FERM BP-3519 And FERM BP-3520 (U.S. Patent No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetikaet (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A) Strains belonging to the genus Escherichia such as, but not limited to.

The TDH-6 strain lacks the thrC gene, is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. B-3996 was deposited on 19 November 1987 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) under the deposit number RIA 1867. . This stock was also deposited on April 7, 1987 at Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) under accession number B-3996 Has been.

E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it. The B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter. VKPM B-5318 was assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318. Has been deposited internationally.

The thrA gene encoding aspartokinase homoserine dehydrogenase I of Escherichia coli has been clarified (nucleotide numbers 337 to 2799, “GenBank accession” NC_000913.2, “gi”: “49175990”). The thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12. The thrB gene encoding homoserine kinase of Escherichia coli has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 49175990). The thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12. The thrC gene encoding the threonine synthase of Escherichia coli has been elucidated (nucleotide numbers 3734-5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. All three of these genes function as a single threonine operon. To increase the expression of the threonine operon, the attenuator region that affects transcription is preferably removed from the operon (WO2005 / 049808, WO2003 / 097839).

The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.

The rhtA gene is present on the 18th minute of the E. 染色体 coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system. The rhtA gene is identical to ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene. The unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant to homoserine and threonine). It has also been found that the rhtA23 mutation is a G → A substitution at position -1 relative to the ATG start codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).

The E. coli asd gene has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene. (See White, TJ et al., Trends Genet., 5, 185 (1989)). The asd gene of other microorganisms can be obtained similarly.

In addition, the aspC gene of E.coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC_000913.1, gi: 16128895) and can be obtained by PCR. The aspC gene of other microorganisms can be obtained similarly.

Examples of L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to L-lysine analogs. L-lysine analogues inhibit the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially desensitized when L-lysine is present in the medium. Examples of L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and the like. . Mutant strains resistant to these lysine analogs can be obtained by subjecting bacteria belonging to the genus Escherichia to normal artificial mutation treatment. Specific examples of bacterial strains useful for the production of L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is released.

Examples of L-lysine-producing bacteria or parent strains for inducing them include strains in which one or more activities of L-lysine biosynthetic enzymes are enhanced. Examples of such enzymes include dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat.No. 6,040,160). ), Phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase gene, diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate deacylase (dapE) and aspartase (aspA) ( EP 1253195 A), but is not limited to these. Among these enzymes, dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and Succinyl diaminopimelate deacylase is particularly preferred. The parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Patent No. 5,830,716), ybjE gene (WO2005 / 073390), or The expression level of these combinations may be increased.

Examples of L-lysine-producing bacteria or parent strains for deriving the same include reduction or loss of the activity of enzymes that catalyze reactions that branch off from the L-lysine biosynthetic pathway to produce compounds other than L-lysine. There are also stocks. Examples of enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malate enzyme ( WO2005 / 010175).

A preferred L-lysine-producing bacterium includes Escherichia coli WC196ΔcadAΔldcC / pCABD2 (WO2006 / 078039). This strain was constructed by disrupting the cadA and ldcC genes encoding lysine decarboxylase and introducing plasmid pCABD2 (US Pat. No. 6,040,160) containing a lysine biosynthesis gene from WC196 strain. The WC196 strain was obtained from the W3110 strain derived from E. coli K-12, and encodes aspartokinase III in which feedback inhibition by L-lysine was released by replacing threonine at position 352 with isoleucine. After the wild type lysC gene on the chromosome of the W3110 strain was replaced with a mutant lysC gene (US Pat. No. 5,661,012), it was bred by conferring AEC resistance (US Pat. No. 5,827,698). The WC196 strain was named Escherichia coli AJ13069. On December 6, 1994, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center, 305-8566 茨 Ibaraki, Japan Deposited as FERM P-14690 at Tsukuba City Higashi 1-chome 1-1 1 Chuo 6), transferred to international deposit based on the Budapest Treaty on September 29, 1995, and assigned FERM BP-5252 (US Pat. No. 5,827,698). WC196ΔcadAΔldcC itself is also a preferred L-lysine-producing bacterium. WC196ΔcadAΔldcC was named AJ110692, and was deposited internationally on October 7, 2008, at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center (1-6 Chuo, 1-chome, 1-chome, Tsukuba, Ibaraki, Japan, 305-8566) The accession number is FERM BP-11027.

pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine. A mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes (International Publication Nos. WO95 / 16042 and WO01 / 53459).

Examples of L-cysteine producing bacteria L-cysteine producing bacteria or parent strains for deriving them include E. coli JM15 (US Pat. No. 6,218,168) transformed with a different cysE allele encoding a feedback inhibition resistant serine acetyltransferase. , Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663) having an overexpressed gene encoding a protein suitable for excretion of a substance toxic to cells, cysteine desulfohydrase activity These include strains belonging to the genus Escherichia such as E. coli strains that have been reduced (JP11155571A2) and E. coli W3110 (WO0127307A1) that have increased the activity of the transcriptional regulator of the positive cysteine regulon encoded by the cysB gene. It is not limited.

Examples of L-leucine-producing bacteria L-leucine-producing bacteria or parent strains for inducing them include leucine-resistant E. coil strains (eg, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)) or β E. coli strains resistant to leucine analogs such as -2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (Japanese Patent Publication No. 62-34397 and JP-A-8-70879), Examples include, but are not limited to, strains belonging to the genus Escherichia such as E. coli strains and E. coli H-9068 (JP-A-8-70879) obtained by the genetic engineering method described in WO96 / 06926. .

The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-leucine biosynthesis. As an example of such a gene, a gene of leuABCD operon represented by a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase which is preferably desensitized to feedback inhibition by L-leucine can be mentioned. . Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (EP 1239041 A2).

Examples of L-histidine-producing bacteria L-histidine-producing bacteria or parent strains for inducing them include E. coli 24 strain (VKPM B-5945, RU2003677), E. coli 80 strain (VKPM B-7270, RU2119536) E. coli NRRL B-12116-B12121 (U.S. Pat.No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat.No. 6,344,347), E. coli. Examples include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli H-9341 (FERM BP-6674) (EP1085087) and E. coli AI80 / pFM201 (US Pat. No. 6,258,554).

Examples of L-histidine-producing bacteria or parent strains for inducing them include strains in which expression of one or more genes encoding L-histidine biosynthetic enzymes are increased. Examples of such genes include ATP phosphoribosyltransferase gene (hisG), phosphoribosyl AMP cyclohydrolase gene (hisI), phosphoribosyl-ATP pyrophosphohydrolase gene (hisI), phosphoribosylformimino-5- Examples include aminoimidazole carboxamide ribotide isomerase gene (hisA), amide transferase gene (hisH), histidinol phosphate aminotransferase gene (hisC), histidinol phosphatase gene (hisB), and histidinol dehydrogenase gene (hisD). It is done.

L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine, and therefore L-histidine-producing ability is feedback-inhibited by the ATP phosphoribosyltransferase gene (hisG). Can be efficiently increased by introducing mutations that confer resistance to (Russian Patent Nos. 2003677 and 2119536).

