JP2008086305A - L-amino acid-producing bacterium and method for producing l-amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bacterial strain producing L-amino acids in a good efficiency and a method for producing the L-amino acids in a good efficiency by using the bacterial strain. <P>SOLUTION: This method for producing the L-amino acids is provided by culturing a microorganism belonging to Enterobacteriaceae, having a production capacity of the L-amino acids and modified so as to enhance an iron transporter activity by the enhancement or the like of the expression of ≥1 kinds of genes selected from tonB gene, fepA gene and fecA gene. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing an L-amino acid using a microorganism, and particularly to a method for producing an L-amino acid such as L-lysine, L-threonine, L-glutamic acid. L-lysine and L-threonine are industrially useful L-amino acids as additives for animal feeds, health food ingredients, amino acid infusions, etc., and L-glutamic acid as a seasoning.

L-amino acids are industrially produced by fermentation using microorganisms belonging to the genera Brevibacterium, Corynebacterium, Escherichia and the like. For example, as a manufacturing method of L-lysine, the method described in patent documents 1-8 can be mentioned, for example. On the other hand, in these production methods, strains isolated from the natural world or artificial mutants of the strains, and microorganisms modified to enhance the activity of L-amino acid biosynthetic enzymes by recombinant DNA technology, etc. It is used.

As a method for improving the L-amino acid production ability, a method for modifying an L-amino acid uptake system or excretion system is known. As a method for enhancing the L-amino acid excretion system, for example, L-lysine, a method for producing L-lysine using a strain of Corynebacterium microorganism with enhanced expression of L-arginine excretion gene (LysE) (for example, A method for producing L-arginine (see, for example, Patent Document 9) is known. In addition, rhtA, B, C genes (for example, see Patent Document 11) or yfiK, yahN genes, etc. (for example, see Patent Document 12), which are genes suggested to be involved in L-amino acid excretion, ybjE A method for producing an L-amino acid using a microorganism belonging to the family Enterobacteriaceae with enhanced expression of a gene (Patent Document 13) and a yhfK gene (Patent Document 14) has also been reported.

  In addition, it is known that amino acid-producing ability is improved by enhancing the expression of a sugar uptake gene serving as a fermentation substrate. For example, a method for producing an L-amino acid using an Escherichia bacterium having enhanced expression of the ptsG gene (Patent Document 15), or a method for producing an L-amino acid using an Escherichia bacterium having enhanced expression of the ptsH, ptsI, or crr genes (Patent Document 16) is known.

The fepA gene and fecA gene are genes encoding membrane proteins known as iron transporters, and the tonB gene is a gene encoding proteins that control the activities of these iron transporters (Non-Patent Documents 1-4). reference). However, it has never been known that enhancing the activity of these gene products has an effect on L-amino acid production.
European Patent No. 0643135 European Patent No. 0733712 European Patent Application Publication No. 1477565 European Patent Application Publication No. 0796912 European Patent Application Publication No. 0837134 International publication 01/53459 pamphlet European Patent Application Publication No. 1170376 (International Publication 2005/010175 Pamphlet) International Publication No. 97/23597 Pamphlet US Patent Application Publication No. 2003-0113899 JP 2000-189177 A European Patent Application Publication No. 1016710 International publication 2005/073390 pamphlet International publication 2005/085419 pamphlet WO03 / 04670 pamphlet International Publication No. 03/04674 Specification J Bacteriol. 1990; 172 (5): 2736-46 J. Bacteriol, 2003, vol.185, No. 6, p1870-1885 Mol Microbiol. 2005; 58 (5): 1226-1237 J. Bacteriol 2001, vol.183, No. 20, p5885-5895

  An object of the present invention is to provide a strain capable of efficiently producing an L-amino acid and to provide a method for efficiently producing an L-amino acid using the strain.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies. As a result, it was found that L-amino acid productivity was improved by amplifying genes involved in the tonB system in L-amino acid-producing bacteria. Furthermore, it has been found that L-amino acids can be efficiently produced by using a microorganism modified with a gene involved in the tonB system, and the present invention has been completed.

That is, the present invention is as follows.
(1) A microorganism belonging to the family Enterobacteriaceae that has L-amino acid-producing ability and has been modified so that the activity of the iron transporter is enhanced, the tonB gene involved in the tonB system, the fepA gene, and fecA A microorganism modified so that the expression level of one or more genes selected from the group consisting of genes is increased.
(2) Iron transporter activity is enhanced by increasing the copy number of one or more genes selected from the group consisting of tonB gene, fepA gene, and fecA gene, or by modifying the expression regulatory sequence of the gene. (1) Microorganisms.
(3) The tonB gene has a protein having the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in SEQ ID NO: 2, and iron transporter activity The microorganism according to (1) or (2), which is a gene encoding a protein having a function of regulating
(4) The fepA gene has a protein having the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in SEQ ID NO: 4, and has an iron transporter activity The microorganism according to (1) or (2), which is a gene encoding a protein having
(5) The fecA gene has a protein having the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in SEQ ID NO: 10, and has an iron transporter activity The microorganism according to (1) or (2), which is a gene encoding a protein having
(6) The microorganism according to (1) or (2), wherein the tonB gene is the DNA described in (a) or (b) below:
(A) DNA comprising the base sequence shown in SEQ ID NO: 1,
(B) a protein having a function of hybridizing with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or a probe that can be prepared from the base sequence under stringent conditions and having a function of controlling iron transporter activity DNA to encode.
(7) The microorganism according to (1) or (2), wherein the fepA gene is the DNA described in (c) or (d) below:
(C) DNA comprising the base sequence shown in SEQ ID NO: 3,
(D) A DNA that hybridizes under stringent conditions with a probe complementary to the nucleotide sequence shown in SEQ ID NO: 3 or that can be prepared from the nucleotide sequence and encodes a protein having iron transporter activity.
(8) The microorganism according to (1) or (2), wherein the fecA gene is the DNA described in (e) or (f) below:
(E) a DNA comprising the base sequence shown in SEQ ID NO: 9,
(F) A DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 9 or a probe that can be prepared from the base sequence, and that encodes a protein having iron transporter activity.
(9) The L-amino acid is L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L- The microorganism according to any one of (1) to (8), which is one or more L-amino acids selected from cysteine and L-glutamic acid.
(10) The microorganism according to any one of (1) to (9), wherein the microorganism belonging to the family Enterobacteriaceae is a microorganism selected from Escherichia bacteria, Pantoea bacteria, and Enterobacter bacteria. (11) culturing the microorganism of any one of (1) to (10) in a medium, generating and accumulating L-amino acid in the medium or in the microbial cell, and recovering the L-amino acid from the medium or microbial cell A process for producing an L-amino acid characterized by
(12) The L-amino acid is L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L- (11) The production method of (11), which is one or more L-amino acids selected from cysteine and L-glutamic acid.

  By using the microorganism of the present invention, basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L -Aliphatic amino acids such as leucine and L-glycine, amino acids that are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, L-phenylalanine, L-tyrosine and L-tryptophan Fermentative production of aromatic amino acids such as L-cysteine, L-cystine, and sulfur-containing amino acids such as L-methionine, and acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine, and L-asparagine .

  Hereinafter, the present invention will be described in detail.

