WO2015005405A1

WO2015005405A1 - Method for producing useful substance

Info

Publication number: WO2015005405A1
Application number: PCT/JP2014/068366
Authority: WO
Inventors: 優山上; 吉彦原; 啓太福井; 直也小泉
Original assignee: 味の素株式会社
Priority date: 2013-07-09
Filing date: 2014-07-09
Publication date: 2015-01-15
Also published as: JP2016163540A

Abstract

Provided is a method for producing a desired substance that is biosynthesized using isocitric acid as a precursor. A desired substance can be produced by: culturing a microorganism in a culture medium, wherein the microorganism is so modified that the activity of an enzyme capable of synthesizing malyl-CoA from L-malic acid, the activity of malyl-CoA lyase and the activity of isocitrate lyase can be increased; and then collecting the desired substance from the culture medium.

Description

Method for producing useful substances

The present invention relates to a method for producing useful substances using microorganisms.

L-glutamic acid is mainly produced by fermentation using a so-called coryneform bacterium belonging to the genus Brevibacterium, Corynebacterium, Microbacterium, or L-glutamic acid-producing bacteria or mutants thereof (non- Patent Document 1). Examples of the method for producing L-glutamic acid by fermentation using other strains include, for example, a method using microorganisms such as Bacillus, Streptomyces, and Penicillium (Patent Document 1), Pseudomonas, Arthrobacter. , A method using microorganisms belonging to the genus Serratia, Candida, etc. (Patent Document 2), a method using microorganisms belonging to the genus Bacillus, Pseudomonas, Serratia, Aerobacter Aerogenes (currently Enterobacter Aerogenes) (Patent Documents) 3) A method using a mutant strain of Escherichia coli (Patent Document 4) and a method using a microorganism belonging to the genus Klebsiella, Erwinia, Pantothea, Enterobacter and the like (Patent Documents 5 to 7) are known.

In addition, various techniques for improving the ability of microorganisms to produce L-glutamic acid by increasing the activity of L-glutamic acid biosynthetic enzymes by recombinant DNA techniques are known. For example, introduction of a gene encoding citrate synthase derived from Escherichia coli or Corynebacterium glutamicum into bacteria belonging to the genus Corynebacterium or Brevibacterium was effective in improving L-glutamic acid production ability (Patent Document 8). Furthermore, introduction of a citrate synthase gene derived from a coryneform bacterium into an enteric bacterium belonging to the genus Enterobacter, Klebsiella, Serratia, Erbinia, or Escherichia is effective in improving L-glutamic acid production ability. It has been reported (Patent Document 7).

L-glutamic acid amino acids other than L-glutamic acid, such as ornithine and citrulline (Non-patent Documents 2 to 4), L-glutamine (Patent Document 9), L-proline (Patent Document 10), and L-arginine (Patent Document) 11 and 12) are also produced by fermentation using microorganisms as described above, like L-glutamic acid.

The technique for improving the L-glutamic acid-producing ability as described above mainly enhances the activity of glycolytic and TCA cycle enzymes. However, in the L- glutamic acid production via glycolysis and the TCA cycle, to accompany decarboxylation by pyruvate dehydrogenase and isocitrate dehydrogenase, the CO ₂ of always one molecule to generate a one molecule of L- glutamic acid Released. Therefore, in order to further improve the productivity of L-glutamic acid, it is considered necessary to reduce this decarboxylation.

As an attempt to reduce decarboxylation and increase the ability to produce L-glutamic acid, for example, a method using D-xylulose-5-phosphate phosphoketolase and / or fructose-6-phosphate phosphoketolase (Patent Document 13); α- Method using ketoglutarate synthase (Patent Document 14); Method using malate thiokinase, malyl CoA lyase, glyoxylate carboligase, and 2-hydroxy-3-oxopropionate reductase (Patent Document 15); glyoxylate cycle (Patent Document 16) has been reported.

U.S. Pat.No. 3,220,929 U.S. Pat.No. 3,563,857 Japanese Patent Publication No. 32-9393 JP-A-5-244970 Japanese Unexamined Patent Publication No. 2000-106869 JP 2000-189169 A JP 2000-189175 A Japanese Patent Publication No.7-121228 JP 2002-300887 A European Patent No. 1172433 JP 2000-287693 A JP 2001-046082 A WO2006 / 016705 pamphlet WO2008 / 114721 pamphlet WO2013 / 018734 pamphlet WO2011 / 099006 pamphlet

An object of the present invention is to develop a novel technique for improving the ability of microorganisms to produce a target substance and to provide an efficient method for producing the target substance.

As a result of intensive studies to solve the above problems, the present inventors modify microorganisms so that the activities of an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase are increased. As a result, it was found that the ability to produce target substances of microorganisms can be improved, and the present invention has been completed.

That is, the present invention can be exemplified as follows.
[1]
A method for producing a target substance, comprising:
Culturing a microorganism having the ability to produce a target substance in a medium to produce and accumulate the target substance in the medium or in the cells of the microorganism, and collecting the target substance from the medium or the cells,
The microorganism has been modified to increase the activity of an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase activity, and isocitrate lyase activity;
The method, wherein the target substance is a substance biosynthesized with isocitrate as a precursor.
[2]
The method, wherein the enzyme that synthesizes malyl CoA from L-malate is one or more enzymes selected from the group consisting of malate thiokinase, succinyl CoA synthase, and succinyl CoA: malate CoA transferase.
[3]
The above method having one or more characteristics selected from the group consisting of the following (A) to (C):
(A) by increasing the expression of a gene encoding an enzyme that synthesizes malyl-CoA from L-malate, the activity of the enzyme that synthesizes malyl-CoA from L-malate was increased;
(B) Increased expression of the gene encoding malyl-CoA lyase increased malyl-CoA lyase activity;
(C) Isocitrate lyase activity was increased by increasing the expression of the gene encoding isocitrate lyase.
[4]
The method, wherein the expression of the gene is increased by increasing the copy number of the gene and / or modifying the expression regulatory sequence of the gene.
[5]
The expression of the gene encoding malate thiokinase has increased,
The gene encoding malate thiokinase is one or more genes selected from the group consisting of the mtkA gene and the mtkB gene;
The mtkA gene is DNA selected from the group consisting of (A) to (D) below:
The above method, wherein the mtkB gene is DNA selected from the group consisting of the following (E) to (H):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 76, 80, or 84;
(B) an amino acid sequence represented by SEQ ID NO: 76, 80, or 84, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and a complex with other subunits DNA encoding a subunit that forms and exhibits malate thiokinase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 75, 79, or 83;
(D) A base sequence complementary to the base sequence shown in SEQ ID NO: 75, 79, or 83 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and complexed with other subunits A DNA encoding a subunit that forms and exhibits malate thiokinase activity;
(E) a DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 78, 82, or 86;
(F) an amino acid sequence represented by SEQ ID NO: 78, 82, or 86, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and a complex with other subunits DNA encoding a subunit that forms and exhibits malate thiokinase activity;
(G) a DNA comprising the base sequence represented by SEQ ID NO: 77, 81, or 85;
(H) a base sequence complementary to the base sequence shown in SEQ ID NO: 77, 81, or 85 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and complexed with other subunits A DNA encoding a subunit that forms and exhibits malate thiokinase activity;
[6]
The expression of the gene encoding succinyl-CoA synthase has increased,
The gene encoding succinyl CoA synthase is one or more genes selected from the group consisting of sucC gene and sucD gene;
The sucC gene is a DNA selected from the group consisting of the following (A) to (D):
The above method, wherein the sucD gene is DNA selected from the group consisting of (E) to (H) below:
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 88, 92, 96, or 184;
(B) an amino acid sequence represented by SEQ ID NO: 88, 92, 96, or 184, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and combined with other subunits DNA encoding a subunit that forms a body and exhibits succinyl-CoA synthase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 87, 91, 95, or 183;
(D) a base sequence complementary to the base sequence shown in SEQ ID NO: 87, 91, 95, or 183 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and with other subunits DNA encoding a subunit that forms a complex and exhibits succinyl-CoA synthase activity;
(E) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 90, 94, 98, or 186;
(F) an amino acid sequence represented by SEQ ID NO: 90, 94, 98, or 186, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and combined with other subunits DNA encoding a subunit that forms a body and exhibits succinyl-CoA synthase activity;
(G) a DNA comprising the base sequence represented by SEQ ID NO: 89, 93, 97, or 185;
(H) a base sequence complementary to the base sequence shown in SEQ ID NO: 89, 93, 97, or 185 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and with other subunits DNA encoding a subunit that forms a complex and exhibits succinyl-CoA synthase activity.
[7]
The expression of the gene encoding succinyl CoA: malate CoA transferase has increased,
The gene encoding the succinyl CoA: malate CoA transferase is one or more genes selected from the group consisting of smtA gene, smtB gene, and smt gene;
The smtA gene is a DNA selected from the group consisting of (A) to (D) below:
The smtB gene is DNA selected from the group consisting of (E) to (H) below:
The above method, wherein the smt gene is DNA selected from the group consisting of (I) to (L) below:
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 100 or 104;
(B) in the amino acid sequence shown in SEQ ID NO: 100 or 104, comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids, and forming a complex with other subunits Succinyl CoA: a DNA encoding a subunit exhibiting malate CoA transferase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 99 or 103;
(D) It hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 99 or 103 or a probe that can be prepared from the complementary sequence, and forms a complex with other subunits. A DNA encoding a subunit exhibiting succinyl CoA: malate CoA transferase activity;
(E) a DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 102 or 106;
(F) an amino acid sequence represented by SEQ ID NO: 102 or 106, which comprises an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and forms a complex with other subunits Succinyl CoA: a DNA encoding a subunit exhibiting malate CoA transferase activity;
(G) a DNA comprising the base sequence represented by SEQ ID NO: 101 or 105;
(H) It hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 101 or 105 or a probe that can be prepared from the complementary sequence, and forms a complex with other subunits. A DNA encoding a subunit exhibiting succinyl CoA: malate CoA transferase activity;
(I) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 108 or 110;
(J) a protein comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in SEQ ID NO: 108 or 110, and having succinyl CoA: malate CoA transferase activity The encoding DNA;
(K) a DNA comprising the base sequence represented by SEQ ID NO: 107 or 109;
(L) a protein that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 107 or 109 or a probe that can be prepared from the complementary sequence and that has succinyl CoA: malate CoA transferase activity DNA encoding
[8]
The method, wherein the gene encoding the malyl-CoA lyase is DNA selected from the group consisting of the following (A) to (D):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 112, 114, or 116;
(B) a protein having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence shown in SEQ ID NO: 112, 114, or 116, and having malyl-CoA lyase activity The encoding DNA;
(C) DNA comprising the base sequence shown in SEQ ID NO: 111, 113, or 115;
(D) a protein that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 111, 113, or 115, or a probe that can be prepared from the base sequence, and that has malyl-CoA lyase activity DNA encoding
[9]
The above method, wherein the gene encoding isocitrate lyase is DNA selected from the group consisting of the following (A) to (D):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 118, 120, 122, 168, 170, or 172;
(B) the amino acid sequence shown in SEQ ID NO: 118, 120, 122, 168, 170, or 172, comprising an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions or additions, and isocitrate DNA encoding a protein having lyase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 117, 119, 121, 167, 169, or 171;
(D) hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 117, 119, 121, 167, 169, or 171 or a probe that can be prepared from the complementary sequence, and DNA encoding a protein having acid lyase activity.
[10]
The method, wherein the microorganism retains a gene encoding a mutant succinyl CoA synthase, thereby increasing succinyl CoA synthase activity and / or malate thiokinase activity.
[11]
The gene encoding the mutant succinyl CoA synthase is a succinyl CoA synthase having a mutation corresponding to one or more mutations selected from the group consisting of the following (A) to (E) in the wild type succinyl CoA synthase: Said method, which is a gene encoding:
(A) a mutation in which proline at position 124 in SEQ ID NO: 90 is substituted with alanine;
(B) a mutation in which tyrosine at position 157 in SEQ ID NO: 90 is substituted with glycine;
(C) a mutation in which valine at position 161 in SEQ ID NO: 90 is substituted with alanine;
(D) a mutation in which glutamic acid at position 97 in SEQ ID NO: 90 is substituted with aspartic acid;
(E) A mutation in which glycine at position 271 in SEQ ID NO: 88 is substituted with alanine.
[12]
The method as described above, wherein the microorganism is further modified so that malate synthase activity is decreased.
[13]
The method, wherein the microorganism is further modified to enhance the recruitment pathway of the TCA cycle.
[14]
The target substance biosynthesized with isocitrate as a precursor is composed of L-glutamic acid, L-glutamine, L-proline, L-arginine, L-ornithine, L-citrulline, itaconic acid, and γ-aminobutyric acid Said method is one or more substances selected from.
[15]
The method, wherein the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.
[16]
The method, wherein the microorganism is a coryneform bacterium or a bacterium belonging to the family Enterobacteriaceae.
[17]
The method, wherein the coryneform bacterium is Corynebacterium glutamicum.
[18]
The method as described above, wherein the bacterium belonging to the family Enterobacteriaceae is Pantoea ananatis or Escherichia coli.

Hereinafter, the present invention will be described in detail.

<1> Microorganism of the Present Invention The microorganism of the present invention has the ability to produce a target substance and has increased activity of an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase activity, and isocitrate lyase activity. It is a microorganism modified so as to.

<1-1> Microorganism having ability to produce target substance In the present invention, the “target substance” is a substance biosynthesized with isocitrate as a precursor. Substances biosynthesized with isocitrate as a precursor include L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, L-arginine, itaconic acid, and γ-aminobutyric (gamma-aminobutyric acid; GABA). In the present invention, all amino acids are L-amino acids unless otherwise specified. In the present invention, one kind of target substance may be produced, or two or more kinds of target substances may be produced.

“The ability to produce a target substance” refers to the ability to produce and accumulate in a cell or medium to such an extent that the target substance can be recovered from the cell or medium when the microorganism of the present invention is cultured in the medium. The microorganism having the ability to produce the target substance may be a microorganism that can accumulate a larger amount of the target substance in the medium than the unmodified strain. Non-modified strains include wild strains and parent strains. The microorganism having the ability to produce the target substance may be a microorganism capable of accumulating the target substance in an amount of preferably 0.5 g / L or more, more preferably 1.0 g / L or more. The target substance produced by the microorganism of the present invention may be one kind, or two or more kinds.

Examples of microorganisms include bacteria and yeasts. Of these, bacteria are preferred.

Examples of the bacteria include bacteria belonging to the family Enterobacteriaceae and coryneform bacteria. Examples of the bacterium include Alicyclobacillus genus bacteria and Bacillus genus bacteria.

The bacteria belonging to the family Enterobacteriaceae include Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photolabdas Examples include bacteria belonging to genera such as (Photorhabdus), Providencia, Salmonella, Morganella, and the like. Specifically, according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) Bacteria classified in the family Enterobacteriaceae can be used.

The Escherichia bacterium is not particularly limited, but includes bacteria classified into the genus Escherichia by classification known to microbiologists. Examples of Escherichia bacteria include, for example, Neidhardt et al. (Backmann, B. J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F. D. Nehard (ed.), “Escherichia, coli, and Salmonella, Cellular, and Molecular, Biology / Second Edition, American, Society, for Microbiology, Press, Washington, DC). Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Specific examples of Escherichia coli include Escherichia coli W3110 (ATCC11027325) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild-type strain K12.

The bacteria belonging to the genus Enterobacter are not particularly limited, but include bacteria classified into the genus Enterobacter by classification known to microbiologists. Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Specific examples of Enterobacter agglomerans include the Enterobacter agglomerans ATCC12287 strain. Specific examples of Enterobacter aerogenes include Enterobacter aerogenes ATCC13048, NBRC12010 (BiotechonolonBioeng.eng2007 Mar 27; 98 (2) 340-348), AJ110637 (FERM BP-10955) . Examples of Enterobacter bacteria include those described in European Patent Application Publication No. EP0952221. Some Enterobacter agglomerans are classified as Pantoea agglomerans.

The Pantoea bacterium is not particularly limited, and examples include bacteria classified into the Pantoea genus by classification known to microbiologists. Examples of the genus Pantoea include Pantoea 、 ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specifically, for example, Pantoea Ananatis LMG20103, Pantoea Ananatis AJ13355 (FERM13BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), SC17 (FERM BP-11091) and SC17 (0) strain (VKPM B-9246). Certain types of Enterobacter agglomerans were recently reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc. based on 16S rRNA nucleotide sequence analysis (Int. J. Syst. Bacteriol) ., 43, 162-173 (1993)). In the present invention, the Pantoea bacterium also includes a bacterium reclassified as Pantoea in this way.

Examples of the genus Erwinia include Erwinia amylovora and Erwinia carotovora. Examples of Klebsiella bacteria include Klebsiella planticola.

Examples of coryneform bacteria include bacteria belonging to genera such as Corynebacterium genus, Brevibacterium genus, and Microbacterium genus.

Specific examples of coryneform bacteria include the following species.
Corynebacterium acetoacidophilum
Corynebacterium acetoglutamicum
Corynebacterium alkanolyticum
Corynebacterium callunae
Corynebacterium glutamicum
Corynebacterium lilium
Corynebacterium melassecola
Corynebacterium thermoaminogenes (Corynebacterium efficiens)
Corynebacterium herculis
Brevibacterium divaricatum
Brevibacterium flavum
Brevibacterium immariophilum
Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum
Brevibacterium saccharolyticum
Brevibacterium thiogenitalis
Corynebacterium ammoniagenes (Corynebacterium stationis)
Brevibacterium album
Brevibacterium cerinum
Microbacterium ammoniaphilum

Specific examples of coryneform bacteria include the following strains.
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium alkanolyticum ATCC 21511
Corynebacterium callunae ATCC 15991
Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734
Corynebacterium lilium ATCC 15990
Corynebacterium melassecola ATCC 17965
Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC 13868
Brevibacterium divaricatum ATCC 14020
Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC 14068
Brevibacterium lactofermentum ATCC 13869
Brevibacterium roseum ATCC 13825
Brevibacterium saccharolyticum ATCC 14066
Brevibacterium thiogenitalis ATCC 19240
Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872
Brevibacterium album ATCC 15111
Brevibacterium cerinum ATCC 15112
Microbacterium ammoniaphilum ATCC 15354

In addition, the corynebacteria belonging to the genus Brevibacterium has been classified as a genus of corynebacteria, but bacteria integrated into the genus corynebacteria (Int. J. Syst. Bacteriol., 41, 255 (1991)) are also available. included. Corynebacterium stationis, which was previously classified as Corynebacterium ammoniagenes, includes bacteria that have been reclassified as Corynebacterium stationis by 16S rRNA sequencing (Int. J Syst. Evol. Microbiol., 60, 874-879 (2010)).

Although it does not restrict | limit especially as a bacterium of the genus Bacillus, The bacterium classified into the genus Bacillus by the classification | category known to the expert of microbiology is mentioned. Examples of Bacillus bacteria include the following species.
Bacillus subtilis
Bacillus amyloliquefaciens
Bacillus pumilus
Bacillus licheniformis
Bacillus megaterium
Bacillus brevis
Bacillus polymixa
Bacillus stearothermophilus

Specific examples of Bacillus subtilis include Bacillus subtilis 168 Marburg strain (ATCC 6051) and Bacillus subtilis PY79 strain (Plasmid, 1984, 12, 1-9). Specific examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T strain (ATCC 842 23842) and Bacillus amyloliquefaciens N strain (ATCC 23845).

These strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States 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.

The microorganism of the present invention may be inherently capable of producing a target substance, or may be modified so as to have the ability to produce a target substance. A microorganism having the ability to produce a target substance can be obtained, for example, by imparting the ability to produce the target substance to the microorganism as described above, or by enhancing the ability to produce the target substance of the microorganism as described above. .

Giving or enhancing the ability to produce a target substance can be performed by a method that has been conventionally employed for breeding amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (Amino Acid Fermentation, Society Publishing Center, Inc., 1986). (May 30, 1st edition issued, see pages 77-100). Examples of such methods include acquisition of auxotrophic mutant strains, acquisition of analog-resistant strains of the target substance, acquisition of metabolic control mutant strains, and recombinant strains with enhanced activity of the target substance biosynthesis enzyme. Creation is mentioned. In the breeding of the target substance-producing bacterium, the auxotrophy, analog resistance, metabolic control mutation and other properties imparted may be single, or two or more. In addition, the target substance biosynthetic enzyme whose activity is enhanced in breeding the target substance-producing bacteria may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with enhancing the activity of biosynthetic enzymes.

An auxotrophic mutant, an analog resistant strain, or a metabolically controlled mutant having the ability to produce the target substance is subjected to normal mutation treatment of the parent strain or wild strain, and the auxotrophic, analog It can be obtained by selecting a substance that exhibits resistance or metabolic control mutation and has the ability to produce a target substance. Normal mutation treatments include X-ray and ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), etc. Treatment with a mutagen is included.

Also, the production ability of the target substance can be imparted or enhanced by enhancing the activity of an enzyme involved in the biosynthesis of the target substance. Enhancing enzyme activity can be performed, for example, by modifying a microorganism so that expression of a gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like. A detailed method for enhancing the enzyme activity will be described later.

Further, the production ability of the target substance can be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target substance to produce a compound other than the target substance. The “enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target substance to produce a compound other than the target substance” includes an enzyme involved in the decomposition of the target substance. A method for reducing the enzyme activity will be described later.

Hereinafter, specific examples of the target substance-producing bacteria and the method for imparting or enhancing the target substance-producing ability will be given. In addition, the modification | reformation for providing or enhancing the property which the microbe producing the target substance and the ability to produce the target substance exemplified below may be used singly or in combination as appropriate.

<L-glutamic acid producing bacteria>
Examples of the method for imparting or enhancing L-glutamic acid-producing ability include a method of modifying a microorganism so that the activity of one or more enzymes selected from L-glutamic acid biosynthetic enzymes is increased. . Such enzymes include, but are not limited to, glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconite hydratase (acnA, acnB), citrate synthase (GltA), methyl citrate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate kinase (pykA, pykF), pyruvate dehydrogenase (aceEF, lpdA), phosphoenolpyruvate Synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisli Acid aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda) ), Transhydrogenase and the like. The parentheses after the enzyme name are gene names (the same applies to the following description). Among these enzymes, it is preferable to enhance the activity of one or more enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase.

Strains belonging to the family Enterobacteriaceae that have been modified to increase expression of the citrate synthase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A, and EP952221A Can be mentioned. Examples of strains belonging to the family Enterobacteriaceae that have been modified to increase the expression of the Entner-Doudoroff pathway genes (edd, eda) include those disclosed in EP1352966B. Examples of coryneform bacteria modified to increase the expression of the glutamate synthetase gene (gltBD) include those disclosed in WO99 / 07853.

Examples of the method for imparting or enhancing L-glutamic acid-producing ability include, for example, one selected from an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid. The method of modifying microorganisms so that the activity of the above enzyme may fall is also mentioned. Examples of such enzymes include, but are not limited to, α-ketoglutarate dehydrogenase (sucA, odhA), succinate dehydrogenase (sdhABCD), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxyacid synthase (ilvG ), Acetolactate synthase (such as ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamate decarboxylase (gadAB), 1-pyrroline-5-carboxylate dehydrogenase (putA), etc. Can be mentioned. Among these enzymes, for example, it is preferable to reduce or eliminate α-ketoglutarate dehydrogenase activity.

Methods for deleting or reducing the α-ketoglutarate dehydrogenase activity in Escherichia bacteria are described in JP-A-5-244970 and JP-A-7-203980. A method for deleting or reducing the α-ketoglutarate dehydrogenase activity in coryneform bacteria is described in WO95 / 34672. Furthermore, a method for deleting or reducing α-ketoglutarate dehydrogenase activity in enteric bacteria such as Pantoea bacteria, Enterobacter bacteria, Klebsiella bacteria, Erwinia bacteria, and the like is disclosed in US Pat. No. 6,1975,592, US Pat. This is disclosed in US Pat. No. 6,314,419 and US Pat. No. 8,812,151. Further, a method for reducing α-ketoglutarate dehydrogenase activity and succinate dehydrogenase activity in coryneform bacteria and Pantoea bacteria is disclosed in WO2008 / 075483.