Specific examples of strains having the ability to produce L-histidine include E. coli FERM-P 5038 and 5048 introduced with a vector carrying a DNA encoding an L-histidine biosynthetic enzyme (Japanese Patent Laid-Open No. 56-005099). E. coli strain (EP1016710A) introduced with a gene for amino acid transport, E. coli 80 strain (VKPM B-7270) to which resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin was imparted , Russian Patent No. 2119536).

Examples of L-glutamic acid-producing bacteria L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, strains belonging to the genus Escherichia such as E. coli VL334thrC + (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotroph having a mutation in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K12 strain (VKPM B-7). As a result, L-isoleucine-requiring L-glutamic acid-producing bacterium VL334thrC + (VKPM B-8961) was obtained.

Examples of L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, strains with enhanced activity of one or more L-glutamic acid biosynthetic enzymes. Examples of such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), Methyl citrate synthase (prpC), phosphoenolpyruvate carbocilase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase ( eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triphosphate isomerase (tpiA), fructose bisphosphate aldolase ( fbp), phosphofructokinase ( pfkA, pfkB), glucose phosphate isomerase (pgi) and the like. Of these enzymes, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase are preferred.

Examples of strains modified to increase expression of citrate synthetase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A and EP952221A.

Examples of L-glutamic acid-producing bacteria or parent strains for deriving the same are those in which the activity of an enzyme that catalyzes the synthesis of compounds other than L-glutamic acid by diverging from the biosynthetic pathway of L-glutamic acid is reduced or absent Stocks are also mentioned. Examples of such enzymes include isocitrate triase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), Examples include acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), and the like. Bacteria belonging to the genus Escherichia lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity, and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945. .

Specific examples include the following.
E. coli W3110sucA :: Kmr
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA :: Kmr is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter also referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacteria include those belonging to the genus Escherichia and having resistance to an aspartic acid antimetabolite. These strains may be deficient in α-ketoglutarate dehydrogenase, for example, E. coli AJ13199 (FERM BP-5807) (US Patent No. 5.908,768), and FFRM with reduced L-glutamate resolution. P-12379 (US Pat. No. 5,393,671); AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) and the like.

An example of an L-glutamic acid-producing bacterium of Pantoae ananatis is Pantoea ananatis AJ13355 strain. This strain was isolated from the soil of Iwata City, Shizuoka Prefecture as a strain that can grow on a medium containing L-glutamic acid and a carbon source at a low pH. Pantoea Ananatis AJ13355 was commissioned on February 19, 1998 at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center (address: 1st, 1st, 1st, 1-chome, Tsukuba, Ibaraki, Japan, 305-8566). Deposited under the number FERM644P-16644, transferred to an international deposit under the Budapest Treaty on 11 January 1999 and given the accession number FERM BP-6614. The strain was identified as Enterobacter agglomerans at the time of its isolation and deposited as Enterobacter グロ agglomerans AJ13355, but recently, Pantoea ananatis (Pantoea ananatis) was analyzed by 16S rRNA sequence analysis. ).

Further, examples of L-glutamic acid-producing bacteria of Pantoae ananatis include bacteria belonging to the genus Pantoea in which α-ketoglutarate dehydrogenase (αKGDH) activity is deficient or αKGDH activity is reduced. Such strains include AJ13356 (US Pat. No. 6,331,419) in which the αKGDH-E1 subunit gene (sucA) of AJ13355 strain is deleted, and sucA derived from SC17 strain selected from AJ13355 strain as a low mucus production mutant. There is SC17sucA (US Pat. No. 6,596,517) which is a gene-deficient strain. AJ13356 was founded on February 19, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-chome, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 No. 6) was deposited under the deposit number FERM P-16645, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6616. AJ13355 and AJ13356 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification. The SC17sucA strain has been assigned the private number AJ417, and was deposited at the National Institute of Advanced Industrial Science and Technology as the accession number FERM BP-08646 on February 26, 2004.

Furthermore, as L-glutamic acid-producing bacteria of Pantoae ananatis, SC17sucA / RSFCPG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain can be mentioned. The SC17sucA / RSFCPG + pSTVCB strain is a plasmid RSFCPG containing the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppsA), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum. The AJ13601 strain was selected from the SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain. On August 18, 1999, AJ13601 shares were registered with the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (305-1856, Ibaraki, Japan, 1st-chome, 1st-chome, 1st-chome, 1st-centre, 6th). Deposited as 17516, transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the deposit number FERM BP-7207.

Examples of L-phenylalanine producing bacteria L-phenylalanine producing bacteria or parent strains for deriving them include E. coli AJ12739 (tyrA :: Tn10, tyrR) lacking chorismate mutase-prefenate dehydrogenase and tyrosine repressor ( VKPM B-8197) (WO03 / 044191), E. coli HW1089 (ATCC 55371) (US Pat.No. 5,354,672) carrying a mutant pheA34 gene encoding chorismate mutase-prefenate dehydratase with desensitized feedback inhibition, Strains belonging to the genus Escherichia such as E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Pat.No. 4,407,952) However, it is not limited to these. In addition, E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K that retains the gene encoding chorismate mutase-prefenate dehydratase whose feedback inhibition has been released. -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 named AJ 12604 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579) can also be used (EP 488424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1, WO03 / 044192).

Examples of L-tryptophan-producing bacteria L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 lacking the tryptophanyl-tRNA synthetase encoded by the mutant trpS gene (DSM10123) (U.S. Pat.No. 5,756,345), E. coli having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase not subject to feedback inhibition by tryptophan. SV164 (pGH5) (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614) E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, U.S. Pat.No. 6,319,696) with increased ability to produce phosphoenolpyruvate Strains include belonging to Erihia genus, but is not limited thereto. L-tryptophan-producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1).

Examples of L-tryptophan-producing bacteria or parent strains for inducing them include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), 3-deoxy-D-arabinohepturonic acid-7-phosphorus Acid synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolate pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC ), Prephenate dehydratase, chorismate mutase and tryptophan synthase (trpAB). One or more strains having enhanced activity are also included. Prefenate dehydratase and chorismate mutase are encoded by the pheA gene as a bifunctional enzyme (CM-PD). Among these enzymes, phosphoglycerate dehydrogenase, 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase, 3-dehydroquinate synthase, shikimate dehydratase, shikimate kinase, 5-enolic acid Pyruvylshikimate 3-phosphate synthase, chorismate synthase, prefenate dehydratase, chorismate mutase-prefenate dehydrogenase are particularly preferred. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying a desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase with desensitized feedback inhibition Examples include a transformant obtained by introducing the plasmid pGH5 (WO 94/08031) into E.coli SV164.

Examples of L-tryptophan-producing bacteria or parent strains for deriving the same include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (Japanese Patent Laid-Open Nos. 57-71397 and 1994 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of the isocitrate triase-malate synthase operon (WO2005 / 103275).

Examples of L-proline-producing bacteria L-proline-producing bacteria or parent strains for deriving them include E. coli 702ilvA (VKPM B-8012) (EP 1172433) that lacks the ilvA gene and can produce L-proline Strains belonging to the genus Escherichia, but are not limited thereto.

