JP2007117076A - L-amino acid producing bacteria and method for producing l-amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide bacterial strains and a method for efficiently producing L-amino acids using microorganism by fermentation. <P>SOLUTION: The method for efficiently producing the L-amino acids, especially L-lysine, L-threonine and L-glutamic acid is to culture the microorganism belonging to Enterobacteriaceae modified to enhance activity by increasing copy number of the gene of the β-glucoside PTS, especially bacteria belonging to Escherichia, Pantoea, in a medium, producing and accumulating the L-amino acids in the medium or the bacterial cells and harvesting the L-amino acids from the medium or the bacterial cells. <P>COPYRIGHT: (C)2007,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 L-amino acids that are industrially useful 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 1-4 can be mentioned, for example. On the other hand, examples of the method for producing L-arginine include the method described in Patent Document 5, 6 or 14. In these production methods, a strain isolated from the natural world or an artificial mutant of the strain, and a microorganism modified so that the activity of a basic L-amino acid biosynthetic enzyme is enhanced 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. For example, with respect to the uptake system, there is a method of enhancing L-amino acid-producing ability by deleting or reducing the uptake system of L-amino acids into cells. For example, a method of deleting the gluABCD operon or a part thereof and deleting or reducing the uptake of L-glutamic acid (for example, see Patent Document 7) is known.

Further, as a method for modifying the excretion system, a method of deleting or weakening an excretion system of an intermediate or substrate of an L-amino acid biosynthesis system, and a method of strengthening an excretion system of L-amino acid are known. As the former method, when the target amino acid is L-glutamic acid, the α-ketoglutarate permease gene is mutated or destroyed to attenuate the discharge of α-ketoglutarate, which is an intermediate of the target substance (for example, Patent Document 8) is known.

As a method for strengthening the latter L-amino acid excretion system, for example, a strain of Corynebacterium microorganism having enhanced expression of L-lysine and L-arginine excretion gene (LysE) (for example, see Non-patent Document 1) is used. A method for producing L-lysine used (for example, see Patent Document 9) or a method for producing L-arginine (for example, see Patent Document 10) is known. Moreover, the expression of rhtA, B, C gene (for example, refer to Patent Document 11) or yfiK, yahN gene and the like (for example, refer to Patent Document 12), which are genes suggested to be involved in L-amino acid excretion. A method for producing an L-amino acid using Escherichia bacterium with enhanced N is also reported.

In addition to the modification of the L-amino acid biosynthetic pathway and the incorporation of the L-amino acid and the modification of the excretion system shown above, as one of the methods for improving the L-amino acid production ability, It may be modified. For example, a phosphoenolpyruvate: sugar phosphate transfer system (hereinafter also referred to as PTS: phosphotransferase) is widely known as a transporter that takes in sugar, and PTS is a common system EI (ptsI) that does not depend on a substrate. ), HPr (encoded by ptsH) and substrate-specific component EII. It is known that glucose-specific EII is encoded by ptsG and crr, and that the crr gene has an operon structure with ptsH and ptsI. An amino acid production method using an Escherichia bacterium with enhanced ptsG gene (Patent Document 13) and an amino acid production method using an Escherichia bacterium with enhanced ptsH, ptsI, and crr genes are already known (Patent Document 14). .

In addition to the glucose PTS shown above, bglF is known as a gene encoding a β-glucoside-specific phosphotransferase (PTS), but to date, a gene encoding a PTS other than glucose PTS is known. There are no reports of enhancement and use for amino acid production. (Non-patent documents 2 to 4)

European Patent Application No. 0857784 Japanese Patent Application Laid-Open No. 11-192088 International Publication No. 00/53726 Pamphlet International Publication No. WO96 / 17930 Pamphlet European Patent Application Publication No. 0999267 European Patent Application Publication No. 1170358 European Patent Application No. 1038970 International Publication No. 01/005959 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 No. 03/04670 Specification International Publication No. 03/04674 Specification Journal of Molecular Microbiology Biotechnology 1999 Nov; 1 (2): 327-36 Journal of Bacteriol 1999 p462-468 Vol.181, No.2 Biochemistry 1998,37,17040-17047 Biochemistry 1998,37,8714-8723

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.

As a result of intensive studies in order to solve the above problems, the present inventors have efficiently used L-amino acids by using microorganisms belonging to the family Enterobacteriaceae modified so as to enhance β-glucoside PTS activity. It has been found that it can be manufactured, and the present invention has been completed.

That is, the present invention is as follows.
(1) A microorganism belonging to the family Enterobacteriaceae having an ability to produce L-amino acid and modified so as to enhance β-glucoside PTS activity is cultured in a medium, and the L-amino acid is cultured in the medium or A method for producing an L-amino acid, which is produced and accumulated in the body, and L-amino acid is collected from the culture medium or cells.
(2) Enterobacteriaceae wherein the expression of the gene encoding β-glucoside PTS is enhanced by increasing the copy number of the gene encoding β-glucoside PTS or by modifying the expression regulatory sequence of the gene The manufacturing method as described in (1) using the microorganisms which belong to.
(3) The production method according to (1) or (2), wherein the gene encoding β-glucoside PTS is the DNA described in (a) or (b) below:
(A) DNA comprising the base sequence shown in SEQ ID NO: 5,
(B) a DNA that hybridizes under stringent conditions with the base sequence shown in SEQ ID NO: 5 or a probe that can be prepared from the base sequence, and that encodes a protein having β-glucoside PTS activity. (4) The production method according to any one of (1) to (3), wherein the microorganism is an Escherichia bacterium or a Pantoea bacterium.
(5) The L-amino acid according to any one of (1) to (4), wherein the L-amino acid is one or more amino acids selected from the group consisting of L-lysine, L-threonine, and L-glutamic acid. Manufacturing method.

  By using the microorganism of the present invention, L-amino acids, particularly L-lysine, L-threonine and L-glutamic acid can be efficiently produced by fermentation.

  Hereinafter, the present invention will be described in detail.

<1> Microorganism of the Present Invention The microorganism of the present invention is obtained by culturing a microorganism belonging to the family Enterobacteriaceae, which has L-amino acid-producing ability and has been modified to enhance β-glucoside PTS activity, in a medium. , L-amino acid is produced and accumulated in the medium or cells, and the L-amino acid is collected from the medium or cells. 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 L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline basic amino acid, L-isoleucine, L-alanine, L-valine, L-leucine L-glycine aliphatic amino acid, L-threonine, L-serine hydroxymonoaminocarboxylic acid amino acid, L-proline cyclic amino acid, L-phenylalanine, L-tyrosine, L-tryptophan aromatic amino acid, Examples thereof include 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> Grant of L-amino acid-producing ability Examples of methods for imparting L-amino acid-producing ability and microorganisms imparted with L-amino acid-producing ability that can be used in the present invention are given below. However, as long as it has L-amino acid producing ability, it is not limited thereto.

  The microorganism used in the present invention is a microorganism belonging to the family Enterobacteriaceae, such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erbinia, Salmonella, Morganella, etc., and produces L-amino acids If it has the capability to do, it will not specifically limit. Specifically, those belonging to the family Enterobacteriaceae according to the classification described in the NCBI (National Center for Biotechnology Information) database can be used. (Http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Tree&id=1236&lvl=3&keep=1&srchmode=1&unlock). As parent strains of the family Enterobacteriaceae used for modification, it is desirable to use Escherichia bacteria, Enterobacter bacteria, Pantoea bacteria, among others.

  The parent strain of the genus Escherichia used for obtaining the genus Escherichia of the present invention is not particularly limited, but specifically, it is a book by Neidhardt et al. (Neidhardt, FC et al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology , Washington DC, 1029 table 1) can be used. Among them, for example, Escherichia coli is mentioned. Specific examples of Escherichia coli include Escherichia coli W3110 (ATCC 27325) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild type K12 strain.

  These can be obtained, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). . The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.

  Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and the like, and Pantoea ananatis is an example of the Pantoea bacterium. In recent years, Enterobacter agglomerans has been reclassified as Pantoea agglomerans, Pantoea ananatis, or Pantoea stewartii by 16S rRNA nucleotide sequence analysis. There is. 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 Can be used. These strains were identified as Enterobacter agglomerans at the time of isolation, and deposited as Enterobacter agglomerans, but as described above, they have been reclassified as Pantoea Ananatis by 16S rRNA sequencing etc. .