For example, to reduce the α-ketoglutarate dehydrogenase activity, the sucA (odhA) gene encoding the E1o subunit of the enzyme may be modified. Examples of strains with reduced α-ketoglutarate dehydrogenase activity include the following strains.
Brevibacterium lactofermentum strain ΔS (International pamphlet No. 95/34672)
Brevibacterium lactofermentum AJ12821 (FERM BP-4172; see French patent publication 9401748)
Brevibacterium flavum AJ12822 (FERM BP-4173; see French Patent No. 9401748)
Corynebacterium glutamicum AJ12823 (FERM BP-4174; see French Patent Publication 9401748)
Corynebacterium glutamicum ATCC13869 OAGN, OA2-2, OAGN2-2 (see International Publication Pamphlet 2006/028298)
Escherichia coli W3110sucA :: Kmr
Escherichia coli AJ12624 (FERM BP-3853)
Escherichia coli AJ12628 (FERM BP-3854)
Escherichia coli AJ12949 (FERM BP-4881)
Brevibacterium lactofermentum strain ΔS (see pamphlet of International Publication No. 95/34672)
Pantoea Ananatis AJ13601 (FERM BP-7207 European Patent Publication No. 1078989)
Pantoea Ananatis AJ13356 (FERM BP-6615 US Patent 6,331,419)
Pantoea Ananatis SC17sucA (FERM BP-8646 WO2005 / 085419)
Klebsiella Planticola AJ13410 (FERM BP-6617 U.S. Patent No. 6,197,559)

In addition, examples of L-glutamic acid-producing bacteria or parent strains for inducing them include Pantoea ananatis AJ13355 strain (FERM-6BP-6614) and SC17 strain (FERM BP-11091). The AJ13355 strain is a strain isolated as a strain capable of growing on a medium containing L-glutamic acid and a carbon source at low pH from soil in Iwata City, Shizuoka Prefecture. The SC17 strain is a strain selected from the AJ13355 strain as a low mucus production mutant (US Pat. No. 6,596,517). On February 4, 2009, SC17 shares were registered at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently the National Institute of Technology and Evaluation, Patent Biological Depositary Center, Postal Code: 292-0818, Address: Kisarazu City, Chiba Prefecture) Deposited at Kazusa Kamashika 2-5-8 (room 120) and given the accession number FERM BP-11091. Pantoea Ananatis AJ13355 was founded on February 19, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Biological Depositary Center, Postal Code: 292-0818, Address: Kisarazu City, Chiba Prefecture Kazusa Kamashika 2-5-8 ず 120) was deposited under the deposit number FERM P-16644, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6614. Yes.

In addition, examples of L-glutamic acid-producing bacteria or parent strains for inducing them include bacteria belonging to the genus Pantoea in which α-ketoglutarate dehydrogenase (αKGDH) activity is deficient or reduced. Such strains include AJ13356 (US Pat. No. 6,331,419) which is a deletion of the αKGDH-E1 subunit gene (sucA) of AJ13355 strain, and SC17sucA (US Pat. No. 6,596,517) which is a sucA gene deletion strain of SC17 strain. ). AJ13356 was founded on February 19, 1998 at the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Patent Biological Deposit Center, Postal Code: 292-0818, Address: Kazusa-Kama, Kisarazu City, Chiba Prefecture No. 2-5-8 120) was deposited under the accession number FERM P-16645, transferred to an international deposit under the Budapest Treaty on 11 January 1999, and assigned the accession number FERM BP-6616. The SC17sucA strain was also granted the private number AJ417. On February 26, 2004, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently the National Institute of Technology and Evaluation, Patent Biological Depositary Center, ZIP Code: 292 -0818, Address: 2-5-8 120, Kazusa-Kamashita, Kisarazu City, Chiba Prefecture), deposited under the accession number FERM BP-08646.

AJ13355 was identified as Enterobacter agglomerans at the time of its isolation, but has recently been reclassified as Pantoea Ananatis by 16S rRNA sequencing. Therefore, AJ13355, AJ13356, and AJ13601 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification.

Moreover, examples of L-glutamic acid-producing bacteria or parent strains for inducing them include SC17sucA / RSFCPG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain. The SC17sucA / RSFCPG + pSTVCB strain is different from the SC17sucA strain in that the plasmid RSFCPG containing the citrate synthase gene (gltA), the phosphoenolpyruvate carboxylase gene (ppsA), and the glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum. The AJ13601 strain was selected from the SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain. On August 18, 1999, AJ13601 shares were submitted to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Patent Biological Depositary Center, zip code: 292-0818, address: Kazusa-kama, Kisarazu City, Chiba Prefecture. No. 2-5-8 120) was deposited under the accession number FERM P-17516, transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the accession number FERM BP-7207.

Examples of L-glutamic acid-producing bacteria or parent strains for inducing them include strains in which both α-ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity are reduced or deficient (JP 2010) -041920). Specific examples of such strains include, for example, a pantoea ananatis NA1 sucAsdhA double-deficient strain and a Corynebacterium glutamicum 140ATCC14067 odhAsdhA double-deficient strain (Corynebacterium glutamicum 8L3GΔSDH strain) (Japanese Patent Laid-Open No. 2010-041920).

In addition, examples of L-glutamic acid-producing bacteria or parent strains for inducing them include auxotrophic mutants. Specific examples of the auxotrophic mutant include E. coli VL334thrC ⁺ (VKPM B-8961) (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotroph having a mutation in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). E. coli VL334thrC ⁺ is an L-isoleucine-requiring L-glutamic acid-producing bacterium obtained by introducing a wild type allele of the thrC gene into VL334. The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K12 strain (VKPM B-7).

In addition, examples of L-glutamic acid-producing bacteria or parent strains for inducing them also include strains resistant to aspartic acid analogs. These strains may be deficient in α-ketoglutarate dehydrogenase activity, for example. Specific examples of strains resistant to aspartate analogs and lacking α-ketoglutarate dehydrogenase activity include, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5,908,768), and L-glutamic acid. E. coli FFRM P-12379 (US Pat. No. 5,393,671) and E. coli AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) are known.

As a method for imparting or enhancing L-glutamic acid-producing ability, for example, a bacterium is modified so that the activity of D-xylulose-5-phosphate-phosphoketolase and / or fructose-6-phosphate phosphoketolase is increased. There is also a method to do (Special Table 2008-509661). Either one or both of D-xylulose-5-phosphate-phosphoketolase activity and fructose-6-phosphate phosphoketolase activity may be enhanced. In the present specification, D-xylulose-5-phosphate phosphoketolase and fructose-6-phosphate phosphoketolase may be collectively referred to as phosphoketolase.

D-xylulose-5-phosphate-phosphoketolase activity is the consumption of phosphoric acid to convert xylulose-5-phosphate into glyceraldehyde-3-phosphate and acetyl phosphate, and one molecule of H ₂ O Means the activity of releasing. This activity is measured by the method described in Goldberg, M. et al. (Methods Enzymol., 9,515-520 (1966)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.

In addition, fructose-6-phosphate phosphoketolase activity means that phosphoric acid is consumed, fructose 6-phosphate is converted into erythrose-4-phosphate and acetyl phosphate, and one molecule of H ₂ O is released. Means activity. This activity is measured by the method described in Racker, E (Methods Enzymol., 5, 276-280 (1962)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.

In addition, as a method for imparting or enhancing L-glutamic acid-producing ability, for example, an yhfK gene (WO2005 / 085419) or a ybjL gene (WO2008 / 133161), which are L-glutamic acid excretion genes, can be mentioned.

Examples of methods for imparting or enhancing L-glutamic acid-producing ability for coryneform bacteria include methods for imparting resistance to organic acid analogs and respiratory inhibitors, and methods for imparting sensitivity to cell wall synthesis inhibitors. It is done. For example, a method of imparting monofluoroacetic acid resistance (Japanese Patent Laid-Open No. 50-113209), a method of imparting adenine resistance or thymine resistance (Japanese Patent Laid-Open No. 57-065198), and a method of weakening urease (Japanese Patent Laid-Open No. 52-038088) , A method of imparting resistance to malonic acid (Japanese Patent Laid-Open No. 52-038088), a method of imparting resistance to benzopyrones or naphthoquinones (Japanese Patent Laid-Open No. 56-1889), a method of imparting HOQNO resistance (Japanese Patent Laid-Open No. 140895), method for imparting resistance to α-ketomalonic acid (Japanese Patent Laid-Open No. 57-2689), method for imparting resistance to guanidine (Japanese Patent Laid-Open No. 56-35981), method for imparting sensitivity to penicillin (Japanese Patent Laid-Open No. 4-88994) Etc.

Specific examples of such resistant bacteria include the following strains.
Brevibacterium flavum AJ3949 (FERM BP-2632: see JP-A-50-113209)
Corynebacterium glutamicum AJ11628 (FERM P-5736; see JP 57-065198)
Brevibacterium flavum AJ11355 (FERM P-5007; see JP 56-1889)
Corynebacterium glutamicum AJ11368 (FERM P-5020; see JP 56-1889)
Brevibacterium flavum AJ11217 (FERM P-4318; see JP-A-57-2689)
Corynebacterium glutamicum AJ11218 (FERM P-4319; see JP-A-57-2689)
Brevibacterium flavum AJ11564 (FERM P-5472; see JP 56-140895 A)
Brevibacterium flavum AJ11439 (FERM P-5136; see JP 56-35981 A)
Corynebacterium glutamicum H7684 (FERM BP-3004; see JP 04-88994 A)
Brevibacterium lactofermentum AJ11426 (FERM P-5123; see JP-A-56-048890)
Corynebacterium glutamicum AJ11440 (FERM P-5137; see JP-A-56-048890)
Brevibacterium lactofermentum AJ11796 (FERM P-6402; see JP-A-58-158192)

Examples of a method for imparting or enhancing L-glutamic acid producing ability for coryneform bacteria include a method for enhancing expression of the yggB gene and a method for introducing a mutant yggB gene having a mutation introduced into the coding region ( WO2006 / 070944). The yggB gene encodes a mechanosensitive channel. The yggB gene of Corynebacterium glutamicum ATCC13032 corresponds to a complementary sequence of the sequences 1,336,091 to 1,337,692 in the genome sequence registered in the NCBI database under GenBank Accession No. NC_003450, and is also called NCgl1221. The YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession No. NP_600492. In addition, the nucleotide sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the YggB protein encoded by the same gene are shown in SEQ ID NOs: 250 and 251, respectively.

Examples of the mutant yggB gene used herein include the yggB gene having the following mutations. The YggB protein encoded by the mutant yggB gene is also referred to as a mutant YggB protein. The yggB gene not having the mutation and the YggB protein encoded by the same gene are also referred to as a wild-type yggB gene and a wild-type YggB protein, respectively. Examples of the wild type YggB protein include a protein having the amino acid sequence shown in SEQ ID NO: 251.

(1) C-terminal side mutation The C-terminal side mutation is a mutation introduced into a part of the base sequence of the region encoding the sequence of amino acid numbers 419 to 533 of SEQ ID NO: 251. The C-terminal mutation is not particularly limited as long as the mutation is introduced into at least a part of the base sequence of the above region, but preferably has an insertion sequence (hereinafter also referred to as “IS”) or a transposon inserted therein. The C-terminal mutation may be any of those accompanied by amino acid substitution (missense mutation), those having a frameshift mutation introduced by insertion of the IS or the like, and those having a nonsense mutation introduced.

Examples of the C-terminal mutation include a mutation (2A-1 type mutation) in which a base sequence is inserted at a position encoding the 419th valine residue of the wild type YggB protein. The 2A-1 type mutation may cause, for example, deletion or substitution of some or all of amino acid residues at positions 419 to 533 of the wild type YggB protein. Specifically, as a mutant yggB gene having a 2A-1 type mutation, for example, IS is inserted after “G” at position 1255 of SEQ ID NO: 250, and the original wild-type YggB protein (SEQ ID NO: 251) is inserted. Also, a yggB gene encoding a mutant YggB protein having a short full-length 423 amino residues can be mentioned (Japanese Patent Laid-Open No. 2007-222163).

Also, examples of the C-terminal mutation include a mutation that substitutes a proline residue existing at positions 419 to 533 of the wild type YggB protein with another amino acid. Such proline residues include wild-type YggB protein at positions 424, 437, 453, 457, 462, 469, 484, 489, 497, 515, 529, and 533. Of proline residues.

(2) Mutation of transmembrane region It is estimated that the YggB protein encoded by the yggB gene has five transmembrane regions. In the amino acid sequence of the wild-type YggB protein of SEQ ID NO: 251, the transmembrane regions are amino acid numbers 1 to 23 (first transmembrane region), 25 to 47 (second transmembrane region), and 62 to 84 (third membrane), respectively. This corresponds to the region of through region), 86 to 108 (fourth membrane penetration region), and 110 to 132 (fifth membrane penetration region). The yggB gene may have a mutation in the region encoding these transmembrane regions. The mutation in the transmembrane region is preferably a mutation including substitution, deletion, addition, insertion or inversion of one or several amino acids, and is not accompanied by a frameshift mutation and a nonsense mutation. As a mutation in the transmembrane region, one or several amino acids (for example, Cys-Ser-Leu) are inserted between the leucine residue at position 14 and the tryptophan residue at position 15 in the amino acid sequence shown in SEQ ID NO: 251. A mutation that replaces the alanine residue at position 100 with another amino acid residue (for example, an amino acid having a hydroxyl group in the side chain (Thr, Ser, or Tyr), preferably Thr), an alanine residue at position 111 And other amino acid residues (for example, amino acids having a hydroxyl group in the side chain (Thr, Ser, or Tyr), preferably Thr), and the like. As a mutant yggB gene having such a transmembrane region mutation, specifically, for example, a yggB gene (A1 mutation) in which TTCATTGTG is inserted next to “G” at position 44 of SEQ ID NO: 250, YggB gene (19 type mutation) in which “G” at position 298 in number 250 is replaced with “A”, and yggB gene (in L30 type mutation) in which “C” at position 332 in SEQ ID NO: 250 is replaced with “T” Is mentioned.

When the wild-type YggB protein has an amino acid sequence other than the amino acid sequence shown in SEQ ID NO: 251, the mutant yggB gene is mutated into a region encoding an amino acid residue corresponding to the amino acid residue at the above position in SEQ ID NO: 251. As long as it has. In any wild type YggB protein, which amino acid residue is the “amino acid residue corresponding to the amino acid residue at the above position in SEQ ID NO: 251” is determined based on the amino acid sequence of the wildtype YggB protein and SEQ ID NO: 251. It can be determined by alignment with the amino acid sequence.

<Γ-Aminobutyric acid producing bacteria>
γ-aminobutyric acid (GABA) is produced by decarboxylation of L-glutamic acid. Therefore, in order to impart or enhance GABA production ability, for example, the above-described method for imparting or enhancing L-glutamic acid production ability can be used as appropriate. Further, the ability to produce GABA can be imparted or enhanced by modifying the microorganism so that the activity of glutamate decarboxylase is increased (Japanese Patent Laid-Open No. 2001-054390).

<L-glutamine producing bacteria>
Examples of the method for imparting or enhancing L-glutamine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-glutamine biosynthesis enzymes is increased. . Examples of such an enzyme include, but are not limited to, glutamate dehydrogenase (gdhA) and glutamine synthetase (glnA). The activity of glutamine synthetase may be enhanced by disrupting the glutamine adenylyltransferase gene (glnE) or the PII regulatory protein gene (glnB) (EP1229121).

The method for imparting or enhancing L-glutamine production ability is, for example, selected from an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamine to produce a compound other than L-glutamine. Alternatively, a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned. Such an enzyme is not particularly limited, and includes glutaminase.

Examples of L-glutamine-producing bacteria or parent strains for inducing them include coryneform bacteria (EP1229121, EP1424398) with enhanced activity of glutamate dehydrogenase (gdhA) and / or glutamine synthetase (glnA), and coryneforms with reduced glutaminase activity Type bacteria (Japanese Patent Laid-Open No. 2004-187684). The L-glutamine-producing bacterium or the parent strain for inducing it is a strain belonging to the genus Escherichia having a mutant glutamine synthetase in which the tyrosine residue at position 397 of glutamine synthetase is substituted with another amino acid residue. (US Patent Application Publication No. 2003-0148474).

Further, as a method for imparting or enhancing L-glutamine production ability for coryneform bacteria, a method for imparting 6-diazo-5-oxo-norleucine resistance (Japanese Patent Laid-Open No. 3-232497), purine analog resistance and methionine sulfoxide resistance Examples thereof include a method for imparting resistance (Japanese Patent Laid-Open No. 61-202694) and a method for imparting resistance to α-keto maleic acid (Japanese Patent Laid-Open No. 56-151495).

Specific examples of coryneform bacteria having the ability to produce L-glutamine include the following strains.
Brevibacterium flavum AJ11573 (FERM P-5492, JP 56-161495)
Brevibacterium flavum AJ11576 (FERM BP-10381, JP 56-161495)
Brevibacterium flavum AJ12212 (FERM P-8123, JP-A-61-202694)

<L-proline producing bacteria>
Examples of L-proline-producing bacteria or parent strains for inducing them include bacteria that retain γ-glutamyl kinase that has been desensitized to feedback inhibition by L-proline, and bacteria that have weakened the L-proline degradation system. A method for modifying bacteria using DNA encoding γ-glutamyl kinase desensitized to feedback inhibition by L-proline is described in Dandekar and Uratsu (J. Bacteriol. 170, 12: 5943-5945 (1988)). It is disclosed. Examples of a method for obtaining a bacterium with a weakened L-proline degradation system include a method of introducing a mutation that reduces the enzyme activity into the proline dehydrogenase gene. Specific examples of bacteria having L-proline-producing ability include Escherichia coli NRRL B-12403 strain and NRRL B-12404 strain (British Patent 2075056), Escherichia coli VKPM B-8012 strain (US Patent Publication 2002-0058315) Escherichia coli mutant carrying the plasmid disclosed in German Patent 3127361, and Escherichia coli mutant carrying the plasmid disclosed in Bloom FR et al. (The 15th Miami winter symposium, 1983, p. 34) Strains.

Specific examples of bacteria having L-proline producing ability include 3,4-dehydroxyproline, azatidine-2-carboxylate resistant strain Escherichia coli 702 (VKPMB-8011), and 702 ilvA deficiency. Examples thereof include 702ilvA strain (VKPMB-8012 strain), E.Ecoli having enhanced activity of protein encoded by b2682, b2683, b1242 or b3434 gene (Japanese Patent Laid-Open No. 2002-300874).

<L-arginine producing bacteria>
Examples of the method for imparting or enhancing L-arginine-producing ability include a method of modifying a microorganism so that the activity of one or more enzymes selected from L-arginine biosynthesis enzymes is increased. . Examples of such enzymes include, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine Examples include transaminase (argD), acetylornithine deacetylase (argE) ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH) carbamoyl phosphate synthase (carAB). As the N-acetylglutamate synthase (argA) gene, for example, 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 eliminated is used. Yes (European Application Publication No. 1170361).

Microorganisms capable of producing L-arginine include α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S- (2-aminoethyl) -cysteine, α-methylserine, β-2-thienylalanine. Or Escherichia coli mutants having resistance to sulfaguanidine (see JP-A-56-106598) and the like. The microorganism having L-arginine-producing ability includes Escherichia, which is an L-arginine-producing bacterium having a mutation resistant to feedback inhibition by L-arginine and having a highly active N-acetylglutamate synthase.・ Kori 237 strain (Russian patent application No. 2000117677) is also included. The stock was deposited on April 10, 2000 at the Russian National Collection of Industrial Microorganisms (VKPM) and GNII Genetika, under the accession number VKPM B-7925. 2001 It was transferred to an international deposit under the Budapest Treaty on May 18, 2000. Further, as microorganisms having L-arginine-producing ability, Escherichia coli 382 strain (Japanese Patent Laid-Open No. 2002-017342), which is an L-arginine-producing bacterium having improved acetic acid utilization ability, is a derivative of 237 strains. Can be mentioned. Escherichia coli 382 stock was deposited on April 10, 2000 in the Russian National Collection of Industrial Microorganisms (VKPM) under the accession number VKPM B-7926.

Microorganisms capable of producing L-arginine include coryneform bacteria wild strains; coryneform bacteria resistant to drugs such as sulfa drugs, 2-thiazolealanine or α-amino-β-hydroxyvaleric acid; In addition, coryneform bacteria having L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine or L-tryptophan requirement (Japanese Patent Laid-Open No. 54-44096); ketomalonic acid, fluoromalonic acid or Coryneform bacterium resistant to monofluoroacetic acid (JP-A-57-18989); Coryneform bacterium resistant to argininol (JP-A 62-24075); or X-guanidine (X is fatty acid or fatty chain) And coryneform bacteria (Japanese Patent Laid-Open No. 2-186995) having resistance to such derivatives. Coryneform bacteria having the ability to produce L-arginine include mutants resistant to 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, 6-azacytosine and the like; Mutants resistant to arginine hydroxamate and 2-thiouracil, mutants resistant to arginine hydroxamate and 6-azauracil (JP 49-126819); mutants resistant to histidine analog or tryptophan analog (JP No. 52-114092), a mutant having a requirement for at least one of methionion, histidine, threonine, proline, isoleucine, lysine, adenine, guanine or uracil (or uracil precursor) (see JP-A-52-99289) ); Mutants resistant to arginine hydroxamate (Japanese Patent Publication No. 51-6754); Mutants that are succinic acid-requiring or resistant to nucleobase analogs (Japanese Patent Laid-Open No. 58-9692); mutants that lack arginine resolution, are resistant to arginine antagonists and canavanine, and require lysine Arginine, arginine hydroxamate, homoarginine, D-arginine, and canavanine resistant, or arginine hydroxamate and 6-azauracil resistant mutants (JP-A-53-143288); and canavanine Examples include resistant mutant strains (Japanese Patent Laid-Open No. 53-3586).

Specific examples of coryneform bacteria having the ability to produce L-arginine include the following strains.
Brevibacterium flavum AJ11169 (FERM BP-6892)
Brevibacterium lactofermentum AJ12092 (FERM BP-6906)
Brevibacterium flavum AJ11336 (FERM BP-6893)
Brevibacterium flavum AJ11345 (FERM BP-6894)
Brevibacterium lactofermentum AJ12430 (FERM BP-2228)

Examples of L-arginine-producing bacteria or parent strains for inducing them include strains lacking ArgR, an arginine repressor (US Patent Application Publication No. 2002-0045223), and strains that have increased intracellular glutamine synthetase activity. (US Patent Application Publication No. 2005-0014236).

<L-citrulline-producing bacteria and L-ornithine-producing bacteria>
L-citrulline and L-ornithine share a biosynthetic pathway with L-arginine. Thus, N-acetylglutamate synthase (argA), N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), and / or acetylornithine By increasing the enzyme activity of deacetylase (argE), the ability to produce L-citrulline and / or L-ornithine can be imparted or enhanced (WO 2006-35831).

<Itaconic acid producing bacteria>
Examples of itaconic acid-producing bacteria include C. glutamicum R / cad002 strain (JP 2008-182936), E. coli PCI 516 strain and PCI 519 strain (both US2010 / 0285546 A1), Aspergillus terreus AtCAD strain (JP 2013-51900). ).

Further, the microorganism having the target substance-producing ability may be modified so that the activity of the protein involved in sugar metabolism or energy metabolism is increased. The activity of these proteins can be increased, for example, by increasing the expression of genes encoding these proteins.