<1> Microorganism of the Present Invention The microorganism of the present invention is a microorganism belonging to the family Enterobacteriaceae that has been modified so as to be capable of producing an L-amino acid and to enhance the activity of an iron transporter, and is involved in the tonB system. It is a microorganism characterized in that it has been modified so that the expression level of the gene is increased. Here, the L-amino acid-producing ability refers to the ability to produce and accumulate L-amino acids in the medium or in the cells when the microorganism of the present invention is cultured in the medium. The microorganism of the present invention may be capable of producing a plurality of L-amino acids. The microorganism having L-amino acid-producing ability may be inherently L-amino acid-producing ability, but the following microorganisms can be obtained by using a mutation method or recombinant DNA technology, It may be modified so as to have L-amino acid production ability.

The type of L-amino acid is not particularly limited, but basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L- Aliphatic amino acids such as leucine and L-glycine, amino acids which are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, L-phenylalanine, L-tyrosine and L-tryptophan Aromatic amino acids, sulfur-containing amino acids such as L-cysteine, L-cystine and L-methionine, and acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine. The microorganism of the present invention may be capable of producing two or more amino acids.

<1-1> Granting L-amino acid-producing ability Below, a method for imparting L-amino acid-producing ability and a microorganism imparted with L-amino acid-producing ability that can be used to obtain the microorganism of the present invention are exemplified. To do. However, as long as it has L-amino acid producing ability, it is not limited thereto.
As the parent strain of the microorganism of the present invention, microorganisms belonging to the family Enterobacteriaceae, represented by Escherichia bacteria, Pantoea bacteria, Enterobacter bacteria, can be used. Other microorganisms belonging to the family Enterobacteriaceae include the genus Enterobacter, Klebsiella, Serratia, Erwinia, Salmonella, Morganella, etc. Examples include microorganisms belonging to the family Enterobacteriaceae belonging to γ-proteobacteria.

  As a bacterium belonging to the genus Escherichia, a book by Nitehard et al. ((Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC), such as Escherichia coli, etc. Examples of the wild strain of Escherichia coli include, for example, the K12 strain or a derivative thereof, Examples include Escherichia coli MG1655 (ATCC No. 47076), W3110 (ATCC No. 27325), etc. To obtain these, for example, you can obtain a sale from the American Type Culture Collection (ATCC). Yes (address PO Box 1549, Manassas, VA 20108, 1, United Statesof America).

Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and the like, and Pantoea ananatis is an example of Pantoea bacteria. In recent years, Enterobacter agglomerans has been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc., by base sequence analysis of 16S rRNA. There is something. In the present invention, any substance belonging to the genus Enterobacter or Pantoea may be used as long as it is classified into the family Enterobacteriaceae. When breeding Pantoea ananatis using genetic engineering techniques, Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) and derivatives thereof Etc. can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans, but as described above, they have been reclassified as Pantoea Ananatis by 16S rRNA sequence analysis, etc. .

Hereinafter, a method for imparting L-amino acid-producing ability to the parent strain as described above will be described.
In order to confer L-amino acid-producing ability, acquisition of an auxotrophic mutant, analog resistant strain or metabolically controlled mutant, creation of a recombinant strain with enhanced expression of L-amino acid biosynthetic enzyme, etc. Conventionally, methods that have been adopted for breeding Escherichia bacteria and the like can be applied (see Amino Acid Fermentation, Japan Society for Publishing Press, 30 May 1986, first edition, pages 77 to 100). Here, in the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation may be used singly or in combination of two or more. In addition, L-amino acid biosynthesis 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.

  An auxotrophic mutant having L-amino acid-producing ability, an L-amino acid analog-resistant strain, or a metabolically controlled mutant is obtained by subjecting a parent strain or a wild strain to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl. -N'-nitro-N-nitrosoguanidine (NTG), treated with a mutagen such as ethyl methanesulfonate (EMS), etc., among the obtained mutant strains, auxotrophy, analog resistance, or metabolism It can be obtained by selecting those that show regulatory mutations and have the ability to produce L-amino acids.

  As the L-lysine analog, for example, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, norleucine and the like can be used. As the L-arginine analog, arginine hydroxamate, homoarginine, D-arginine, canavanine, and the like can be used.

  Specific examples of L-lysine analog-resistant strains or metabolic control mutants having L-lysine-producing ability include Escherichia coli AJ11442 strain (FERM BP-1543, NRRL B-12185; JP 56-18596 A and US Patent No. 4346170), Escherichia coli VL611 strain (Japanese Patent Laid-Open No. 2000-189180) and the like. In addition, as an L-lysine-producing bacterium of Escherichia coli, WC196 strain (see International Publication No. 96/17930 pamphlet) can also be used. The WC196 strain was bred by imparting AEC (S- (2-aminoethyl) -cysteine) resistance to the W3110 strain derived from Escherichia coli K-12. This strain was named Escherichia coli AJ13069, and on December 6, 1994, the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, 305-8566 Japan) No. 1 in Higashi 1-chome, 1-chome, Tsukuba City, Ibaraki Prefecture, Japan, as deposit number FERM P-14690, transferred to an international deposit based on the Budapest Treaty on September 29, 1995, with deposit number FERM BP-5252 Has been granted.

L-amino acid-producing ability can also be imparted by enhancing the expression of the L-amino acid biosynthesis enzyme gene.
For example, L-lysine-producing ability can be imparted by enhancing dihydrodipicolinate synthase activity and aspartokinase gene expression as follows. That is, a gene fragment encoding dihydrodipicolinate synthase and a gene fragment encoding aspartokinase are ligated with a vector functioning in a host microorganism used for the production of L-lysine, preferably a multicopy vector, to produce recombinant DNA. And then transform the host. As a result of the increase in the copy number of the gene encoding dihydrodipicolinate synthase and the gene encoding aspartokinase in the host cell by transformation, the activities of these enzymes are enhanced. Hereinafter, dihydrodipicolinate synthase may be abbreviated as DDPS, aspartokinase as AK, and aspartokinase III as AKIII.

The gene encoding DDPS and the gene encoding AK are not particularly limited as long as they can express DDPS activity and AK activity in the host microorganism. For example, Escherichia coli, Methylophilus methylotrophus, Coryne Examples include genes such as bacteria and glutamicum. A gene encoding DDPS derived from bacteria belonging to the genus Escherichia (dapA, Richaud, F. et al. J. Bacteriol., 297 (1986)) and a gene encoding AKIII (lysC, Cassan, M., Parsot, C., Cohen , GN and Patte, JC, J. Biol. Chem., 261, 1052 (1986)), the base sequences have been elucidated. Therefore, primers were synthesized based on the base sequences of these genes, and Escherichia -These genes can be obtained by PCR using a chromosomal DNA of a microorganism such as Koli K-12 as a template. Hereinafter, dapA and lysC derived from Escherichia coli will be described as examples, but the gene encoding DDPS and the gene encoding AK are not limited thereto.

  It is known that wild-type DDPS derived from Escherichia coli is subject to feedback inhibition by L-lysine, and wild-type AKIII derived from Escherichia coli is known to undergo inhibition and feedback inhibition by L-lysine. Therefore, when dapA and lysC are used, it is preferable that these genes are mutant genes whose encoded DDPS and AK are not subject to feedback inhibition by L-lysine. Incidentally, DDPS derived from Corynebacterium microorganisms may not be subject to feedback inhibition by L-lysine from the beginning, and DDPS and AK used in the present invention do not necessarily need to be mutated.

Examples of DNA encoding a mutant DDPS that is not subject to feedback inhibition by L-lysine include DNA encoding DDPS having a sequence in which the histidine residue at position 118 is substituted with a tyrosine residue. As a DNA encoding mutant AKIII that is not subject to feedback inhibition by L-lysine, the threonine residue at position 352 is replaced with an isoleucine residue, the glycine residue at position 323 is replaced with an asparagine residue, Examples include DNA encoding AKIII having a sequence in which methionine is replaced with isoleucine (see US Pat. Nos. 5661012 and 6040160 for these variants). Mutant DNA can be obtained by site-specific mutagenesis such as PCR.
The expression enhancement of the L-lysine biosynthetic gene as described above can be achieved by methods such as transformation using plasmids or homologous recombination, as with the tonB gene, fepA gene, and fecA gene described later.