  Hereinafter, a method for imparting L-amino acid-producing ability to microorganisms belonging to the family Enterobacteriaceae 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 coryneform bacteria or bacteria belonging to the genus Escherichia can be applied (see Amino Acid Fermentation, Japan Society Publishing Center, May 30, 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.

  In order to obtain an auxotrophic mutant having L-amino acid-producing ability, an analog-resistant strain of L-amino acid, or a metabolically controlled mutant, the parent strain or the wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, In addition, among the mutants obtained by treatment with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine, the mutant exhibits auxotrophy, analog resistance, or metabolic control mutation, and L- It can be obtained by selecting one having amino acid-producing ability. An L-amino acid-producing bacterium can also be obtained by enhancing the enzyme activity of an L-amino acid biosynthetic enzyme by gene recombination.

  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, WC196 strain (see International Publication No. 96/17930 pamphlet) can also be used as an L-lysine producing bacterium of Escherichia coli. 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.

  Moreover, L-lysine producing bacteria can also be constructed by increasing the enzyme activity of the L-lysine biosynthesis system. Examples of genes encoding L-lysine biosynthesis enzymes include dihydrodipicolinate synthase gene (dapA), aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB), and diaminopimelate decarboxylase gene (lysA). , Diaminopimelate dehydrogenase gene (ddh) (international publication No. 96/40934 pamphlet), phosphoenolpyruvate carboxylase gene (ppc) (Japanese Patent Laid-Open No. 60-87788), aspartate aminotransferase gene (aspC) ( Japanese Patent Publication No.6-102028), diaminopimelate epimerase gene (dapF) (Japanese Patent Laid-Open No. 2003-135066), aspartate semialdehyde dehydrogenase gene (asd) (WO 00/61723 pamphlet), etc. Gene of enzyme of acid pathway or homoaconite hydratase gene ( It includes genes such as enzymes of aminoadipic acid pathway Open Patent Laid-open No. 2000-157276) and the like.

  For example, L-lysine-producing ability can be imparted by introducing a gene encoding an L-lysine biosynthetic enzyme into a host as follows. That is, a gene fragment encoding an L-lysine biosynthetic gene is ligated with a vector that functions in a host microorganism used for the production of L-lysine, preferably a multi-copy vector, to produce a recombinant DNA. Transform. Transformation increases the copy number of the gene encoding the L-lysine biosynthetic enzyme in the host cell and enhances the expression level. As a result, the activities of these enzymes are enhanced.

  The gene encoding the L-lysine biosynthetic enzyme is not particularly limited as long as it is a gene that can be expressed in the host microorganism, and examples thereof include genes derived from Escherichia coli and genes derived from coryneform bacteria. It is done. Since the entire genome sequence of both Escherichia coli and Corynebacterium glutamicum has been clarified, primers are synthesized based on the base sequences of these genes, and chromosomal DNA of microorganisms such as Escherichia coli K-12 is obtained. These genes can be obtained by PCR using a template.

  The plasmid used for gene cloning may be any plasmid that can autonomously replicate in the family Enterobacteriaceae. Specifically, pBR322, pTWV228 (Takara Bio), pMW119 (Nippon Gene), pUC19, pSTV29 ( Takara Bio Inc.), RSF1010 (Gene vol.75 (2), p271-288, 1989). In addition, phage DNA vectors can also be used.

  In order to prepare a recombinant DNA by ligating the target gene to the above vector, the vector is cleaved with a restriction enzyme that matches the end of the DNA fragment containing the target gene. Ligation is usually performed using a ligase such as T4 DNA ligase. The target genes may be loaded on separate vectors or on the same vector. 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), etc. Any method can be used for introducing the recombinant DNA prepared as described above into a microorganism as long as sufficient transformation efficiency can be obtained. For example, electroporation (Canadian Journal of Microbiology) , 43. 197 (1997)). As a plasmid prepared by such a method, a Lys production plasmid pCABD2 (International Publication No. WO01 / 53459 pamphlet) carrying dapA, dapB and LysC genes can be mentioned.

  In addition, enhancement of expression of a gene encoding an L-lysine biosynthetic enzyme can also be achieved by introducing multiple copies of the target gene onto the chromosomal DNA of the microorganism. In order to introduce the gene of interest in multiple copies on the chromosomal DNA of a microorganism, homologous recombination is performed using a sequence present in multiple copies on the chromosomal DNA as a target. Such site-directed mutagenesis by gene replacement using homologous recombination has already been established, and includes a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383). Or Japanese Patent Laid-Open No. 05-007491). 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. The L-lysine biosynthesis gene may be linked in tandem next to the gene originally present on the chromosome. Alternatively, as disclosed in JP-A-2-109985, a target gene can be mounted on a transposon, transferred, and introduced in multiple copies on chromosomal DNA. In any method, as a result of the increase in the copy number of the target gene in the transformed strain, the enzyme activity of the L-lysine biosynthesis system is enhanced.

  The activity enhancement of L-lysine biosynthetic enzymes can be achieved not only by the above gene amplification but also by replacing expression control sequences such as a promoter of the target gene with a strong one (Japanese Patent Laid-Open No. 1-215280). reference). 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. By substituting these promoters, the enzyme activity is amplified by enhancing expression of the target gene. 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).

  It can also be achieved by modifying factors involved in regulating the expression of the target gene, such as operators and repressors (Hamilton et al ,; J Bacteriol. 1989 Sep; 171 (9): 4617-22.). As disclosed in International Publication No. WO00 / 18935, it is possible to introduce a base substitution of several bases into the promoter region of the target gene and modify it to be more powerful. 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. The expression regulatory region such as the promoter of the target gene can be determined using a promoter search vector or gene analysis software such as GENETYX. The replacement of the expression regulatory sequence can be performed, for example, in the same manner as the gene replacement using the above-described temperature sensitive plasmid.

  Furthermore, the L-lysine-producing bacterium of the present invention functions negatively for the activity of an enzyme that catalyzes a reaction that produces a compound other than L-lysine by branching from the biosynthetic pathway of L-lysine, and L-lysine production. The enzyme activity may be reduced or deficient. Examples of such enzymes include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), and malic enzyme, and strains in which the activity of the enzyme is reduced or deleted are disclosed in WO 95/23864 and WO96 / 17930. , WO 2005/010175 pamphlet and the like.

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 or the entire region of the gene (Journal of biological Chemistry 272: 8611-8617 (1997). In addition, a gene encoding a mutant enzyme in which the coding region is deleted is constructed, The enzyme activity can also be reduced or eliminated by replacing a normal gene on the chromosome with the gene by homologous recombination or the like, or 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, transformed into a microorganism belonging to the family Enterobacteriaceae with the DNA containing the gene, and the mutant gene and chromosome By causing recombination with the above gene, the target gene on the chromosome can be replaced with a mutant form. Such gene replacement using homologous recombination is a method called “Red-driven integration” (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640- 6645) Red driven integration method and excision system derived from λ phage (J. Bacteriol. 2002 Sep; 184 (18): 5200-3. Interactions between integral and excisionase in the phage lambda excisive nucleoprotein complex. Cho EH, Gumport RI, Gardner JF.) (See WO2005 / 010175) and other methods using linear DNA and methods using temperature-sensitive replication origin plasmids (Proc. Natl. Acad. Sci. USA, 2000) , vol. 97, No. 12, p6640-6645, US Pat. No. 6,303,383, or 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 method for enhancing the enzyme activity involved in L-lysine biosynthesis and the method for reducing the enzyme activity can be similarly applied to breeding other L-amino acid-producing bacteria. Hereinafter, methods for breeding other L-amino acid-producing bacteria will be described.

  Examples of the L-glutamic acid-producing bacterium used in the present invention include microorganisms belonging to the family Enterobacteriaceae modified so that expression of a gene encoding an enzyme involved in L-glutamic acid biosynthesis is enhanced. Examples of enzymes involved in L-glutamate biosynthesis include glutamate dehydrogenase (hereinafter also referred to as “GDH”), glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconite hydratase, and citrate synthase (hereinafter referred to as “CS”). ), Phosphoenolpyruvate carboxylase (hereinafter also referred to as “PEPC”), pyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, glycerin Aldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, furtose bisphosphate aldolase, phosphofructokinase, glucose phosphate iso Hydrolase, etc., and the like. Among these enzymes, one or more of CS, PEPC and GDH are preferable, and all three are more preferable.