Proteins involved in sugar metabolism include proteins involved in sugar uptake and glycolytic enzymes. Examples of genes encoding proteins involved in sugar metabolism include glucose 6-phosphate isomerase gene (pgi; WO 01/02542 pamphlet), phosphoenolpyruvate synthase gene (pps; European Patent Publication No. 877090) , Phosphoenolpyruvate carboxylase gene (ppc; WO 95/06114 pamphlet), pyruvate carboxylase gene (pyc; WO 99/18228 pamphlet, European application 1092776), phosphoglucomutase gene (Pgm; WO 03/04598 pamphlet), fructose diphosphate aldolase gene (pfkBfbp; WO 03/04664 pamphlet), pyruvate kinase gene (pykF; WO 03/008609 pamphlet), transaldolase gene ( talB; WO03 / 008611 pamphlet), fumarase gene (fum International publication 01/02545 pamphlet), non-PTS sucrose uptake gene (csc; European application publication 149911 pamphlet), sucrose utilization gene (scrAB operon; international publication 90/04636 pamphlet) .

Examples of genes encoding proteins involved in energy metabolism include a transhydrogenase gene (pntAB; US Pat. No. 5,830,716), a cytochrome bo type oxidase (cyoB; European Patent Application Publication No. 1070376) Is mentioned.

In addition, when glycerol is used as a carbon source, in order to increase the utilization of glycerol, microorganisms that have the ability to produce a target substance have a reduced expression of glpR gene (EP1715056) or glpA, glpB, glpC, glpD Modified to enhance expression of glycerol metabolic genes (EP1715055A) such as, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, tpiA, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC genes May be.

Further, the microorganism having the target substance-producing ability may be modified so that the activity of discharging the target substance from the cells of the microorganism is increased. The activity to excrete the target substance can be increased, for example, by increasing the expression of a gene encoding a protein that excretes the objective substance. For example, genes encoding proteins that excrete various amino acids include b2682 gene and b2683 gene (ygaZH gene) (EP 1239041 A2).

The gene used for breeding a microorganism having the ability to produce the target substance is not limited to the gene exemplified above or a gene having a known base sequence, as long as it encodes a protein having the original function maintained. It may be a variant. For example, in a gene used for breeding a microorganism capable of producing a target substance, one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of a known protein. It may also be a gene encoding a protein having an amino acid sequence. Regarding the variants of genes and proteins, the description regarding the enzyme for synthesizing malyl CoA from L-malic acid, malyl CoA lyase and isocitrate lyase, and conservative variants of genes encoding them can be applied mutatis mutandis.

<1-2> Enhancement of malyl-CoA pathway The microorganism of the present invention has been modified to increase the activities of the enzyme that synthesizes malyl-CoA from L-malate, malyl-CoA lyase, and isocitrate-lyase. Hereinafter, an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase may be collectively referred to as “enzyme of the present invention”. The bacterium of the present invention modifies a microorganism having the ability to produce a target substance as described above so that the activities of an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase are increased. Can be obtained. In addition, the microorganism of the present invention imparts or enhances the ability to produce a target substance after modifying the microorganism so that the activities of the enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase are increased. Can also be obtained. The microorganism of the present invention has acquired the ability to produce a target substance by being modified to increase the activities of an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase. It may be. In the present invention, the modification for constructing the microorganism of the present invention can be performed in any order.

“An enzyme that synthesizes malyl-CoA from L-malic acid” refers to a protein having an activity of catalyzing the reaction of binding L-malic acid and CoA to convert it to malyl-CoA. Enzymes that synthesize malyl-CoA from L-malate include malate thiokinase, succinyl-CoA synthase, and succinyl-CoA: malate-CoA transferase. In the present invention, the activity of one or more enzymes selected from enzymes that synthesize malyl-CoA from L-malic acid can be increased. That is, for example, the activity of any one of malate thiokinase, succinyl CoA synthase, and succinyl CoA: malate CoA transferase may be increased, or all the activities may be increased. The activity of a protein can be increased, for example, by increasing the expression of a gene encoding the protein. A detailed method for increasing the activity of the protein will be described later.

“Malatothiokinase” refers to an enzyme (EC 6.2.1.9) that reversibly catalyzes the reaction of producing malyl-CoA from L-malic acid and CoA. The activity that catalyzes this reaction is also referred to as “malate thiokinase activity”. The above reaction is known to be reversible in vivo and in vitro, that is, it is known that malate thiokinase can also catalyze the reverse reaction of the above reaction. Malate thiokinase is also called malyl-CoA synthase, malate-CoA ligase, or malyl-coenzyme A synthase.

Malate thiokinase is known to function as a complex composed of a plurality of subunits, usually a complex composed of an α subunit and a β subunit. The α subunit is encoded by the mtkB gene, and the β subunit is encoded by the mtkA gene. The mtkA gene and the mtkB gene are usually present continuously in the genome.

Genes encoding malate thiokinase are assimilated pathways of C1 carbon sources such as methane (J. Bacteriol., 176 (23), 7398-7404 (1994)) and 3-hydroxypropionic acid pathway (Arch. Microbiol., 151, 252-256 (1989)). In general, the mclA gene encoding malyl-CoA lyase described later is present in the vicinity of the mtkAB gene encoding malate thiokinase on the genome. A biological species in which the mkAB gene and the mclA gene are close to each other on the genome can be identified by, for example, NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).

Specific examples of genes encoding malate thiokinase include, for example, Methylobacterium extorquens and other Methylobacterium genus bacteria, Mesorhizobium loti and other Mesorhizobium genus bacteria, Granulibacter bacteria such as Granulibacter bethesdensis, Roseobacter bacteria such as Roseobacter denitrificans, Morella bacteria such as Moorella thermoacetica, Hierobium bacteria such as Moorella thermoacetica・ Hyphomicrobium bacteria such as Hyphomicrobium methylovorum, Chloroflexus aurantiacus bacteria such as Chloroflexus aurantiacus, Nitrosomonas europa Examples include mtbAB genes of bacteria belonging to the genus Nitrosomonas such as ea) and bacteria belonging to the genus Methylococcus capsulatus such as Methylococcus capsulatus.

The complete nucleotide sequence of the genomic DNA of Methylobacterium Extrugens AM1 strain is known (GenBank accession number NC_012808.1), and the mtkAB gene encoding the malate thiokinase of Methylobacterium Extruences AM1 strain is known. Base sequences have also been reported. That is, the mtkA gene of Methylobacterium Extrusence AM1 strain corresponds to base numbers 1803549 to 1804721 of the genome sequence of Methylobacterium Extrusence AM1 strain described in GenBank accession number NC_012808.1. Further, the mtkB gene of Methylobacterium Extrusence AM1 strain corresponds to nucleotide numbers 1804744 to 1805634 of the genome sequence of Methylobacterium Extrusence AM1 strain described in GenBank accession number NC_012808.1. The nucleotide sequence of the mtkA gene of Methylobacterium extremens AM1 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 75 and 76, respectively. The nucleotide sequence of the mtkB gene of Methylobacterum extruens AM1 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 77 and 78, respectively.

The complete nucleotide sequence of the genomic DNA of Mesozobium roti MAFF303099 strain is known (GenBank accession number NC_002678.2), and the base sequence of the mtkAB gene encoding malate thiokinase of Mesozobium roti MAFF303099 strain has also been reported. That is, the mtkA gene and mtkB gene of Mesozobium roti MAFF303099 strain correspond to the base numbers 1110720 to 1111904 and base numbers 1111919 to 1112818 of the genome sequence (GenBank accession number NC_002678.2) of the Mesozobium roti MAFF303099 strain, respectively. The nucleotide sequence of the mtkA gene of Mesozobium roti MAFF303099 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 79 and 80, respectively. The nucleotide sequence of the mtkB gene of Mesozobium loti MAFF303099 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 81 and 82, respectively.

The complete nucleotide sequence of the genomic DNA of Granulinobacter bethesdensis CGDNIH1 strain is known (GenBank accession number NC_008343.1), and the nucleotide sequence of the mtkAB gene encoding malate thiokinase of Granulibacter bethesdensis CGDNIH1 strain has also been reported. Yes. That is, the mtkA gene and the mtkB gene of the Granulibacter bethesdensis CGDNIH1 strain are the nucleotide numbers 55236 to 56405 and the base numbers 56421 to 57717 of the genomic sequence of the Granulibacter bethesdensis CGDNIH1 strain (GenBank accession number NC_008343.1), respectively. It corresponds to. The nucleotide sequence of the mtkA gene of Granulibacter bethesdensis strain CGDNIH1 and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOs: 83 and 84, respectively. SEQ ID NOs: 85 and 86 show the base sequence of the mtkB gene of Granulibacter bethesdensis strain CGDNIH1 and the amino acid sequence of the protein encoded by the gene, respectively.

As the malate thiokinase gene, any gene can be used as long as it encodes a protein that functions in the host. For example, Hyphomicrobium methylovolum, Hyphomicrobium denitrificans, Rhizobium sp. NGR234 strain, Granulibacter bethesdensis, It has been reported that genes encoding malate thiokinase from Monas europia (Nitrosomonas europaea) and Methylococcus capsulatus are expressed and function in E. coli, Pantoea ananatis, and Corynebacterium glutamicum (WO2013 / 018734).

The α subunit and β subunit of malate thiokinase have high homology with the α subunit and β subunit of succinyl CoA synthase described later. As shown in Examples described later, the present inventors have discovered that succinyl CoA synthase has malate thiokinase activity. That is, malate thiokinase activity can also be increased by increasing succinyl CoA synthase activity.

The increase in the activity of malate thiokinase can be confirmed, for example, by preparing a crude enzyme solution from a microorganism before modification and a microorganism after modification, and comparing the malate thiokinase activity. The activity of malate thiokinase can be measured, for example, according to the method of Louis (Louis B. Hersh J Biol Chem. 1973 Nov 10; 248 (21): 7295-303.). Specifically, L-malic acid is added to a reaction solution containing phenylhydrazine, CoA, ATP, malyl-CoA lyase, and a crude enzyme solution that reacts quickly with glyoxylic acid to give a color, and the resulting glyoxylate phenyl is produced. By measuring the amount of hydrazine spectrophotometrically, malate thiokinase activity can be measured. This method utilizes the fact that malyl-CoA produced by malate thiokinase is decomposed into acetyl-CoA and glyoxylic acid by malyl-CoA lyase.

“Succinyl-CoA synthase” means that succinyl-CoA is converted from succinic acid and coenzyme A (hereinafter referred to as “CoA”), with the reaction involving hydrolysis of nucleotide 3-phosphate such as ATP or GTP into nucleotide 2-phosphate and inorganic phosphate. An enzyme that catalyzes the reaction that occurs (EC 6.2.1.5 or EC 6.2.1.4). The activity that catalyzes this reaction is also referred to as “succinyl-CoA synthase activity”. It is known that the above reaction is reversible in vivo and in vitro, that is, succinyl CoA synthase is also known to be able to catalyze the reverse reaction of the above reaction. Succinyl CoA synthase is also referred to as succinyl CoA ligase, succinyl coenzyme A synthase, succinate thiokinase, succinic thiokinase, succinate phosphorylating enzyme, or P-enzyme.

Succinyl CoA synthase is known to function as a complex composed of a plurality of subunits, usually a complex composed of an α subunit and a β subunit. The α subunit is encoded by the sucD gene and the β subunit is encoded by the sucC gene. The sucC gene and sucD gene usually exist continuously on the genome.

The gene encoding succinyl CoA synthase has been recognized in various organisms. Genes encoding succinyl CoA synthase include, for example, KEGG (Kyoto Encyclopedia of Genes and Genomes; http://www.genome.jp/kegg/), NCBI (National Center for Biotechnology Information; http: //www.ncbi. nlm.nih.gov/gene/) and BRENDA (BRaunschweig ENzyme DAtabase; http://www.brenda-enzymes.info/). The succinyl-CoA synthase gene can be used without particular limitation as long as it encodes a protein that functions in the host. For example, from the viewpoint of succinyl-CoA production efficiency, a succinyl-CoA synthase gene used in the host microorganism Also good.

Specific examples of genes encoding succinyl-CoA synthase include, for example, Escherichia bacteria such as Escherichia coli, Pantoea bacteria such as Pantoea ananatis, Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium, Examples include the sucCD gene of Corynebacterium bacteria such as ammoniagenes.

The entire base sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the base sequence of the sucCD gene encoding the succinyl CoA synthase of Escherichia coli MG1655 strain has also been reported. That is, the sucC gene corresponds to base numbers 762237 to 763403 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3. The sucD gene corresponds to nucleotide numbers 763403 to 764272 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3. The nucleotide sequence of the sucC gene of Escherichia coli MG1655 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 87 and 88, respectively. The nucleotide sequence of the sucD gene of Escherichia coli MG1655 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 89 and 90, respectively.

The entire base sequence of the genomic DNA of Pantoea ananatis AJ13355 strain is known (GenBank accession number NC_017531.1), and the base sequence of the sucCD gene encoding the succinyl CoA synthase of Pantoea ananatis AJ13355 strain has also been reported. That is, the sucC gene corresponds to base numbers 610188 to 611354 of the genome sequence of Pantoea ananatis AJ13355 strain described in GenBank accession number NC_017531.1. The sucD gene corresponds to nucleotide numbers 611354 to 612229 of the genome sequence of Pantoea ananatis AJ13355 described in GenBank accession number NC_017531.1. The nucleotide sequence of the sucC gene of Pantoea ananatis AJ13355 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 91 and 92, respectively. The nucleotide sequence of the sucD gene of Pantoea ananatis AJ13355 strain and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOs: 93 and 94, respectively.

The entire nucleotide sequence of the genomic DNA of Corynebacterium glutamicum ATCC13032 is known (GenBank accession number NC_003450.3), and the nucleotide sequence of the sucCD gene encoding the succinyl CoA synthase of Corynebacterium glutamicum ATCC13032 has also been reported. Yes. That is, the sucC gene corresponds to the complementary sequence of nucleotide numbers 2723582 to 2726578 of the genome sequence of Corynebacterium glutamicum ATCC13032 described in GenBank accession number NC_003450.3. The sucD gene corresponds to the complementary sequence of nucleotide numbers 2724476 to 2725360 of the genome sequence of Corynebacterium glutamicum ATCC13032 described in GenBank accession number NC_003450.3. The nucleotide sequence of the sucC gene of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 95 and 96, respectively. The nucleotide sequence of the sucD gene of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 97 and 98, respectively. The nucleotide sequence of the sucC gene of Corynebacterium glutamicum 2256 strain (ATCC 13869) and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 183 and 184, respectively. The nucleotide sequence of the sucD gene of Corynebacterium glutamicum 2256 strain (ATCC 13869) and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 185 and 186, respectively.

In addition, succinyl CoA synthase activity and / or malate thiokinase activity may be increased by introducing a mutation into succinyl CoA synthase. Examples of the mutation that increases at least malate thiokinase activity include the following mutations.
・ A mutation where the proline at position 124 of the α subunit encoded by the sucD gene of Escherichia coli is replaced with alanine ・ A mutation where the tyrosine at position 157 of the α subunit encoded by the sucD gene of Escherichia coli is replaced with glycine Mutation in which the valine at position 161 of the α subunit encoded by the sucD gene of Escherichia coli is replaced with alanine. Glutamic acid at position 97 of the α subunit encoded by the sucD gene of Escherichia coli is replaced with aspartic acid. Mutation in which glycine at position 271 of the β subunit encoded by the sucC gene of Escherichia coli is substituted with alanine

Only one of these mutations may be introduced, or two or more thereof may be introduced. For example, a mutant succinyl CoA synthase gene in which the valine at position 161 of the α subunit encoded by the sucD gene of Escherichia coli is replaced with alanine, and the glycine at position 271 of the β subunit encoded by the sucC gene is replaced with alanine May be constructed.

The succinyl CoA synthase having no mutation is also referred to as “wild-type succinyl CoA synthase”, and the gene encoding it is also referred to as “wild-type succinyl CoA synthase gene”. The succinyl CoA synthase having the mutation is also referred to as “mutant succinyl CoA synthase” and the gene encoding it is also referred to as “mutant succinyl CoA synthase gene”.

The wild-type succinyl-CoA synthase is not limited to the wild-type succinyl-CoA synthase of Escherichia coli as exemplified above, and may be a conservative variant thereof. In addition, the position of the mutation in the above description of the mutation is relative, and the position may be moved back and forth by amino acid deletion, insertion, or addition. For example, “the valine at position 161 of the α subunit” means an amino acid residue corresponding to the valine residue at position 161 in SEQ ID NO: 90, and one amino acid residue on the N-terminal side from position 161 is deleted. In this case, the 160th amino acid residue from the N-terminus is “valine at position 161 of the α subunit”. In addition, when one amino acid residue is inserted on the N-terminal side from the 161st position, the 162nd amino acid residue from the N-terminal is assumed to be “the valine at the 161st position of the α subunit”.

The amino acid residue to be mutated in an arbitrary amino acid sequence can be determined by aligning the arbitrary amino acid sequence with the amino acid sequence of SEQ ID NO: 90 or 88. The alignment can be performed using, for example, known gene analysis software. Specific software includes DNA Solutions from Hitachi Solutions and GENETYX from Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et) al., Journal of molecular biology, 198 (2), 327-37. 1987).

The increase in the activity of succinyl-CoA synthase can be confirmed, for example, by preparing a crude enzyme solution from a microorganism before modification and a microorganism after modification, and comparing the succinyl-CoA synthase activity. The activity of succinyl-CoA synthase can be measured, for example, according to the method of Williamson (John R. Williamson, barBarbara E. Corkey Methods in Enzymology, edited by Colowich JM. New York: Academic, 1969, p. 434-514.). Specifically, succinic acid is added to a reaction solution containing CoA, ATP, phosphoenolpyruvate, pyruvate kinase, lactate dehydrogenase, NADH, and crude enzyme solution, and the amount of NADH consumed is analyzed spectroscopically. By measuring, succinyl-CoA synthase activity can be measured.

“Succinyl CoA: malate CoA transferase” refers to an enzyme (EC-2.8.3.-) that catalyzes the reaction of producing succinic acid and malyl CoA from succinyl CoA and L-malic acid. The activity that catalyzes this reaction is also referred to as “succinyl CoA: malate CoA transferase activity”. Succinyl CoA: malate CoA transferase is also referred to as succinyl CoA (S) -malate CoA transferase, or L-carnitine dehydrotase / bile acid-inducible protein family.

As succinyl CoA: malate CoA transferase, one that functions as a complex composed of a plurality of subunits is known. Such a succinyl CoA: malate CoA transferase is usually composed of a subunit encoded by the smtA gene and a subunit encoded by the smtB gene. The smtA gene and the smtB gene are usually present continuously in the genome.

As a gene encoding such a succinyl CoA: malate CoA transferase, specifically, for example, chloroflexus bacteria such as Chloroflexus aurantiacus, and Accucumubacter phosphatis, etc. The SmtAB gene and its homologue of Accubacter bacterium. The protein encoded by the smtA gene and the protein encoded by the smtB gene are highly homologous to each other. For example, the amino acid homology of the protein encoded by the smtA gene of Chloroflexus aurantiax and the protein encoded by the smtB gene Is 59%. It has been reported that the smtAB gene of Chloroflexus aurantix is expressed and functions in E. において coli (Friedmann S et al. (2006) J Bacteriol. 188 (7): 2646-55.).

Also, examples of the succinyl CoA: malate CoA transferase include those encoded by a single gene. Such succinyl CoA: malate CoA transferase is not particularly limited as long as it is an enzyme classified into CoA-transferase family III (CaiB / BaiF) and has succinyl CoA: malate CoA transferase activity.

Specific examples of a gene encoding such a succinyl CoA: malate CoA transferase include, for example, a genus Magnetospirillum bacterium such as Magnetospirillum magneticum, and a rhodospirillum rubrum such as Rhodospirillum rubrum. The smtB gene homologue of genus bacteria is mentioned. Such a gene encoding succinyl CoA: malate CoA transferase is also referred to as “smt gene”.

The entire nucleotide sequence of the genomic DNA of Chloroflexus aurantix J-10-fl strain is known (GenBank accession number NC_010175.1), and the succinyl CoA of Chloroflexus aurantix J-10-fl: Malate CoA The nucleotide sequence of smtAB gene encoding transferase (hereinafter also referred to as “Ca_smtAB gene”) has been reported. That is, the Ca_smtA gene and the Ca_smtB gene are respectively a complementary sequence of nucleotide numbers 224515 to 225882 and a complementary sequence of 223035 to 224252 of the genomic sequence of the Chloroflexus aurantix Ax J-10-fl strain (GenBank accession number NC_010175.1). It corresponds to. The nucleotide sequence of the Ca_smtA gene and the amino acid sequence (YP_001633822) of the protein encoded by the same gene are shown in SEQ ID NOs: 99 and 100, respectively. The nucleotide sequence of the Ca_smtB gene and the amino acid sequence (YP_001633821) of the protein encoded by the same gene are shown in SEQ ID NOs: 101 and 102, respectively.

The complete nucleotide sequence of the genomic DNA of Accumulactor phosphatis (candidate) clade IIAstr .. UW-1 strain (Candidatus Accumulibacter phosphatis clade IIAstr. UW-1) is known (GenBank accession number NC_013194.1). Accumubacter phosphatis (candidate) clade IIAstr .. UW-1 succinyl CoA: malate CoA transferase gene is also referred to as Ca_smtA gene and Ca_smtB homolog (hereinafter referred to as “Ap_smtA gene” and “Ap_smtB gene”, respectively) 2 are collectively referred to as “Ap_smtAB gene”). The Ap_smtA gene and the Ap_smtB gene correspond to the base numbers 2888316 to 2889563 and 2889587 to 2890813 of the genome sequence (GenBank accession number NC_013194.1) of the Accumulactor phosphatis (candidate strain) clade IIAstr .. UW-1 strain, respectively. . The nucleotide sequence of the Ap_smtA gene and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 103 and 104, respectively. The nucleotide sequence of Ap_smtB gene and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 105 and 106, respectively.

The complete nucleotide sequence of the genomic DNA of Rhodospirillum rubrum ATCC-11170 strain is known (GenBank accession number NC_007643.1). Examples of the succinyl CoA: malate CoA transferase gene of Rhodospirillum rubrum ATCC 11170 strain include a gene homologous to the Ca_smtB gene (hereinafter also referred to as “Rr_smt gene”). The Rr_smt gene corresponds to the complementary sequence of base numbers 2965790 to 2967016 of the genomic sequence of the Rhodospirillum rubrum ATCC 17011170 strain (GenBank accession number NC_007643.1). The base sequence of the Rr_smt gene and the amino acid sequence (YP_427637) of the protein encoded by the same gene are shown in SEQ ID NOs: 107 and 108, respectively.

The complete nucleotide sequence of the genomic DNA of Magnetospirillum magneticumum AMB-1 strain is known (GenBank accession number NC_007626.1). Examples of the succinyl CoA: malate CoA transferase gene of Magnetospirillum magneticumum AMB-1 include a gene homologous to the Ca_smtB gene (hereinafter also referred to as “Mm_smt gene”). The Mm_smt gene corresponds to the complementary sequence of nucleotide numbers 2307230 to 2308438 of the genome sequence of the Spirrum magnetiumum AMB-1 strain (GenBank accession number NC_007626.1). The nucleotide sequence of the Mm_smt gene and the amino acid sequence (YP_421496) of the protein encoded by the gene are shown in SEQ ID NOs: 109 and 110, respectively.

The increase in the activity of succinyl CoA: malate CoA transferase can be confirmed, for example, by preparing a crude enzyme solution from the microorganism before modification and the microorganism after modification, and comparing the succinyl CoA: malate CoA transferase activity. . The activity of succinyl CoA: malate CoA transferase can be measured, for example, according to the method of Friedmann (Friedmann Set et al. (2006) J Bacteriol. 188 (7): 2646-55.). Specifically, glyoxylate phenylhydrazine is produced by adding L-malic acid to a reaction solution containing phenylhydrazine, succinyl CoA, malyl CoA lyase, and a crude enzyme solution, which reacts with glyoxylic acid and develops color. Can be measured spectrophotometrically to determine the succinyl CoA: malate CoA transferase activity.