The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-proline biosynthesis. An example of a gene preferable for L-proline-producing bacteria includes a proB gene (German Patent No. 3127361) encoding glutamate kinase that is desensitized to feedback inhibition by L-proline. Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) gene (EP1239041 gene A2).

Examples of bacteria belonging to the genus Escherichia having the ability to produce L-proline include NRRL B-12403 and NRRL B-12404 (British Patent No. 2075056), VKPM B-8012 (Russian Patent Application 2000124295), German Patent No. 3127361 And E. coli strains such as the plasmid variants described in Bloom FR et al (The 15th Miami winter symposium, 1983, p.34).

Examples of L-arginine producing bacteria L-arginine producing bacteria or parent strains for inducing them include E. coli strain 237 (VKPM B-7925) (US Patent Application Publication 2002/058315 A1) and mutant N- Derivatives carrying acetylglutamate synthase (Russian patent application No. 2001112869), E. coli 382 strain (VKPM B-7926) (EP1170358A1), arginine producing strain introduced with argA gene encoding N-acetylglutamate synthetase Examples include, but are not limited to, strains belonging to the genus Escherichia, such as (EP1170361A1).

Examples of L-arginine-producing bacteria or parent strains for inducing them include strains in which expression of one or more genes encoding L-arginine biosynthetic enzymes are increased. Examples of such genes include N-acetylglutamylphosphate reductase gene (argC), ornithine acetyltransferase gene (argJ), N-acetylglutamate kinase gene (argB), acetylornithine transaminase gene (argD), ornithine carbamoyltransferase gene ( argF), arginosuccinate synthetase gene (argG), arginosuccinate lyase gene (argH), carbamoylphosphate synthetase gene (carAB).

Examples of L-valine-producing bacteria L-valine-producing bacteria or parent strains for inducing the same include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). Not. It is preferable to remove the region of the ilvGMEDA operon necessary for attenuation so that the expression of the operon is not attenuated by the produced L-valine. Furthermore, it is preferred that the ilvA gene of the operon is disrupted and the threonine deaminase activity is reduced.
Examples of L-valine-producing bacteria or parent strains for deriving them also include mutants having aminoacyl t-RNA synthetase mutations (US Pat. No. 5,658,766). For example, E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase can be used. E. coli VL1970 was registered with Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on June 24, 1988 under the accession number VKPM B-4411. It has been deposited.
Furthermore, a mutant strain (WO96 / 06926) that requires lipoic acid for growth and / or lacks H + -ATPase can be used as a parent strain.

Examples of L-isoleucine-producing bacteria and L-isoleucine-producing bacteria or parent strains for inducing them include mutants resistant to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate Mutants having resistance to isoleucine analogs such as the above, and mutants having resistance to DL-ethionine and / or arginine hydroxamate (Japanese Patent Laid-Open No. 5-130882), but are not limited thereto. Furthermore, a recombinant strain transformed with a gene encoding a protein involved in L-isoleucine biosynthesis such as threonine deaminase and acetohydroxy acid synthase can also be used as a parent strain (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).

L-tyrosine-producing bacteria Examples of tyrosine-producing bacteria include Escherichia bacteria (European Patent Application Publication No. 1616940) having a desensitized prefenate dehydratase gene (tyrA) that is not inhibited by tyrosine.

When breeding the bacterium used in the present invention by genetic recombination, the gene to be used is not limited to the gene having the genetic information described above or a gene having a known sequence, but variants of those genes, that is, encoded proteins As long as these functions are not impaired, genes having conservative mutations such as homologues and artificially modified variants of those genes can also be used. That is, it may be a gene encoding a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the amino acid sequence of a known protein.

Here, “one or several” differs depending on the position of the protein in the three-dimensional structure of the amino acid residue and the type of amino acid residue, but specifically, preferably 1 to 20, more preferably 1 to 10 Means, more preferably 1-5. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the microorganism from which the gene is derived. Also included by Such a gene can be modified, for example, by site-directed mutagenesis so that the amino acid residue at a specific site of the encoded protein contains substitutions, deletions, insertions or additions. Can be obtained by:

Furthermore, the gene having a conservative mutation as described above has a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more with respect to the entire encoded amino acid sequence. And a gene encoding a protein having a function equivalent to that of a wild-type protein.
In addition, each codon in the gene sequence may be replaced with a codon that is easy to use in the host into which the gene is introduced.

The gene having a conservative mutation may be one obtained by a method usually used for mutation treatment such as treatment with a mutation agent.

A gene is a DNA that hybridizes with a probe complementary to a known gene sequence or a probe that can be prepared from the complementary sequence under stringent conditions and encodes a protein having a function equivalent to that of a known gene product. Also good. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. As an example, DNAs having high homology, for example, 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, are hybridized to each other. Conditions under which DNAs with low homology do not hybridize, or conditions for washing of ordinary Southern hybridization, 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably The conditions include washing once at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, and 0.1% SDS, more preferably 2 to 3 times.

As the probe, a part of the complementary sequence of the gene can also be used. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

The above description regarding gene variants also applies to the following rng genes and other genes described herein.

<1-2> Decrease in Ribonuclease G Activity Next, modifications that reduce the activity of ribonuclease G in bacteria belonging to the family Enterobacteriaceae will be described.

In the present invention, “ribonuclease G (RNaseG) activity” refers to an activity of degrading RNA serving as a substrate for RNaseG.
Examples of RNA serving as a substrate for RNaseG include RNA transcribed from the gene eno (GenBank Accession No. X82400) encoding enolase and the gene adhE (GenBank Accession No. M33504) encoding alcohol dehydrogenase. . In the activity measurement, for example, RNA can be extracted from a strain in which RNA synthesis is suppressed by rifampicin, and the activity can be indirectly known by measuring the degradation half-life of mRNA of the eno gene or adhE gene. Further, the activity can be determined by isolating and purifying RNaseG and measuring the cleavage reaction of an artificial substrate such as an oligoribonucleotide containing an RNaseG cleavage site. Such an activity measurement method has already been disclosed (J. Biol. Chem., 275, 8726-8732, 2000).

The phrase “modified so that the RNaseG activity is reduced” means that the RNaseG activity per bacterial cell is lower than that of an unmodified strain, for example, a strain belonging to the wild type Enterobacteriaceae. For example, the case where the number of RNaseG molecules per cell decreases, the case where the RNaseG activity per molecule decreases, and the like are applicable. The comparison of the RNaseG activity per cell can be performed, for example, by comparing the RNaseG activity contained in the cell extract of bacteria cultured under the same conditions. The “decrease” in activity includes a case where the activity is completely lost. Examples of wild-type bacteria belonging to the genus Escherichia that serve as a comparative control include Escherichia coli MG1655 strain.

The decrease in the activity of RNaseG is achieved by inactivating the gene (rng) encoding RNaseG. “Inactivation” of the rng gene means that the gene is modified by genetic recombination or a mutation is introduced into the gene so that the activity of RNaseG encoded by the gene is reduced or eliminated. .