  As a microorganism belonging to the family Enterobacteriaceae modified so that expression of citrate synthase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene is enhanced by the method as described above, U.S. Patent No. 6,197,559, Examples thereof include microorganisms described in 6,331,419 specification and European Patent 0999282 specification.

  In addition, a microorganism belonging to the family Enterobacteriaceae, which has been modified so that the 6-phosphogluconate dehydratase activity, the 2-keto-3-deoxy-6-phosphogluconate aldolase activity, or both of these activities are enhanced, is used. May be. (European Patent Application Publication No. 1352966)

  In addition, microorganisms belonging to the family Enterobacteriaceae having L-glutamic acid-producing ability include microorganisms having a reduced or deficient activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce other compounds. May be used. Examples of the enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid include 2-oxoglutarate dehydrogenase, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxyacid synthase Acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase and the like. Among these, it is particularly preferable to reduce or eliminate 2-oxoglutarate dehydrogenase activity.

Methods for deleting or reducing 2-oxoglutarate dehydrogenase activity of microorganisms belonging to the family Enterobacteriaceae are described in US Pat. No. 5,573,945, US Pat. No. 6,197,559, and US Pat. No. 6,331,419. Specific examples of microorganisms belonging to the family Enterobacteriaceae in which 2-oxoglutarate dehydrogenase activity is deficient or reduced include:
Pantoea Ananatis AJ13601 (FERM BP-7207)
Klebsiella Planticola AJ13410 (FERM BP-6617)
Pantoea Ananatis AJ13355 (FERM BP-6614)
Escherichia coli AJ12949 (FERM BP-4881)
Etc.

  A preferable L-tryptophan-producing bacterium used in the present invention is a bacterium in which one or more of anthranilate synthase activity, phosphoglycerate dehydrogenase activity or tryptophan synthase activity are enhanced. Since anthranilate synthase and phosphoglycerate dehydrogenase are subjected to feedback inhibition by L-tryptophan and L-serine, respectively, the enzyme activity can be enhanced by retaining a desensitized mutant enzyme. Specifically, for example, the anthranilate synthase gene (trpE) and / or the phosphoglycerate dehydrogenase gene (serA) is mutated so as not to be subjected to feedback inhibition, and the obtained mutant gene is converted to the Enterobacteriaceae family. Bacteria that retain the desensitizing enzyme can be obtained by introducing them into microorganisms belonging to. More specifically, such a bacterium is a plasmid pGH5 having a mutant serA encoding a desensitized phosphoglycerate dehydrogenase in Escherichia coli SV164 holding a desensitized anthranilate synthase (International Publication No. No. 94/08031 pamphlet) can be mentioned.

  A bacterium into which a recombinant DNA containing a tryptophan operon has been introduced is also a suitable L-tryptophan-producing bacterium. Specific examples include Escherichia coli into which a tryptophan operon containing a gene encoding a desensitized anthranilate synthase has been introduced (JP 57-71397, JP 62-244382, U.S. Pat. No. 4,371,614). Moreover, L-tryptophan production ability can also be improved or imparted by enhancing expression of a gene (trpBA) encoding tryptophan synthase among tryptophan operons. Tryptophan synthase consists of α and β subunits and is encoded by trpA and trpB, respectively.

  Furthermore, as an L-tryptophan-producing bacterium, a strain Escherichia coli AGX17 (pGX44) [NRRL B-12263] strain having an L-phenylalanine and L-tyrosine auxotrophic trait, and an AGX6 (carrying a plasmid pGX50 containing a tryptophan operon) pGX50) aroP [NRRL B-12264] strain (see U.S. Pat. No. 4,371,614).

  Preferred examples of the L-threonine-producing bacterium used in the present invention include microorganisms belonging to the family Enterobacteriaceae with enhanced L-threonine biosynthetic enzymes. The genes encoding L-threonine biosynthetic enzymes include aspartokinase III gene (lysC), aspartate semialdehyde dehydrogenase gene (asd), and aspartokinase encoded by the thr operon. Examples thereof include a ze I gene (thrA), a homoserine kinase gene (thrB), and a threonine synthase gene (thrC). In parentheses are abbreviations for the genes. Two or more of these genes may be introduced. The L-threonine biosynthesis gene may be introduced into a bacterium belonging to the genus Escherichia in which threonine degradation is suppressed. Examples of bacteria belonging to the genus Escherichia 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 L-threonine biosynthetic enzymes 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 as not to receive 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 is inhibited by isoleucine and threonine in the culture medium. The expression is suppressed by attenuation. This modification can be achieved by removing the leader sequence of the attenuation region or the attenuator. (See WO02 / 26993 pamphlet, Biotechnology Letters vol24, No.21, November 2002, WO2005 / 049808 pamphlet)

  A unique promoter exists upstream of the threonine operon, but it may be replaced with a non-native promoter (see pamphlet of WO 98/04715) or expression of a gene involved in threonine biosynthesis A threonine operon may be constructed such that is governed by a lambda phage repressor and promoter. (Refer to European Patent No. 0593792) In order to modify Escherichia bacteria so as not to receive feedback inhibition by L-threonine, a strain resistant to α-amino-β-hydroxyvaleric acid (AHV) is used. It can also be obtained by selecting.

  The threonine operon modified so as not to receive feedback inhibition by L-threonine is increased in the number of copies in the host or linked to a strong promoter to improve the expression level. It is preferable. The increase in the number of copies can be achieved not only by plasmid amplification but also by transferring the threonine operon onto the chromosome by transposon, Mu-fuzzy, etc.

  Further, aspartokinase III gene (lysC) is desirably a gene modified so as not to be subjected to feedback inhibition by L-lysine. The lysC gene modified so as not to receive such feedback inhibition can be obtained by the method described in US Pat. No. 5,932,453.

  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 that control gene expression, and sugar uptake genes. Examples of these genes effective for L-threonine production include transhydronase (pntAB) gene (European Patent 733712), phosphoenolpyruvate carboxylase gene (pepC) (International Publication 95/06114). No. pamphlet), phosphoenolpyruvate synthase gene (pps) (European Patent No. 877090), pyruvate carboxylase gene of Coryneform bacteria or Bacillus genus bacteria (International Publication No. 99/18228 pamphlet, European application) Publication No. 1092776).

  It is also preferable to enhance the expression of a gene imparting resistance to L-threonine and a gene conferring resistance to L-homoserine, or imparting L-threonine resistance and L-homoserine resistance to the host. Examples of genes that confer resistance include rhtA gene (Res Microbiol. 2003 Mar; 154 (2): 123-35.), RhtB gene (European Patent Application Publication No. 0994190), rhtC gene (European Patent Application Publication No. No. 1013765) yfiK, yeaS gene (European Patent Application Publication No. 1016710). For methods for imparting L-threonine resistance to a host, methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

  Further, Escherichia coli VKPM B-3996 strain (see US Pat. No. 5,175,107) can also be exemplified. This VKPM B-3996 stock was registered with the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika on November 19, 1987. And have been deposited. In addition, the VKPM B-3996 strain is a threonine biosynthetic gene (threonine operon) in a broad vector plasmid pAYC32 having a streptomycin resistance marker (see Chistorerdov, AY, Tsygankov, YDPlasmid, 1986, 16, 161-167). : ThrABC) plasmid pVIC40 (WO90 / 04636 pamphlet) is obtained. In this pVIC40, feedback inhibition of aspartokinase I-homoserine dehydrogenase I encoded by thrA in the threonine operon by L-threonine is released.

  Further, Escherichia coli B-5318 strain (see European Patent No. 0593792) can also be exemplified. B-5318 shares were registered with the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika on November 19, 1987 under the registration number VKPM B-5318. It has been deposited. This VKPM B-5318 strain is an isoleucine non-requiring strain, and is an attenuator region which is an inherent transcriptional regulatory region downstream of the N-terminal part of the temperature-sensitive C1 repressor, PR promoter and Cro protein of lambda phage. A threonine operon lacking the threonine, that is, a gene involved in threonine biosynthesis, is located, and a recombinant plasmid DNA constructed so that expression of the gene involved in threonine biosynthesis is controlled by a lambda phage repressor and promoter is retained.

  A preferred L-histidine-producing bacterium used in the present invention is Escherichia coli FERM-P5038,5048 strain into which a vector incorporating a DNA carrying genetic information involved in L-histidine biosynthesis is introduced (Japanese Patent Laid-Open No. 56-005099). ), A strain introduced with the amino acid excretion gene Rht (European Patent Publication No. 1016710), sulfaguanidine, D, L-1,2,4-triazole-3-alanine, 80 strains of Escherichia coli imparted with streptomycin resistance (VKPM B-7270 Russian Patent Publication No. 2119536).