  Wide host range plasmids RSFD80, pCAB1, and pCABD2 are known as plasmids containing mutant dapA encoding mutant DDPS and mutant lysC encoding mutant AKIII (US Pat. No. 6,040,160). The Escherichia coli JM109 strain (US Pat. No. 6,040,160) transformed with this plasmid was named AJ12396, and this strain was established on 28 October 1993 at the Institute of Biotechnology, Ministry of International Trade and Industry (METI). Deposited at the National Institute of Advanced Industrial Science and Technology (AIST) as deposit number FERM P-13936, transferred to an international deposit based on the Budapest Treaty on November 1, 1994, with the deposit number of FERM BP-4859 It is deposited with. RSFD80 can be obtained from AJ12396 strain by a known method.

  The L-lysine-producing ability may be imparted by enhancing expression of genes encoding enzymes involved in L-lysine biosynthesis other than DDPS and AK. Examples of such enzymes include dihydrodipicolinate reductase (dapB: gene name in parentheses below) (International Publication 01/53459 pamphlet), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (above, international Publication 96/40934 pamphlet), phosphoenolpyruvate carboxylase (pepC) (Japanese Patent Laid-Open No. 60-87788), aspartate aminotransferase (aspC) (Japanese Patent Publication No. 6-2002028), diaminopimelate epimerase gene (dapF) ) (Japanese Patent Laid-Open No. 2003-135066), aspartate semialdehyde dehydrogenase (asd) (International Publication No. 00/61723 pamphlet), tetrahydrodipicolinate succinylase gene (dapD), succinyl diaminopimelate deacylase (dapE), etc. Diaminopimelate pathway enzymes or homoaconitic hydrata Examples thereof include enzymes of the aminoadipate pathway such as sesase (JP 2000-157276 A). In addition, the literature regarding the L-lysine production strain in which the gene expression of each enzyme was enhanced is shown in parentheses. The enhancement of gene expression of these enzymes may be combined with the enhancement of DDPS and AK gene expression.

Further, in the present invention, in addition to a gene encoding a biosynthetic enzyme unique to L-lysine, sugar uptake, sugar metabolism (glycolytic system), TCA cycle, pentose phosphate cycle, each enzyme in the replenishment pathway, energy metabolism Expression may be enhanced by using a gene involved in the target gene as a target gene. In addition, a gene conferring resistance to each amino acid or an amino acid excretion gene may be enhanced as a target gene, or an uptake gene of each by-product may be enhanced as a target gene. These gene enhancements are effective for all L-amino acids.

  Examples of genes involved in sugar metabolism include genes encoding glycolytic enzymes and sugar uptake genes. Glucose 6-phosphate isomerase gene (pgi; WO 01/02542 pamphlet), phosphoenolpyruvate Synthase gene (pps; European application 877090), phosphoglucomutase gene (pgm; WO 03/04598 pamphlet), fructose diphosphate aldolase gene (pfkB; WO 03/04664 pamphlet), pyruvate Kinase gene (pykF; WO 03/008609 pamphlet), transaldolase gene (talB; WO 03/008611 pamphlet), fumarase gene (fum; WO 01/02545 pamphlet), phosphoenolpyruvate synthase gene ( pps; European Application Publication No. 877090 pamphlet), non-PTS sucrose incorporation Child gene (csc; European application 149911 pamphlet), sucrose utilization gene (scrAB operon; WO90 / 04636 pamphlet), PTS glucose uptake gene (ptsG, ptsH, ptsI, crr; WO03 / 04670, 03/04674 pamphlet, European patent application 1254957), galactose proton symporter gene (galP; US application publication 2004-214294), D-xylose permease gene (xylE; international publication 2006) / 043730 pamphlet), a gene involved in maltose transport (malK).

As the enzyme of TCA cycle, citrate synthase gene (gltA; WO 03/008607 pamphlet), isocitrate dehydrogenase gene (icd; WO 03/008607 pamphlet), 2-ketoglutarate dehydrogenase gene (sucAB; WO03 / 008614 pamphlet), succinate dehydrogenase gene (sdh; WO01 / 02544 pamphlet), and glutamate dehydrogenase (gdh; WO00 / 53726 pamphlet).

Examples of enzymes of the pentose phosphate cycle include glucose 6-phosphate dehydrogenase gene (zwf; International Publication No. 03/008607 pamphlet) and ribose 5-phosphate isomerase gene (rpiB; International Publication No. 03/008607 pamphlet).

The recruitment pathway is a pathway called anaplerotic pathways, and the enzymes of the recruitment pathway include phosphoenolpyruvate carboxylase gene (pepC; US Pat. No. 5,876,983), pyruvate carboxylase gene (pyc; European Patent Publication No. 1092776) ), Malate dehydrogenase gene (mdh; WO 01/02546 pamphlet), phosphoenolpyruvate carboxykinase (pckA; WO04 / 090125), and the like.

Examples of genes involved in energy metabolism include a transhydrogenase gene (pntAB; US Pat. No. 5,830,716) and a cytochromoe bo type oxidase gene (cyoB European Patent Application Publication 1070376). In addition, as a gene conferring resistance to various amino acids, rhtB gene (US Patent 6,887,691 specification), rhtC gene (European Patent Application Publication No. 1013765 specification), yedA gene (European Patent Application Publication EP1449917 specification), yddG gene (European patent application publication 1449918), ygaZH gene (European patent application publication 1290441), yahN, yfiK, yeaS gene (European patent application publication 1016710), rhtA gene (Res Microbiol. 2003) Mar; 154 (2): 123-35.), YbjE gene (International Publication 2005/073390 pamphlet).

Furthermore, the microorganism of the present invention may have a reduced or deficient activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-lysine to produce a compound other than L-lysine. Examples of such enzymes include homoserine dehydrogenase, lysine decarboxylase, and malic enzyme, and strains in which the activity of the enzyme is reduced or deleted are WO 95/23864, WO 96/17930, WO 2006/038695, It is described in the pamphlet of International Publication No. 2005/010175.
In order to reduce or eliminate lysine decarboxylase activity, it is preferable to reduce the expression of both the cadA gene and ldcC gene encoding lysine decarboxylase. The decrease in the expression of both genes can be performed, for example, according to the method described in Example 2 described later.
As the cadA gene, a DNA having the nucleotide sequence of SEQ ID NO: 17 (cadA gene derived from Escherichia coli), a complementary sequence of the nucleotide sequence of SEQ ID NO: 17, or a probe that can be prepared from this complementary sequence under stringent conditions Examples thereof include DNA that hybridizes and encodes a protein having lysine decarboxylase activity.
The ldcC gene gene includes DNA having the nucleotide sequence of SEQ ID NO: 11 (ldcC gene derived from Escherichia coli), a complementary sequence of the nucleotide sequence of SEQ ID NO: 11, or a probe that can be prepared from this complementary sequence under stringent conditions. And a DNA encoding a protein having a lysine decarboxylase activity.
The “stringent conditions” are as described later.