Examples of the rng gene include Escherichia coli rng gene registered in GenBank (complementary strand of base numbers 3394348 to 3395817 of GenBank Accession No. NC_000913.2: SEQ ID NO: 1). The amino acid sequence of RNaseG encoded by this rng gene is shown in SEQ ID NO: 2. The rng gene can be cloned by synthesizing a synthetic oligonucleotide based on these sequences and performing a PCR reaction using Escherichia coli chromosome as a template. When the rng gene is deleted by homologous recombination, it has a certain degree of homology with the rng gene on the chromosome, for example, 80% or more, preferably 90% or more, more preferably 95% or more. Genes can also be used. A gene that hybridizes with a rng gene on a chromosome under stringent conditions can also be used. The stringent conditions include, for example, a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably 1 to 2 times. The condition of washing three times is mentioned.

Specifically, inactivation of the rng gene is achieved, for example, by deleting a part or all of the coding region of the rng gene on the chromosome, or by inserting another sequence into the coding region. These techniques are also called gene disruption.
The rng gene can also be inactivated by reducing the expression of the rng gene, for example, by modifying the expression regulatory sequence such as the promoter of the rng gene or Shine-Dalgarno (SD) sequence. Decreased expression includes reduced transcription and reduced translation. Moreover, gene expression can also be reduced by modifying non-translated regions other than the expression regulatory sequences.

Furthermore, the entire target gene may be deleted, including sequences before and after the target gene on the chromosome. Inactivation of the rng gene can be achieved by introducing an amino acid substitution (missense mutation) into the coding region of the rng gene on the chromosome, introducing a stop codon (nonsense mutation), or adding or deleting one or two bases. (Journal of Biological Chemistry 272: 8611-8617 (1997) Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 266, 20833- 20839 (1991)).

The modification of each gene is preferably performed by gene recombination. Specifically, the gene recombination method uses homologous recombination to delete the expression regulatory sequence of the target gene on the chromosome, for example, the promoter region, the coding region, or a part or all of the non-coding region. Or insertion of other sequences into these regions.

The modification of the expression regulatory sequence is preferably 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. When the coding region is deleted, if the function of the protein produced by each gene is reduced or deleted, the region to be deleted is any of the N-terminal region, internal region, and C-terminal region. It may be the entire code area. Usually, the longer region to be deleted can surely inactivate the target gene. Moreover, it is preferable that the upstream and downstream reading frames of the region to be deleted do not match.

When inserting other sequences into the coding region, the insertion position may be any region of the target gene, but the longer the sequence to be inserted, the more reliably the target gene can be inactivated. The sequences before and after the insertion site preferably do not match the reading frame. Other sequences are not particularly limited as long as they reduce or eliminate the function of the protein encoded by the target gene. Examples include antibiotic resistance genes and transposons carrying genes useful for L-glutamic acid production. It is done.

In order to modify a target gene on a chromosome as described above, for example, a deletion type gene is prepared by deleting a partial sequence of the target gene and modifying it so as not to produce a protein that functions normally. This can be accomplished by replacing the target gene on the chromosome with the deleted gene by transforming bacteria with the contained DNA and causing homologous recombination between the deleted gene and the target gene on the chromosome. Even if the protein encoded by the deletion-type target gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function decreases or disappears. Gene disruption by gene replacement using such homologous recombination has already been established, and a method called “Red-driven integration” (Datsenko, K. A, and Wanner, B. L. Proc Natl. Acad. Sci. U S A. 97: 6640-6645 (2000)), or Red-driven integration method and λ phage-derived excision system (Cho, E. H., Gumport, R. I., Gardner , J. F. J. Bacteriol. 184: 5200-5203 (2002)), a method using linear DNA such as a method (see WO2005 / 010175), a plasmid containing a temperature-sensitive replication origin, There are a method using a plasmid capable of conjugation transfer and a method using a suicide vector which does not have an origin of replication in the host (US Pat. No. 6,303,383 or Japanese Patent Laid-Open No. 05-007491).

It can be confirmed that the amount of transcription of the target gene is reduced by comparing the amount of mRNA transcribed from the target gene with a wild strain or an unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold spring spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The decrease in the amount of transcription may be any as long as it is reduced compared to the wild strain or the unmodified strain, but for example, at least 75% or less, 50% or less, 25% or less, compared to the wild strain or the unmodified strain, Alternatively, it is desirable that the concentration is reduced to 10% or less, and it is particularly preferable that no expression occurs.

Confirmation that the amount of the protein encoded by the target gene has decreased can be confirmed by Western blotting using an antibody that binds to the protein (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001) )). The decrease in the amount of protein may be any as long as it is lower than that of the wild strain or non-modified strain, but for example, at least 75% compared to the wild strain or non-modified strain compared to the wild strain or non-modified strain. In the following, it is desirable to decrease to 50% or less, 25% or less, or 10% or less, and it is particularly preferable that no protein is produced (the activity is completely lost).

It is also possible to obtain a gene encoding RNaseG having a low activity by mutating the rng gene. For example, the expression of the adhE gene encoding alcohol dehydrogenase depends on the function of the rng gene (Biochem. Biophys. Res. Commun., 295 (2002) 92-97), so that adhE and a reporter gene such as β-galactosidase are combined. The activity-reduced rng gene can also be screened by allowing the plasmid expressing the fusion protein to coexist in the cell with the mutant rng gene and measuring β-galactosidase activity.

In order to reduce the activity of RNaseG, in addition to the above-described genetic manipulation methods, for example, Escherichia bacteria are irradiated with ultraviolet light, or normal mutation such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrite Examples include a method of selecting a strain that has been treated with the mutagen used in the treatment and has reduced RNaseG activity. Examples of mutant strains with reduced RNaseG activity include strains in which the maturation activity at the 5 'end of 16S rRNA remains but only mRNA degradation activity has decreased, such as mutant DC430 strain and GM1430 strain (Biochem. Biobios. Res. Commun., 289 (5), 1301-1306, 201).

<2> Method for Producing L-Amino Acid of the Present Invention In the method for producing L-amino acid of the present invention, a medium containing ethanol as a carbon source, belonging to the family Enterobacteriaceae, having L-amino acid producing ability, Then, the bacterium modified so that the activity of RNaseG is decreased, L-amino acid is produced and accumulated in the culture, and L-amino acid is collected from the culture.

The ethanol to be used may be used at any concentration that is suitable for producing L-amino acids. When used as a sole carbon source in the medium, ethanol may be any ethanol as long as it is contained as much as a carbon source in the medium, but is 0.001 w / v% or more, preferably 0.05 w / v% or more, more preferably It is desirable to contain 0.1 w / v% or more. The ethanol concentration in the medium is preferably 20 w / V% or less, preferably 10 w / v% or less, more preferably 2 w / v% or less.

When used as a fed-batch medium, ethanol is desirably contained in the medium in an amount of 0.001 w / v% or more, preferably 0.05 w / v% or more, more preferably 0.1 w / v% or more. It is preferable to contain v% or less, preferably 5 w / v% or less, more preferably 1 w / v% or less.
The concentration of ethanol can be measured by various methods, but measurement by an enzyme method is simple and general (Swift, R. 2003. Addiction 98: 73-80).

Furthermore, other carbon sources may be added to the medium used in the method of the present invention in addition to ethanol. Preferred are sugars such as glucose, fructose, sucrose, lactose, galactose, molasses, starch hydrolysate, polyhydric alcohols such as glycerol, and organic acids such as fumaric acid, citric acid, and succinic acid. . One carbon source may be used, or a mixture of two or more carbon sources may be used.