"Malyl CoA lyase" refers to an enzyme (EC IV.3.2.24) that reversibly catalyzes the reaction of generating acetyl CoA and glyoxylic acid from malyl CoA. The activity that catalyzes this reaction is also referred to as “malyl CoA lyase activity”. Malyl CoA lyase is also referred to as malyl coenzyme A lyase, or (3S) -3-carboxy-3-hydroxypropanoyl CoA glyoxylate lyase.

Specific examples of genes encoding malyl-CoA lyase include, for example, Methylobacterium extorquens and other Methylobacterium genus bacteria, Mesorhizobium loti and other Mesozobium genus bacteria, Granulibacter bacteria such as Granulibacterlibbethesdensis, Roseobacter bacteria such as Roseobacter denitrificans, Morellaum bacteria such as Moorella thermoacetica,・ Hyphomicrobium bacteria such as Hyphomicrobium methylovorum, Chloroflexus aurantiacus such as Chloroflexus aurantiacus, Nitrosomonas europaea Of Nitrosomonas bacteria include mclA gene Methylococcus bacteria such as Methylococcus Kyapusuratasu (Methylococcus capsulatus).

The mclA gene encoding the malyl-CoA lyase of Methylobacterium extremens AM1 is equivalent to the base numbers 1808790 to 1809764 of the genome sequence of Methylobacterium extremens AM1 described in GenBank accession number NC_012808.1 To do. The base sequence of the mclA gene of Methylobacterium extremens AM1 strain and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 111 and 112, respectively.

The mclA gene encoding the malyl-CoA lyase of Mesozobium roti MAFF303099 corresponds to nucleotide numbers 1109744 to 1110700 of the genome sequence of Mesozobium roti MAFF303099 (GenBank accession number NC_002678.2). The nucleotide sequence of the mclA gene of Mesozobium roti MAFF303099 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 113 and 114, respectively.

The DNA sequence of the mclA gene encoding the malyl-CoA lyase of Granulibacter bethesdensis CGDNIH1 strain corresponds to the base numbers 60117 to 61112 of the genomic sequence of the Granulibacter bethesdensis CGDNIH1 strain (GenBank accession number NC_008343.1). The nucleotide sequence of the mclA gene of Granulibacter bethesdensis CGDNIH1 strain and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 115 and 116, respectively.

The increase in the activity of malyl CoA lyase can be confirmed, for example, by preparing a crude enzyme solution from a microorganism before modification and a microorganism after modification, and comparing the malyl CoA lyase activity. The activity of malyl-CoA lyase can be measured, for example, according to the method of Louis (Louis B. Hersh J Biol Chem. 1973 Nov 10; 248 (21): 7295-303.). Specifically, L-malic acid is added to a reaction solution containing phenylhydrazine, CoA, ATP, malate thiokinase, and a crude enzyme solution that reacts quickly with glyoxylic acid to give a color, and the resulting glyoxylate phenyl is produced. By measuring the amount of hydrazine spectroscopically, the malyl CoA lyase activity can be measured. This method utilizes the fact that malyl-CoA produced by malate thiokinase is decomposed into acetyl-CoA and glyoxylic acid by malyl-CoA lyase. Alternatively, malyl-CoA lyase activity can be similarly measured by using malyl-CoA instead of CoA, ATP, malate thiokinase and L-malic acid.

“Isocitrate lyase” refers to an enzyme (EC IV 4.1.3.1) that reversibly catalyzes the reaction of isocitrate to produce glyoxylic acid and succinic acid. The activity that catalyzes the same reaction is also referred to as “isocitrate lyase activity”. Isocitrate lyase is also called isocytolase, isocitrate, isocitrate, threo-Ds-isocytolate glyoxylate lyase, or isocytolate glyoxylate lyase.

The gene encoding isocitrate lyase has been recognized in various organisms. Examples of genes encoding isocitrate lyase include KEGG (Kyoto Encyclopedia of Genes and Genomes; http://www.genome.jp/kegg/), NCBI (National Center for Biotechnology Information; http: //www.ncbi. nlm.nih.gov/gene/) and BRENDA (BRaunschweig ENzyme DAtabase; http://www.brenda-enzymes.info/). The isocitrate lyase gene can be used without particular limitation as long as it encodes a protein that functions in the host. For example, from the viewpoint of isocitrate lyase production efficiency, an isocitrate lyase gene endogenous to the host microorganism is used. May be.

Specific examples of genes encoding isocitrate lyase include aceA gene of Escherichia bacteria such as Escherichia coli, Pantoea bacteria such as Pantoea ananatis, and Corynebacterium bacteria such as Corynebacterium glutamicum Is mentioned.

The aceA gene encoding the isocitrate lyase of Escherichia coli MG1655 strain corresponds to nucleotide numbers 4215132 to 4216436 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3. The nucleotide sequence of the aceA gene of Escherichia coli MG1655 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 117 and 118, respectively.

AceA gene encoding isocitrate lyase of Pantoea ananatis AJ13355 strain corresponds to base numbers 4068278 to 4069579 of the genome sequence of Pantoea ananatis AJ13355 strain described in GenBank accession number NC_017531.1. The base sequence of the aceA gene of Pantoea ananatis AJ13355 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 119 and 120, respectively.

Also, for example, some Corynebacterium bacteria have two copies of isocitrate lyase gene (hereinafter also referred to as “ICL1 gene” and “ICL2 gene”). The ICL1 gene (Cgl2331) of Corynebacterium glutamicum ATCC13032 strain corresponds to nucleotide numbers 2470741 to 2472039 of the genome sequence of Corynebacterium glutamicum ATCC13032 described in GenBank accession number NC_003450.3. The ICL2 gene (Cgl0097) of Corynebacterium glutamicum ATCC13032 corresponds to the complementary sequence of base numbers 106392 to 105838 of the genome sequence of Corynebacterium glutamicum ATCC13032 described in GenBank accession number NC_003450.3. The nucleotide sequence of the ICL1 gene of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 121 and 122, respectively. The nucleotide sequence of the ICL2 gene of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 167 and 168, respectively. In addition, the nucleotide sequence of the ICL1 gene of Corynebacterium glutamicum 2256 strain (ATCC 13869) and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 169 and 170, respectively. The nucleotide sequence of the ICL2 gene of Corynebacterium glutamicum 2256 strain (ATCC 13869) and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 171 and 172, respectively.

The aceA gene usually forms an operon consisting of the aceBAK gene. As described later, the activity of malate synthase encoded by aceB is preferably weakened. Therefore, when enhancing the activity of isocitrate lyase, for example, as described in the Examples, the aceA gene expression was enhanced by deleting the aceB gene from the aceBAK operon and simultaneously introducing a strong promoter. May be.

The increase in the activity of isocitrate lyase can be confirmed, for example, by preparing a crude enzyme solution from the microorganism before modification and the microorganism after modification, and comparing the activity of isocitrate lyase. The activity of isocitrate lyase can be measured, for example, according to the method of Hoyt et al. (Hoyt JC et al. (1988) Biochim Biophys Acta. 14; 966 (1): 30-5.). Specifically, isocitrate is added to a reaction solution containing phenylhydrazine that reacts quickly with glyoxylic acid and color and the crude enzyme solution, and the amount of glyoxylate phenylhydrazine produced is measured spectroscopically. Thus, the isocitrate lyase activity can be measured. The activity of isocitrate lyase can be measured, for example, according to the method of Mackintosh et al. (Mackintosh, C et al. (1988) Biochem. J. 250, 25-31). Specifically, by adding glyoxylic acid and succinic acid to a reaction solution containing NADP, isocitrate dehydrogenase and crude enzyme solution, the amount of NADPH produced is measured spectroscopically, thereby isocitrate lyase activity. It can be measured.

As long as the original function is maintained, the enzyme of the present invention is a variant of the above-exemplified enzyme of the present invention, for example, a protein encoded by various mtkAB, sucCD, smtAB, smt, mclA, or aceA genes. Also good. Such variants may be referred to as “conservative variants”. Examples of the conservative variants include homologues and artificial modifications of the above-exemplified enzymes of the present invention, for example, proteins encoded by various mtkAB, sucCD, smtAB, smt, mclA, or aceA genes.

“The original function is maintained” means that the variant of the protein has an activity corresponding to the activity of the original protein. That is, “original function is maintained” for malate thiokinase means that the protein has malate thiokinase activity, and “original function is maintained” for succinyl-CoA synthase. The protein has succinyl-CoA synthase activity, and `` the original function is maintained '' for succinyl-CoA: malate CoA transferase means that the protein has succinyl-CoA: malate-CoA transferase activity, and malyl-CoA “The original function is maintained” for lyase means that the protein has malyl CoA lyase activity, and “the original function is maintained” for isocitrate lyase means that the protein is isocitrate. Having lyase activity. When a protein functions as a complex composed of a plurality of subunits, “the original function is maintained” for each subunit means that each subunit forms a complex with the remaining subunits. , The complex may have a corresponding activity. That is, for example, “the original function is maintained” for each subunit of malate thiokinase means that each subunit forms a complex with the remaining subunit, and that the complex exhibits malate thiokinase activity. It may be to have.

The gene encoding the homologue of the enzyme of the present invention exemplified above can be easily obtained from a public database by BLAST search or FASTA search using the base sequence of the gene encoding the enzyme of the present invention exemplified above as a query sequence. be able to. Further, the gene encoding the homologue of the enzyme of the present invention exemplified above is obtained by PCR using, for example, a bacterial or yeast chromosome as a template and oligonucleotides prepared based on these known gene sequences as primers. be able to.

The gene encoding the conservative variant of the enzyme of the present invention exemplified above may be, for example, the following gene. That is, as long as the gene encoding the enzyme of the present invention encodes a protein maintaining the original function, one or several amino acids at one or several positions are substituted or deleted in the above amino acid sequence. Alternatively, it may be a gene encoding a protein having an inserted or added amino acid sequence. In this case, the corresponding activity is usually 60% or more, preferably 70% or more, more preferably 80% or more with respect to the protein before one or several amino acids are substituted, deleted, inserted or added. More preferably, 90% or more can be maintained. The “one or several” is different depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically 1 to 50, 1 to 40, 1 to 30 It means 1 to 20, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

The substitution, deletion, insertion, or addition of one or several amino acids described above is a conservative mutation that maintains the protein function normally. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the organism from which the gene is derived. Also included by

Furthermore, the gene having a conservative mutation as described above is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99%, based on the entire amino acid sequence. It may be a gene encoding a protein having a homology of at least% and maintaining the original function. In the present specification, “homology” means “identity”.

The gene encoding the enzyme of the present invention hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to the whole or a part of the above base sequence, and maintains the original function. It may be a DNA encoding the prepared protein. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more between DNAs having homology. Is hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC And 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, salt concentration and temperature corresponding to 0.1% SDS, and conditions of washing once, preferably 2 to 3 times.

As described above, the probe used for the hybridization may be a part of a gene complementary sequence. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, as the probe, a DNA fragment having a length of about 300 bp can be used. 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.

In addition, the gene encoding the enzyme of the present invention may be one in which any codon is replaced with an equivalent codon as long as it encodes a protein that maintains the original function. For example, the gene encoding the enzyme of the present invention may be modified so as to have an optimal codon according to the codon usage frequency of the host to be used.

The percentage sequence identity between two sequences can be determined using, for example, a mathematical algorithm. Non-limiting examples of such mathematical algorithms include Myers and Miller (1988) CABIOS 4: 11 17 algorithm, Smith et aldv (1981) Adv. Appl. Math. 2: 482 local homology algorithm, Needleman and Wunsch (1970) J. Mol. Biol. 48: 443 453 homology alignment algorithm, Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444 2448 similarity search method, Karlin and Altschul 類似 (1993) Proc. Natl. Acad. Sci. USA 90: 5873 5877, an improved algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264.

Using a program based on these mathematical algorithms, sequence comparison (alignment) for determining sequence identity can be performed. The program can be appropriately executed by a computer. Such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligents, Mountain View, Calif.), The ALIGN program (Version 2.0), and Wisconsin Genetics Software Package, Version 8 (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA) GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignment using these programs can be performed using initial parameters, for example. Regarding the CLUSTAL program, Higgins et al. (1988) Gene 73: 237 244 (1988), Higgins et al. (1989) CABIOS 5: 151 153, Corpet et al. (1988) Nucleic Acids Res. 16: 10881 90, Huang et al. (1992) CABIOS 8: 155 65 and Pearson et al. (1994) Meth. Mol. Biol. 24: 307 331.

In order to obtain a nucleotide sequence having homology with the nucleotide sequence encoding the protein of interest, specifically, for example, a BLAST nucleotide search can be performed with the BLASTN program, score = 100, word length = 12. In order to obtain an amino acid sequence having homology with the protein of interest, specifically, for example, a BLAST protein search can be performed with the BLASTX program, score = 50, word length = 3. Refer to http://www.ncbi.nlm.nih.gov for BLAST nucleotide search and BLAST protein search. In addition, Gapped BLAST (BLAST 2.0) can be used to obtain an alignment with a gap added for the purpose of comparison. Also, PSI-BLASTＡ (BLAST 2.0) can be used to perform iterative searches that detect distant relationships between sequences. For Gapped BLAST and PSI-BLAST, see Altschul et al. (1997) Nucleic Acids Res. 25: 3389. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, for example, the initial parameters of each program (eg, BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. The alignment may be performed manually.

The sequence identity between two sequences is calculated as the ratio of residues that match between the two sequences when the two sequences are aligned for maximum matching.

In addition, the description regarding the said gene and protein conservative variant is applicable mutatis mutandis to arbitrary proteins, such as alpha-ketoglutarate synthase, and the gene which codes them.

<1-3> Other Modifications The microorganism of the present invention may further have other modifications. Other modifications can be appropriately selected according to the type of the target substance and the type of microorganism.

For example, the microorganism of the present invention may be modified so that the activity of malate synthase is reduced. “Malate synthase” refers to an enzyme that reversibly catalyzes the following reaction (EC 2.3.3.9).
acetyl-CoA + glyoxylate + H ₂ O → (S) -malate + coenzyme A + H ⁺

The malate synthase activity can be lowered, for example, by destroying a gene encoding malate synthase, as described later. Examples of the gene encoding malate synthase include the aceB gene and the glcB gene. The nucleotide sequence of the aceB gene of Escherichia coli MG1655 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 123 and 124, respectively. The nucleotide sequence of the glcB gene of Escherichia coli MG1655 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 173 and 174, respectively. The base sequence of the aceB gene of Pantoea ananatis AJ13355 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 125 and 126, respectively. The nucleotide sequence of the aceB gene of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 127 and 128, respectively. The nucleotide sequence of the aceB gene of Corynebacterium glutamicum 2256 strain (ATCC 13869) and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 175 and 176, respectively. The decrease in the activity of malate synthase is, for example, a method in which the degradation of the thioester bond of acetyl CoA dependent on glyoxylic acid is measured with a decrease in absorbance at 232 nm (Dixon, GH, Kornberg, HL, 1960, Biochem. , 1; 41: p217-233) and can be confirmed by measuring malate synthase activity.

In addition, the microorganism of the present invention may be modified so that the activity of PTS-glucose-enzyme II-BC is reduced. “PTS glucose enzyme II BC” refers to glucose-specific phosphotransferase system (PTS). The PTS glucose enzyme II BC activity can be decreased, for example, by disrupting the gene encoding PTS glucose enzyme II BC, as described later. PTS glucose enzyme II BC is encoded by the ptsG gene. The nucleotide sequence of the ptsG gene of Escherichia coli MG1655 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 129 and 130, respectively. The base sequence of the ptsG gene of Pantoea ananatis AJ13355 strain and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 131 and 132, respectively.

For example, some Corynebacterium bacteria have two copies of the ptsG gene (hereinafter also referred to as “ptsG1 gene” and “ptsG2 gene”). The base sequence of the ptsG1 gene (Cgl1360) of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 133 and 134, respectively. The nucleotide sequence of the ptsG2 gene (Cgl2642) of Corynebacterium glutamicum ATCC13032 and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 177 and 178, respectively. In addition, the base sequence of the ptsG1 gene of Corynebacterium glutamicum 2256 strain (ATCC 13869) and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 179 and 180, respectively. The base sequence of the ptsG2 gene of Corynebacterium glutamicum strain 2256 (ATCC 13869) and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 181 and 182, respectively.

In addition, the microorganism of the present invention may be modified so that the activity of one or more enzymes selected from enzymes involved in glyoxylate degradation is reduced. Enzymes involved in glyoxylate degradation include glyoxylate reductase, glyoxylate carboligase, and 2-keto-3-deoxygluconate 6-phosphate aldolase.

Glyoxylate reductase is encoded by ghrA gene (E. coli), ghrB gene (E. coli) (Nunez et al, Biochem. J. 54: 707-715 (2001)) and ycdW gene (P. ananatis). And catalyzes the following reactions:
glyoxylate + NADPH + H ⁺ → glycolate + NADP ⁺ (EC 1.1.1.79)

Glyoxylate carboligase is encoded by the gcl gene and catalyzes the following reaction.
2 glyoxylate + H ⁺ → CO ₂ + tartronate semialdehyde (EC 4.1.1.47)

2-keto-3-deoxygluconate 6-phosphate aldolase is encoded by the eda gene and catalyzes the following reaction.
glyoxylate + pyruvate → 4-Hydroxy-2-oxoglutarate

In addition, the microorganism of the present invention may be modified so that the biosynthetic system of a substance derived from pyruvate or acetyl CoA accompanied by NADH oxidation is weakened. Attenuating the biosynthetic system of substances derived from pyruvate or acetyl-CoA with the oxidation of NADH can be achieved by reducing the activity of one or more enzymes of the biosynthetic system. Examples of the biosynthetic enzymes include the following enzymes (WO2009 / 072562).
・ Lactate dehydrogenase (lactic acid biosynthesis system)
-Alcohol dehydrogenase (ethanol biosynthesis system).
Acetolactate synthase, acetolactate decarboxylase, acetoin reductase (2,3-butanediol biosynthesis system).

“Lactic acid dehydrogenase” refers to an enzyme that catalyzes the reaction of producing lactic acid from pyruvic acid using NADH or NADPH as an electron donor. The activity that catalyzes this reaction is also referred to as “lactate dehydrogenase activity”. Lactate dehydrogenase consists of L-type lactate dehydrogenase (L-LDH; EC 1.1.1.27) that produces L-lactic acid and D-type lactate dehydrase (D-LDH; EC1.1.1.28) that produces D-lactic acid. Any of these activities may be reduced. Lactate dehydrogenase (LDH) activity can be lowered, for example, by destroying a gene encoding lactate dehydrogenase (LDH gene), as described later. As the LDH gene of Escherichia coli, the base sequence of the D-LDH gene (ldhA) is shown in SEQ ID NO: 135, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 136. As the Pantoea ananatis LDH gene, the base sequence of the D-LDH gene (ldhA) is shown in SEQ ID NO: 137, and the amino acid sequence encoded by the gene is shown in SEQ ID NO: 138. The base sequence of the L-LDH gene (ldh) of Corynebacterium glutamicum ATCC13032 is shown in SEQ ID NO: 187, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 188. The base sequence of the L-LDH gene (ldh) of Corynebacterium glutamicum 2256 strain (ATCC 13869) is shown in SEQ ID NO: 189, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 190. The decrease in lactate dehydrogenase activity can be confirmed, for example, by measuring the lactate dehydrogenase activity by a known method (L. Kanarek and R. L. Hill, J. Biol. Chem. 239, 4202 1 (1964)). A specific method for producing an enterobacterial mutant having reduced lactate dehydrogenase activity is described in Alam, K. Y., Clark, D. P. 1989. J. Bacteriol. 171: 6213-6217 Methods and the like.

“Alcohol dehydrogenase” refers to an enzyme that catalyzes a reaction to produce alcohol from aldehyde using NADH or NADPH as an electron donor (EC 1.1.1.1, EC 1.1.1.2, or EC 1.1.1.71). The activity that catalyzes this reaction is also referred to as “alcohol dehydrogenase activity”. Alcohol dehydrogenase (ADH) activity can be reduced by, for example, destroying a gene encoding alcohol dehydrogenase (ADH gene), as described later. As the ADH gene of Escherichia coli, the base sequence of the adhE gene is shown in SEQ ID NO: 139, and the amino acid sequence encoded by the same gene is shown in SEQ ID NO: 140. As the ADH gene of Pantoea ananatis, the base sequence of the adhE gene is shown in SEQ ID NO: 141, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 142. As the ADH gene of Corynebacterium glutamicum, the base sequence of the adhE gene of Corynebacterium glutamicum ATCC13032 is shown in SEQ ID NO: 191, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 192. The decrease in alcohol dehydrogenase activity is determined by, for example, measuring alcohol dehydrogenase activity by a known method (Lutstorf, UM, Schurch, PM & von Wartburg, JP, Eur. J. Biochem. 17, 497-508 (1970)). Can be confirmed. As a specific method for producing an enterobacterial mutant with reduced alcohol dehydrogenase activity, Sanchez, A. M., Bennett, G. N., San, K.-Y., Biotechnol. Prog. 21, 358 -365 (2005).

“Acetolactate synthase” refers to an enzyme that catalyzes the reaction of producing acetolactate and CO ₂ from two molecules of pyruvate (EC 2.2.1.6). The activity that catalyzes this reaction is also referred to as “acetolactic acid synthase activity”. As acetolactate synthase (AHAS), AHAS I to III isozymes are known, but the activity of any isozyme may be reduced. The acetolactate synthase activity can be lowered, for example, by destroying a gene encoding acetolactate synthase, as described later. Examples of the gene encoding acetolactate synthase include the ilvB gene encoding the active subunit of AHAS I, the ilvG gene encoding the active subunit of AHAS II, and the ilvI gene encoding the active subunit of AHAS III. The nucleotide sequences of the ilvB and ilvI genes of E. coli MG1655 are shown in SEQ ID NOs: 193 and 195, respectively, and the amino acid sequences of proteins encoded by the genes are shown in SEQ ID NOs: 194 and 196, respectively. The nucleotide sequences of the ilvG and ilvI genes of Pantoea ananatis AJ13355 are shown in SEQ ID NOs: 197 and 199, respectively, and the amino acid sequences of the proteins encoded by the same genes are shown in SEQ ID NOs: 198 and 200, respectively. The nucleotide sequence of the ilvB gene of Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO: 201, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 202. The decrease in acetolactate synthase activity is measured, for example, by a known method (FC Stormer and HE Umbarger, Biochem. Biophys. Res. Commun., 17, 5, 587-592 (1964)). Can be confirmed.

“Acetolactate decarboxylase” refers to an enzyme that catalyzes the reaction of decarboxylating acetolactate to produce acetoin (EC 4.1.1.5). The activity that catalyzes this reaction is also referred to as “acetolactic acid decarboxylase activity”. The acetolactate decarboxylase activity can be reduced, for example, by destroying a gene encoding acetolactate decarboxylase, as described later. The base sequence of the acetolactate decarboxylase gene (budA) of Pantoea ananatis AJ13355 is shown in SEQ ID NO: 203, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 204. For example, E. coli and Corynebacterium glutamicum do not have acetolactate decarboxylase. The decrease in the acetolactate decarboxylase activity is, for example, by measuring the acetolactate decarboxylase activity by a known method (Juni E., J. Biol. Chem., 195 (2): 715-726 (1952)). Can be confirmed.

“Acetoin reductase” refers to an enzyme that catalyzes the reaction of producing 2,3-butanediol from acetoin using NADH or NADPH as an electron donor (EC 1.1.1.4). The activity that catalyzes the same reaction is also referred to as “acetoin reductase activity”. The acetoin reductase activity can be reduced by, for example, destroying a gene encoding acetoin reductase, as described later. The base sequence of the acetoin reductase gene (budC) of Pantoea ananatis AJ13355 is shown in SEQ ID NO: 205, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 206. The nucleotide sequence of the acetoin reductase gene (butA) of Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO: 207, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 208. For example, E. coli does not have acetoin reductase. The decrease in acetoin reductase activity can be confirmed, for example, by measuring acetoin reductase activity by a known method (K. Blomqvist et al., J Bacteriol., 175, 5, 1392-1404 (1993)). it can.