As a method for reducing or eliminating these enzyme activities, a mutation that reduces or eliminates the activity of the enzyme in the cell may be introduced into the gene of the enzyme on the chromosome by a usual mutation treatment method. For example, it can be achieved by deleting a gene encoding an enzyme on a chromosome by genetic recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. Introducing amino acid substitutions (missense mutations) into the chromosomal enzyme-encoding region, introducing stop codons (nonsense mutations), and introducing frameshift mutations that add or delete one or two bases This can also be achieved by deleting a part of the gene (Journal of biological Chemistry 272: 8611-8617 (1997). The enzyme activity can also be reduced or eliminated by substituting the normal gene on the chromosome with the gene by introducing a transposon or IS factor into the gene.

  For example, in order to introduce a mutation that reduces or eliminates the activity of the above enzyme by genetic recombination, the following method is used. A partial gene of the target gene is modified to produce a mutant gene that does not produce a normally functioning enzyme, and the DNA containing the gene is transformed into a microorganism belonging to the family Enterobacteriaceae. By causing recombination with the above gene, the target gene on the genome can be replaced with a mutant. Such gene replacement using homologous recombination is a method called “Red-driven integration” (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97 : 6640-6645 (2000)) Combined with Red driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (WO2005 / 010175) and a method using a plasmid containing a temperature-sensitive replication origin (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640). -6645 (2000); U.S. Pat. No. 6,303,383; JP-A-05-007491). In addition, site-directed mutagenesis by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have replication ability on the host.

  The above-described methods for enhancing gene expression of enzymes involved in L-lysine biosynthesis and methods for reducing enzyme activity can be similarly applied to genes encoding other L-amino acid biosynthetic enzymes. .

Hereinafter, L-amino acid producing bacteria to which L-amino acid producing ability other than L-lysine is imparted will be exemplified.

Examples of L-threonine-producing bacteria L-threonine-producing bacteria or parent strains for inducing them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. 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. coli FERM BP-3519 and FERM BP-3520 (US Pat.No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 Examples include, but are not limited to, strains belonging to the genus Escherichia, such as (EP 1149911 A).

  The TDH-6 strain lacks the thrC gene and 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 shares were deposited with the accession number RIA 1867 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) on November 19, 1987 . The strain was also deposited with the accession number B-3996 on 7 April 1987 at Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia). 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 has been deposited with Lucian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under the accession number VKPM B-5318.

Preferably, the bacterium used in the present invention is further modified so that expression of one or more of the following genes is increased.
-A mutant thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine-A thrB gene encoding homoserine kinase-A thrC gene encoding threonine synthase-A rhtA gene encoding a putative transmembrane protein-Aspartate- asd gene encoding β-semialdehyde dehydrogenase-aspC gene encoding aspartate aminotransferase (aspartate transaminase)

The thrA gene encoding aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (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 the 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 threonine synthase from 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, which is 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 at 18 minutes of the E. coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system. The rhtA gene is ORF1 (ybiF gene, nucleotide numbers 764 to 1651,
GenBank accession number AAA218541, gi: 440181), and located between the pexB gene and the ompX gene. The unit that expresses the protein encoded by ORF1 is called the 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 asd gene of E. coli 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 base sequence of the gene. Yes (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-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 (for example, 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. Examples of such genes include the leuABCD operon gene represented by a mutated leuA gene (US Pat. No. 6,403,342) that preferably encodes isopropylmalate synthase which is desensitized to feedback inhibition by L-leucine. . 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 deriving the same also 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. 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 into which a vector holding a DNA encoding an L-histidine biosynthetic enzyme has been introduced (Japanese Patent Laid-Open No. 56-005099). E. coli strain into which amino acid transport gene was introduced (EP1016710A), E. coli imparted resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin
80 shares (VKPM B-7270, 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 auxotrophic strain having mutations 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 deriving the same include, but are not limited to, strains in which expression of one or more genes encoding L-glutamic acid biosynthetic enzymes are increased. Examples of such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (g
ltA), phosphoenolpyruvate carbosylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phospho Glyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triphosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phospho Examples include fructokinase (pfkA, pfkB) and glucose phosphate isomerase (pgi).

  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.

  As an example of an L-glutamic acid-producing bacterium or a parent strain for deriving it, the activity of an enzyme that catalyzes the synthesis of a compound 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, such as E. coli AJ13199 (FERM BP-5807) (US Pat. 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.

Examples of L-glutamic acid producing bacteria include bacteria lacking α-ketoglutarate dehydrogenase activity or belonging to the genus Pantoea with reduced α-ketoglutarate dehydrogenase activity, which can be obtained as described above. it can. An example of such a strain is Pantoea ananatis AJ13356 (US Pat. No. 6,331,419). Pantoea ananatis AJ13356 was founded on 19 February 1998 at the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, 305-8566, Japan 1 Deposited under No. 6) under the accession number FERM P-16645, transferred to an international deposit under the Budapest Treaty on 11 January 1999, and assigned the accession number FERM BP-6616. Pantoea ananatis AJ13356 is deficient in α-ketoglutarate dehydrogenase activity due to disruption of the αKGDH-E1 subunit gene (sucA). When this strain was isolated, Enterobacter agglomera
ns and deposited as Enterobacter agglomerans AJ13356. However, it was reclassified as Pantoea ananatis based on the base sequence of 16S rRNA. AJ13356 is deposited as Enterobacter agglomerans with the above depository institution, but is described as Pantoea ananatis in this specification.

Examples of L-phenylalanine-producing bacteria and L-phenylalanine-producing bacteria or parent strains for inducing them include E. coli AJ12739 (tyrA :: Tn10, tyrR) (VKPM B-8197), E carrying a mutant pheA34 gene .coli HW1089
(ATCC 55371) (U.S. Pat.No. 5,354,672), 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) ) And other strains belonging to the genus Escherichia, but are not limited thereto. In addition, E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K-12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli Also used K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579) named AJ 12604 Yes (EP 488424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia having an 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 L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 lacking 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 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 the same include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and a kind of activity of an enzyme selected from tryptophan synthase (trpAB) Also included are strains with increased above. 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 desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase desensitized to feedback inhibition Examples include a transformant obtained by introducing plasmid pGH5 (WO 94/08031) into E. coli SV164.

Examples of L-tryptophan-producing bacteria or parent strains for inducing the same include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (JP-A 57-71397, JP 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) (EP1239041 A2).

  Examples of bacteria belonging to the genus Escherichia having L-proline producing ability 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 those 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 237 strain (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 deriving the same also 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 operon expression 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 the same 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 launched on June 24, 1988 at Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow
117545, Russia) under the deposit number VKPM B-4411.
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 having resistance 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).

<1-2> Enhancement of iron transporter activity The microorganism of the present invention can be obtained by modifying a microorganism having the ability to produce an L-amino acid as described above so as to enhance the iron transporter activity. However, L-amino acid-producing ability may be imparted after modification so as to enhance iron transporter activity. As described later, the enhancement of iron transporter activity can be achieved by modifying the expression level of genes involved in the tonB system to be increased. The expression of an endogenous gene may be enhanced by modifying a regulatory region, or the expression of an exogenous gene may be enhanced by introducing a plasmid containing the gene. Furthermore, these may be combined.