Although ethanol and other carbon sources can be mixed in any ratio, the ratio of ethanol in the carbon source is 20% by weight or more, more preferably 30% by weight or more, more preferably 37% by weight. It is desirable. In particular, in the case of using a bacterium in which pyruvate synthase activity or pyruvate: NADP ⁺ oxidoreductase activity is not enhanced, the ratio of ethanol is preferably within the above range from the viewpoint of the yield of amino acid produced.

In the present invention, when a bacterium with enhanced pyruvate synthase activity or pyruvate: NADP ⁺ oxidoreductase activity is used, the mixing ratio of ethanol and other carbon source is preferably higher in ethanol concentration, 80% Above, preferably 90% or more, more preferably 100%.

In the present invention, ethanol may be contained at a constant concentration in all the steps of the culture, may be added only to the fed-batch medium or only to the initial medium, and if other carbon sources are satisfied, There may be a period of ethanol shortage for a certain period of time. The term “temporary” means that ethanol may be insufficient for a time of 10% or less, or 20% or less, and a maximum of 30% of the entire fermentation time.

As other components to be added to the medium, a normal medium containing a nitrogen source, inorganic ions and other organic components as required can be used in addition to the carbon source. As the nitrogen source contained in the culture medium of the present invention, ammonium salts such as ammonia, ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, ammonium acetate, urea, or nitrates can be used, and are used for pH adjustment. Ammonia gas and aqueous ammonia can also be used as a nitrogen source. Moreover, peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean hydrolyzate and the like can also be used. Only one of these nitrogen sources may be included in the medium, or two or more thereof may be included. These nitrogen sources can be used for both the initial medium and the fed-batch medium. In addition, the same nitrogen source may be used for both the initial culture medium and the feed medium, or the nitrogen source of the feed medium may be changed to the initial culture medium.

The medium of the present invention preferably contains a phosphate source and a sulfur source in addition to a carbon source and a nitrogen source. As the phosphoric acid source, phosphoric acid polymers such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate and pyrophosphoric acid can be used. The sulfur source may be any one containing sulfur atoms, but sulfates such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione are desirable. However, ammonium sulfate is desirable.

The medium may contain a growth promoting factor (a nutrient having a growth promoting effect) in addition to the carbon source, nitrogen source, and sulfur source. Examples of the growth promoting factor include trace metals, amino acids, vitamins, nucleic acids, and peptone, casamino acid, yeast extract, soybean protein degradation products, and the like containing these. Examples of trace metals include iron, manganese, magnesium, calcium, and vitamins include vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12. These growth promoting factors may be contained in the initial culture medium or in the fed-batch medium.

In addition, it is preferable to supplement the medium with nutrients required in the case of using an auxotrophic mutant strain that requires amino acids for growth. In particular, L-lysine-producing bacteria that can be used in the present invention have many L-lysine biosynthetic pathways as described later, and L-lysine resolution is weakened. It is desirable to add one or more selected from homoserine, L-isoleucine, and L-methionine. The initial medium and fed-batch medium may have the same or different medium composition. In addition, the initial culture medium and the fed-batch medium may have the same or different medium composition. Furthermore, when the feeding of the feeding medium is performed in multiple stages, the composition of each feeding medium may be the same or different.

The culture is preferably carried out by aeration culture at a fermentation temperature of 20 to 45 ° C, particularly preferably 33 to 42 ° C. Here, the oxygen concentration is adjusted to 5 to 50%, preferably about 10%. Further, it is preferable to perform aeration culture while controlling the pH to 5 to 9. When the pH falls during the culture, for example, calcium carbonate can be added or neutralized with an alkali such as ammonia gas or ammonia water. By culturing preferably for about 10 to 120 hours under such conditions, a significant amount of L-amino acid is accumulated in the culture solution. The concentration of the accumulated L-amino acid is higher than that of the wild strain, and any concentration can be used as long as it can be collected and recovered from the medium, but it is 50 g / L or more, preferably 75 g / L or more, more preferably 100 g / L or more. is there.

When the target amino acid is a basic amino acid, the pH during the culture is controlled to 6.5 to 9.0, and the pH of the medium at the end of the culture is controlled to 7.2 to 9.0. A culture period in which the pressure in the tank is controlled to be positive, or carbon dioxide gas or a mixed gas containing carbon dioxide gas is supplied to the medium so that bicarbonate ions and / or carbonate ions are present in the medium at least 20 mM or more. And may be produced by a method of fermenting the bicarbonate ion and / or carbonate ion with a counter ion of a cation mainly composed of a basic amino acid, and recovering the target basic amino acid ( JP 2002-065287, US Patent Application Publication No. 2002025564, EP1813677A).

In the above aspect, both the control so that the pressure in the fermenter during the fermentation may be positive and the supply of carbon dioxide or a mixed gas containing carbon dioxide to the medium may be performed. In any case, it is preferable that there is a culture period in which bicarbonate ions and / or carbonate ions in the medium are preferably present at 20 mM or more, more preferably 30 mM or more, and particularly preferably 40 mM or more. The pressure in the fermenter, the supply amount of carbon dioxide or a mixed gas containing carbon dioxide, or the limited supply amount is measured, for example, by measuring bicarbonate ions or carbonate ions in the medium, or by measuring pH or ammonia concentration. Can be determined.

In the above embodiment, the pH of the medium during the culture is controlled to 6.0 to 9.0, preferably 6.5 to 8.0, and the pH of the medium at the end of the culture is controlled to 7.2 to 9.0. To do. According to the said aspect, compared with the conventional method, it becomes possible to hold down the pH of the culture medium for making the quantity of bicarbonate ion and / or carbonate ion which are required as a counter ion exist in a culture medium. When the pH is controlled with ammonia, ammonia is supplied to increase the pH, which can serve as an N source for basic amino acids. Examples of cations other than basic amino acids contained in the medium include K, Na, Mg, Ca and the like derived from medium components. These are preferably 50% or less of the total cations.

Moreover, in order to make the fermenter internal pressure during fermentation positive, for example, the supply air pressure may be set higher than the exhaust pressure. By making the pressure in the fermenter positive, the carbon dioxide gas generated by fermentation dissolves in the culture solution to produce bicarbonate ions or carbonate ions, which can be counter ions of basic amino acids. Specifically, the pressure in the fermenter is 0.03 to 0.2 MPa, preferably 0.05 to 0.15 MPa, more preferably 0.1 to 0.3 MPa in terms of gauge pressure (differential pressure with respect to atmospheric pressure). Can be mentioned. In addition, carbon dioxide gas may be dissolved in the culture solution by supplying carbon dioxide gas or a mixed gas containing carbon dioxide gas to the culture solution. For example, pure carbon dioxide or a mixed gas containing 5% by volume or more of carbon dioxide may be blown. Furthermore, you may adjust so that a fermenter internal pressure may become positive, supplying carbon dioxide or the mixed gas containing carbon dioxide to a culture solution.

In addition, the above-mentioned method for dissolving bicarbonate ions and / or carbonate ions in the medium may be used alone or in combination.