  As the microorganism having the ability to produce L-histidine, a microorganism in which the expression level of a gene encoding an enzyme of the L-histidine biosynthesis pathway is enhanced may be used. L-histidine biosynthesis enzymes include ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrase phosphoribosyl L-ATP pyrophosphohydrolase (hisIE), phosphoribosyl Formimino 5-aminoinidazole carboxamide ribotide isomerase phosphoribosylformimino-5-aminoimidazole carboxamide ribotide Isomerase (hisA), amide transferase amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB) And histidinol dehydrogenase (hisD).

  Preferred L-cysteine-producing bacteria of the present invention retain bacteria with reduced cystathionine-β-lyase activity (Japanese Patent Laid-Open No. 2003-169668) and serine acetyltransferase with reduced feedback inhibition by L-cysteine. Examples include bacteria belonging to the genus Escherichia (Japanese Patent Laid-Open No. 11-155571).

  Preferred L-proline-producing bacteria of the present invention are 3,4-dehydroxyproline and azatidine-2-carboxylate resistant strains such as Escherichia coli 702 (VKPMB-8011) and 702 ilvA-deficient strains. 702ilvA strain (VKPMB-8012 strain) is mentioned (Japanese Patent Laid-Open No. 2002-300874).

  As L-phenylalanine producing bacteria, L-phenylalanine producing bacteria include AJ12739 (tyrA :: Tn10, tyrR) (VKPM B-8197) lacking tyrA and tyrR, and yddG and yedA amplified strains that are phenylalanine excretion genes ( International Publication No. 03/044192 pamphlet). In addition, L-tryptophan, L-phenylalanine, and L-tyrosine are aromatic amino acids and have a common biosynthetic system. As a gene encoding a common biosynthetic enzyme, deoxyarabino-heptulonic acid phosphorus Acid synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydratase, shikimate kinase (aroL), 5-enolic acid pyruvin shikimate 3-phosphate synthase (aroA), chorismate synthase (aroC) Can be mentioned. (European Application Publication No. 763127)

  Examples of L-arginine-producing bacteria include α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S- (2-aminoethyl) -cysteine, α-methylserine, β-2-thienylalanine, or sulfur. Examples include Escherichia coli mutants having resistance to faguanidine (see JP-A-56-106598). In addition, Escherichia coli 237 strain (Russian patent application), which is an N-acetylglutamate synthase having a mutation resistant to feedback inhibition by L-arginine and having high activity, and an L-arginine-producing bacterium having the same enzyme 20000176777) is also a suitable L-arginine producing strain. The stock was deposited with the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika on April 10, 2000 under the number VKPM B-7925. It was transferred to an international deposit on May 18 under the Budapest Treaty. Escherichia coli 382 strain (Japanese Patent Laid-Open No. 2002-017342), which is an L-arginine-producing bacterium having an improved acetic acid assimilation ability, which is a derivative of 237 strains, can also be used. Escherichia coli 382 was deposited with the Russian National Collection of Industrial Microorganisms (VKPM) on April 10, 2000 under the number VKPM B-7926.

  Moreover, as a microorganism having L-arginine-producing ability, a microorganism having an improved expression level of a gene encoding an enzyme involved in L-arginine biosynthesis can be used. For example, L-arginine biosynthetic enzymes include N-acetylglutamate synthase (argA), N-acetylglutamyl phosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetyl One or more selected from ornithine transaminase (argD), acetylornithine deacetylase (argE) ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH) carbamoyl phosphate synthase (carAB) Is mentioned. The parentheses after these enzyme names are the names of genes encoding each enzyme. Among them, N-acetylglutamate synthase (argA) is more preferably a mutant gene in which feedback inhibition by L-arginine in which the amino acid sequence corresponding to the 15th to 19th positions of the wild type is substituted is released. (European Application Publication No. 1170361)

  As an L-leucine-producing bacterium, a bacterium belonging to the genus Escherichia in which the branched chain amino acid transaminase encoded by the ilvE gene is inactivated and the activity of the aromatic amino acid transaminase encoded by the tyrB gene is enhanced (JP 2004-024259) Escherichia coli H-9068 (ATCC21530), Escherichia coli H-9070 (FERM BP-4704), Escherichia coli H-9072 resistant to 4-azaleucine or 5,5,5-trifluoroleucine (FERM BP-4706) (US Pat. No. 5,744,331), Escherichia coli strain desensitized for feedback inhibition of isopropylmalate synthase by L-leucine (European Patent No. 1067191), β-2 Using Escherichia coli AJ11478 (US Pat. No. 5,763,231) having resistance to thienylalanine and β-hydroxyleucine Rukoto can also.

  Examples of L-isoleucine-producing bacteria include 6-dimethylaminopurine-resistant Escherichia coli mutant strains capable of producing L-isoleucine (JP-A-5-304969), and L-isoleucine hydroxamate-resistant Escherichia coli mutant strains. (JP-A-5-130882), thioisoleucine-resistant Escherichia coli mutant (JP-A-5-130882), DL-ethionine-resistant Escherichia coli mutant (JP-A-5-130882), arginine There is a mutant strain resistant to hydroxamate (Japanese Patent Laid-Open No. 5-130882). As a recombinant Escherichia bacterium, a gene encoding threonine deaminase or acetohydroxy acid synthase, which is an L-isoleucine biosynthetic enzyme, is used as a plasmid. Strains enhanced with 2-458, JP-A No. 2-42988, JP-A No. 8-47397 JP), and the like.

  Examples of L-valine-producing bacteria include Escherichia coli VL1970 strain (US Pat. No. 5,658,766). In addition, as described in WO96 / 06926, L-valine-producing bacteria having a mutation that requires lipoic acid for growth or / and a mutation that lacks H + -ATPase, or at least ilvG, ilvM, Escherichia bacteria that express each gene of ilvE and ilvD and into which a DNA fragment containing the ilvGMEDA operon has been introduced into the cells can also be suitably used as the parent strain of the present invention. In addition, since the ilvGMEDA operon is subjected to expression regulation (attenuation) of the operon by L-valine and / or L-isoleucine and / or L-leucine, in order to release the expression suppression by the produced L-valine, It is preferable that a region necessary for nuation is removed or mutated (US Pat. No. 5,998,178). The operon preferably does not express threonine deaminase activity. Escherichia coli VL1970 with the ileS17 mutation, which is detained as described above, is the Lucian National Collection of Industrial Microorganisms (VKPM) Depositary GNIIgenetika (Russian National Collection of Industrial Microorganisms (VKPM) Depositary, GNIIgenetika) (address: 1, Dorozhny Proezd., 1, 113545, Moscow, Russia), with a registration number of VKPM B-4411.

<1-2> Activity enhancement of β-glucoside PTS
The microorganism of the present invention can be obtained by modifying a microorganism belonging to the family Enterobacteriaceae having the ability to produce L-amino acids as described above so that the enzyme activity of β-glucoside PTS is enhanced. it can. However, L-amino acid-producing ability may be imparted after modification that increases the enzyme activity of β-glucoside PTS first. The enzyme activity of β-glucoside `PTS can be achieved by modifying the expression level of bglF gene (described later) that encodes β-glucoside PTS. The expression of the bglF gene may be enhanced, or the expression of the exogenous bglF gene may be enhanced by introducing a plasmid containing the bglF gene, increasing the copy number by amplification of the bglF gene on the chromosome, or the like. Furthermore, these may be combined.

In the present invention, β-glucoside PTS means an activity (permease) for transferring a phosphate group of phosphoenolpyruvate (hereinafter referred to as PEP) to β-glucoside (phosphotransferase) and simultaneously incorporating it into Cytoplasm. Here, β-glucoside is a glycoside having β-D-glucose as a sugar component, for example, salicin bonded with salicyl alcohol and glycid, or arbutin bonded with hydroquinone and glycid, Means a sugar derivative in which various compounds such as alcohol, phenol and anthocyanin are bonded to the reducing group of β-D-glucose. Further, the β-glucoside PTS of the present invention may have not only β-glucoside but also a function of transferring a phosphate group to glucose at the same time. ^{(E.coli & Salmonella 2 nd Edition Americsn} society for Microbiology)

  Enhancement of the enzyme activity of β-glucoside PTS can be confirmed by measuring phosphorylation activity in vitro by the method of Chen et al. (Biochemistry 1998 37: 8714-8723). (EC 2.7.1.69) Confirmation that bglF expression is improved compared to parental strains, eg, wild-type and non-modified strains, is confirmed by comparing the amount of bglF mRNA with wild-type or non-modified strains. I can do it. 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. Enhancing enzyme activity can be confirmed by the increase in the amount of the target protein compared to the unmodified strain and the wild strain, and can be detected, for example, by Western blotting using an antibody. (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)).