Here, the iron transporter means a membrane protein having a function of incorporating iron into the cell, and “modified so that the activity of the iron transporter is increased” means a wild strain or an unmodified strain. On the other hand, the case where the number of molecules of iron transporter per cell increases or the case where the activity per molecule of iron transporter is improved is applicable. The activity of the iron transporter is modified so as to be improved to 150% or more per cell, preferably 200% or more, and more desirably 300% or more per cell compared to the wild strain or the unmodified strain. preferable. Here, as microorganisms belonging to the wild-type Enterobacteriaceae as control, Escherichia coli MG1655 strain (ATCC No. 47076), W3110 strain (ATCC No. 27325), Pantoea Ananatis AJ13335 strain (FERM BP-6615) Etc. The activity of taking iron into cells can be measured by measuring the uptake of labeled Fe ³⁺ . (J. Bacteriol, May. 2003 p1870-1885)

  Enhancement of iron transporter activity can be achieved by modification to enhance the expression of genes involved in the tonB system. Confirmation that the expression of a gene involved in the tonB system is improved as compared with the parent strain, for example, a wild strain or an unmodified strain, can be confirmed by comparing the amount of mRNA with that of a wild type or an unmodified strain. Examples of the method for confirming the expression level include Northern hybridization and RT-PCR (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The expression level may be any as long as it is increased compared to the wild strain or the unmodified strain, but for example, 1.5 times or more, more preferably 2 times or more compared to the wild strain or the non-modified strain, It is desirable that it rises 3 times or more. The enhancement of the expression level of the gene involved in the tonB system can also be confirmed by the increase in the amount of the target protein compared to the non-modified strain and the wild strain. It can be detected by blotting. (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)).

  Examples of genes involved in the tonB system include tonB gene, fepA gene, fecA gene, and homologs thereof. The tonB system is a system that takes up iron through the membrane protein TonB. TonB controls the activities of these transporters by transferring electrons to FepA and FecA, which are iron transporters. (J. Bacteriol Oct 2001 vol.183, No. 20, p5885-5895) Here, as the Escherichia coli gene, for example, the tonB gene of SEQ ID NO: 1 (base number 1309113..1309832 of GenBank Accession No. NC_000913) ), The fepA gene of SEQ ID NO: 3 (complementary strand of base number 609477..611717 of GenBank Accession No. NC_000913), and the fecA gene of SEQ ID NO: 9 (complementary strand of GenBank Accession No. NC_000913.2: 4512376..4514700) Can be mentioned.

  Furthermore, homologues of the above genes are based on homology with the genes exemplified above based on γ-proteobacteria such as Escherichia, Enterobacter, Klebsiella, Serratia, Erbinia, Yersinia, etc., Corynebacterium It may be cloned from coryneform bacteria such as glutamicum, Brevibacterium lactofermentum, Pseudomonas bacteria such as Pseudomonas aeruginosa, Mycobacterium bacteria such as Mycobacterium tuberculosis, etc. In the case of the tonB gene, SEQ ID NOs: 5 and 6, and in the case of the fepA gene, those that can be amplified using the synthetic oligonucleotides shown in SEQ ID NOs: 7 and 8 may be used.

The homology between the amino acid sequence and the base sequence is, for example, the algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed (see http://www.ncbi.nlm.nih.gov).

  A homologue of a gene involved in the tonB system is a mutant gene derived from other microorganisms or a natural or artificial variant, and has a similar structure to a gene selected from the group consisting of the Escherichia coli tonB gene, fepA gene, and fecA gene. It means a gene having a function of improving iron transporter activity when introduced into a host or amplified. Homologs of genes involved in the tonB system (tonB gene, fepA gene, fecA gene) are 80% or more, preferably 90% or more, more preferably, relative to the entire amino acid sequence of SEQ ID NO: 2, 4, or 10, respectively. It means that which encodes a protein having a homology of 95%, particularly preferably 98% or more, and having a function as an iron transporter activity regulator (tonB) or an iron transporter (fepA, fecA). The function as an iron transporter activity regulator or an iron transporter can be confirmed by expressing these genes in a host cell and examining the transport of iron through the membrane (for example, the above-mentioned Non-patent documents 1 to 4). In addition, if it is a homolog, it can complement the function of the wild-type gene when a corresponding wild-type gene-disrupted strain is prepared and introduced into the disrupted strain, for example, whether iron uptake reduced by gene disruption can be recovered. It can be confirmed by examining whether.

In addition, the tonB gene, fepA gene, and fecA gene used in the present invention are not limited to wild type genes, and the functions of the encoded TonB protein, FepA protein, and FecA protein, that is, iron transporter activity regulator (TonB) or iron As long as the function as a transporter (FepA, FecA) is not impaired, one or several amino acid substitutions, deletions, insertions at one or more positions in the amino acid sequence of SEQ ID NO: 2, 4, or 10 or It may be a mutant or artificial modification that encodes a protein having a sequence including an addition or the like. Here, “1 or several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically 1 to 20, preferably 1 to 10, more preferably 1 to 10. Mean 5. The above-mentioned substitution is preferably conservative substitution, and conservative mutation refers to Phe, Trp, Tyr when the substitution site is an aromatic amino acid, and Leu, Ile when the substitution site is a hydrophobic amino acid. In the case of a polar amino acid between Val, between Gln and Asn, in the case of a basic amino acid, between Lys, Arg and His, and in the case of an acidic amino acid, between Asp and Glu, In the case of an amino acid having a hydroxyl group, it is a mutation that substitutes between Ser and Thr. Conservative substitutions include Ala to Ser or Thr substitution, Arg to Gln, His or Lys substitution, Asn to Glu, Gln, Lys, His or Asp substitution, Asp to Asn, Glu or Gln. Substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln, Lys or Asp substitution, Gly to Pro substitution, Substitution from His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe, Lys to Asn, Glu, Gln, His Or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala, Thr to Ser or Ala Substitution, 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 substitution, deletion, insertion, addition, or inversion as described above is naturally caused by individual differences in microorganisms that retain the tonB gene, fepA gene, and fecA gene, and cases based on differences in species. Also included are those caused by the resulting mutation.

  The tonB gene, fepA gene, and fecA gene are DNAs that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, 3, or 9, respectively, or a probe that can be prepared from the nucleotide sequence. Alternatively, it may be DNA encoding a protein having a function as an iron transporter activity regulator (tonB) or iron transporter (fepA, fecA). Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify this condition, for example, DNAs with high homology, for example, 80%, preferably 90% or more, more preferably 95%, particularly preferably 98% or more are present. DNAs having homology hybridize to each other, DNAs having lower homology to each other do not hybridize, or normal Southern hybridization washing conditions at 60 ° C., 1 × SSC, 0.1% SDS, Preferably, 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., salt concentration and temperature corresponding to 0.1 × SSC, 0.1% SDS, more preferably 1 to 3 times. The conditions to wash | clean are mentioned.

  The modification for enhancing the expression of the tonB gene, the fepA gene, and the fecA gene can be performed, for example, by increasing the copy number of these genes in a cell using a gene recombination technique. For example, a DNA fragment containing these genes may be ligated with a vector that functions in a host microorganism, preferably a multicopy vector, to produce a recombinant DNA, which is introduced into the microorganism and transformed.

When using the Escherichia coli tonB gene and fepA gene as the tonB gene and fepA gene, primers prepared based on the nucleotide sequences of SEQ ID NOs: 1 and 3, respectively, for example, primers shown in SEQ ID NOs: 5, 6 or 7, 8 The tonB and fepA genes were obtained by PCR using the chromosomal DNA of Escherichia coli as a template (PCR: polymerase chain reaction; see White, TJ et al., Trends Genet. 5, 185 (1989)). can do. The other tonB and fepA genes of other microorganisms are primers based on oligonucleotides prepared based on the sequence information of tonB, fepA, or tonB, fepA or TonB and FepA proteins of other microorganisms, respectively. Can be obtained from the chromosomal DNA or chromosomal DNA library of the microorganism by the PCR method described above or the hybridization method using the oligonucleotide prepared based on the sequence information as a probe. Chromosomal DNA can be obtained from microorganisms that are DNA donors, for example, the method of Saito and Miura (H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Biotechnology Experiments, Japan. Biomedical Society edition, pages 97-98, Bafukan, 1992).
The fecA gene can be obtained in the same manner.