In the conventional method, a sufficient amount of ammonium sulfate or ammonium chloride is usually added to the medium as a counter anion of the basic amino acid to be produced, and a sulfate or hydrolyzate of protein or the like as a nutrient component is added to the medium. The culture medium contains sulfate ions and chloride ions. Therefore, the concentration of carbonate ion, which is weakly acidic, is extremely low during the culture, and is in ppm. The above aspect is characterized in that the sulfate ions and chloride ions are reduced, and carbon dioxide released by the microorganisms during fermentation is dissolved in the medium in the fermentation environment to form counter ions. Therefore, in the above-described embodiment, it is not necessary to add sulfate ions or chloride ions to the culture medium in an amount necessary for growth. Preferably, an appropriate amount of ammonium sulfate or the like is fed to the medium at the beginning of the culture, and the feed is stopped during the culture. Or you may feed ammonium sulfate etc., maintaining the balance with the dissolved amount of the carbonate ion or bicarbonate ion in a culture medium. Alternatively, ammonia may be fed to the medium as a nitrogen source for basic amino acids. Ammonia can be supplied to the medium alone or with other gases.

The concentration of bicarbonate ions and / or other anions other than carbonate ions contained in the medium is preferably low as long as it is an amount necessary for the growth of microorganisms. Such anions include chloride ions, sulfate ions, phosphate ions, ionized organic acids, hydroxide ions, and the like. The total molar concentration of these other ions is preferably usually 900 mM or less, more preferably 700 mM or less, particularly preferably 500 mM or less, still more preferably 300 mM or less, and particularly preferably 200 mM or less.

In the above embodiment, one of the purposes is to reduce the amount of sulfate ion and / or chloride ion used, and the sulfate ion or chloride ion contained in the medium, or the total of these, is usually 700 mM. Hereinafter, it is preferably 500 mM or less, more preferably 300 mM or less, still more preferably 200 mM or less, and particularly preferably 100 mM or less.

Normally, when ammonium sulfate is added to the medium as a counter ion source for basic amino acids, carbon dioxide in the culture medium is released by sulfate ions. On the other hand, in the above embodiment, it is not necessary to add an excessive amount of ammonium sulfate to the medium, so that carbon dioxide gas can be easily dissolved in the fermentation broth.

In the above embodiment, it is preferable to control the total ammonia concentration in the medium to such an extent that “the production of basic amino acids is not inhibited”. Such conditions include, for example, preferably 50% or more, more preferably 70% or more, particularly preferably 90%, as compared to the yield and / or productivity in the case of producing a basic amino acid under optimum conditions. Conditions for obtaining the above yield and / or productivity are included. Specifically, the total ammonia concentration in the medium is preferably 300 mM or less, more preferably 250 mM, and particularly preferably 200 mM or less. The degree of ammonia dissociation decreases with increasing pH. Undissociated ammonia is more toxic to bacteria than ammonium ions. Therefore, the upper limit of the total ammonia concentration also depends on the pH of the culture solution. That is, the higher the pH of the culture solution, the lower the allowable total ammonia concentration. Therefore, the total ammonia concentration that “does not inhibit the production of basic amino acids” is preferably set for each pH. However, the total ammonia concentration range allowed at the highest pH during the culture may be the upper limit range of the total ammonia concentration throughout the culture period.

On the other hand, the total ammonia concentration as a nitrogen source necessary for the growth of microorganisms and the production of basic substances decreases the productivity of target substances by microorganisms due to the lack of a nitrogen source that does not continuously deplete ammonia during culture. There is no particular limitation as long as it is not set, and it can be set as appropriate. For example, the ammonia concentration may be measured over time during the culture, and a small amount of ammonia may be added to the medium when the ammonia in the medium is depleted. The concentration when ammonia is added is not particularly limited. For example, the total ammonia concentration is preferably 1 mM or more, more preferably 10 mM or more, and particularly preferably 20 mM or more.

In the present invention, in order to keep L-amino acid accumulation at a certain level or more, the culture of microorganisms may be performed separately in seed culture and main culture, and the seed culture is performed in a flask culture or a batch culture. The main culture may be performed by fed-batch culture or continuous culture, and both seed culture and main culture may be performed by batch culture.

In the present invention, when fed-batch culture or continuous culture is performed, the feed medium may be fed intermittently so that the feed of ethanol or other carbon source is temporarily stopped. In addition, it is preferable to stop the feeding of the fed-batch medium at a maximum of 30% or less, desirably 20% or less, particularly desirably 10% or less of the feeding time. When the fed-batch culture is fed intermittently, the fed-batch medium is added for a certain period of time, and the second and subsequent additions are performed when the carbon source in the fermentation medium is depleted in the addition stop period preceding the addition stage. Control to start when a rise in pH or an increase in dissolved oxygen concentration is detected by the computer, so that the substrate concentration in the culture tank may always be automatically maintained at a low level (US Pat. No. 5,912,113). book).

The fed-batch medium used for fed-batch culture is preferably a medium containing ethanol or other carbon source and a nutrient (growth promoting factor) that has a growth promoting effect, and is controlled so that the concentration of ethanol in the fermentation medium is below a certain level. May be. Here, the term “below a certain concentration” means preparing a medium to be added so that the concentration is 10 w / v% or less, preferably 5 w / v% or less, and more preferably 1 w / v% or less.

The L-amino acid can be collected from the culture solution by combining an ion exchange resin method, a precipitation method and other known methods. In addition, when L-amino acid accumulates in the microbial cells, for example, the microbial cells are crushed by ultrasonic waves and the microbial cells are removed by centrifugation, and the L-amino acid is removed from the supernatant obtained by ion exchange resin method or the like. Can be recovered. The recovered L-amino acid may be a free L-amino acid or a salt containing sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, or potassium salt.

Further, the L-amino acid collected in the present invention may contain microbial cells, medium components, moisture, and microbial metabolic byproducts in addition to the target L-amino acid. The purity of the collected L-amino acid is 50% or more, preferably 85% or more, particularly preferably 95% or more. , 238,714, US2005 / 0025878).

If L-amino acid is precipitated in the medium, it can be recovered by centrifugation or filtration. The L-amino acid precipitated in the medium may be isolated together after crystallization of the L-amino acid dissolved in the medium.

Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1] Construction of L-lysine-producing bacterium with reduced ribonuclease G activity <1-1> Giving ethanol-assimilating ability to WC196ΔcadAΔldcC strain An alcohol dehydrogenase gene (adhE *) was introduced. As the mutant alcohol dehydrogenase gene, a gene derived from MG1655 :: PL _-tac adhE * (WO2008 / 010565) was used. The MG1655 :: PL _L-tac adhE * strain contains a chloramphenicol resistance gene (cat) and a DNA fragment linked to the mutant adhE gene controlled by the PL-tac promoter in the genome of Escherichia coli MG1655 strain. It is a strain obtained by insertion. In order to be able to remove the cat gene from the genome, the cat gene was replaced with a DNA fragment (att-tet) linking the attachment site of lambda phage and the tetracycline resistance gene.