In addition, the microorganism of the present invention may be modified so that the acetic acid biosynthesis system is weakened. Specifically, the microorganism of the present invention may be modified so that, for example, the activity of one or more enzymes selected from the following enzymes is reduced (US2007-0054387, WO2005 / 052135, WO99 / 53035). , WO2006 / 031424, WO2005 / 113745, WO2005 / 113744).
・ Phosphotransacetylase ・ Acetate kinase ・ Pyruvate oxidase ・ Acetyl CoA hydrolase

The phosphotransacetylase (PTA) activity can be lowered by, for example, destroying a gene encoding a phosphotransacetylase (PTA gene) as described later. As the PTA gene of Escherichia coli, the base sequence of the pta gene is shown in SEQ ID NO: 143, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 144. As the PTA gene of Pantoea ananatis, the base sequence of the pta gene is shown in SEQ ID NO: 145, and the amino acid sequence encoded by this gene is shown in SEQ ID NO: 146. The decrease in the phosphotransacetylase activity can be confirmed by measuring the phosphotransacetylase activity by a known method (Klotzsch, H.R., Meth. Enzymol. 12, 381-386 (1969)).

In addition, the microorganism of the present invention may be modified so that pyruvate / formate lyase (PFL) activity decreases. The pyruvate / formate lyase activity can be lowered, for example, by destroying a gene (PFL gene) encoding pyruvate / formate lyase as described later. As the PFL gene of Escherichia coli, the base sequences of the pflB, pflD, and tdcE genes are shown in SEQ ID NOs: 147, 149, and 151, respectively, and the amino acid sequences of the proteins encoded by the same genes are shown in SEQ ID NOs: 148, 150, and 152, respectively. . As the PFL gene of Pantoea ananatis, the base sequence of the pflB gene is shown in SEQ ID NO: 153, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 154. The decrease in pyruvate / formate lyase activity was confirmed by measuring pyruvate / formate lyase activity by a known method (Knappe, J. & Blaschkowski, HP, Meth.molEnzymol. 41, (508-518 (1975)). Can be confirmed.

In addition, the microorganism of the present invention may be modified so that the recruitment pathway of the TCA cycle is enhanced. Specifically, the microorganism of the present invention may be modified so that, for example, the activity of one or more enzymes selected from the following enzymes is increased (JP-A-11-196888, JP-A-2006- 320208, WO99 / 53035, WO2005 / 021770, Hong SH, Lee SY. Biotechnol Bioeng. 74 (2): 89-95 (2001), Millard, CS, Chao, YP, Liao, JC, Donnelly, MI Appl. Environ Microbiol. 62: 1808-1810 (1996), Pil Kim, Maris Laivenieks, Claire Vieille, and J. Gregory Zeikus. Appl. Environ. Microbiol. 70: 1238-1241 (2004)).
・ Pyruvate carboxylase ・ Phosphoenolpyruvate carboxylase ・ Phosphoenolpyruvate carboxykinase

Enzyme activity can be increased, for example, by increasing the expression of a gene encoding the enzyme, as described later. Examples of genes encoding pyruvate carboxylase include coryneform bacteria such as Corynebacterium glutamicum and Brevibacterium flavum, Bacillus stearothermophilus, Rhizobium etli, Saccharomyces Examples include yeast PC genes such as Saccharomyces cerevisiae and Schizosaccharomyces pombe (WO2009 / 072562). Examples of the gene encoding phosphoenolpyruvate carboxykinase include the pckA gene (GenBank Accession No. YP_001343536.1) of Actinobacillus succinogenes and the pckA gene (GenBank Accession of Haemophilus influenzae). YP_248516.1), pckA gene of Pasteurella multocida (GenBank Accession No. NP_246481.1), Manheimia succiniciproducens pckA gene (GenBank Accession No. YP_089485.1)・ The pckA gene of Yersinia pseudotuberculosis (GenBank Accession No. YP_072243), the pckA gene of Vibrio cholerae (GenBank Accession No. ZP_01981004.1), Selenomonas luminantumrum (Selenomonasrum) ntium) pckA gene (GenBank Accession No. AB016600) (WO2009 / 072562). Examples of genes encoding phosphoenolpyruvate carboxylase include coryneform bacteria such as Corynebacterium glutamicum and Brevibacterium flavum, Escherichia bacteria such as Escherichia coli, and Rhodopseudomonas palustris. An example is the ppc gene. Enzyme activity can also be increased, for example, by reducing or eliminating feedback inhibition. For example, the activity of phosphoenolpyruvate carboxylase (PEPC) is inhibited by L-malate, an intermediate product of the succinic acid biosynthetic pathway (Masato Yano and Katsura Izui, Eur. Biochem. FEBS, 247, 74-81 , 1997). Inhibition by L-malic acid can be reduced, for example, by introducing a desensitizing mutation by substitution of one amino acid into PEPC. Specific examples of the desensitizing mutation by substitution of one amino acid include a mutation that substitutes the 620th amino acid of PEPC protein derived from Escherichia coli from lysine to serine (the same document).

The microorganism of the present invention may be modified so that the activity of α-ketoglutarate dehydrogenase (also referred to as “α-KGDH”) decreases. “Α-ketoglutarate dehydrogenase” refers to an enzyme that catalyzes a reaction in which α-ketoglutarate (2-oxoglutarate) is oxidatively decarboxylated to produce succinyl-CoA. The activity that catalyzes this reaction is also referred to as “α-ketoglutarate dehydrogenase activity”. α-KGDH is also called oxoglutarate dehydrogenase or 2-oxoglutarate dehydrogenase.

The above reactions include α-KGDH (E1o; EC 1.2.4.2), dihydrolipoamide S-succinyltransferase (E2o; EC 2.3.1.61), dihydrolipoamide dehydrogenase (E3; Catalyzed by three enzymes of EC 1.8.1.4). That is, these three types of enzymes each catalyze the following reaction, and the α-KGDH activity specifically refers to an activity that catalyzes a reaction that combines these three reactions.
E1o: 2-oxoglutarate + [dihydrolipoyllysine-residue succinyltransferase] lipoyllysine → [dihydrolipoyllysine-residue succinyltransferase] S-succinyldihydrolipoyllysine + CO ₂
E2o: CoA + enzyme N6- (S-succinyldihydrolipoyl) lysine → succinyl-CoA + enzyme N6- (dihydrolipoyl) lysine
E3: protein N6- (dihydrolipoyl) lysine + NAD ⁺ → protein N6- (lipoyl) lysine + NADH + H ⁺

In the Enterobacteriaceae family, for example, Pantoea ananatis, these three types of subunit proteins, E1o, E2o, and E3, which have enzyme activities, form a complex. Each subunit is encoded by the sucA, sucB, and lpdA genes, which are present downstream of the succinate dehydrogenase iron-sulfur protein gene (sdhB) (US Pat. No. 6,331,419). . In this patent, these genes are described as genes of Enterobacter agglomerans AJ13355, but this strain was later reclassified as Pantoea ananatis. The nucleotide sequences of the sucA, sucB, and lpdA genes of Pantoea ananatis AJ13355 are shown in SEQ ID NOs: 155, 157, and 159, respectively, as genes encoding the enterobacterial α-KGDH. In addition, amino acid sequences of SucA, SucB, and LpdA proteins encoded by the same gene are shown in SEQ ID NOs: 156, 158, and 160, respectively. In addition, the SucA, SucB, and LpdA proteins encoded by the sucA, sucB, and lpdA genes, which are α-KGDH genes of Escherichia coli, are disclosed as GenBank NP_415254, NP_415255, and NP_414658, respectively.

In coryneform bacteria, the E1o subunit is encoded by the odhA gene (also called the sucA gene; registered as NCgl1084 of GenBank Accession No. It is coded. On the other hand, the E2o subunit is encoded by the odhA gene as a bifunctional protein together with the E1o subunit (see Usuda, Y. et al., Microbiology 1996. 142: 3347-3354), or different from the odhA gene It is presumed to be encoded by a gene registered as NCgl2126 of GenBank Accession No. NC_003450. Therefore, in the present invention, the odhA gene is a gene that encodes the E1o subunit, but may also encode E2o. The nucleotide sequence of the odhA gene of Brevibacterium lactofermentum ATCC 13032 and the amino acid sequence of the E1o subunit encoded by the same gene (WO2006 / 028298) are shown in SEQ ID NOs: 161 and 162, respectively. Further, the nucleotide sequence of the lpd gene and the amino acid sequence of the E3 subunit encoded by the gene (WO2006 / 028298) are shown in SEQ ID NOs: 163 and 164, respectively. Further, the nucleotide sequence of NCgl2126 of GenBank 上記 Accession No. NC_003450 and the amino acid sequence of the protein encoded by the same sequence are shown in SEQ ID NOs: 165 and 166, respectively.

The decrease in α-KGDH activity was confirmed by measuring α-KGDH activity by a known method (Shiio, I. and Ujigawa-Takeda, K. 1980. Agric. Biol. Chem. 44: 1897-1904) can do.

It should be noted that the genes used for these other modifications are not limited to the above-exemplified genes or genes having a known base sequence, as long as they encode proteins having the original functions maintained, and may be variants thereof. Regarding the variants of genes and proteins, the above-mentioned description of the enzyme for synthesizing malyl CoA from L-malate, malyl CoA lyase, isocitrate lyase, and conservative variants of the genes encoding them can be applied mutatis mutandis.

<1-4> Technique for Increasing Protein Activity A technique for increasing protein activity is described below.

"" Protein activity increases "means that the activity per cell of the protein is increased relative to unmodified strains such as wild strains and parental strains. Note that “increasing protein activity” is also referred to as “enhancing protein activity”. “Protein activity increases” specifically means that the number of molecules per cell of the protein is increased and / or the function per molecule of the protein compared to an unmodified strain. Is increasing. That is, “activity” in the case of “increasing protein activity” means not only the catalytic activity of the protein, but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. “Protein activity increases” means not only to increase the activity of the protein in a strain that originally has the activity of the target protein, but also to the activity of the protein in a strain that does not originally have the activity of the target protein. Including granting. As a result, as long as the activity of the protein increases, the activity of the target protein originally possessed by the microorganism may be reduced or eliminated, and then the activity of a suitable target protein may be imparted.

The activity of the protein is not particularly limited as long as it is increased compared to the non-modified strain. For example, the protein activity is increased 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain. Good. In addition, when the non-modified strain does not have the activity of the target protein, it is sufficient that the protein is generated by introducing a gene encoding the protein. For example, the protein has an enzymatic activity. It may be produced to the extent that it can be measured.

Modification that increases the activity of the protein is achieved, for example, by increasing the expression of the gene encoding the protein. Note that “increasing gene expression” is also referred to as “enhanced gene expression”. The expression of the gene may be increased 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain. In addition, “increasing gene expression” means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not hold the target gene and expressing the gene.

An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.

Increase in gene copy number can be achieved by introducing the gene into the chromosome of the host microorganism. Introduction of a gene into a chromosome can be performed, for example, using homologous recombination (Miller I, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy of the gene may be introduced, or two copies or more may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies on the chromosome as a target. Examples of sequences having many copies on a chromosome include repetitive DNA sequences (inverted DNA) and inverted repeats present at both ends of a transposon. Alternatively, homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for production of the target substance. Homologous recombination is, for example, the Red-driven integration method (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97: 6640-6645 (2000) ), A method using a linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, a method using a suicide vector that does not have a replication origin and functions in a host, or a phage It can be performed by the transduction method used. The gene can also be randomly introduced onto the chromosome using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1).

Confirmation of the introduction of the target gene on the chromosome was performed using Southern hybridization using a probe having a sequence complementary to all or part of the gene, or a primer prepared based on the sequence of the gene. It can be confirmed by PCR.

Also, the increase in gene copy number can be achieved by introducing a vector containing the target gene into the host microorganism. For example, a DNA fragment containing a target gene is linked to a vector that functions in the host microorganism to construct an expression vector for the gene, and the host microorganism is transformed with the expression vector to increase the copy number of the gene. be able to. A DNA fragment containing a target gene can be obtained, for example, by PCR using a genomic DNA of a microorganism having the target gene as a template. As the vector, a vector capable of autonomous replication in a host microorganism cell can be used. The vector is preferably a multicopy vector. In order to select a transformant, the vector preferably has a marker such as an antibiotic resistance gene. Moreover, the vector may be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, pBR322, and pSTV29 (all available from Takara Bio Inc.) ), PMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pACYC vector A broad host range vector RSF1010. Specific examples of vectors capable of autonomous replication in coryneform bacteria include, for example, pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., .48, 2901- 2903 (1984)); plasmids having improved drug resistance genes; plasmid pCRY30 described in JP-A-3-210184; plasmid pCRY21 described in JP-A-2-72876 and US Pat. No. 5,185,262. PCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; JP-A-57-134500 Examples include pCG1 described in the publication; pCG2 described in JP-A-58-35197; pCG4 and pCG11 described in JP-A-57-183799.

When a gene is introduced, the gene only needs to be retained in the microorganism of the present invention so that it can be expressed. Specifically, the gene may be introduced so as to be expressed under the control of a promoter sequence that functions in the microorganism of the present invention. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a native promoter of a gene to be introduced or a promoter of another gene. As the promoter, for example, a stronger promoter as described later may be used.

A transcription terminator can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the bacterium of the present invention. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a terminator specific to the gene to be introduced, or may be a terminator of another gene. Specific examples of the terminator include T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

The vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8, Genetic Engineering, Kyoritsu Shuppan, 1987”, and these can be used.

In addition, when two or more genes are introduced, each gene may be retained in the microorganism of the present invention so that it can be expressed. For example, all the genes may be held on a single expression vector, or all may be held on a chromosome. Moreover, each gene may be separately hold | maintained on several expression vector, and may be separately hold | maintained on the single or several expression vector and chromosome. Further, an operon may be constructed by introducing two or more genes. “When two or more genes are introduced” means, for example, when a gene encoding each of two or more enzymes is introduced, each of two or more subunits constituting a single enzyme is encoded. And a combination thereof.

The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The introduced gene may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR using, for example, a primer designed based on the base sequence of the gene, and using a genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. The introduced gene may be totally synthesized based on the base sequence of the same gene (Gene, 60 (1), 115-127 (1987)).

When the protein functions as a complex composed of a plurality of subunits, all of the plurality of subunits may be modified or only a part may be modified as long as the activity of the protein increases as a result. . That is, for example, when the activity of a protein is increased by increasing the expression of a gene, the expression of a plurality of genes encoding those subunits may be enhanced, or only a part of the expression may be enhanced. Also good. Usually, it is preferable to enhance the expression of all of a plurality of genes encoding these subunits. That is, for example, when the malate thiokinase activity is increased by increasing the expression of the malate thiokinase gene, the expression of either the mtkA gene or the mtkB gene may be enhanced, or the expression of both may be enhanced. Although good, it is preferred to enhance the expression of both. In addition, for example, when increasing the succinyl-CoA synthase activity by increasing the expression of the succinyl-CoA synthase gene, the expression of one of the sucC gene and the sucD gene may be enhanced, or the expression of both may be enhanced. Although good, it is preferred to enhance the expression of both. In addition, for example, when the succinyl CoA: malate CoA transferase activity is increased by increasing the expression of the succinyl CoA: malate CoA transferase gene, the expression of either one of the smtA gene and the smtB gene may be enhanced. Although expression may be enhanced, it is preferred to enhance both expressions. In addition, each subunit constituting the complex may be derived from one organism or two or more different organisms as long as the complex has the function of the target protein. That is, for example, genes derived from the same organism encoding a plurality of subunits may be introduced into the host, or genes derived from different organisms may be introduced into the host.

Moreover, the increase in gene expression can be achieved by improving the transcription efficiency of the gene. Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter. By “stronger promoter” is meant a promoter that improves transcription of the gene over the native wild-type promoter. Examples of stronger promoters include the known high expression promoters T7 promoter, trp promoter, lac promoter, thr promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter. Can be mentioned. In addition, as a stronger promoter that can be used in coryneform bacteria, artificially redesigned P54-6 promoter (Appl. Microbiol. Biotechnolo., 53, 674-679 (2000)), acetic acid in coryneform bacteria Pta, aceA, aceB, adh, amyE promoter that can be induced by ethanol, pyruvic acid, etc., cspB, SOD, tuf promoter (Journal of Biotechnology 104 (2003) 311- 323, Appl Environ Microbiol. 2005 Dec; 71 (12): 8587-96.). As a more powerful promoter, a highly active promoter of a conventional promoter may be obtained by using various reporter genes. For example, the activity of the promoter can be increased by bringing the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 00/18935). Examples of the highly active promoter include various tac-like promoters (Katashkina JI et al. Russian Patent application 2006134574) and pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotickpromoters in biotechnology. Biotechnol. Annu. Rev.,. 1, 105-128 (1995)).

Moreover, the increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. By “a stronger SD sequence” is meant an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence. As a stronger SD sequence, for example, RBS of gene 10 derived from phage T7 can be mentioned (Olins P. O. et al, Gene, 1988, 73, 227-235). Furthermore, substitution of several nucleotides in the spacer region between the RBS and the start codon, particularly the sequence immediately upstream of the start codon (5'-UTR), or insertion or deletion, contributes to mRNA stability and translation efficiency. It is known to have a great influence, and the translation efficiency of a gene can be improved by modifying them.

In the present invention, promoters, SD sequences, and sites that affect gene expression such as a spacer region between the RBS and the start codon are also collectively referred to as “expression control regions”. The expression regulatory region can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression control regions can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (WO2005 / 010175).

Improvement of gene translation efficiency can also be achieved, for example, by codon modification. In Escherichia coli, etc., there is a clear codon bias among the 61 amino acid codons found in the population of mRNA molecules, and the abundance of a tRNA seems to be directly proportional to the frequency of use of the corresponding codon. (Kane, JF, Curr. Opin. Biotechnol., 6 (5), 494-500 (1995)). That is, if a large amount of mRNA containing an excessive rare codon is present, translation problems may occur. Recent studies suggest that, inter alia, clusters of AGG / AGA, CUA, AUA, CGA, or CCC codons can reduce both the amount and quality of the synthesized protein. Such a problem can occur particularly during the expression of heterologous genes. Therefore, when performing heterologous expression of a gene, the translation efficiency of the gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently. Codon substitution can be performed, for example, by a site-specific mutagenesis method in which a target mutation is introduced into a target site of DNA. As site-directed mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, rlErlich, H. A. Eds., Stockton press (1989); Carter, P., ethMeth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, H. J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth In Enzymol., 154, 367 (1987)). Alternatively, gene fragments in which codons have been replaced may be fully synthesized. The frequency of codon usage in various organisms can be found in the “Codon Usage Database” (http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)) Is disclosed.

Also, the increase in gene expression can be achieved by amplifying a regulator that increases gene expression or by deleting or weakening a regulator that decreases gene expression.

The techniques for increasing gene expression as described above may be used alone or in any combination.

Further, the modification that increases the activity of the protein can be achieved, for example, by enhancing the specific activity of the protein. Specific activity enhancement also includes the reduction and elimination of feedback inhibition. Proteins with enhanced specific activity can be obtained by searching for various organisms, for example. Alternatively, a highly active protein may be obtained by introducing a mutation into a conventional protein. The introduced mutation may be, for example, a substitution, deletion, insertion or addition of one or several amino acids at one or several positions of the protein. Mutation can be introduced by, for example, the site-specific mutation method as described above. Moreover, you may introduce | transduce a variation | mutation by a mutation process, for example. Mutation treatments include X-ray irradiation, UV irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like. Alternatively, DNA may be directly treated with hydroxylamine in vitro to induce random mutations. The enhancement of specific activity may be used alone or in any combination with the above-described method for enhancing gene expression.

The method of transformation is not particularly limited, and a conventionally known method can be used. For example, as reported for Escherichia coli K-12, recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162) and methods for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E .., 1997. Gene 1: 153-167) can be used. Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, can be made into protoplasts or spheroplasts that readily incorporate recombinant DNA into recombinant DNA. Introduction method (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M. J., Ward, J. M. and Hopwood, O. A. 1978. Nature 274: 398-400; Hinnen, A., Hicks, J. B. and Fink, G. R. 1978. Proc. Natl.Acad. Sci. USA 75: 1929-1933) can also be applied. Alternatively, an electric pulse method (Japanese Patent Laid-Open No. 2-207791) as reported for coryneform bacteria can also be used.

The increase in protein activity can be confirmed by measuring the activity of the protein.

The increase in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has increased. An increase in gene expression can be confirmed by confirming that the transcription amount of the gene has increased, or by confirming that the amount of protein expressed from the gene has increased.

It can be confirmed that the transcription amount of the gene has increased by comparing the amount of mRNA transcribed from the gene with an unmodified strain such as a wild strain or a parent strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, etc. ), 2001). The amount of mRNA may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain.

Confirmation of the increase in the amount of protein can be performed by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein may be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain.

The above-described techniques for increasing the activity of a protein include enhancing the activity of an arbitrary protein such as an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase, and any gene such as any of them. It can be used to enhance the expression of a gene encoding a protein.

<1-5> Technique for reducing protein activity A technique for reducing protein activity will be described below.

“Protein activity decreases” means that the activity per cell of the protein is decreased compared to wild-type strains and parental unmodified strains, and the activity is completely lost. including. Specifically, “the activity of the protein is decreased” means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. In other words, “activity” in the case of “decrease in protein activity” means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost. The activity of the protein is not particularly limited as long as it is lower than that of the non-modified strain. For example, it is 50% or less, 20% or less, 10% or less, 5% or less, or 0, compared to the non-modified strain. %.

The modification that reduces the activity of the protein is achieved, for example, by reducing the expression of a gene encoding the protein. “Gene expression decreases” includes the case where the gene is not expressed at all. In addition, “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”. Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to an unmodified strain.

The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Reduction of gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter or Shine-Dalgarno (SD) sequence. In the case of modifying the expression control sequence, the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted. In addition, reduction of gene expression can be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like.

Further, the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein. Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted. The region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved. Usually, the longer region to be deleted can surely inactivate the gene. Moreover, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.

In addition, gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a frameshift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

Also, gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. Moreover, it is preferable that the reading frames of the sequences before and after the insertion site do not match. The other sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples thereof include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

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

Further, the modification that reduces the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatments include X-ray irradiation, UV irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like.

When the protein functions as a complex composed of a plurality of subunits, all of the plurality of subunits may be modified or only a part may be modified as long as the activity of the protein decreases as a result. . That is, for example, all of a plurality of genes encoding these subunits may be destroyed, or only a part of them may be destroyed. In addition, when a plurality of isozymes are present in a protein, as long as the activity of the protein is reduced as a result, all the activities of the plurality of isozymes may be reduced, or only a part of the activities may be reduced. That is, for example, all of a plurality of genes encoding these isozymes may be destroyed, or only a part of them may be destroyed.

The decrease in the activity of the protein can be confirmed by measuring the activity of the protein.

The decrease in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has decreased. The decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased, or confirming that the amount of protein expressed from the gene has decreased.

It can be confirmed that the amount of transcription of the gene has been reduced by comparing the amount of mRNA transcribed from the same gene with that of the unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold spring spring Laboratory Laboratory, Cold spring Harbor (USA), 2001)). The amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.

Confirmation that the amount of the protein has decreased can be performed by Western blotting using an antibody (Molecular cloning (Cold spring Spring Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.

It can be confirmed that the gene has been destroyed by determining part or all of the nucleotide sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.

The above-described technique for reducing the activity of a protein can be used to reduce the activity of an arbitrary protein, such as malate synthase, or to reduce the expression of an arbitrary gene, such as a gene encoding the arbitrary protein.