  Next, the tonB gene, fepA gene, and fecA gene amplified by the PCR method are connected to a vector DNA that can function in the cells of the host microorganism to prepare recombinant DNA. Examples of the vector capable of functioning in the host microorganism cell include a vector capable of autonomous replication in the host microorganism cell.

  Examples of vectors that can autonomously replicate in Escherichia coli cells include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184 (pHSG and pACYC are available from Takara Bio Inc.), RSF1010 (Gene vol.75 (2), p271 -288, 1989), pBR322, pMW219, pMW119 (pMW is available from Nippon Gene), pSTV28, pSTV29 (Takara Bio). In addition, phage DNA vectors can also be used.

In order to prepare recombinant DNA by ligating these genes to the above vector, the vector is cleaved with a restriction enzyme that matches the ends of DNA fragments containing the tonB gene, fepA gene, and fecA gene. Ligation is usually performed using a ligase such as T4 DNA ligase. For methods such as DNA cleavage, ligation, chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation, setting of oligonucleotides used as primers, etc., ordinary methods well known to those skilled in the art should be adopted. Can do. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition",
Cold Spring Harbor Laboratory Press, (1989).

In order to introduce the recombinant DNA prepared as described above into a microorganism, it may be carried out according to the transformation methods reported so far. For example, the electroporation method (Canadian Journal of Microbiology, 43. 197 (1997)) can be mentioned. In addition, as reported for Escherichia coli K-12, a method for increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol. , 53, 159 (1970)), and a method for preparing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)) can also be used.

  On the other hand, increasing the copy number of tonB gene, fepA gene and fecA gene can also be achieved by making these genes exist in multiple copies on the chromosomal DNA of the microorganism. In order to introduce these genes in multiple copies onto the chromosomal DNA of a microorganism, homologous recombination is performed using a sequence present in multiple copies on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is also possible to mount a tonB gene, a fepA gene, and a fecA gene on a transposon, transfer them, and introduce multiple copies onto the chromosomal DNA. Confirmation that these genes have been transferred onto the chromosome can be confirmed by Southern hybridization using a part of these genes as a probe.

Furthermore, the enhancement of the expression of tonB gene, fepA gene, and fecA gene is not limited to the amplification of the gene copy number described above, but as described in International Publication No. 00/18935 pamphlet. Replacing expression control sequences such as gene promoters with strong ones, amplifying regulators that increase the expression of these genes, and deleting or weakening regulators that reduce the expression of these genes Is also achieved. For example, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter and the like are known as strong promoters (WO 98/004715 pamphlet).
Moreover, it is possible to introduce a nucleotide substitution into the promoter region of tonB gene, fepA gene, and fecA gene to modify it to be more powerful (EP1033407 specification). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128). Furthermore, it is known that substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon, has a great influence on the translation efficiency of mRNA, These can be modified. These promoter substitutions or modifications enhance the expression of tonB gene, fepA gene, and fecA gene.

  Furthermore, in order to enhance the activity of the protein encoded by the tonB gene, fepA gene, and fecA gene, a mutation that increases the activity of the iron transport system may be introduced into these genes. Mutations that increase the activity of the iron transport system include mutations in the promoter sequence that increase the transcription amount of tonB, fepA, and fecA genes, and high specific activities of TonB, FepA, and FecA proteins. Mutations in the coding region of these genes. Moreover, you may introduce | transduce the mutation which raises the activity which controls the expression of these genes positively.

<2> Method for Producing L-Amino Acid In the method for producing L-amino acid of the present invention, the microorganism of the present invention is cultured in a medium, and L-amino acid is produced and accumulated in the medium or in the microbial cells. A method for producing L-amino acid from a body.

  As the medium to be used, a medium conventionally used in the fermentation production of L-amino acids using microorganisms can be used. That is, a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required can be used. Here, as the carbon source, saccharides such as glucose, sucrose, lactose, galactose, fructose and starch hydrolysate, alcohols such as glycerol and sorbitol, organic acids such as fumaric acid, citric acid and succinic acid are used. be able to. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used. As an organic trace nutrient source, it is desirable to contain an appropriate amount of a required substance such as vitamin B1, L-homoserine or a yeast extract. In addition to these, a small amount of potassium phosphate, magnesium sulfate, iron ion, manganese ion or the like is added as necessary. The medium used in the present invention may be a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic ions, and other organic trace components as required.

  The culture is preferably carried out under aerobic conditions for 1 to 7 days, the culture temperature is 24 ° C. to 37 ° C., and the pH during the culture is preferably 5 to 9. In addition, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment. The L-amino acid can be recovered from the fermentation broth by combining an ion exchange resin method, a precipitation method and other known methods. In addition, when L-amino acid accumulates in the microbial cell, for example, the microbial cell is crushed by ultrasonic waves, and the microbial cell is removed by centrifugation. Can be recovered.

In addition, when producing a basic amino acid, the pH during culture is controlled to 6.5 to 9.0, and the pH of the medium at the end of the culture is 7.2 to 9.0. The fermenter pressure is controlled to be positive, or carbon dioxide gas or a mixed gas containing carbon dioxide gas is supplied to the culture medium, and at least 2 g / L or more of bicarbonate ions and / or carbonate ions are present in the culture medium In this method, fermentation is performed by using a method in which bicarbonate ions and / or carbonate ions are used as counter ions of a cation mainly composed of basic amino acids, and the target basic amino acid is recovered. Also good. (See JP 2002-065287, International Publication 2006/038695 pamphlet)

[Example]
Hereinafter, the present invention will be described more specifically with reference to examples. Unless otherwise specified, Wako Pure Chemicals or those manufactured by Nacalai Tesque were used as reagents. The composition of the medium used in each example is as shown below. The pH of each medium was adjusted with NaOH or HCl.

(L medium)
Bacto Trypton (Difco) 10g / L
Yeast extract (Difco) 5g / L
Sodium chloride 10g / L
pH7.0
Steam sterilization was performed at 120 ° C. for 20 minutes.

[L agar medium]
L Medium Bactoagar 15g / L
Steam sterilization was performed at 120 ° C. for 20 minutes.

[Escherichia bacterium L-lysine production medium]
Glucose 40g / L
Ammonium sulfate 24g / L
Potassium dihydrogen phosphate 1.0g / L
Magnesium sulfate heptahydrate 1.0g / L
Iron sulfate heptahydrate 0.01g / L
Manganese sulfate heptahydrate 0.01g / L
Yeast extract 2.0g / L
Pharmacopeia calcium carbonate 50g / L (disinfection)
The pH was adjusted to 7.0 with potassium hydroxide, and the above sterilization was performed at 115 ° C. for 10 minutes.
However, glucose and MgSO ₄ · 7H ₂ O were sterilized separately.
Pharmacopoeia calcium carbonate was heat-sterilized at 180 ° C.
As antibiotics, chloramphenicol 25 mg / L or ampicillin 100 mg / L was added.

[Escherichia bacterium L-Thr production medium]
Glucose 40g / L
Ammonium sulfate 16g / L
Potassium dihydrogen phosphate 1.0g / L
Magnesium sulfate heptahydrate 1.0g / L
Iron sulfate 4/7 hydrate 0.01g / L
Manganese sulfate 4.7 salt / 0.01g / L
Pharmacopeia calcium carbonate 30g / L (disinfection)
The pH was adjusted to 7.5 with potassium hydroxide, and the above sterilization was performed at 115 ° C. for 10 minutes.
However, glucose and MgSO ₄ · 7H ₂ O were sterilized separately.
Calcium carbonate was sterilized by dry heat at 180 ° C.
As antibiotics, streptomycin 100 mg / L and ampicillin 100 mg / L were added.