Replacement of the cat gene with the att-tet gene is a method called “Red-driven integration” originally developed by Datsenko and Wanner described in WO2005 / 010175 (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645). According to the “Red-driven integration” method, a synthetic oligonucleotide designed with a part of the target gene on the 5 ′ side of the synthetic oligonucleotide and a part of the antibiotic resistance gene on the 3 ′ side is used as a primer. Using the obtained PCR product, a gene-disrupted strain can be constructed in one step. In addition, a lambda phage-derived excision system (J. Bacteriol. 2002 Sep; 184 (18): 5200-3. Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex. Cho EH, Gumport RI, Gardner JF.) Thus, the antibiotic resistance gene incorporated in the gene disruption strain can be removed. The primers of SEQ ID NOs: 5 and 6 were used as primers for replacing the cat gene with the att-tet gene. Thus, MG1655-att-tet-PL _-tac adhE * strain in which the cat gene of MG1655 :: PL _-tac adhE * was replaced with the att-tet gene was obtained.

In order to confer ethanol-assimilating properties to L-lysine-producing bacteria, MG1655-att-tet-P _L-tac adhE * was used as a donor, P1 transduction was performed on L-lysine-producing bacteria WC196ΔcadAΔldcC, and WC196ΔcadAΔldcC-att-tet -P _L-tac adhE * strain was obtained.

Next, a helper plasmid pMW-intxis-ts (US Patent Application Publication 20060141586) was used to remove the att-tet gene introduced upstream of the P _L-tac promoter. pMW-intxis-ts is a plasmid carrying a gene encoding λ phage integrase (Int) and gene encoding excisionase (Xis) and having temperature-sensitive replication ability.

Competent cells of the WC196ΔcadAΔldcC-att-tet-PL _-tac adhE * strain obtained above were prepared according to a conventional method, transformed with the helper plasmid pMW-intxis-ts, and 50 mg / L at 30 ° C. Plates were plated on LB agar medium containing ampicillin, and ampicillin resistant strains were selected. In order to remove the pMW-intxis-ts plasmid, it was cultured on LB agar medium at 42 ° C, and the resulting colonies were tested for ampicillin resistance and tetracycline resistance. Att-tet and pMW-intxis-ts were removed. We obtained tetracycline and ampicillin sensitive strains, which are P _L-tac adhE * introduced strains. This strain was named WC196ΔcadAΔldcC PL _-tac adhE * strain.

<1-2> against WC196ΔcadAΔldcC P _L-tac adhE * ribonuclease G non-producing strains from stock (rng gene-deficient strain WC196ΔcadAΔldcΔrng P _L-tac adhE * stock) Construction of strain MG1655, by the method "Red-driven integration" The deletion of the rng gene was performed. As a primer for deletion of the rng gene, the primers of SEQ ID NOs: 7 and 8 can be used. As a result, the MG1655Δrng :: Cm strain was obtained. Using the MG1655Δrng :: Cm strain as a donor, P1 transduction was performed on the _L- lysine-producing WC196ΔcadAΔldcC PL _-tac adhE * strain to obtain a ribonuclease G non-producing strain WC196ΔcadAΔldcCP _L-tac adhE * Δrng :: Cm strain.

WC196ΔcadAΔldcC P _L-tac adhE * strain and WC196ΔcadAΔldcC P _L-tac adhE * Δrng :: Cm strain are routinely used in the Lys production plasmid pCABD2 (International Publication No. WO01 / 53459 pamphlet) carrying the dapA, dapB and lysC genes. Then, WC196ΔcadAΔldcC P _L-tac adhE * / pCABD2 strain and WC196ΔcadAΔldcC P _L-tac adhE * Δrng :: Cm / pCABD2 were obtained. After culturing these strains in L medium containing 20 mg / L streptomycin at 37 ° C. so that the final OD600≈0.6, an equal amount of 40% glycerol solution and the same amount as the culture solution were added and stirred. Aliquots were stored at -80 ° C. This is called glycerol stock.

[Example 2] Evaluation of L-lysine producing ability of a ribonuclease G non-producing strain Melt the glycerol stock of the above strain, apply 100 μL of each uniformly onto an L plate containing 20 mg / L of streptomycin, and maintain at 37 ° C. For 15 hours. Suspend the obtained cells in 0.85% saline, inoculate 5 mL of fermentation medium containing 20 mg / L streptomycin in a large test tube (inner diameter 18 mm) so that the initial OD = 0.25, The cells were cultured for 16 hours at 37 ° C. in a reciprocal shaking culture apparatus under the condition of stirring at 120 rpm. After the cultivation, the amount of lysine accumulated in the medium was measured by a known method (Sakura Seiki Biotech Analyzer AS210).
The composition of the fermentation medium is shown below.

[L-lysine fermentation medium composition]
Ethanol 10 ml / L
(NH ₄ ) ₂ SO ₄ 24 g / L
K ₂ HPO ₄ 1.0 g / L
MgSO ₄・ 7H ₂ O 1.0 g / L
FeSO ₄・ 7H ₂ O 0.01 g / L
MnSO ₄・ 5H ₂ O 0.01 g / L
Yeast Extract 2.0 g / L
CaCO ₃ (Japanese Pharmacopoeia) 30 g / L
Distilled water Final volume 1L
The pH was adjusted to 5.7 with KOH and autoclaved at 115 ° C. for 10 minutes. However, MgSO ₄ · 7H ₂ O was sterilized separately, and ethanol was sterilized by filter filtration. CaCO ₃ was sterilized by dry heat at 180 ° C. for 2 hours.
20 mg / L streptomycin was added as an antibiotic.

The results are shown in Table 1. Yield (%) indicates L-lysine yield (w / w) from ethanol. As can be seen from Table 1, the WC196ΔcadAΔldcC P _L-tac adhE * Δrng :: Cm / pCABD2 strain contains a larger amount of L-lysine than the WC196ΔcadAΔldcC P _L-tac adhE * / pCABD2 strain which does not lack the rng gene. Accumulated.