The bglF gene of the present invention refers to the bglF gene of Escherichia coli and homologs thereof.
Examples of the bglF gene of Escherichia coli include a gene encoding a protein having the amino acid sequence of SEQ ID NO: 6. The bglF gene derived from Escherichia coli MG1655 is registered in Genbank NP_418178 (SEQ ID NO: 5), and the sequence of W3110 is registered in Genbank PTV3B_ECOLI [P08722] (SEQ ID NO: 5). The bglF gene of Escherichia coli is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.

  The bglF gene homologue is a gene derived from other microorganisms, showing a high similarity in the bglF gene structure of Escherichia coli, improving L-amino acid production when introduced into the host, and exhibiting β-glucoside PTS activity Say. For example, bglF gene homologs include bglF gene from Erwinia carotovora (GenbankAccession No. YP_050260), bglF gene from Streptococcus pyogenes (Genbank Accession No. YP_059817), bglF gene from Streptococcus agalactiae (NP_735260), Photorhabdus lumines. NP_927931.) Gene. Further, the bglF gene is based on the homology with the genes exemplified above, coryneform bacteria such as Corynebacterium glutamicum, Brevibacterium lactofermentum, pseudomonas bacteria such as Pseudomonas aeruginosa, mycobacteria It may be cloned from Mycobacterium bacteria such as Um. Tuberculosis. For example, the bglF gene can be cloned using the synthetic oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2.

  In addition, the gene encoding β-glucoside PTS used in the present invention is not limited to the wild type gene, and as long as the function of the encoded β-glucoside PTS protein, ie, β-glucoside PTS activity is not impaired, SEQ ID NO: 6 Even if it is a mutant or an artificial modification that encodes a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or a plurality of positions in the amino acid sequence of Good. Here, “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically 2 to 20, preferably 2 to 10, more preferably 2 to 5 Means. One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations that maintain β-glucoside PTS activity. A conservative mutation 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 the case of 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. A typical conservative mutation is a conservative substitution. As a substitution regarded as a conservative substitution, the above substitution is preferably a conservative substitution, and as a conservative substitution, a substitution from ala to ser or thr, arg to gln, his or lys, 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, gly to pro, 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 Substitution, phe to trp, tyr, met, ile or leu, ser to thr or ala, thr to ser or ala, trp to phe or tyr, tyr to his, to phe or trp Conversion, and, met from val, and substitution of ile or leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutant or variant) such as those based on individual differences or species differences of microorganisms that retain the bglF gene. ) Is also included. Such a gene is modified by the site-directed mutagenesis, for example, by modifying the nucleotide sequence shown in SEQ ID NO: 5 so that the amino acid residue at a specific site of the encoded protein includes substitution, deletion, insertion or addition. Can be obtained by

  Furthermore, the bglF gene encodes a protein having 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 amino acid sequence of SEQ ID NO: 6, In addition, a sequence encoding β-glucoside PTS can be used. In addition, since the degeneracy of the gene differs depending on the host to be introduced, it may be replaced with a codon that can be easily used in the host into which bglF is introduced. Similarly, as long as the bglF gene has the function of β-glucoside PTS, the N-terminal side and the C-terminal side may be extended or deleted. For example, the length to be extended / deleted is 50 or less, preferably 20 or less, more preferably 10 or less, and particularly preferably 5 or less in terms of amino acid residues. More specifically, the amino acid sequence of SEQ ID NO: 6 may be extended or deleted by 5 amino acids from the 50 amino acids on the N-terminal side and 5 amino acids from the 50 amino acids on the C-terminal side.

  It can also be obtained by a conventionally known mutation treatment as follows. As the mutation treatment, a method having an in vitro treatment with a gene having the base sequence shown in SEQ ID NO: 5 with hydroxylamine or the like, and a microorganism carrying the gene, such as an Escherichia bacterium, with ultraviolet light or N-methyl-N′-nitro- Examples thereof include a method of treating with a mutagen that is usually used for mutagenesis such as N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS). Whether or not these genes encode a protein having β-glucoside PTS activity can be determined by, for example, expressing these genes in appropriate cells and improving β-glucoside uptake, or Chen et al. This can be confirmed by examining phosphorylation activity in vitro by the method (Biochemistry 1998 37: 8714-8723).

  In addition, the bglF gene includes DNA that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 5 or a probe that can be prepared from these sequences, and that encodes a protein having β-glucoside PTS. 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 quantify these conditions clearly, for example, DNAs with high homology, for example, DNAs having 50% or more homology hybridize and DNA with lower homology than that. 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably, conditions that do not hybridize to each other or washing conditions of normal Southern hybridization, The conditions include washing 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, the base sequence of SEQ ID NO: 5 or a part of the sequence can also be used. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the base sequence of SEQ ID NO: 5 as a primer and a DNA fragment containing the base sequence of SEQ ID NO: 5 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 modification for enhancing the expression of the bglF gene can be performed, for example, by increasing the copy number of the bglF gene in the cell using a gene recombination technique. For example, a DNA fragment containing the bglF gene may be ligated with a vector that functions in a host microorganism, preferably a multi-copy vector, to produce a recombinant DNA, which is introduced into the microorganism and transformed.

  When the Escherichia coli bglF gene is used as the bglF gene, a primer prepared based on the nucleotide sequence of SEQ ID NO: 5, for example, the primers shown in SEQ ID NOs: 1 and 2, and using Escherichia coli chromosomal DNA as a template The bglF gene can be obtained by PCR (PCR: polymerase chain reaction; see White, TJ et al., Trends Genet. 5, 185 (1989)). The bglF gene of other microorganisms belonging to the family Enterobacteriaceae is also a PCR method using as a primer an oligonucleotide prepared based on the known bglF gene in the microorganism or the sequence information of the bglF gene or BglF protein of other types of microorganisms, 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. 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).

Next, a recombinant DNA is prepared by connecting the bglF gene amplified by the PCR method to a vector DNA that can function in the cells of the host microorganism. Examples of the vector capable of functioning in the host microorganism cell include a vector capable of autonomous replication in the host microorganism cell.
The vectors that can replicate autonomously in Escherichia coli cells include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184 (pHSG and pACYC are available from Takara Bio Inc.), RSF1010, pBR322, pMW219 (pMW from Nippon Gene) And pSTV29 (available from Takara Bio Inc.).

  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, a method for increasing DNA permeability by treating recipient cells with calcium chloride as reported for Escherichia coli K-12 (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)). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, are introduced into the DNA-receptor cells in a protoplast or spheroplast state that readily incorporates recombinant DNA. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nature, 274, 398 (1978); Hinnen, A Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). Further, transformation of coryneform bacteria can also be performed by the electric pulse method (Sugimoto et al., Japanese Patent Laid-Open No. 2-207791).

  On the other hand, increasing the copy number of the bglF gene can also be achieved by making multiple copies of the bglF gene on the chromosomal DNA of the microorganism. In order to introduce the bglF gene in multiple copies on 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. Further, it may be linked in tandem beside the bglF gene present on the chromosome, or may be redundantly integrated on an unnecessary gene on the chromosome. These gene introductions can be achieved using a temperature sensitive vector or using an integration vector.

  Alternatively, as disclosed in JP-A-2-109985, a bglF gene can be mounted on a transposon, transferred, and introduced in multiple copies onto chromosomal DNA. Confirmation that the bglF gene has been transferred onto the chromosome can be confirmed by Southern hybridization using a part of the bglF gene as a probe.