<1> Construction of plasmids for amplifying tonB and fepA genes
In order to confirm the amplification effect that contributes to L-lysine production of tonB gene and fepA gene alone, a vector for amplifying each gene alone was constructed. The entire nucleotide sequence of the chromosome of Escherichia coli (Escherichia coli K-12 strain) has already been clarified (Science, 277, 1453-1474 (1997)), and the tonB gene (GenBank Accession) No. NC_000913 base numbers 1309113 to 1309832: SEQ ID NO: 2), a primer for amplifying each gene based on the base sequence of the fepA gene (complementary strand of base numbers 609477 to 611717 of NCBI Accession No. NC_000913: SEQ ID NO: 4) Designed. Primers for amplifying each gene are shown in SEQ ID NOs: 5 to 8 (tonB: SEQ ID NOs: 5, 6; fepA: SEQ ID NOs: 7, 8). Using these primers, PCR was performed using the chromosomal DNA of Escherichia coli MG1655 strain as a template. Chromosomal DNA was obtained using Bacterial Genomic DNA purifiation kit (Edge Bio Systems). For the PCR, a probest DNA polymerase (manufactured by Takara Bio Inc.) was used, and 25 cycles of 96 ° C. for 20 seconds, 65 ° C. for 20 seconds, and 72 ° C. for 2 minutes were performed.
The purified tonB gene and fepA gene were respectively ligated to vectors pSTV28 (Takara Bio) and pMW119 (Nippon Gene) digested with SmaI to construct tonB amplification plasmid pStonB and fepA amplification plasmid pSfepA.

<2> Effect of tonB and fepA amplification in Escherichia bacterium L-lysine producing strain <2-1> Construction of cadA, ldcC gene-disrupted strain encoding lysine decarboxylase A lysine decarboxylase non-producing strain was constructed. The lysine decarboxylase is encoded by the cadA gene (Genbank Accession No. NP_418555. SEQ ID NO: 17) and the ldcC gene (Genbank Accession No. NP_414728. SEQ ID NO: 11) (see International Publication WO96 / 17930 pamphlet). Here, WC196 strain was used as the parent strain.

  Deletion of cadA and ldcC genes encoding lysine decarboxylase was performed by a method called “Red-driven integration” originally developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645) and λ 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.). 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. Furthermore, by combining the excision system derived from λ phage, the antibiotic resistance gene incorporated in the gene disruption strain can be removed (Japanese Patent Laid-Open No. 2005-058227).

(1) Disruption of cadA gene
As a PCR template, plasmid pMW118-attL-Cm-attR (Japanese Patent Laid-Open No. 2005-058827) was used. pMW118-attL-Cm-attR is a plasmid in which attL and attR genes, which are attachment sites of λ phage, and a cat gene, which is an antibiotic resistance gene, are inserted into pMW118 (Takara Bio Inc.). Inserted in order

  Synthetic oligonucleotides shown in SEQ ID NOS: 13 and 14 having the sequences corresponding to both ends of attL and attR at the 3 ′ end of the primer and the 5 ′ end of the primer corresponding to a part of the target gene cadA gene are used as primers. PCR was performed.

The amplified PCR product was purified on an agarose gel and introduced by electroporation into Escherichia coli WC196 strain containing plasmid pKD46 having a temperature-sensitive replication ability. Plasmid pKD46 (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645)
Is a DNA fragment (GenBank / EMBL accession number J02459) with a total of 2154 bases of λ phage, including the gene encoding the red recombinase (γ, β, exo gene) of the λRed homologous recombination system controlled by the arabinose-inducible ParaB promoter. , 31088-33241). Plasmid pKD46 is required for integrating the PCR product into the chromosome of WC196 strain.

  A competent cell for electroporation was prepared as follows. That is, Escherichia coli WC196 strain cultured overnight at 30 ° C. in LB medium containing 100 mg / L ampicillin, 5 mL SOB medium (molecular molecular) containing ampicillin (20 mg / L) and L-arabinose (1 mM). Cloning: Dilute 100-fold with Laboratory Manual Second Edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)). The resulting dilution was allowed to grow by aeration at 30 ° C. until the OD600 was about 0.6, then concentrated 100 times, and washed three times with 10% glycerol so that it could be used for electroporation. did. Electroporation was performed using 70 μL of competent cells and approximately 100 ng of PCR product. The cell after electroporation was added with 1 mL of SOC medium (Molecular Cloning: Laboratory Manual Second Edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) and cultured at 37 ° C. for 2.5 hours. Then, it was plated on an L-agar medium containing Cm (chloramphenicol) (25 mg / L) at 37 ° C., and a Cm resistant recombinant was selected. Next, in order to remove the pKD46 plasmid, it was subcultured twice at 42 ° C. on an L-agar medium containing Cm, and the resulting colony was tested for ampicillin resistance. I got it.

  The deletion of the mutant cadA gene that could be identified by the chloramphenicol resistance gene was confirmed by PCR. The obtained cadA-deficient strain was named WC196ΔcadA :: att-cat strain.

  Next, in order to remove the att-cat gene introduced into the cadA gene, the above-described helper plasmid pMW-intxis-ts (Japanese Patent Laid-Open No. 2005-058827) was used. 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.

The WC196ΔcadA :: att-cat strain competent cell obtained above was prepared according to a conventional method, transformed with the helper plasmid pMW-intxis-ts, and L-agar containing 50 mg / L ampicillin at 30 ° C. Plated on the medium and ampicillin resistant strains were selected.
Next, in order to remove the pMW-intxis-ts plasmid, the cells were passaged twice at 42 ° C. on L-agar medium, and the resulting colonies were tested for ampicillin resistance and chloramphenicol resistance. Chloramphenicol and ampicillin sensitive strains, which are cadA-disrupted strains from which cat and pMW-intxis-ts have been removed, were obtained. This strain was named WC196ΔcadA.

(2) Deletion of ldcC gene of WC196ΔcadA strain
Deletion of the ldcC gene in the WC196ΔcadA strain was performed using the primers of SEQ ID NOS: 15 and 16 as the ldcC disruption primer in accordance with the above method. Thus, WC196ΔcadAΔldcC, which is a cadA ldcC-disrupted strain, was obtained.

<2> Introduction of plasmid for lys production of WC196ΔcadAΔldcC strain
The WC196ΔcadAΔldcC strain was transformed with the Lys production plasmid pCABD2 (International Publication No. WO01 / 53459 pamphlet) carrying the dapA, dapB and LysC genes according to a conventional method to obtain a WC196ΔcadAΔldcC / pCABD2 strain (WC196LC / pCABD2).