[Explanation of Sequence Listing]
SEQ ID NO: 1: Escherichia coi ribonuclease G gene (rng) nucleotide sequence SEQ ID NO: 2: Escherichia coi ribonuclease G amino acid sequence SEQ ID NO: 3: Escherichia coli alcohol dehydrogenase gene (adhE) nucleotide sequence SEQ ID NO: 4: Escherichia coli Amino acid sequence of alcohol dehydrogenase of SEQ ID NO: 5: primer for replacing cat gene with att-tet gene SEQ ID NO: 6: primer for replacing cat gene with att-tet gene SEQ ID NO: 7: for deletion of rng gene Primer SEQ ID NO: 8: primer for deletion of rng gene SEQ ID NO: 9: nucleotide sequence of Chlorobium tepidum pyruvate synthase gene SEQ ID NO: 10: amino acid sequence of Chlorobium tepidum pyruvate synthase SEQ ID NO: 11: pyruvate synthase gene of Escherichia coli (ydbK) nucleotide sequence SEQ ID NO: 12: Escherichia coli pyruvate syn Tyrosine (YdbK) amino acid sequence SEQ ID NO: 13: Methanococcus maripaludis pyruvate synthase γ subunit gene (porA) nucleotide sequence SEQ ID NO: 14: Methanococcus maripaludis pyruvate synthase γ subunit amino acid sequence SEQ ID NO: 15: Methanococcus maripaludis Pyruvate synthase δ subunit gene (porB) nucleotide sequence SEQ ID NO: 16: Methanococcus maripaludis pyruvate synthase δ subunit amino acid sequence SEQ ID NO: 17: Methanococcus maripaludis pyruvate synthase α subunit gene (porC) nucleotide sequence Number 18: amino acid sequence of pyruvate synthase α subunit of Methanococcus maripaludis SEQ ID NO: 19: nucleotide sequence of pyruvate synthase β subunit gene (porD) of Methanococcus maripaludis SEQ ID NO: 20: pyruvate synthase β subunit of Methanococcus maripaludis Minoic acid sequence SEQ ID NO: 21: base sequence of the pyruvate synthase porE gene of Methanococcus maripaludis SEQ ID NO: 22: amino acid sequence of the pyruvate synthase PorE of Methanococcus maripaludis SEQ ID NO: 23: base sequence of the pyruvate synthase porF gene of Methanococcus maripaludis : Methanococcus maripaludis pyruvate synthase PorF amino acid sequence SEQ ID NO: 25: Euglena gracilis pyruvate: NADP ⁺ oxidoreductase gene nucleotide sequence SEQ ID NO: 26: Euglena gracilis pyruvate: NADP ⁺ oxidoreductase amino acid sequence SEQ ID NO: 27: Escherichia coli of flavodoxin-NADP ⁺ reductase gene (fpr) the nucleotide sequence SEQ ID NO: 28: amino acid sequence SEQ ID NO: 29 Escherichia coli of flavodoxin-NADP ⁺ reductase gene (fpr) encoded: salts of Escherichia coli ferredoxin gene (fdx) SEQ ID NO: 30: amino acid sequence encoded by the ferredoxin gene (fdx) of Escherichia coli SEQ ID NO: 31: base sequence of the ferredoxin gene (yfhL) of Escherichia coli SEQ ID NO: 32: amino acid sequence encoded by the ferredoxin gene (yhfL) of Escherichia coli SEQ ID NO: 33: Escherichia coli flavodoxin gene (fldA) nucleotide sequence SEQ ID NO: 34: Escherichia coli flavodoxin gene (fldA) encoded amino acid sequence SEQ ID NO: 35: Escherichia coli flavodoxin gene (fldB) nucleotide sequence SEQ ID NO: 36 : Amino acid sequence encoded by the flavodoxin gene (fldB) of Escherichia coli SEQ ID NO: 37: nucleotide sequence of ferredoxin I gene of Chlorobium tepidum 38: amino acid sequence encoded by ferredoxin I gene of Chlorobium tepidum SEQ ID NO: 39: ferredoxin of Chlorobium tepidum Base sequence of gene II SEQ ID NO: 40: Chlor Amino acid sequence encoded by the ferredoxin II gene of obium tepidum SEQ ID NO: 41: Chlorobium tepidum pyruvate synthase gene amplification primer 1
SEQ ID NO: 42: Chlorobium tepidum pyruvate synthase gene amplification primer 2
SEQ ID NO: 43: Escherichia coli pyruvate synthase gene amplification primer 1
SEQ ID NO: 44: Escherichia coli pyruvate synthase gene amplification primer 2
SEQ ID NO: 45: Euglena gracilis pyruvate: NADP ⁺ oxidoreductase gene amplification primer 1
SEQ ID NO: 46: Euglena gracilis pyruvate: NADP ⁺ oxidoreductase gene amplification primer 2
SEQ ID NO: 47: Primer 1 for amplification of Escherichia coli flavodoxin-NADP ⁺ reductase gene
SEQ ID NO: 48: Escherichia coli flavodoxin-NADP ⁺ reductase gene amplification primer 2
SEQ ID NO: 49: Escherichia coli fdx gene amplification primer 1
SEQ ID NO: 50: Primer 2 for amplifying the fdx gene of Escherichia coli
SEQ ID NO: 51: Nucleotide sequence of the gene (b2463) encoding the NADP-type malic enzyme of Escherichia coli SEQ ID NO: 53: Base sequence of the gene (sfcA) encoding the NAD-type malic enzyme of Escherichia coli SEQ ID NO: 55: Pilvin of Escherichia coli Nucleotide sequence number of acid dehydrogenase Ep1 subunit gene (aceE) SEQ ID NO: 56: Amino acid sequence number of pyruvate dehydrogenase Ep1 subunit of Escherichia coli SEQ ID NO: 57: Nucleotide sequence number of pyruvate dehydrogenase E2 subunit gene (aceF) of Escherichia coli 58: amino acid sequence of the pyruvate dehydrogenase E2 subunit of Escherichia coli SEQ ID NO: 59: base sequence of the pyruvate dehydrogenase E3 subunit gene (lpdA) of Escherichia coli SEQ ID NO: 60: amino acid sequence of the pyruvate dehydrogenase E3 subunit of Escherichia coli

According to the present invention, L-amino acid can be efficiently produced using ethanol as a carbon source.

Claims

Bacteria belonging to the family Enterobacteriaceae and having the ability to produce L-amino acids are cultured in a medium containing ethanol as a carbon source, L-amino acids are produced and accumulated in the culture, and L-amino acids are collected from the culture. A method for producing an L-amino acid, wherein the bacterium is a bacterium modified so that the activity of ribonuclease G is reduced.
The method according to claim 1, wherein the activity of ribonuclease G is reduced by inactivation of the rng gene encoding ribonuclease G.
The method according to claim 2, wherein the rng gene is DNA encoding the amino acid sequence of SEQ ID NO: 2 or a variant thereof.
The method according to any one of claims 1 to 3, wherein the bacterium is modified so as to assimilate ethanol aerobically.
5. The method of claim 4, wherein the bacterium retains an adh gene that has been modified to be expressed under the control of a non-native promoter that functions under aerobic conditions, thereby aerobically assimilating ethanol. .
The method according to claim 4 or 5, wherein the bacterium is modified so as to retain a mutated adhE gene, whereby ethanol can be assimilated aerobically.
The method according to claim 6, wherein the mutant adhE gene encodes a protein having the amino acid sequence of SEQ ID NO: 4 or a conservative variant thereof except that the glutamic acid residue at position 568 is substituted with another amino acid residue. .
The L-amino acid is L-lysine, L-glutamic acid, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, L The method according to any one of claims 1 to 7, which is one or more L-amino acids selected from the group consisting of -proline and L-cysteine.
The L-amino acid is L-lysine, and the bacterium is dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, The activity of one or more enzymes selected from the group consisting of tetrahydrodipicolinate succinylase and succinyldiaminopimelate deacylase is enhanced and / or the activity of lysine decarboxylase is weakened The method of claim 8.
The L-amino acid is L-threonine, and the bacterium is one or more selected from the group consisting of aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase. The method according to claim 8, wherein the activity of said enzyme is enhanced.
The method according to any one of claims 1 to 10, wherein the bacterium belonging to the family Enterobacteriaceae is an Escherichia bacterium, an Enterobacter bacterium, or a Pantoea bacterium.
The method according to claim 11, wherein the bacterium is Escherichia coli.
The method according to any one of claims 1 to 12, wherein ethanol is contained in the medium in an amount of 0.001 w / v% or more.