In addition to the amplification of the gene copy number described above, the enhancement of the expression of the bglF gene can be performed by regulating the expression of the promoter of the bglF gene on the chromosomal DNA or on the plasmid as described in International Publication No. 00/18935 pamphlet. Replacing sequences with strong ones, bringing the -35 and -10 regions closer to consensus sequences, amplifying regulators that increase bglF expression, and deleting or weakening regulators that reduce bglF expression Can also be achieved. For example, lac promoter, lac _uv5 promoter, trp promoter, trc promoter, araBA promoter, T7 promoter, φ10 promoter and the like are known as strong promoters. It is also possible to introduce a base substitution into the promoter region of the bglF gene and modify it to be more powerful. 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. Expression control regions such as the promoter of bglF can also be determined using a promoter search vector or gene analysis software such as GENETYX. These promoter substitutions or modifications enhance bglF gene expression. For example, a method using a temperature-sensitive plasmid or a Red driven integration method (WO2005 / 010175) can be used to replace the expression regulatory sequence.

  In order to enhance the activity of the protein encoded by the bglF gene, a mutation that increases β-glucoside PTS activity may be introduced into the bglF gene. Mutations that increase the activity of the protein encoded by the bglF gene (BglF protein) include mutations in the promoter sequence that increase the transcription amount of the bglF gene and bglF that increases the specific activity of the BglF protein. Examples include mutations in the coding region of the gene.

<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. Among these, it is more preferable to use a medium using glucose as a sugar source. 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 recovery of L-lysine or L-arginine from the fermentation broth can be usually carried out by combining ion exchange resin method, 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.

  Moreover, it can also culture | cultivate, making L-glutamic acid precipitate in a culture medium using the liquid culture medium adjusted to the conditions which L-glutamic acid precipitates. Examples of conditions for precipitation of L-glutamic acid include pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0. Can do.

  The method for collecting L-glutamic acid from the culture solution after completion of the culture may be performed according to a known recovery method. For example, it is collected by removing microbial cells from the culture solution and then concentrating crystallization or ion exchange chromatography. When culturing under conditions where L-glutamic acid is precipitated, L-glutamic acid precipitated in the culture solution can be collected by centrifugation or filtration. In this case, L-glutamic acid dissolved in the medium may be crystallized and then isolated together.

[Example]
Hereinafter, the present invention will be described more specifically with reference to examples.
[Reference example]

Construction of cadA, ldcC gene-disrupted strain encoding lysine decarboxylase <1-1> Construction of cadA, ldcC gene-disrupted strain encoding lysine decarboxylase First, a lysine decarboxylase non-producing strain was constructed. The method of destruction is the Red driven integration method described in WO2005 / 01075 and the excision system derived from lambda phage (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.). Lysine decarboxylase is encoded by the cadA gene (Genbank Accession No. NP_418555. SEQ ID NO: 42) and the ldcC gene (Genbank Accession No. NP_414728. SEQ ID NO: 44) (see International Publication WO96 / 17930 pamphlet). Here, WC196 strain was used as the parent strain. 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.

  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 into the gene disruption strain can be removed.

(1) Disruption of cadA gene

  The plasmid pMW118-attL-Cm-attR was used as the PCR template. 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. (See WO2005 / 01075) The attL sequence is shown in SEQ ID NO: 11, and the attR sequence is shown in SEQ ID NO: 12.

  The synthetic oligonucleotides shown in SEQ ID NOs: 46 and 47 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 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 gene encoding the Red recombinase of the λRed homologous recombination system controlled by the arabinose-inducible ParaB promoter. A DNA fragment (GenBank / EMBL Accession No. J02459, 31088th to 33241th) of λ phage including (γ, β, exo genes) in total is included. 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 in LB medium containing 100 mg / L ampicillin at 30 ° C., 5 mL SOB medium (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. The resulting colony was tested for ampicillin resistance, and an ampicillin sensitive strain from which pKD46 had been removed was obtained. 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 was used. pMW-intxis-ts is equipped with a gene encoding lambda phage integrase (Int) (SEQ ID NO: 13) and a gene encoding excisionase (Xis) (SEQ ID NO: 15), and has temperature-sensitive replication ability. It has a plasmid. When pMW-intxis-ts is introduced, attL (SEQ ID NO: 11) or attR (SEQ ID NO: 12) on the chromosome is recognized and recombination occurs, and the gene between attL and attR is excised, and the attL or attR sequence on the chromosome The only remaining structure.

  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: 48 and 49 as ldcC disruption primers in accordance with the above procedure. Thus, WC196ΔcadAΔldcC, which is a cadA ldcC-disrupted strain, was obtained.

(3) Preparation of PCR template and helper plasmid
PCR template pMW118-attL-Cm-attR and helper plasmid pMW-intxis-ts were prepared as follows.

(3-1) pMW118-attL-Cm-attR
The construction of pMW118-attL-Cm-attR was based on pMW118-attL-Tc-attR. The following four DNA fragments were ligated.

  1) E. coli strain W3350 (including λ prophage) using oligonucleotides P1 and P2 (SEQ ID NOs: 17 and 18) as primers (these primers additionally contain recognition sites for BglII and EcoRI endonucleases) ) BglII-EcoRI DNA fragment (120 bp) containing attL obtained by PCR amplification of the corresponding sequence of the chromosome (SEQ ID NO: 11).

  2) E. coli strain W3350 (including λ prophage) using oligonucleotides P3 and P4 (SEQ ID NOs: 19 and 20) as primers (these primers additionally contain recognition sites for PstI and HindIII endonucleases) ) PstI-HindIII DNA fragment (182 bp) containing attR obtained by PCR amplification of the corresponding sequence of the chromosome (SEQ ID NO: 12).

3) Large BglII-HindIII fragment (3916 bp) of pMW118-ter_rrnB. pMW118-ter_rrnB was obtained by linking the following three fragments.
A large fragment (2359 bp) containing the AatII-EcoRIpol fragment of pMW118 obtained by cleaving pMW118 with EcoRI restriction endonuclease, treating with Klenow fragment of DNA polymerase I, and then cleaving with AatII restriction endonuclease.

  Obtained by PCR amplification of the corresponding sequence of the pUC19 plasmid using oligonucleotides P5 and P6 (SEQ ID NOs: 21 and 22) as primers (these primers additionally contain recognition sites for AatII and BglII endonucleases). In addition, an AatII-BglII small fragment (1194 bp) of pUC19 containing the bla gene of ampicillin resistance (ApR).

  Using oligonucleotides P7 and P8 (SEQ ID NOs: 23 and 24) as primers (these primers additionally contain recognition sites for BglII and PstI endonucleases), the corresponding region of the chromosome of E. coli MG1655 strain was PCR BglII-PstIpol small fragment (363 bp) of transcription terminator ter_rrnB obtained by amplification.

  4) A small EcoRI-PstI fragment (1388 bp) of pML-Tc-ter_thrL from the tetracycline resistance gene and transcription terminator ter_thrL (SEQ ID NO: 29). pML-Tc-ter_thrL is obtained as follows.

  pML-MSC (2001 # 5) was cleaved with XbaI and BamHI restriction endonucleases, and the large fragment (3342 bp) was ligated with the XbaI-BamHI fragment (68 bp) containing the terminator ter_thrL. The XbaI-BamHI fragment uses the oligonucleotides P9 and P10 (SEQ ID NOs: 25 and 26) as primers (these primers additionally contain recognition sites for XbaI and BamHI endonucleases) and the chromosome of E. coli MG1655 strain The corresponding region was obtained by PCR amplification. The product of this ligation reaction was designated as plasmid pML-ter_thrL.

  pML-ter_thrL was digested with KpnI and XbaI restriction endonucleases, treated with Klenow fragment of DNA polymerase I, and then a small EcoRI-Van91I fragment (1317 bp) of pBR322 containing the tetracycline resistance gene (with EcoRI and Van91I restriction endonucleases). pBR322 was treated with DNA polymerase I Klenow fragment. The product of this ligation reaction was designated as plasmid pML-Tc-ter_thrL.

As described above, pMW118-attL-Tc-attR was obtained.
pMW118-attL-Cm-attR is a large BamHI-XbaI fragment (4413 bp) of pMW118-attL-Tc-attR, promoter PA2 (early promoter of T7 phage), chloramphenicol resistance (CmR) cat gene, It was constructed by ligating a BglII-XbaI artificial DNA fragment (1162 bp) containing transcription terminators ter_thrL and attR. An artificial DNA fragment (SEQ ID NO: 30) was obtained as follows.

  ML-MSC (2001 # 5) was cut with KpnI and XbaI restriction endonucleases and then ligated with a small KpnI-XbaI fragment (120 bp) containing the promoter PA2 (early promoter of T7 phage). The KpnI-XbaI fragment uses oligonucleotides P11 and P12 (SEQ ID NOs: 27 and 28) as primers (these primers additionally contain recognition sites for KpnI and XbaI endonucleases) and the corresponding region of T7 phage DNA Was obtained by PCR amplification. The product of this ligation reaction was designated as plasmid pML-PA2-MCS.