<2-2> Effect of tonB amplification in Escherichia bacterium L-lysine producing strain
The WC196LC / pCABD2 strain was transformed with the tonB amplification plasmid pStonB used in Example 1 and the comparative control plasmid pSTV28 (manufactured by Takara Bio Inc.) to obtain a chloramphenicol resistant strain. After confirming that the predetermined plasmid had been introduced, the pStonB introduced strain was named WC196LC / pCABD2 / pStonB strain, and the pSTV28 introduced strain was named WC196LC / pCABD2 / pSTV28 strain.
Cultivate the WC196LC / pCABD2 / pStonB and WC196LC / pCABD2 / pSTV28 strains in L medium containing 50 mg / L chloramphenicol at 37 ° C. so that the final OD600≈0.6, and then the same volume as the culture solution A 40% glycerol solution was added and stirred, and then dispensed in appropriate amounts to obtain a glycerol stock, which was stored at -80 ° C. This is called glycerol stock.
The glycerol stocks of these strains were thawed, and 100 μL of each was spread evenly on an L plate containing 25 mg / L chloramphenicol and 20 mg / L streptomycin, and cultured at 37 ° C. for 24 hours. The cells growing on the plate are suspended in 2 to 3 mL of fermentation medium, and 1 mL of OD620≈13.5 OD is added to a fermentation medium containing 25 mg / L chloramphenicol and 20 mg / L streptomycin (Escherichia spp.) In a 500 mL Sakaguchi flask. Bacterial L-lysine production medium) was inoculated into 20 mL and cultured at 37 ° C. for 48 hours on a reciprocating shaker. After culture, the amount of L-lysine accumulated in the medium was measured using Biotech Analyzer AS210 (Sakura Seiki).
Table 1 shows the L-lysine accumulation and yield at 24 hours. As can be seen from Table 1, the WC196LC / pCABD2 / pStonB strain has higher L-lysine accumulation at 24 hours and improved L-lysine productivity than the WC196LC / pCABD2 / pSTV28 strain into which no gene is introduced. There was found.

<2-3> Effect of fepA amplification on Escherichia bacterium L-lysine producing strain The WC196LC / pCABD2 strain was transformed with the fepA amplification plasmid pSfepA used in Example 1 to obtain an ampicillin resistant strain. It was confirmed that a predetermined plasmid pSfepA for fepA amplification was introduced, and the pSfepA-introduced strain was named WC196LC / pCABD2 / pSfepA strain.
In the same manner as in <2-2>, glycerol stocks of the WC196LC / pCABD2 / pSfepA strain and the comparative control WC196LC / pCABD2 strain were prepared. However, 100 mg / L ampicillin was used instead of 50 mg / L chloramphenicol.
The glycerol stocks of these strains were thawed and each 100 μL was evenly spread on an L plate containing 100 mg / L ampicillin and 20 mg / L streptomycin and cultured at 37 ° C. for 24 hours. The cells grown on the plate were inoculated into the fermentation medium in the same manner as in <2-2>, and cultured for 48 hours, and the amount of L-lysine accumulated in the medium was measured using Biotech Analyzer AS210 (Sakura Seiki). . However, 100 mg / L ampicillin was used instead of 50 mg / L chloramphenicol.
Table 2 shows L-lysine accumulation and yield at 24 hours. As can be seen from Table 2, it was found that L-lysine accumulation was higher at 24 hours and L-lysine productivity was improved compared to the WC196LC / pCABD2 / pSfepA strain and the WC196LC / pCABD2 strain without gene transfer. .

Effect of fepA amplification on L-threonine-producing bacterium belonging to the genus Escherichia B-5318 strain was used as an L-threonine-producing bacterium. B-5318 shares were registered with the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika on May 3, 1990 under the registration number VKPM B-5318. It has been deposited. Construction of a fepA amplified strain from an L-threonine-producing bacterium was performed using the plasmid described in Example 1.

The B5318 strain was transformed with the fepA amplification plasmid pSfepA used in Example 1 to obtain an ampicillin resistant strain. After confirming that the predetermined plasmid was introduced, the pSfepA-introduced strain was named B5318 / pSfepA strain.
100 strains of B5318 / pSfepA and B5318 / pMW strain introduced with a control plasmid for comparison
After culturing at 37 ° C. so that the final OD600≈0.6 in L medium containing mg / L ampicillin and 100 mg / L streptomycin, after adding 40% glycerol solution equal to the culture solution and stirring, Appropriate amounts were dispensed into glycerol stocks and stored at -80 ° C. This is called glycerol stock.
Glycerol stocks of these strains were thawed, 100 μl of each was evenly spread on L plates containing 100 mg / L ampicillin and 100 mg / L streptomycin, and cultured at 37 ° C. for 24 hours. Suspend the cells growing on the plate in 6 mL of physiological saline, and add 0.5 mL of OD620 = 3.0 bacterial solution to a 500 mL Sakaguchi flask fermentation medium containing 100 mg / L ampicillin and 100 mg / L streptomycin (Escherichia L -Threonine production medium) was inoculated into 20 mL and cultured at 37 ° C. for 24 hours in a reciprocating shake culture apparatus. After culture, the amount of L-threonine accumulated in the medium was measured using high performance liquid chromatography.
Table 3 shows the L-threonine accumulation at 24 hours. As can be seen from Table 3, the B5318 / pSfepA strain was found to have higher L-threonine accumulation and improved L-threonine productivity than the B5318 / pMW strain.

Claims (12)

A microorganism belonging to the family Enterobacteriaceae having an L-amino acid-producing ability and modified so as to enhance the activity of an iron transporter, comprising a tonB gene, a fepA gene, and a fecA gene involved in the tonB system A microorganism modified so as to increase the expression level of one or more genes selected from the group. The iron transporter activity is enhanced by increasing the copy number of one or more genes selected from the group consisting of the tonB gene, the fepA gene, and the fecA gene, or by modifying the expression regulatory sequence of the gene. Item 2. The microorganism according to Item 1. The tonB gene has a protein having the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in SEQ ID NO: 2, and controls iron transporter activity The microorganism according to claim 1 or 2, which is a gene encoding a protein having a function. A protein in which the fepA gene has the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in SEQ ID NO: 4 and has an iron transporter activity The microorganism according to claim 1 or 2, which is a gene coding for. A protein in which the fecA gene has the amino acid sequence shown in SEQ ID NO: 10, or a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in SEQ ID NO: 10 and having iron transporter activity The microorganism according to claim 1 or 2, which is a gene coding for. The microorganism according to claim 1 or 2, wherein the tonB gene is the DNA described in (a) or (b) below:
(A) DNA comprising the base sequence shown in SEQ ID NO: 1,
(B) a protein having a function of hybridizing with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or a probe that can be prepared from the base sequence under stringent conditions and having a function of controlling iron transporter activity DNA to encode. The microorganism according to claim 1 or 2, wherein the fepA gene is the DNA described in (c) or (d) below:
(C) DNA comprising the base sequence shown in SEQ ID NO: 3,
(D) A DNA that hybridizes under stringent conditions with a probe complementary to the nucleotide sequence shown in SEQ ID NO: 3 or that can be prepared from the nucleotide sequence and encodes a protein having iron transporter activity. The microorganism according to claim 1 or 2, wherein the fecA gene is the DNA described in (e) or (f) below:
(E) a DNA comprising the base sequence shown in SEQ ID NO: 9,
(F) A DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 9 or a probe that can be prepared from the base sequence, and that encodes a protein having iron transporter activity. The L-amino acid is L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L The microorganism according to any one of claims 1 to 8, which is one or more L-amino acids selected from -glutamic acid. The microorganism according to any one of claims 1 to 9, wherein the microorganism belonging to the family Enterobacteriaceae is a microorganism selected from Escherichia bacteria, Pantoea bacteria, and Enterobacter bacteria. Culturing the microorganism according to any one of claims 1 to 10 in a medium, producing and accumulating L-amino acid in the medium or in the fungus body, and recovering the L-amino acid from the medium or the fungus body. A process for producing a characteristic L-amino acid. The L-amino acid is L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L The production method according to claim 11, which is one or more L-amino acids selected from -glutamic acid.