<2> Method for Producing the Target Substance of the Present Invention The method of the present invention comprises culturing the microorganism of the present invention in a medium, producing and accumulating the target substance in the medium or in the cells of the microorganism, and the medium or fungus. A method for producing a target substance, which includes collecting the target substance from the body. In the present invention, one kind of target substance may be produced, or two or more kinds of target substances may be produced.

The medium to be used is not particularly limited as long as the microorganism of the present invention can grow and the target substance is produced. As the medium, for example, a normal medium used for culturing microorganisms such as bacteria can be used. As the medium, for example, a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other components selected from various organic components and inorganic components as necessary can be used. The type and concentration of the medium component may be appropriately set according to various conditions such as the type of microorganism used and the type of target substance to be produced.

The carbon source is not particularly limited as long as the microorganism of the present invention can be assimilated to produce a target substance. Specifically, as the carbon source, for example, glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolyzate, saccharides such as biomass hydrolyzate, acetic acid, fumaric acid, citric acid, Examples thereof include organic acids such as succinic acid and malic acid, alcohols such as glycerol, crude glycerol and ethanol, and fatty acids. In addition, as a carbon source, a plant-derived raw material can be used suitably. Examples of plants include corn, rice, wheat, soybean, sugar cane, beet, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, seeds, plants containing them, and degradation products of these plant organs. The form of use of the plant-derived raw material is not particularly limited, and for example, any form such as a raw product, juice, pulverized product, or product can be used. Moreover, pentoses such as xylose, hexoses such as glucose, or a mixture thereof can be obtained from plant biomass and used, for example. Specifically, these saccharides can be obtained by subjecting plant biomass to treatment such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass is hydrolyzed in advance to release pentose, and then cellulose is hydrolyzed to produce hexose. Good. In addition, xylose may be supplied by conversion from hexose, for example, by allowing the microorganism of the present invention to have a conversion path from hexose such as glucose to xylose. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

The concentration of the carbon source in the medium is not particularly limited as long as the microorganism of the present invention can grow and the target substance is produced. The concentration of the carbon source in the medium is preferably as high as possible as long as the production of the target substance is not inhibited. The initial concentration of the carbon source in the medium may be, for example, usually 5 to 30% (W / V), preferably 10 to 20% (W / V). Moreover, you may add a carbon source additionally according to consumption of the carbon source accompanying progress of fermentation.

Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein degradation product, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.

Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid. As the phosphoric acid source, one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.

Specific examples of the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.

Other various organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these. As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

Moreover, when using an auxotrophic mutant strain that requires an amino acid or the like for growth, it is preferable to supplement nutrients required for the medium. In addition, for example, when L-glutamic acid is produced by coryneform bacteria, it is preferable to limit the amount of biotin in the medium, or to add a surfactant or penicillin to the medium. In order to suppress foaming during culture, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the medium.

Culture conditions are not particularly limited as long as the microorganism of the present invention can grow and the target substance is produced. The culture can be performed, for example, under normal conditions used for culture of microorganisms such as bacteria. The culture conditions may be appropriately set according to various conditions such as the type of microorganism used and the type of target substance to be produced.

Cultivation can be performed using a liquid medium. When culturing, the microorganism of the present invention cultured in a solid medium such as an agar medium may be directly inoculated into a liquid medium. The medium may be inoculated. That is, the culture may be performed separately for seed culture and main culture. The amount of the microorganism of the present invention contained in the medium at the start of culture is not particularly limited. For example, a seed culture solution having an OD660 of 4 to 8 may be added at 0.1 to 30% by mass, preferably 1 to 10% by mass with respect to the medium for main culture at the start of culture.

Culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. In addition, when culture is performed separately for seed culture and main culture, the culture conditions for seed culture and main culture may or may not be the same. For example, both seed culture and main culture may be performed by batch culture. Further, for example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.

Cultivation may be performed under aerobic conditions, microaerobic conditions, or anaerobic conditions. The culture is preferably performed under microaerobic conditions or anaerobic conditions. The aerobic condition means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of the oxygen membrane electrode, and preferably 1.5 ppm or more. The microaerobic condition means that oxygen is supplied to the culture system, but the dissolved oxygen concentration in the liquid medium is less than 0.33 ppm. Anaerobic conditions refer to conditions where oxygen is not supplied to the culture system. The culture may be performed under the conditions selected above during the entire period, or may be performed under the conditions selected above only during a part of the period. That is, “culturing under aerobic conditions” means that the culture is performed under aerobic conditions during at least a part of the whole period of culture. Further, “culturing under microaerobic conditions” means that the culture is performed under microaerobic conditions during at least a part of the entire culture period. Further, “culturing under anaerobic conditions” means that culturing is performed under anaerobic conditions in at least a part of the entire period of culture. The “partial period” may be, for example, a period of 50% or more, 70 or more, 80% or more, 90% or more, 95% or more, or 99% or more of the entire culture period. In addition, "the whole period of culture | cultivation" may mean the whole period of main culture, when culture | cultivation is performed by dividing into seed culture and main culture. Specifically, culture under aerobic conditions can be performed by aeration culture or shaking culture. In addition, the dissolved oxygen concentration in the liquid medium can be reduced by means such as reducing the aeration volume and stirring speed, culturing without sealing the container and aerated with an inert gas containing carbon dioxide gas. Achieving air or anaerobic conditions.

The pH of the medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the medium can be adjusted as necessary. The pH of the medium is adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. can do.

The medium may contain carbonate ions, bicarbonate ions, carbon dioxide gas, or a combination thereof. These components can be supplied, for example, by metabolism of the microorganisms of the present invention or from carbonates and / or bicarbonates used for pH adjustment. Moreover, these components can also be supplied from carbonic acid, bicarbonate, a salt thereof, or carbon dioxide as required. Specific examples of the carbonate or bicarbonate salt include, for example, calcium carbonate, magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, and potassium bicarbonate. Carbonate ions and / or bicarbonate ions may be added, for example, at a concentration of 0.001 to 5M, preferably 0.1 to 3M, more preferably 1 to 2M. When carbon dioxide gas is contained, for example, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 10 g of carbon dioxide gas per liter of the solution may be contained.

The culture temperature may be, for example, 20 to 45 ° C., preferably 25 to 37 ° C. The culture period may be, for example, 1 hour or more, 4 hours or more, 10 hours or more, or 15 hours or more, and may be 168 hours or less, 120 hours or less, 90 hours, or 72 hours or less. Specifically, the culture period may be, for example, 10 hours to 120 hours. The culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the microorganism of the present invention is lost.

By culturing the microorganism of the present invention under such conditions, the target substance accumulates in the microbial cells and / or in the medium.

In addition, when producing L-glutamic acid, it is also possible to carry out the culture while precipitating L-glutamic acid in the medium using a liquid medium adjusted to conditions under which L-glutamic acid is precipitated. The conditions under which L-glutamic acid precipitates are, for example, pH 5.0 to 3.0, preferably pH 4.9 to 3.5, more preferably pH 4.9 to 4.0, and particularly preferably around pH 4.7. (European Patent Application Publication No. 1078989). In addition, culture | cultivation may be performed at the said pH in the whole period, and may be performed at the said pH only for a part of period. The “partial period” may be a period as exemplified above.

The formation of the target substance can be confirmed by a known method used for detection or identification of a compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used in appropriate combination.

The produced target substance can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, and a crystallization method. These methods can be used in appropriate combination. In addition, when the target substance accumulates in the microbial cells, for example, the microbial cells are crushed by ultrasonic waves, etc., and the target substance is removed from the supernatant obtained by removing the microbial cells by centrifugation by an ion exchange resin method or the like. It can be recovered. The target substance to be recovered may be a free form, a salt thereof, or a mixture thereof. That is, the term “target substance” in the present invention may mean a free target substance, a salt thereof, or a mixture thereof. Examples of the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt. For example, the L-glutamic acid may be free L-glutamic acid, sodium L-glutamate (MSG), ammonium L-glutamate, or a mixture thereof. For example, in the case of L-glutamic acid, ammonium L-glutamate in the fermentation broth is crystallized by adding an acid, and equimolar sodium hydroxide is added to the crystals to obtain sodium L-glutamate (MSG). In addition, you may decolorize by adding activated carbon before and after the crystallization (see Industrial crystallization of sodium glutamate, Journal of the Seawater Society of Japan, Vol. 56, No. 5, Tetsuya Kawakita).

If the target substance is precipitated in the medium, it can be recovered by centrifugation or filtration. Further, the target substance precipitated in the medium may be isolated together after crystallization of the target substance dissolved in the medium.

The target substance to be collected may contain, for example, microbial cells, medium components, moisture, and microbial metabolic byproducts in addition to the target substance. The purity of the collected target substance is, for example, 30% (w / w) or higher, 50% (w / w) or higher, 70% (w / w) or higher, 80% (w / w) or higher, 90% ( w / w) or more, or 95% (w / w) or more.

When the target substance is L-glutamic acid, for example, sodium L-glutamate crystals can be used as an umami seasoning. The sodium L-glutamate crystals may be used as a seasoning by mixing with nucleic acids such as sodium guanylate and sodium inosinate having a similar umami taste.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

<Example 1: Preparation of an evaluation strain derived from Escherichia coli MG1655 strain>
In order to show that L-glutamic acid amino acids are produced in Escherichia coli MG1655 strain via succinyl CoA synthase, malate thiokinase, or succinyl CoA: malate CoA transferase, malyl CoA lyase and isocitrate lyase Evaluation stock was constructed.

<1-1> Construction of gltA gene to sdhABCD gene region-deficient strain The entire base sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the base of the gltA gene encoding citrate synthase Sequences have also been reported. That is, the gltA gene corresponds to the complementary sequence of base numbers 752408 to 753691 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3. The sdhCDAB gene encoding succinate dehydrogenase is present adjacent to the gltA gene in the genomic DNA of Escherichia coli MG1655 strain. That is, the sdhCDAB gene corresponds to the base numbers 754400 to 757628 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3.

This deficiency in the region containing gltA gene to sdhCDAB gene is determined by a method called “Red-driven integration” (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. went.

Using the oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 as DNA fragments for deletion of the region containing gltA gene to sdhCDAB gene using pMW118-attL-Km-attR (WO2005 / 010175, JP2005-58227) as a template Amplified by PCR. PMW118-attL-Km-attR is inserted into pMW118 (manufactured by Takara Bio Inc.) in order of attL-Km-attR. Plasmid. The fragment amplified by PCR was purified using Wizard® PCR® Prep® DNA® Purification System (Promega).

The DNA fragment for gene deletion was introduced by electroporation into Escherichia coli MG1655 strain containing plasmid pKD46 having 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. This is a plasmid into which a DNA fragment of 2154 bases in total (including gam, bet, and exo genes) (GenBank / EMBL accession number J02459, base number 31088 to 33241 region) was inserted. The plasmid pKD46 is necessary for integrating the DNA fragment for gene deletion into the genome (chromosome) of the MG1655 strain.

A competent cell for electroporation was prepared as follows. First, Escherichia coli MG1655 strain into which pKD46 was introduced at 30 ° C in L medium (10 g / L Bacto trypton, 5 g / L Bacto Yeast extract, 5 g / L NaCl) containing 100 mg / L ampicillin. Cultured overnight. 50 μL of this culture solution was inoculated into 5 μmL L medium containing 100 μmg / L ampicillin and 10 μmM L-arabinose. This was aerated at 30 ° C until the absorbance at 600 ODnm (OD600) reached about 0.6, then the cells were collected, washed 3 times with 10% glycerol, and concentrated 100 times by electroporation. A competent cell was obtained. Electroporation was performed using 70 μL of competent cells and about 1000 ng of PCR product. The cells after electroporation were added with 1 mL of SOC medium (Molecular Cloning: Laboratory Manual 2nd Edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) and incubated at 37 ° C for 2.5 hours. LBGM9 agarose plate containing Km (kanamycin) (40 mg / L) at 37 ° C (tryptone 1%, yeast extract 0.5%, sodium chloride 1.05%, glucose 0.5%, disodium hydrogen phosphate dodecahydrate 1.72% Km resistant recombinants were selected by plating with potassium dihydrogen phosphate 0.3%, ammonium chloride 0.1%, agarose 2%). The resulting colony was tested for ampicillin resistance, and an ampicillin sensitive strain from which pKD46 had been removed was obtained.

The deletion of the region containing the gltA to sdhCDAB gene of the mutant selected by kanamycin resistance was confirmed by PCR. A strain in which deletion of the region containing the gltA to sdhCDAB gene was confirmed was named EMC0 :: Km.

Next, the kanamycin resistance gene introduced into the gltA to sdhABCD gene region was extracted by a λ phage-derived excision system (Cho, E. H. et al. 2002. J. Bacteriol. 184: 5200-5203) as follows. Removed.

PMW-intxis-sacB, a plasmid with a temperature-sensitive replication ability, which carries a gene encoding lambda phage integrase (Int) and an ecdysase (Xis) to remove drug resistance genes (Cm) and pMW-intxis-sacB (Spc) were constructed.

PMW-intxis-sacB (Cm), a plasmid for removing drug resistance genes carrying the chloramphenicol resistance gene, is RSF-Red-TER (specialized) in the PstI-SphI site of pMW-intxis-ts (WO2007 / 037460). It was constructed by inserting a chloramphenicol resistance gene and a sacB gene derived from kai 2009-232844). Specifically, using RSF-Red-TER as a template, the primers of SEQ ID NO: 37 and SEQ ID NO: 38 were used to amplify a fragment of about 4.0 kb containing chloramphenicol resistance gene and sacB gene by PCR. Purified. On the other hand, pMW-intxis-ts was treated with PstI and SphI, and the ends were smoothed and phosphorylated using BKL kit from Takara. These two fragments were ligated by a ligation reaction, transformed into Escherichia coli DH5α strain, and an L agarose plate (10 g / L Bacto trypton, 5 g containing 25 mg / L chloramphenicol and 100 mg / L ampicillin). DH5α strain carrying pMW-intxis-sacB (Cm) was obtained by selection with / L Bacto Yeast extract, 5 g / L NaCl, 2% agarose). A plasmid pMW-intxis-sacB (Cm) was obtained from the DH5α strain carrying pMW-intxis-sacB (Cm) by a conventional method.

PMW-intxis-sacB (Spc), which is a drug resistance gene removal plasmid carrying a spectinomycin resistance gene, was constructed according to the following procedure. As a spectinomycin resistance gene, a plasmid pDG1726 was prepared from Escherichia coli ECE101 strain commercially available from Bacillus Genetic Stock Center (BGSC), a spectinomycin resistance gene derived from Enterococcus faecalis. It can be obtained by removing it from the plasmid as a cassette. First, the SC17 (0) / RSFRedSpc strain was prepared from the Pantoea ananatis SC17 (0) / RSFRedTER strain (RU patent application 2006134574, WO2008 / 090770, US2010-062496) by the Red driven integration method. Specifically, PCR is performed using the primers shown in SEQ ID NO: 68 and SEQ ID NO: 69, using pDG1726 as a template, and a spectrum having a 50-bp sequence homologous to part of the sequence on RSF-Red-TER at both ends. A tinomycin resistance gene fragment was obtained. The SC17 (0) / RSFRedTER strain is cultured overnight in LB liquid medium, and 1 μmL of this culture is inoculated into 100 μmL of LB liquid medium containing IPTG at a final concentration of 1 μmM and 25 μg / L chloramphenicol. Then, shaking culture was performed at 34 ° C. for 3 hours. After recovering the cells, the cells washed 3 times with 10% glycerol were used as competent cells. The amplified PCR fragment was purified using Promega's Wizard® PCR® Prep, introduced into competent cells by electroporation, and selected on L agarose plate containing 25 μmg / L spectinomycin. (0) / RSFRedSpc strain was obtained. Plasmid RSF-Red-TER (Spc) was obtained from SC17 (0) / RSFRedSpc strain by a conventional method. Subsequently, the ORF of the sacB gene and the spectinomycin resistance gene is amplified by PCR using RSF-Red-TER (Spc) as a template and the synthetic oligonucleotides of SEQ ID NO: 70 and SEQ ID NO: 71, and amplified by a conventional method. The obtained DNA fragment was purified. After purification, both ends were phosphorylated with a BKL kit (TaKaRa). Subsequently, pMW-intxis-ts (WO2007 / 037460) was treated with restriction enzyme with SphI, then the cleaved fragment was smoothed with DNA により Blunting Kit (TaKaRA), and the above phosphorylated DNA fragment was ligated to pMW- intxis-sacB (Spc) was prepared.

By introducing pMW-intxis-sacB (Cm) or pMW-intxis-sacB (Spc), recognizing attL or attR sequences on the genome, causing recombination, cutting out the gene between attL and attR, Only the attL or attR sequence remains.

EMC0 :: Km competent cells were prepared according to conventional methods, transformed with the helper plasmid pMW-intxis-sacB (Cm), and placed on an LBGM9 agarose plate containing 25 mg / L chloramphenicol at 30 ° C. And chloramphenicol resistant strains were selected. Next, the cells were passaged twice at 42 ° C on LBGM9 agarose plates without drugs (antibiotics), and the resulting colonies were tested for chloramphenicol resistance and kanamycin resistance. Acquired shares. The deletion of the kanamycin resistance gene introduced into the region containing the gltA to sdhCDAB gene of the obtained strain was confirmed by PCR. This strain was named EMC0.

<1-2> Enhanced expression of aceA gene and construction of aceB gene and aceK gene-deficient strain The complete nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3) and malate synthase The aceB gene encoding A, the aceA gene encoding isocitrate lyase, and the aceK gene encoding isocitrate dehydrogenase kinase / phosphatase have also been reported. These genes are continuously present on the genome of Escherichia coli MG1655 strain. That is, the aceB gene corresponds to the base numbers 4213501 to 4215102 of the genome sequence of the Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3. The aceA gene corresponds to base numbers 4215132 to 4216436 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3. The aceK gene corresponds to nucleotide numbers 4216619 to 4218355 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

As described below, expression enhancement of aceA gene and deletion of aceB and aceK genes of Escherichia coli MG1655 strain were performed.

As a method for changing the expression intensity of a gene, a method has been developed in which a promoter sequence having various expression intensities is inserted upstream of a target gene (Katashkina JI et al. Russian Federation Patent application 2006134574). In the same document, in the P. ananatis SC17 (0) strain, the kanamycin resistance gene (attL-Km-attR) that can be removed by the excision system derived from λ phage and the -35 region were mutated upstream of the lacZ gene on the genome. Strains in which a tac-like promoter having various expression intensities and a ribosome binding site (RBS) are inserted have been constructed. Therefore, using the genomic DNA of this strain as a template, the kanamycin resistance gene (attL-Km-attR) that can be removed by the excision system derived from λ phage upstream of the target gene by the Red driven integration method and various expression intensities A promoter can be inserted. Among the tac-like promoter sequences reported in the same literature, from the −35 region of the sequence with the highest expression intensity (SEQ ID No. 16 in the same literature) to the start codon including the ribosome binding site (RBS) The DNA sequence is shown in SEQ ID NO: 73.

As described below, aceA gene expression enhancement and aceB gene deletion were simultaneously performed in Escherichia coli MG1655 strain. A synthetic oligonucleotide having a sequence upstream of the aceB gene and an attL sequence (SEQ ID NO: 3), and a synthetic oligonucleotide having a sequence downstream from the start codon of the aceA gene and a sequence corresponding to a part of the tac-like promoter (SEQ ID NO: 4) The PCR was performed using the genomic DNA of the P. ananatis SC17 (0) strain having the tac-like promoter sequence, which has the highest expression intensity, as a template. The amplified PCR product was purified by a conventional method, and then introduced into Escherichia coli MG1655 strain carrying plasmid pKD46 having temperature-sensitive replication ability by electroporation. Strains were selected using kanamycin resistance as an index, and it was confirmed by PCR that the obtained kanamycin resistant strain was deficient in the aceB gene and inserted a tac-like promoter sequence immediately before the aceA gene. This strain was named MG1655 :: ΔaceBP4071-aceA :: Km. Since MG1655 :: ΔaceBP4071-aceA :: Km did not retain the pKD46 plasmid, the pKD46 plasmid was introduced again by a conventional method. In this strain, the aceK gene was deleted by the Red driven integration method described in Example <1-1>. Specifically, PCR was performed using the oligonucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 using pMW118-attL-Tc-attR (WO2005 / 010175, JP 2005-58227) as a template. The amplified PCR product was purified by a conventional method, and then introduced into MG1655 :: ΔaceBP4071-aceA :: Km retaining pKD46 by electroporation. Strains were selected on LBGM9 agarose plates containing 25 mg / L tetracycline hydrochloride using tetracycline resistance as an index. In the tetracycline resistant strain thus obtained, it was confirmed by PCR that the aceB gene was deleted, the tac-like promoter sequence was inserted immediately before the aceA gene, and the aceK gene was deleted. This strain was named MG1655 :: ΔaceB P4071-aceA :: KmΔaceK :: tet.

<1-3> Construction of glcB gene-deficient strain The entire base sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the base sequence of the glcB gene encoding malate synthase G is also reported. Has been. That is, the glcB gene corresponds to the complementary sequence of base numbers 3119656 to 3121827 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

Deletion of the glcB gene encoding malate synthase G of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-Tc-attR as a template using the oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 was used. It was confirmed by PCR that the strain selected using tetracycline resistance as an index was a strain lacking the glcB gene of Escherichia coli MG1655 strain, and this strain was named MG1655 ΔglcB :: tet.

<1-4> Construction of gcl gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the nucleotide sequence of the gcl gene encoding glyoxylate carboligase is also reported. Has been. That is, the gcl gene corresponds to nucleotide numbers 533140 to 533921 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3.

Deletion of the gcl gene encoding glyoxylate carboligase of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, fragments amplified by PCR using the oligonucleotides of SEQ ID NO: 9 and SEQ ID NO: 10 with pMW118-attL-Cm-attR (WO 05/010175) as a template were used. In LBGM9 agarose plate containing 25 mg / L chloramphenicol, it was confirmed by PCR that the gcl gene of Escherichia coli MG1655 strain was selected as a strain selected using chloramphenicol resistance as an index, This strain was named MG1655 Δgcl :: Cm.

<1-5> Construction of maeA gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the maeA gene encoding malate dehydrogenase (NAD-malic enzyme) The nucleotide sequence of is also reported. That is, the maeA gene corresponds to the complementary sequence of base numbers 1551996 to 1536993 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

Deletion of maeA gene encoding malate dehydrogenase (NAD-malic enzyme) of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-Tc-attR as a template using the oligonucleotides of SEQ ID NO: 11 and SEQ ID NO: 12 was used. It was confirmed by PCR that the strain selected using tetracycline resistance as an index was a strain lacking the maeA gene of Escherichia coli MG1655 strain, and this strain was named MG1655 ΔmaeA :: tet.

<1-6> Construction of maeB gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the maeB gene encoding malate dehydrogenase (NADP-malic enzyme) The nucleotide sequence of is also reported. That is, the maeB gene corresponds to a complementary sequence of base numbers 2574120 to 2576399 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

Deletion of the maeB gene encoding malate dehydrogenase (NADP-malic enzyme) of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-Cm-attR as a template using the oligonucleotides of SEQ ID NO: 13 and SEQ ID NO: 14 was used. It was confirmed by PCR that the strain selected using chloramphenicol resistance as an indicator was a strain lacking the maeB gene of Escherichia coli MG1655 strain, and this strain was named MG1655 ΔmaeB :: Cm.

<1-7> Construction of mdh gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the nucleotide sequence of the mdh gene encoding malate dehydrogenase has also been reported. ing. That is, the mdh gene corresponds to the complementary sequence of base numbers 3381352 to 3382290 of the Escherichia coli MG1655 strain genome sequence described in GenBank accession number NC_000913.3.