  The XbaI site was removed from pML-PA2-MCS. The obtained product was designated as plasmid pML-PA2-MCS (XbaI-).

  A small BglII-HindIII fragment (928 bp) of pML-PA2-MCS (XbaI-) containing the promoter PA2 (early promoter of T7 phage) and the chloramphenicol resistant (CmR) cat gene was transcribed with the transcription terminators ter_thrL and attR And a small HindIII-HindIII fragment (234 bp) of pMW118-attL-Tc-attR.

  Using the oligonucleotides P9 and P4 (SEQ ID NOs: 25 and 20) as primers (these primers additionally contain recognition sites for HindIII and XbaI endonucleases), the target artificial DNA is obtained by PCR amplification of the ligation mixture. A fragment (1156 bp) was obtained.

(3-2) pMW-intxis-ts
First, two DNA fragments were amplified using λ phage DNA (Fermentas) as a template. The first fragment was composed of the region of nt 37168-38046 (SEQ ID NO: 39) and contained the cI repressor, promoters Prm and Pr, and the leader sequence of the cro gene. This fragment was obtained by amplification using oligonucleotides P1 ′ and P2 ′ (SEQ ID NOs: 31 and 32) as primers. The second fragment consisted of a region of nt 27801 to 29100 (SEQ ID NO: 40) containing the xis-int gene of λ phage. This fragment was obtained by amplification using oligonucleotides P3 ′ and P4 ′ (SEQ ID NOs: 33 and 34) as primers. All primers had appropriate endonuclease recognition sites.

  The resulting PCR amplified fragment containing the cI repressor was cleaved with ClaI restriction endonuclease, treated with Klenow fragment of DNA polymerase I, and then cleaved with EcoRI restriction endonuclease. The second PCR amplified fragment was cut with EcoRI and PstI restriction endonucleases. On the other hand, plasmid pMWPlaclacI-ts was cleaved with BglII endonuclease, treated with Klenow fragment of DNA polymerase I, and then cleaved with PstI restriction endonuclease. The vector fragment of pMWPlaclacI-ts was eluted from the agarose gel and ligated with the cleaved PCR amplified fragment.

  The plasmid pMWPlaclacI-ts is a derivative of pMWPlaclacI consisting of the following parts. 1) BglII-HindIII artificial DNA fragment containing the lacI gene under the control of the PlacUV5 promoter and bacteriophage T7 gene 10 RBS, 2) using oligonucleotides P5 ′ and P6 ′ (SEQ ID NOs: 35 and 36) as primers (these The primers contain additional AatII and BglII endonuclease recognition sites), AatII-BglII fragment containing ampicillin resistance (ApR) gene obtained by PCR amplification of the corresponding region of pUC19 plasmid, 3) Recombination AatII-HindIII fragment containing the AatII-PvuI fragment of plasmid pMW118-ter_rrnB. Plasmid pMW118-ter_rrnB was constructed as follows. Include the terminator ter_rrnB by PCR amplification of the corresponding region of the chromosome of E. coli MG1655 strain using primers P7 ′ and P8 ′ (SEQ ID NOs: 37 and 38) containing appropriate endonuclease recognition sites as primers. A PstI-HindIII fragment was obtained. Prior to ligation, pMW118 and ter_rrnB fragments (complementary strand, SEQ ID NO: 41) were restricted with PvuI or PstI, treated with Klenow fragment of DNA polymerase I to blunt ends, then cleaved with AatII or HindIII endonuclease did. For construction of the pMWPlaclacI-ts mutant, the AatII-EcoRV fragment of the plasmid pMWPlaclacI was replaced with the AatII-EcoRV fragment of the plasmid pMAN997 containing the pSC101 replicon par, ori and repAts genes.

Construction of plasmid for amplifying bglF The complete nucleotide sequence of Escherichia coli (Escherichia coli K-12 strain) has already been clarified (Science, 277, 1453-1474 (1997)). Based on the base sequence of the bglF gene, using the synthetic oligonucleotide of SEQ ID NO: 1 having a HindIII site as a 5 ′ primer and the synthetic oligonucleotide of SEQ ID NO: 2 having an XbaI site as a 3 ′ primer, PCR was carried out using the chromosomal DNA of Escherichia coli MG1655 strain as a template and treated with restriction enzymes HindIII and XbaI to obtain a gene fragment containing bglF.

  The purified PCR product was ligated to a vector pMW219 (manufactured by Nippon Gene) digested with HindIII and XbaI to construct a plasmid pM-bglF for bglF amplification. This plasmid is under the control of the bglF gene linked to the lac promoter in the forward direction. Further, pM-bglF was digested with HindIII and EcoRI, and the bglF gene fragment was recovered, purified, and ligated to vector pSTV29 (manufactured by Takara Shuzo) digested with HindIII and EcoRI to construct plasmid pS-bglF.

  As with the bglF gene, a ptsG gene expression plasmid was constructed as a comparative example. The sequence of ptsG is described in SEQ ID NO: 7, and the amino acid sequence is described in SEQ ID NO: 8. The sequence of ptsG can be obtained with reference to Genbank Accession No. NP_415619. Using the synthetic oligonucleotide of SEQ ID NO: 3 having a HindIII site as the 5 ′ primer and the synthetic oligonucleotide of SEQ ID NO: 4 having the XbaI site as the 3 ′ primer, the chromosomal DNA of Escherichia coli MG1655 strain was used as a template As described above, PCR was performed, and the fragments were treated with restriction enzymes HindIII and XbaI to obtain a gene fragment containing ptsG. The purified PCR product was ligated to a vector pMW219 digested with HindIII and XbaI to construct a ptsG amplification plasmid pM-ptsG. This plasmid is under the control of the ptsG gene linked to the lac promoter in the forward direction. Further, similarly to bglF, a ptsG gene fragment was excised from pM-ptsG and ligated to vector pSTV29 to construct plasmid pS-ptsG.

Effect of bglF amplification in Escherichia bacterium L-lysine producing strain As a L-lysine producing strain of Escherichia coli, the WC196ΔldcCΔcadA strain of Example 1 was used as a plasmid for producing Lys pCABD2 carrying dapA, dapB and LysC genes (International Publication No. 1). WO01 / 53459 pamphlet) The WC196ΔldcCΔcadA (pCABD2) strain transformed with pCABD2 was used. The WC196ΔldcCΔcadA strain was transformed with the bglF amplification plasmid pM-bglF and the ptsG amplification plasmid pM-ptsG prepared in Example 1 and the control plasmid pMW219 to obtain a kanamycin resistant strain. After confirming that the given plasmid has been introduced, the bglF amplification plasmid pM-bglF introduced strain is WC196ΔldcCΔcadA (pCABD2, pM-bglF), the ptsG amplification plasmid pM-ptsG introduced strain is WC196ΔldcCΔcadA (pCABD2, pM-ptsG ) And the control plasmid pMW219 introduced strain were named WC196ΔldcCΔcadA (pCABD2, pMW219) strain.

  Each of the strains constructed above was cultured at 37 ° C. in L medium containing 25 mg / L kanamycin so that the final OD600 was approximately 0.6, and then the same amount of 40% glycerol solution as the culture solution was added and stirred. Thereafter, an appropriate amount was dispensed and stored at −80 ° C. This is called glycerol stock.

  The glycerol stocks of these strains were thawed, 100 μL of each was uniformly applied to an L plate containing 25 mg / L kanamycin, and cultured at 37 ° C. for 24 hours. Approximately 1/8 amount of the cells on the obtained plate was inoculated into 20 mL of a fermentation medium containing 25 mg / L kanamycin in a 500 mL Sakaguchi flask, and cultured at 37 ° C. for 24 hours on a reciprocal shake culture apparatus. . After cultivation, the amount of lysine accumulated in the medium was measured using Biotech Analyzer AS210 (Sakura Seiki).

Table 1 shows the OD and L-lysine accumulation at 24 hours. As can be seen from Table 1, the WC196ΔldcCΔcadA (pCABD2, pM-bglF) strain accumulated a larger amount of lysine than the WC196ΔldcCΔcadA (pCABD2, pMW219) strain into which the bglF gene was not introduced. In addition, the lysine accumulation amount was confirmed to be improved compared to the WC196ΔldcCΔcadA (pCABD2, pM-ptsG) strain into which the ptsG gene was introduced, suggesting that the enhancement of the bglF gene in lysine production was more effective than the ptsG enhancement.

[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 4/7 hydrate 0.01g / L
Manganese sulfate 4.7 salt / 0.01g / L
Yeast extract 2.0g / L
Pharmacopeia calcium carbonate 30g / L
The pH was adjusted to 7.0 with potassium hydroxide and autoclaved at 115 ° C. for 10 minutes. However, glucose and MgSO 4 · 7H 2 O were sterilized separately.

Effect of bglF amplification in Escherichia bacterium L-glutamic acid producing strain AJ12949 strain was used as an L-glutamic acid producing strain of Escherichia coli. The AJ12949 strain is a strain with a reduced α-ketoglutarate dehydrogenase activity, and was dated December 28, 1993 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 Biodeposition Center, 305 -8566 Deposited under the deposit number FERM P-14039 at 1st, 1st East, Tsukuba City, Ibaraki Prefecture, Japan, and transferred to an international deposit based on the Budapest Treaty on November 11, 1994, with the deposit number FERM BP -4881 is granted.

  The AJ12949 strain was transformed with the bglF amplification plasmid pS-bglF used in Example 1 and the control plasmid pSTV29 to obtain a chloramphenicol resistant strain. After confirming that a predetermined plasmid had been introduced, the bglF amplification plasmid pS-bglF-introduced strain was named AJ12949 (pS-bglF) strain, and the control plasmid pSTV29-introduced strain was named AJ12949 (pSTV29) strain.

  AJ12949 (pS-bglF) strain and AJ12949 (pSTV29) strain were cultured in L medium containing 20 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.

  The glycerol stocks of these strains were thawed, and 100 μL of each was uniformly applied to an L plate containing 20 mg / L chloramphenicol and cultured at 37 ° C. for 24 hours. Approximately 1/8 amount of the cells on the obtained plate was inoculated into 20 mL of a fermentation medium containing 20 mg / L chloramphenicol in a 500 mL Sakaguchi flask, and was cultured at 37 ° C in a reciprocating shake culture apparatus. Incubate for hours. After the culture, the amount of glutamic acid accumulated in the medium was measured using Biotech Analyzer AS210 (Sakura Seiki).

  Table 2 shows the OD and L-glutamic acid accumulation after 42 hours. As can be seen from Table 2, the AJ12949 (pS-bglF) strain accumulated a larger amount of glutamic acid than the AJ12949 (pSTV29) strain into which the bglF gene was not introduced.

[Escherichia bacterium L-glutamic acid production medium]
Glucose 40g / L
Ammonium sulfate 20g / 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
Yeast extract 2.0g / L
Pharmacopeia calcium carbonate 30g / L
The pH was adjusted to 7.0 with potassium hydroxide and autoclaved at 115 ° C. for 10 minutes. However, glucose and MgSO 4 · 7H 2 O were sterilized separately. Further, after the medium temperature became 60 ° C. or lower, a thiamine hydrochloride solution sterilized in advance with a filter DISMIC-25cs 0.2 mm filter (ADVANTEC) was added so that the final concentration was 0.01 g / L.

Effect of bglF amplification on L-threonine-producing strains of the genus Escherichia
B-5318 strain was used as a parent strain of bglF-amplified L-threonine producing bacterium. B-5318 shares were registered with the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika on November 19, 1987 under the registration number VKPM B-5318. It has been deposited. Construction of a bglF-amplified strain from an L-threonine-producing bacterium was performed using the plasmid described in Example 1.

  The B-5318 strain was transformed with the bglF amplification plasmid pS-bglF used in Example 1 and the control plasmid pSTV29 to obtain a chloramphenicol resistant strain. Confirm that the specified plasmid has been introduced, and name the bglF amplification plasmid pS-bglF introduced strain B-5318 (pS-bglF) and the control plasmid pSTV29 introduced strain B-5318 (pSTV29). It was.

  B-5318 (pS-bglF) and B-5318 (pSTV29) strains were cultured in L medium containing 20 mg / L chloramphenicol at 37 ° C. so that the final OD600≈0.6. A 40% glycerol solution in an amount equal to the liquid was added and stirred, and then dispensed in appropriate amounts to obtain a glycerol stock, which was stored at -80 ° C.

  The glycerol stocks of these strains were thawed, and 100 μL of each was uniformly applied to an L plate containing 20 mg / L chloramphenicol and cultured at 37 ° C. for 24 hours. Approximately 1/8 amount of the cells on the resulting plate was inoculated into 20 mL of a fermentation medium containing 20 mg / L chloramphenicol in a 500 mL Sakaguchi flask, and 16 times at 37 ° C. on a reciprocating shaker. Incubate for hours. After culture, the amount of L-threonine accumulated in the medium was measured using high performance liquid chromatography.

  The OD and L-threonine accumulation at 16 hours is shown in the table. As can be seen from the table, the B-5318 (pS-bglF) strain accumulated a larger amount of L-threonine than the B-5318 (pSTV29) strain into which the bglF gene was not introduced.

[Escherichia bacterium L-threonine production medium]
Glucose 60g / L
Ammonium sulfate 16g / L
Potassium dihydrogen phosphate 0.7g / L
Magnesium sulfate heptahydrate 1.0g / L
Iron sulfate heptahydrate 0.01g / L
Manganese sulfate heptahydrate 0.01g / L
Yeast extract 0.5g / L
Thiamine hydrochloride 0.2mg / L
L-isoleucine 0.05g / L
Pharmacopeia calcium carbonate 30g / L
The pH was adjusted to 7.0 with potassium hydroxide and autoclaved at 115 ° C. for 10 minutes. However, glucose and MgSO 4 · 7H 2 O were sterilized separately. The calcium carbonate was sterilized by dry heat at 180 ° C. for 3 hours. After the medium temperature became 60 ° C. or lower, a thiamine hydrochloride solution sterilized in advance with a filter DISMIC-25cs 0.2 mm filter (ADVANTEC) was added to a final concentration of 0.2 mg / L.

Effect of bglF amplification in Pantoea bacterium L-glutamate producing strain
Pantoea ananatis AJ13601 can be used as a parent strain of bglF-amplified L-glutamic acid-producing bacteria. In addition, Pantoea Ananatis AJ13601 stock was registered as a reference number FERM P-17516 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of Economy, Trade and Industry (Postal Code: 305-8586, 1-3-1 Higashi, Tsukuba, Ibaraki) on August 18, 1999. The deposit was transferred to the international deposit under the Budapest Treaty on July 6, 2000, and the accession number FERM BP-7207 was assigned. Construction of a bglF-amplified strain from an L-glutamic acid-producing bacterium can be performed using the plasmid of Example 1.

  The bglF amplified strain is cultured in an L-glutamic acid production medium and cultured in a reciprocating shake culture apparatus. After culturing, the amount of L-glutamic acid accumulated in the medium is measured by a known method to confirm that the accumulation of L-glutamic acid is improved. By such a method, a bglF amplified strain with improved L-glutamic acid production ability can be obtained.

The structures of pMW118-attL-Tc-attR and pMW118-attL-Cm-attR are shown. The structure of pMW-intxis-ts is shown.

Claims (5)

A microorganism belonging to the family Enterobacteriaceae, which has L-amino acid-producing ability and has been modified so that β-glucoside PTS activity is enhanced, is cultured in a medium, and L-amino acid is produced in the medium or in the fungus body. A method for producing an L-amino acid, which is accumulated and collected from the medium or cells. A microorganism belonging to the family Enterobacteriaceae, wherein the expression of the gene encoding β-glucoside PTS is enhanced by increasing the copy number of the gene encoding β-glucoside PTS or by modifying the expression regulatory sequence of the gene The manufacturing method of Claim 1 which is these. The production method according to claim 1 or 2, wherein the gene encoding β-glucoside PTS is the DNA described in (a) or (b) below:
(A) DNA comprising the base sequence shown in SEQ ID NO: 5,
(B) a DNA that hybridizes under stringent conditions with the base sequence shown in SEQ ID NO: 5 or a probe that can be prepared from the base sequence, and that encodes a protein having β-glucoside PTS activity. The method according to any one of claims 1 to 3, wherein the microorganism is an Escherichia bacterium or a Pantoea bacterium. The production method according to any one of claims 1 to 4, wherein the L-amino acid is one or more amino acids selected from the group consisting of L-lysine, L-threonine, and L-glutamic acid.