Deletion of the mdh gene encoding malate dehydrogenase of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-Km-attR as a template using the oligonucleotides of SEQ ID NO: 15 and SEQ ID NO: 16 was used. It was confirmed by PCR that the strain selected using kanamycin resistance as an index was a strain in which the mdh gene of Escherichia coli MG1655 was deleted, and this strain was named MG1655 Δmdh :: Km.

<1-8> Construction of prpC gene, prpD gene, and prpE gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and 2-methylcitrate synthase Nucleotide sequences of the prpC gene encoding, the prpD gene encoding 2-methylcitrate dehydratase, and the prpE gene encoding propionyl-CoA ligase have also been reported. These genes are continuously present on the genome of Escherichia coli MG1655 strain. That is, the prpC gene corresponds to base numbers 349236 to 350405 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3. The prpD gene corresponds to nucleotide numbers 350439 to 351890 of the Escherichia coli MG1655 genome sequence described in GenBank accession number NC_000913.3. The prpE gene corresponds to base numbers 351930 to 353816 of the Escherichia coli MG1655 genome sequence described in GenBank accession number NC_000913.3.

Deletion of the region containing the prpC gene, prpD gene, and prpE gene of Escherichia coli MG1655 strain was performed according to the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-Km-attR as a template using the oligonucleotides of SEQ ID NO: 17 and SEQ ID NO: 18 was used. It was confirmed by PCR that the strain selected using kanamycin resistance as an index was a strain lacking the prpCDE gene of Escherichia coli MG1655 strain, and this strain was named MG1655 ΔprpCDE :: Km.

<1-9> Construction of dctA gene expression enhancement strain The entire base sequence of genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and C4 dicarboxylic acid, orotic acid, and citric acid uptake carrier The nucleotide sequence of the dctA gene coding for is also reported. That is, the dctA gene corresponds to a complementary sequence of base numbers 3680184 to 3681470 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3.

The enhanced expression of the dctA gene encoding the C4 dicarboxylic acid, orotic acid, and citric acid uptake carrier of Escherichia coli MG1655 strain is the same as the enhanced expression of the aceA gene described in Example <1-2>. I went by law. Specifically, using the oligonucleotides of SEQ ID NO: 21 and SEQ ID NO: 22, P. aureus having a tac-like promoter sequence with the highest expression intensity in the above-mentioned document (Katashkina JI et al. Russian Federation Patent application 2006134574). A fragment amplified by PCR using the genomic DNA of ananatis (SC17 (0) strain as a template was used. It was confirmed by PCR that the strain selected using kanamycin resistance as an index was a strain in which a tac-like promoter sequence was inserted immediately before the dctA gene of Escherichia coli MG1655 strain, and this strain was named MG1655 P4071-dctA :: Km It was.

<1-10> Construction of mqo gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the nucleotide sequence of the mqo gene encoding malate: quinone oxidoreductase is also known. It has been reported. That is, the mqo gene corresponds to the complementary sequence of base numbers 2303130 to 2304776 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

Deletion of the mqo gene encoding malate: quinone oxidoreductase of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-Km-attR as a template using oligonucleotides of SEQ ID NO: 19 and SEQ ID NO: 20. It was confirmed by PCR that the strain selected using kanamycin resistance as an index was a strain deficient in the mqo gene of Escherichia coli MG1655 strain, and this strain was named MG1655 Δmqo :: Km.

<1-11> Creation of E. coli MG1655 Evaluated Strain Evaluated Strains EMC0 strain as parent strain, strains constructed in Examples <1-2> to <1-10> as donors and transduction and λ phage origin An evaluation strain was prepared by repeating removal of the drug resistance gene by the excision system.

Transduction uses P1kc phage, Miller et al. Method (Miller, J. H., Experiments in molecular genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1972, Generalized transduction: use of P1 in strain construction According to 201-205), the procedure was as follows.

The P1kc phage used for transduction was prepared by the following procedure. MG1655 :: ΔaceB P4071-aceA :: KmΔaceK :: tet prepared in Example <1-2> was cultured overnight at 37 ° C. in 3 mL of L medium containing 2.5 mM CaCl ₂ . Add 100 μl of culture medium and P1kc phage solution to 3 mL of L medium + soft agar (0.5% agar), and add L agarose plate containing 2.5 mM CaCl ₂ (10 g / L Bacto trypton, 5 g / L Bacto Yeast extract) , 5 g / L NaCl, 2% agarose). After soft agar solidified, it was cultured overnight at 37 ° C. 5 mL of L medium was added onto the soft agar on which plaque was generated, the agar was crushed, and the grown phages were collected. 1 mL of chloroform was added to this L medium, mixed gently, and allowed to stand at room temperature for 15 min. Cells and soft agar are removed by centrifugation (4 ° C, 2,000 xg, 5 min), and the supernatant is treated with a 0.45 µm cellulose acetate type membrane filter manufactured by Advantech Toyo Co., Ltd. It was collected.

Transduction was performed according to the following procedure. A strain bred from Escherichia coli MG1655 strain cultured overnight at 37 ° C. in 3 mL of L medium containing 2.5 mM CaCl ₂ was used as a preculture solution for recipients. 100 μl of the phage suspension diluted 1-fold, 10-fold, or 100-fold with L medium containing 2.5 mM CaCl ₂ was added to 100 μl of this preculture solution, and the mixture was incubated at 37 ° C. for 20 minutes. After the adsorption of the phage particles, 100 μl of 1 M trisodium citrate and 1 mL of L medium were added and incubated at 37 ° C. for 30 minutes. The cell pellet obtained by centrifuging the mixture (4 ° C, 5,000 xg, 1 min) is applied to an LBGM9 agarose plate containing 40 mg / L kanamycin and 25 mg / L tetracycline hydrochloride. Incubated overnight at 37 ° C. Among the formed colonies, a strain in which gene transduction (ΔaceB P4071-aceA :: KmΔaceK :: tet) at the target position was confirmed by PCR was obtained as a transductant. This strain was named EMC1.0 :: Km tet.

After transduction, the drug resistance gene was removed using the λ phage-derived excision system. Specifically, the helper plasmid pMW-intxis-sacB (Cm) was introduced into EMC1.0 :: Km tet and plated on LBGM9 agarose plates containing 25 mg / L chloramphenicol at 30 ° C. A chloramphenicol resistant strain was selected. Next, the cells were passaged twice at 42 ° C. on an LBGM9 agarose plate containing no drug (antibiotic), and the resulting colonies were tested for chloramphenicol resistance, tetracycline resistance, and kanamycin resistance. Cole, tetracycline, and kanamycin sensitive strains were obtained. The deletion of the kanamycin resistance gene introduced into the region containing the aceB gene and the tetracycline resistance gene introduced into the region containing the aceK gene of the obtained strain was confirmed by PCR. This strain was named EMC1.0.

Similarly, the strains prepared in Examples <1-3> to <1-10> (MG1655 ΔglcB :: tet, MG1655 Δgcl :: Cm, MG1655 ΔmaeA :: tet, MG1655 ΔmaeB :: Cm, MG1655 Δmdh :: Km, MG1655 ΔprpCDE :: Km, MG1655 P4071-dctA :: Km, MG1655 Δmqo :: Km) were each prepared as a P1kc phage, and an evaluation strain was prepared by repeating transduction and removal of the drug resistance gene.
Specifically, a strain in which the glcB gene of EMC1.0 was disrupted with a P1kc phage prepared using MG1655 ΔglcB :: tet and the drug resistance gene was removed was named EMC1.1.
A strain in which the gcl gene of EMC1.1 was disrupted with the P1kc phage prepared using MG1655 Δgcl :: Cm and the drug resistance gene was removed was named EMC1.2.
A strain from which the maeA gene of EMC1.2 was disrupted with the P1kc phage prepared using MG1655 ΔmaeA :: tet and the drug resistance gene was removed was named EMC1.3.
A strain from which the maeB gene of EMC1.3 was disrupted with the P1kc phage prepared using MG1655 ΔmaeB :: Cm and the drug resistance gene was removed was named EMC1.4.
A strain from which the mdh gene of EMC1.4 was disrupted with the P1kc phage prepared using MG1655 Δmdh :: Km and the drug resistance gene was removed was named EMC1.5.
A strain in which a tac-like promoter sequence was inserted upstream of the dctA gene of EMC1.5 by the P1kc phage prepared using MG1655 P4071-dctA :: Km and the drug resistance gene was removed was named EMC1.6.
A strain in which the prpCDE gene of EMC1.6 was disrupted with the P1kc phage prepared using MG1655 ΔprpCDE :: Km and the drug resistance gene was removed was named EMC1.62.
A strain in which the mqo gene of EMC1.62 was disrupted by the P1kc phage prepared using MG1655 Δmqo :: Km and the drug resistance gene was removed was named EMC1.65.

Note that the helper plasmid pMW-intxis-sacB (Cm) or pMW-intxis-sacB (Spc) was used to remove the drug resistance gene. If pMW-intxis-sacB (Spc) was used, plate overnight on LBGM9 agarose plates containing 25 μg / L spectinomycin dihydrochloride pentahydrate at 30 ° C. After selecting the mycin resistant strain, it was subcultured twice at 42 ° C. on an LBGM9 agarose plate containing no drug to obtain a strain from which the drug resistant gene was removed.

Table 1 shows the strain names and genotypes of the evaluation strains (also referred to as “EMC family strains”) constructed by the above procedure.

<Example 2: Confirmation of reversibility of isocitrate lyase>
<2-1> Preparation of Evaluated Strains EMC0 and EMC1.0 were respectively applied to LBGM9 agarose plates and grown overnight at 37 ° C. The bacterial cells were collected in 1 mL of sterilized water, and the bacterial cells were washed twice with sterilized water, and then a bacterial cell suspension with an OD600 of 10 diluted to 10 was prepared. Table 2 shows the characteristics of EMC0 and EMC1.0 used as the evaluation strains.

<2-2> Preparation of Evaluation Medium As an evaluation medium, M9 glucose minimal medium (5 g / L glucose, 17.1 g / L disodium hydrogen phosphate, 3.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium chloride) 0.5 g / L NaCl, 0.25 g / L magnesium sulfate heptahydrate) was prepared. A medium supplemented with one or both of glyoxylic acid monohydrate and disodium succinate to a final concentration of 1 g / L was also prepared based on M9 glucose minimal medium. In addition, based on the M9 glucose minimal medium, a medium in which sodium glutamate was added to a final concentration of 1 g / L was also prepared.

<2-3> Inoculation and culture 5 μL of the cell suspension having an OD600 of 10 was inoculated into 5 mL of the evaluation medium prepared as described above. Incubation was performed at 37 ° C and 70 rpm while recording the absorbance at 660 nm (OD660) every 15 minutes using the automatic OD measurement culture device BIO-PHOTORECORDER TN-1506 (ADVANTEC) and its dedicated L-shaped test tube. Performed with shaking.

Table 3 shows the presence or absence of bacterial cell growth when cultured for 40 hours under the above conditions. In Table 3, “++” indicates that the cells grew and the OD660 was 0.8 or more, and “−” indicates that the cells did not grow when the OD660 was 0.05 or less. EMC0 and EMC1.0 show no growth of cells in media containing no additives, media containing only glyoxylic acid, and media containing only succinic acid, and growth of cells in media supplemented with glutamic acid. It was observed. From this, it was confirmed that EMC0 and EMC1.0 have glutamic acid requirement due to gltA deficiency. In addition, in the medium supplemented with glyoxylic acid and succinic acid, EMC0 showed no growth of cells but EMC1.0 showed growth of the cells. From this, it is considered that in the evaluation strain EMC1.0, isocitrate was generated from glyoxylic acid and succinic acid by isocitrate lyase, and glutamic acid requirement was complemented. Therefore, it is considered that isocitrate lyase functions not only in the decomposition direction of isocitrate but also in the synthesis direction of isocitrate, that is, has reversibility.

<Example 3: Construction of expression plasmid of succinyl CoA synthase gene derived from Escherichia coli>
The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the gene encoding the succinyl CoA synthase of Escherichia coli MG1655 strain (hereinafter referred to as “sucCD gene”) A certain base sequence has also been reported. That is, the sucC gene corresponds to nucleotide numbers 762237 to 763403 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3. The sucD gene corresponds to nucleotide numbers 763403 to 764272 of the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3.

The Escherichia coli-derived succinyl-CoA synthase gene expression plasmid is as follows. First, a tac-like promoter sequence is ligated to the sucCD gene on genomic DNA, and then the tac-like promoter amplified by PCR using this genomic DNA as a template and The DNA fragment containing the sucCD gene was constructed by cloning into pMW plasmid and pSTV plasmid.

<3-1> Insertion of tac-like promoter sequence upstream of sucCD gene First, in the same manner as in Example <1-2>, in the genomic DNA of Escherichia coli MG1655 strain, tac was inserted immediately before the sucCD gene. A similar promoter sequence was inserted.

A synthetic oligonucleotide having a sequence upstream of the sucCD gene and an attL sequence (SEQ ID NO: 23), and a synthetic oligonucleotide having a sequence downstream from the start codon of the sucCD gene and a sequence corresponding to a part of the tac-like promoter (SEQ ID NO: 24) As a primer, the genome of the P. ananatis SC17 (0) strain having the tac-like promoter sequence (SEQ ID NO: 73) with the highest expression intensity in the above document (Katashkina JI et al. Russian Federation Patent application 2006134574) PCR was performed using DNA as a template. The amplified PCR product was purified by a conventional method, and then introduced into Escherichia coli MG1655 strain carrying plasmid pKD46 having temperature-sensitive replication ability by electroporation. Next, it was confirmed by PCR that a promoter sequence was inserted immediately before the sucCD gene of Escherichia coli MG1655 strain that became kanamycin resistance. From this strain, genomic DNA having a promoter sequence inserted immediately before the sucCD gene was prepared by a conventional method.

<3-2> Construction of Escherichia coli-derived Succinyl CoA Synthase Gene Expression Plasmid Genomic DNA of Escherichia coli MG1655 strain in which a tac-like promoter sequence is inserted immediately before the sucCD gene constructed in Example <3-1> PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 25 and 26 as templates to amplify a DNA fragment containing a tac-like promoter and a sucCD gene. The obtained DNA fragment was cleaved with BamHI and EcoRI, inserted into the BamHI-EcoRI restriction sites of pMW219 and pSTV29 (manufactured by Takara Bio Inc.), and succinyl CoA synthase gene expression plasmids were constructed, respectively, pMW-STK and pSTV- I named it STK.

<Example 4: Introduction of mutation into succinyl CoA synthase gene derived from Escherichia coli>
<4-1> Site-directed mutagenesis into Escherichia coli-derived succinyl-CoA synthase gene Site-directed mutagenesis into Escherichia coli-derived succinyl-CoA synthase gene was performed as follows. PCR was performed using pSTV-STK as a template and the primer pairs of SEQ ID NOs: The PCR product was treated with the restriction enzyme DpnI to decompose the template, and Escherichia coli JM109 strain competent cells were transformed to obtain transformants that grew on L agarose plates containing chloramphenicol 25 μg / mL. The obtained colonies were cultured overnight at 37 ° C. on an L agarose plate containing chloramphenicol 25 μg / mL, the plasmid was recovered from the grown bacteria by a conventional method, the DNA sequence was confirmed, and the DNA of the sucCD gene Plasmids (Table 4) in which the target mutation was correctly introduced into the sequence were obtained.

<4-2> Introduction of double mutation into succinyl-CoA synthase gene derived from Escherichia coli As described below, the 161st valine of the α subunit (encoded by the sucD gene) is alanine in the sucCD gene of pSTV-STK. And a mutation in which the 271st glycine of the β subunit (encoded by the sucC gene) was replaced with alanine was introduced.

In the sucCD gene linked to the tac-like promoter inserted in the BamHI restriction site and EcoRI restriction site of pSTV-STK, a DNA sequence was designed so that the mutation with the above two amino acid substitutions was introduced. The DNA sequence designed in this way is shown in SEQ ID NO: 67. The DNA sequence of SEQ ID NO: 67 was synthesized in a form inserted into the BamHI restriction site and EcoRI restriction site of the pUC57 plasmid vector. A double mutant sucCD gene in which a tac-like promoter was linked to pSTV29 was inserted from this plasmid using the BamHI restriction site and EcoRI restriction site. The plasmid thus obtained was named pSTV-STK (V161A, G271A: β). This plasmid is also referred to as “pSTV-STK ^** ”.

<Example 5: Construction of expression plasmid of malate thiokinase gene, malyl CoA lyase gene, succinyl CoA: malate CoA transferase gene>
<5-1> Construction of expression plasmids for malate thiokinase gene and malyl-CoA lyase gene Malate thiokinase gene derived from Methylobacterium Extrusence, Mesozobium roti, and Granulibacter bethesdensis by the following procedure And the malyl-CoA lyase gene were obtained by artificial DNA synthesis and an expression plasmid was constructed. Each gene sequence was modified according to the codon usage of Escherichia coli so that it was highly expressed in Escherichia coli. In addition, in each of the organisms from which the gene is derived and Escherichia coli, the combination of codons and corresponding amino acids is said to be the same, so that the codon usage frequency of Escherichia coli is highly expressed in Escherichia coli, etc. Even if each gene sequence is modified according to the above, a protein having the same amino acid sequence as that before modification is expressed.

A tac promoter and an RBS sequence (SEQ ID NO: 72) are ligated upstream of the mtkAB gene encoding malate thiokinase derived from Methylobacterium extruens AM1 strain, a HindIII and SalI restriction site at the 5 ′ end, PstI A DNA sequence carrying an EcoRI restriction site at the 3 'end was designed. A tac promoter and an RBS sequence (SEQ ID NO: 72) were ligated upstream of the mclA gene encoding malyl-CoA lyase derived from Methylobacterium extremens AM1 strain. A DNA sequence carrying an EcoRI restriction site at the 3 'end was designed. At that time, the DNA sequence was modified according to the codon usage frequency of Escherichia coli so that the mtkA, mtkB, and mclA genes were highly expressed in Escherichia coli. Moreover, as a result of modifying the DNA sequence, the DNA sequence to be synthesized was designed so that no HindIII, SalI, PstI, or EcoRI restriction sites other than both ends were generated. The thus designed DNA sequence containing the mclA gene is shown in SEQ ID NO: 39, and the DNA sequence containing the mtkAB gene is shown in SEQ ID NO: 40.

The synthesis of DNA sequences of SEQ ID NO: 39 and SEQ ID NO: 40 was outsourced to Takara Bio Inc. The synthesized DNA sequence containing the tac promoter, the RBS sequence, and the mclA gene derived from Methylobacterium extremens AM1 strain is inserted into the HindIII-EcoRI restriction site of pTWV229, and the resulting plasmid is pTWV-MEX_A I named it. The synthesized DNA sequence containing the tac promoter, the RBS sequence, and the mtkAB gene derived from Methylobacterium extremens AM1 strain is inserted into the HindIII-EcoRI restriction site of pHSG396, and the resulting plasmid is pHSG-MEX_K I named it.

Subsequently, a DNA fragment of about 4.5 kbp that can be opened by restriction treatment of pHSG-MEX_K with HindIII and SalI, and a DNA fragment of about 1.0 kbp that can be obtained by restriction treatment of pTWV-MEX_A with HindIII and SalI Were mixed and ligated, and these DNA fragments were ligated. By this operation, it is possible to obtain a plasmid that simultaneously expresses the mtkAB gene and mclA gene derived from the Methylobacterium extremens AM1 strain. This plasmid was named pHSG396-MEX_AK.

The expression plasmids of mtkAB gene and mclA gene derived from Mesozobium roti MAFF303099 strain and Granulibacter bethesdensis strain CGDNIH1 were prepared by the following method.

The gene derived from each of Mesozobium loti MAFF303099 and Granulibacter bethesdensis CGDNIH1 encodes malyl-CoA lyase in which HindIII restriction site, tac promoter and RBS sequence (SEQ ID NO: 72) are linked in order from the 5 ′ end. A mclA gene, a SalI restriction site, a mtkAB gene encoding malate thiokinase linked to a tac promoter and an RBS sequence (SEQ ID NO: 72), and a DNA sequence having an EcoRI restriction site were designed. At that time, the DNA sequence was modified according to the codon usage frequency of Escherichia coli so that the above-described mtkA, mtkB, and mclA genes derived from each organism were highly expressed in Escherichia coli. Moreover, as a result of modifying the DNA sequence, the DNA sequence to be synthesized was designed so that no HindIII, SalI, or EcoRI restriction sites other than both ends were generated. The DNA sequence containing the mclA gene and mtkAB gene derived from Mesozobium loti MAFF303099 strain, which was designed in this way, is SEQ ID NO: 41, and the DNA sequence containing the mclA gene and mtkAB gene from Granulibacter bethesdensis strain CGDNIH1 is arranged. No. 42.

The synthesis of the DNA sequences of SEQ ID NO: 41 and SEQ ID NO: 42 was outsourced to Genscript. The synthesized DNA fragments of SEQ ID NO: 41 and SEQ ID NO: 42 were respectively inserted into pHSG396 using a HindIII-EcoRI restriction site. The thus obtained mtkAB gene and mclA gene expression plasmids derived from Mesozobium loti MAFF303099 strain are pHSG-MLO_AK, mtkAB gene and mclA gene expression plasmids derived from Granularibacterium bethesdensis CGDNIH1 strain are pHSG-GRA_AK Named.

In order to construct a plasmid that expresses only the mclA gene derived from Mesozobium loti MAFF303099 strain, the tac promoter and RBS sequence existing between the HindIII restriction site and the SalI restriction site of the synthesized DNA fragment of SEQ ID NO: 41 were linked. The mclA gene was inserted between the HindIII and SalI restriction sites of pTWV229. The resulting plasmid was named pTWV-MLO_A.

<5-2> Construction of expression plasmid for succinyl CoA: malate CoA transferase gene According to the following procedure, succinyl CoA derived from Chloroflexus aurantix, Accumulibacter phosphatis, Magnetospirillum magneticum, and Rhodospirillum rubrum: The malate CoA transferase gene was obtained by artificially synthesizing DNA to construct an expression plasmid.

The tac promoter and RBS sequence (SEQ ID NO: 72) are linked upstream of the smtAB gene (Ca_smtAB gene) encoding the succinyl CoA: malate CoA transferase derived from Chloroflexus aurantiax J-10-fl, and the SalI restriction site is A DNA sequence having an EcoRI restriction site at the 3 ′ end at the 5 ′ end was designed. At that time, the DNA sequences of the smtA and smtB genes were modified according to the codon usage frequency of Escherichia coli. Moreover, as a result of modifying the DNA sequence, the DNA sequence to be synthesized was designed so that no HindIII, SalI, EcoRI, SphI, or NdeI restriction sites other than both ends were generated. The DNA sequence designed in this way is shown in SEQ ID NO: 43. The synthesis of the DNA sequence of SEQ ID NO: 43 was entrusted to Genscript, and the synthesized DNA fragment was inserted into pSTV29 using a SalI-EcoRI restriction site. The resulting Ca_smtAB gene expression plasmid was named pSTV-Ca_T.

The tac promoter and RBS sequence (SEQ ID NO: 72) are ligated upstream of the smtAB gene (Ap_smtAB gene) encoding succinyl CoA: malate CoA transferase derived from Accumulactor phosphatis (candidate strain) clade IIAstr. UW-1. A DNA sequence having a BamHI restriction site at the 5 ′ end and an EcoRI restriction site at the 3 ′ end was designed. At that time, the DNA sequences of the smtA and smtB genes were modified according to the codon usage frequency of Escherichia coli. In addition, as a result of modifying the DNA sequence, the DNA sequence to be synthesized was designed so that HindIII, BamHI, EcoRI, SphI, and NdeI restriction sites other than both ends were not generated. The DNA sequence designed in this way is shown in SEQ ID NO: 44. The synthesis of the DNA sequence of SEQ ID NO: 44 was entrusted to Genscript, and the synthesized DNA fragment was inserted into pSTV29 using a BamHI-EcoRI restriction site. The resulting Ap_smtAB gene expression plasmid was named pSTV-Ap_T.

The tac promoter and RBS sequence (SEQ ID NO: 72) are linked upstream of the smt gene (Mm_smt gene) encoding the succinyl CoA: malate CoA transferase derived from Magnetospirillum magneticumum AMB-1, and the SalI restriction site is connected to the 5 'end. In addition, a DNA sequence having an EcoRI restriction site at the 3 ′ end was designed. At that time, the DNA sequence of the Mm_smt gene was modified according to the codon usage frequency of Escherichia coli. In addition, when modifying the DNA sequence, the DNA sequence to be synthesized was designed so that no HindIII, SalI, EcoRI, SphI, or NdeI restriction sites other than both ends were generated. The DNA sequence designed in this way is shown in SEQ ID NO: 45. The synthesis of the DNA sequence of SEQ ID NO: 45 was commissioned to Genscript, and the synthesized DNA fragment was inserted into each of pSTV29 and pTWV229 using a SalI-EcoRI restriction site. The Mm_smt gene expression plasmid derived from pSTV29 thus obtained was named pSTV-Mm_T, and the Mm_smt gene expression plasmid derived from pTWV229 was named pTWV-Mm_T.

The tac promoter and RBS sequence (SEQ ID NO: 72) are linked upstream of the smt gene (Rr_smt gene) encoding the succinyl CoA: malate CoA transferase derived from Rhodospirillum rubrum ATCC 11170 strain, and the SalI restriction site is connected to the 5 'end and EcoRI. A DNA sequence carrying a restriction site at the 3 ′ end was designed. At that time, the DNA sequence of the Rr_smt gene was modified according to the codon usage frequency of Escherichia coli. In addition, when modifying the DNA sequence, the DNA sequence to be synthesized was designed so that no HindIII, SalI, EcoRI, SphI, or NdeI restriction sites other than both ends were generated. The DNA sequence designed in this way is shown in SEQ ID NO: 46. The synthesis of the DNA sequence of SEQ ID NO: 46 was entrusted to Genscript, and the synthesized DNA fragment was inserted into pSTV29 using a SalI-EcoRI restriction site. The resulting Rr_smt gene expression plasmid was named pSTV-Rr_T.

<Example 6: Measurement of glyoxylate production activity using L-malate as a substrate by malate thiokinase and malyl-CoA lyase>
<6-1> Construction of malate thiokinase gene and malyl CoA lyase gene-introduced strain derived from each organism Glyoxylate production activity using malate thiokinase and malyl CoA lyase using L-malate as a substrate (hereinafter referred to as “malate thiokinase and In order to perform the measurement of “linked enzyme activity of malyl-CoA lyase”), strains were constructed in which pHSG-MEX_AK, pHSG-MLO_AK, and pHSG-GRA_AK were respectively introduced into EMC1.65. As a control strain, a strain in which pHSG396 was introduced into EMC1.65 was constructed. Since pHSG-MEX_AK, pHSG-MLO_AK, pHSG-GRA_AK, and pHSG396 are chloramphenicol resistant plasmids, plasmids were introduced using LBGM9 agarose plates containing chloramphenicol 25 μg / mL. A stock selection was made.

<6-2> Preparation of cell extract Each strain was cultured overnight at 30 ° C. in 3 mL of LBGM9 medium containing 25 μg / mL of chloramphenicol. Subsequently, 500 μL of the culture solution was inoculated into 50 mL of LBGM9 medium containing 25 μg / mL of chloramphenicol, shake-cultured, and collected when the OD600 reached 0.4 to 0.6 by centrifugation. The collected cells were washed twice with 100 mM potassium phosphate buffer (pH 7.0) and suspended in 50 mM Tris-HCl (pH 7.5) solution containing 5 mM MgCl ₂ . The suspended cells were sonicated and the supernatant fraction obtained after ultracentrifugation (4 ° C., 53,000 rpm, 1 hour (RP80AT, manufactured by HITACHI-KOKI)) was obtained as a cell extract. The protein concentration of the obtained cell extract was measured by the Bradford method. The collected cells may be stored at -80 ° C.

<6-3> Activity measurement Activity measurement was performed spectroscopically at 30 ° C using a Beckman coulter DU-800 spectrometer. The ligase activity of malate thiokinase and malyl CoA lyase was measured according to the method of Louis (Louis B. Hersh J Biol Chem. 1973 Nov 10; 248 (21): 7295-303.). Specifically, the protein was added to 1 mL of a test solution containing 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl ₂ , 1.5 mM phenylhydrazine, and 1 mM CoA Na, 4 mM ATP Na _2. After mixing 50 to 500 μg of cell extract in an amount, disodium L-malate was added to a final concentration of 20 mM. Table 5 shows the enzyme activity values determined from the increase in absorbance at 324 nm after addition of disodium L-malate and the amount of added cell extract protein.

In the strain into which the malate thiokinase gene and the malyl CoA lyase gene were introduced, ligated enzyme activities of malate thiokinase and malyl CoA lyase were detected. In addition, the highest activity was detected in a strain into which malate thiokinase gene and malyl CoA lyase gene derived from Mesozobium roti MAFF303099 were introduced.

<Example 7: Confirmation of malate thiokinase activity by Escherichia coli succinyl CoA synthase>
<7-1> Construction of Escherichia coli-derived succinyl CoA synthase gene expression enhanced strain A strain in which pTWV-MEX_A was introduced into EMC1.65 was constructed. PSTV-STK was further introduced into this strain to construct a strain in which the expression of the mclA gene derived from Methylobacterium extruens AM1 strain and the succinyl CoA synthase gene derived from Escherichia coli was enhanced. In addition, a control strain in which pSTV29 was further introduced into the strain in which pTWV-MEX_A was introduced into EMC1.65 was constructed. Since pTWV-MEX_A is a plasmid conferring ampicillin and pSTV29 is a plasmid conferring chloramphenicol resistance, an LBGM9 agarose plate containing ampicillin sodium 100 μg / mL and chloramphenicol 25 μg / mL was used. A strain into which the plasmid was introduced was selected.

<7-2> Preparation of Cell Extract A cell extract was prepared in the same manner as in Example <6-2> from the strain constructed in Example <7-1>. However, as the medium, LBGM9 medium containing ampicillin sodium 100 μg / mL and chloramphenicol 25 μg / mL was used.

<7-3> Activity measurement Activity measurement was performed in the same manner as in Example <6-3>. The results are shown in Table 6. As shown in Table 6, ligated enzyme activities of malate thiokinase and malyl CoA lyase were increased by amplifying the expression of the succinyl CoA synthase gene. Thus, succinyl CoA synthase was shown to have malate thiokinase activity.

<Example 8: Improvement of malate thiokinase activity by introducing mutation into succinyl-CoA synthase gene derived from Escherichia coli>
<8-1> Construction of mutant succinyl-CoA synthase gene expression strain derived from Escherichia coli Example <4-1> was introduced into a strain into which pTWV-MEX_A was introduced into EMC1.65 in the same manner as Example <7-1>. > And the expression plasmid of each mutant succinyl-CoA synthase gene derived from Escherichia coli prepared in Example <4-2>, and the mclA gene derived from Methylobacterium extruens AM1 strain and Escherichia coli Strains expressing each mutant succinyl-CoA synthase gene derived from E. coli were constructed.

<8-2> Preparation of cell extract A cell extract was prepared in the same manner as in Example <7-2> from the strain prepared in Example <8-1>. However, as a medium, LBGM9 medium containing ampicillin sodium 100 μg / mL and chloramphenicol 25 μg / mL was used.

<8-3> Activity measurement Activity measurement was performed in the same manner as in Example <6-2>. The results are shown in Table 7. As shown in Table 7, the ligated enzyme activities of malate thiokinase and malyl CoA lyase were improved by introducing a mutation accompanied by amino acid substitution into the succinyl CoA synthase gene. That is, when wild-type succinyl-CoA synthase gene was introduced (pSTV-STK), the activity of 43.8 nmol / min / mg protein (hereinafter referred to as mU) was the 124th proline of the α subunit encoded by the sucD gene. When it is substituted with alanine (pSTV-STK (P124A)), it rises to 52.4 mU, and when the 157th tyrosine of the α subunit encoded by the sucD gene is replaced with glycine (pSTV-STK (T157G)) When the 161st valine of the α subunit encoded by the sucD gene is replaced with alanine (pSTV-STK (V161A)), the level increases to 59.3 mU, and the α subunit encoded by the sucD gene When the 97th glutamic acid was replaced with aspartic acid (pSTV-STK (E97D)), it increased to 95.3 mU, and when the 271st glycine of the β subunit encoded by the sucC gene was replaced with alanine ( pSTV-STK (G271A: β)) increased to 48 mU It was. In addition, when the 161st valine of the α subunit encoded by the sucD gene is substituted with alanine and the 271st glycine of the β subunit encoded by the sucC gene is replaced with alanine (pSTV-STK (V161A, G271A: In β)), it increased to 132 mU. Thus, these mutations have been shown to improve the malate thiokinase activity of succinyl CoA synthase.

<Example 9: Measurement of succinyl CoA: malate CoA transferase activity in smtAB gene-introduced strain>
<9-1> Construction of Ca_smtAB gene-introduced strain and Ap_smtAB gene-introduced strain A strain was constructed by introducing pSTV-Ca_T and pSTV-Ap_T into EMC1.65 strain, respectively. As a control strain, a strain obtained by introducing pSTV29 into EMC1.65 strain was constructed. Since the plasmid derived from pSTV29 is a plasmid conferring chloramphenicol resistance, a strain into which the plasmid was introduced was selected using an LBGM9 agarose plate containing 25 μg / mL of chloramphenicol.

<9-2> Preparation of cell extract The strain prepared in Example <9-1> and the strain obtained by introducing pTWV-MEX_A into EMC1.65 prepared in Example <7-1> Each was cultured in the same manner as in Example <6-2>, and the cells were collected. However, as a medium, when culturing a strain in which pTWV-MEX_A was introduced into EMC1.65, LBGM9 medium containing ampicillin sodium 100 μg / mL was used, and each of pSTV-Ca_T, pSTV-Ap_T, and pSYV29 was introduced. When culturing the strain, LBGM9 medium containing 25 μg / mL of chloramphenicol was used. The collected cells were washed twice with 100 mM potassium phosphate buffer (pH 7.0) and then suspended in a 50 mM MOPS-KOH (pH 7.0) solution containing 4 mM MgCl ₂ . The suspended cells were sonicated and the supernatant fraction obtained after ultracentrifugation (4 ° C., 53,000 rpm for 1 hour (RP80AT, manufactured by HITACHI-KOKI)) was obtained as a cell extract. The protein concentration of the obtained cell extract was measured by the Bradford method. The collected cells may be stored at -80 ° C.

<9-3> Activity measurement The activity of succinyl CoA: malate CoA transferase was determined according to the method of Friedmann (Friedmann S. et al., (2006) J Bacteriol. 188 (7): 2646-55.) It was measured as the glyoxylic acid-forming activity using L-malic acid and Succinyl-CoA as substrates by coupling. Specifically, the cell extract was mixed with 500 μl of a measurement solution containing 200 mM MOPS-KOH (pH 6.5), 5 mM MgCl ₂ , 3.5 mM phenylhydrazine, and 20 mM Malate Na ₂ , and then Succinyl-CoA Na was added. The final concentration was 1 mM. As the cell extract, 25 μg of the protein derived from the pTWV-MEX_mclA-introduced strain and 500 μg of the protein derived from the strain into which each of pSTV29, pSTV-Ca_T, and pSTV-Ap_T was introduced were added. . Table 8 shows the activity of succinyl CoA: malate CoA transferase determined from the increase in absorbance at 324 nm after addition of Succinyl-CoA Na. As a result, it was shown that the succinyl CoA: malate CoA transferase activity increased in the Ca_smtAB gene-introduced strain and the Ap_smtAB gene-introduced strain.

<Example 10: Measurement of succinyl CoA: malate CoA transferase activity in smt gene-introduced strain>
<10-1> Construction of Mm_smt gene introduction strain and Rr_smt gene introduction strain A strain in which pMWV-MEX_A was further introduced into EMC1.65 prepared in Example <7-1> was further introduced. It was constructed. This strain was selected using an LBGM9 agarose plate containing 40 μg / mL kanamycin. Further, a strain was constructed by introducing pSTV-Mm_T and pSTV-Rr_T into this strain. As a control strain, a strain into which pSTV29 was introduced was constructed. These strains were selected using LBGM9 agarose plates containing 100 μg / mL ampicillin, 40 μg / mL kanamycin, and 25 μg / mL chloramphenicol.

<10-2> Preparation of cell extract A cell extract was prepared in the same manner as in Example <6-2>. However, as the medium, LBGM9 medium containing 100 μg / mL ampicillin sodium, 40 μg / mL kanamycin, and 25 μg / mL chloramphenicol was used.

<10-3> Activity Measurement The activity of succinyl CoA: malate CoA transferase was measured in the same manner as in Example <9-3>. The activity values are shown in Table 9. As a result, it was shown that the succinyl CoA: malate CoA transferase activity increased in the Mm_smt gene-introduced strain and the Rr_smt gene-introduced strain.

<Example 11: Introduction of the malyl-CoA lyase gene into the genome>
To express the malyl-CoA lyase gene on the genome, follow the procedure below. The mclA gene (MLO_mclA gene) was introduced.

<11-1> Construction of pTWV-Km-pnlp8F10-MLO_A The pnlp8 promoter (WO2010 / 027045) is a promoter obtained by substituting the nucleotide sequence of the −10 region of the promoter region of the nlpD gene of Escherichia coli MG1655 strain It is a promoter with improved gene expression level.

For the purpose of further improving gene expression, a pnlp8F10 was prepared by replacing the Shine-Dalgarno sequence of the pnlp8 promoter region with the Shine-Dalgarno sequence (F10 factor) of the T7 promoter of the pET16b vector as follows.

Specifically, by using pMW-Km-Pnlp8 (WO2010 / 027045) as a template and PCR using the oligonucleotides of SEQ ID NO: 47 and SEQ ID NO: 48, a DNA fragment containing attL-Km-attR sequence and pnlp8F10 sequence (AttL-Km-attR-pnlp8F10) was obtained. The attL-Km-attR-pnlp8F10 fragment was purified by a conventional method. A DNA fragment was amplified by PCR using the purified attL-Km-attR-pnlp8F10 fragment as a template and the oligonucleotides of SEQ ID NO: 49 and SEQ ID NO: 50. The amplified DNA fragment contains an attL-Km-attR sequence and a pnlp8F10 sequence, a sequence complementary to a part of pTWV229 upstream of the attL-Km-attR sequence, and an MLO_mclA gene sequence downstream of the pnlp8F10 sequence. It has a sequence complementary to 20 bases from the start codon. On the other hand, a DNA fragment was amplified by PCR using pTWV-MLO_A as a template and the oligonucleotides of SEQ ID NO: 51 and SEQ ID NO: 52. The amplified DNA fragment consists of a full-length pTWV-MLO_A sequence that does not contain the tac promoter sequence (SEQ ID NO: 72). These two amplified DNA fragments were mixed and ligated with In-Fusion® HD® Cloning® Kit (Clontech). JM109 was transformed with the ligated product to obtain ampicillin resistant colonies. It was confirmed that the plasmid obtained from the colony-forming strain had a sequence containing the Km resistance gene that can be removed by the λ phage-derived excision system and the MLO_mclA gene expressed by pnlp8F10. This plasmid was named pTWV-Km-pnlp8F10-MLO_A.

<11-2> Replacement of glcB gene on the genome of MG1655 with pnlp8F10-MLO_A A DNA fragment containing the Km resistance gene that can be removed by the phage-derived excision system and the MLO_mclA gene expressed by pnlp8F10 was obtained. Both ends of this DNA fragment contain homologous sequences upstream and downstream of the glcB gene. Using this DNA fragment, a Km resistance gene capable of removing the glcB gene region on the genome of Escherichia coli MG1655 strain by the excision system derived from λ phage by the Red driven integration method described in Example <4-1> The sequence was replaced with a sequence containing the MLO_mclA gene expressed by pnlp8F10. The obtained strain was named MG1655ΔglcB :: pnlp8F10-MLO_A :: Km.

<11-3> Replacement of glcB gene on the genome of EMC1.65 with pnlp8F10-MLO_A Using the P1kc phage obtained from MG1655ΔglcB :: pnlp8F10-MLO_A :: Km by the method described in Example <1-11> Thus, the region lacking the glcB gene on the EMC1.65 genome was replaced with a sequence containing the Km resistance gene that can be removed by the excision system derived from λ phage and the MLO_mclA gene expressed by pnlp8F10. The obtained strain was named EMC1.65A :: Km. According to the method described in Example <1-11>, a strain from which the kanamycin resistance gene of this strain was removed by a λ phage-derived excision system was constructed and named EMC1.65A.

<Example 12: Confirmation of glutamic acid production via an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase <12-1> Construction and preparation of evaluation strains EMC1.65 and EMC1.65A Each of pTWV229 and pTWV-Mm_T was introduced into the strain, and an evaluation strain into which pSTV29 and pSTV-STK ^** were further introduced was constructed.

<12-2> Preparation of Evaluation Medium As an evaluation medium, 5 g / L glycerol, 17.1 g / L disodium hydrogen phosphate, 3.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium chloride, 0.5 g / L NaCl, 0.25 g / L magnesium sulfate heptahydrate, 0.1 g / L lysine hydrochloride, 0.1 g / L diaminopimelic acid, 0.5 g / L methionine, 1 g / L sodium aspartate, 8 g / L L-malic acid A medium containing disodium, 2 g / L disodium succinate, 200 mM MOPS-KOH (pH 7.0), 25 μg / mL chloramphenicol, 100 μg / mL ampicillin sodium was prepared.

<12-3> Inoculation / Culture The evaluation strain constructed in Example <12-1> was placed on an LBGM9 agarose plate medium containing 25 μg / mL of chloramphenicol and 100 μg / mL of ampicillin at 37 ° C. Grow overnight. The cells were collected and suspended in sterilized water to prepare a cell suspension with an OD600 of 10.

5 μL of the above cell suspension was inoculated into 5 μmL of the evaluation medium prepared in Example <12-2>. Culturing was performed for 96 hours while shaking at 30 ° C. and 70 ° C. rpm using an automatic OD measurement culture device BIO-PHOTORECORDER TN-1506 (ADVANTEC) and its dedicated L-shaped test tube. After culturing, the concentration of L-glutamic acid accumulated in the medium was analyzed with Biotech Analyzer AS-310 (Sakura S Eye Co., Ltd.).

Table 10 shows the amount of L-glutamic acid contained in the culture broth after culturing. As a result, the target strain (EMC1.65 / pSTV29, pTWV229), the strain into which only the mutant succinyl-CoA synthase gene was introduced (EMC1.65 / pSTV-STK ^** , pTWV229), and the succinyl-CoA: malate CoA transferase gene only were introduced. In the strains obtained (EMC1.65 / pSTV29, pTWV-Mm_T) and the strain into which only the malyl-CoA lyase gene was introduced (EMC1.65A / pSTV29, pTWV229), no glutamic acid was produced. On the other hand, in addition to the malyl-CoA lyase gene, a strain into which a mutant succinyl-CoA synthase gene was introduced (EMC1.65A / pSTV-STK ^** , pTWV229) and a strain into which a succinyl-CoA: malate CoA transferase gene was introduced (EMC1.65A In / pSTV29, pTWV-Mm_T), glutamic acid accumulated. From these facts, it is considered that glyoxylic acid produced by the enzyme that synthesizes malyl CoA from L-malic acid and malyl CoA lyase is condensed with succinic acid by isocitrate lyase to produce isocitrate, and glutamic acid was produced. It is done.

<Example 13: Construction of a succinic acid-producing strain derived from Escherichia coli MG1655 strain>
From the results of Example <12-3>, glutamic acid is efficiently produced from L-malic acid and succinic acid via an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase. Rukoto has been shown. Therefore, a strain that efficiently produces glutamate through this metabolic pathway is constructed based on sugars such as glucose and alcohols such as glycerol, which are carbon sources commonly used for the production of useful substances. did.

In order to efficiently produce glutamic acid through this metabolic pathway based on sugars such as glucose and alcohols such as glycerol, succinic acid and L-malic acid are produced from these carbon sources via a reductive TCA cycle. It is desirable to use strains that are efficiently produced.

For example, by using a strain bred from Escherichia coli MG1655 strain, L-malic acid is derived from sugars such as glucose and alcohols such as glycerol, which are carbon sources generally used for producing useful substances. And succinic acid can be produced efficiently. For example, WO2006 / 034156 shows that succinic acid can be efficiently generated by blocking biosynthetic pathways such as lactic acid, acetic acid, and ethanol as by-products. Specifically, blocking the lactic acid biosynthetic pathway means, for example, deletion of the ldhA gene encoding lactate dehydrogenase. Specifically, blocking the acetic acid biosynthetic pathway means, for example, deletion of the poxB gene encoding pyruvate oxidase, the ackA gene encoding acetate kinase, and the pta gene encoding phosphotransacetylase. Specifically, blocking the ethanol biosynthetic pathway means, for example, deletion of the adhE gene encoding alcohol dehydrogenase.

In order to produce L-glutamic acid using a strain bred from Escherichia coli MG1655, it is desirable to enhance the expression of the glutamate excretion system. Specifically, enhancing the expression of the glutamate excretion system means, for example, enhancing the expression of the ybjL gene (WO2008 / 133161).

Based on the above findings, a strain capable of efficiently producing succinic acid and L-malic acid was constructed from EMC1.4 prepared in Example 1 as follows.

<13-1> Construction of adhE gene-deficient strain The entire base sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the base sequence of the adhE gene encoding alcohol dehydrogenase has also been reported. Yes. That is, the adhE gene corresponds to the complementary sequence of base numbers 1294669 to 1297344 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

Deletion of the adhE gene encoding the alcohol dehydrogenase of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-tc-attR as a template using the oligonucleotides of SEQ ID NO: 55 and SEQ ID NO: 56 was used. The strain deficient in the adhE gene was named MG1655 ΔadhE :: tet.

<13-2> Construction of ldhA gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the nucleotide sequence of the ldhA gene encoding lactate dehydrogenase has also been reported. Yes. That is, the ldhA gene corresponds to the complementary sequence of base numbers 1439878 to 1440867 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

As a deficient strain of the ldhA gene encoding lactate dehydrogenase of Escherichia coli MG1655 strain, one disclosed in WO2008 / 153116 was used. Specifically, the ldhA gene is a chloramphenicol resistance gene (attL-cm-attR), which can be removed by the λ phage-derived excision system, created in the process of creating the MG1655ΔsucAΔldhA strain described in WO / 2008/153116. A substituted strain was used. This strain was named MG1655 ΔsucAΔldhA :: cm.

<13-3> Construction of poxB gene-deficient strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the nucleotide sequence of the poxB gene encoding pyruvate oxidase has also been reported. ing. That is, the poxB gene corresponds to the complementary sequence of nucleotide numbers 908554 to 910272 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

Deletion of the poxB gene encoding pyruvate oxidase of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-tc-attR as a template using the oligonucleotides of SEQ ID NO: 57 and SEQ ID NO: 58 was used. The strain lacking the poxB gene was named MG16551ΔpoxB :: tet.

<13-4> Construction of ybjL gene expression-enhanced strain The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the nucleotide sequence of the ybjL gene encoding the glutamate excretion carrier is also reported. Has been. That is, the ybjL gene corresponds to the complementary sequence of nucleotide numbers 887357 to 889042 of the genome sequence of Escherichia coli MG1655 described in GenBank accession number NC_000913.3.

The gene expression enhancement of the ybjL gene <WO2008 / 133161> encoding the glutamate excretion carrier of Escherichia coli MG1655 strain was performed by the Red driven integration method described in Example <1-2>. Specifically, using the oligonucleotides of SEQ ID NO: 59 and SEQ ID NO: 60, P. aureus having a tac-like promoter sequence having the highest expression intensity in the above-mentioned document (Katashkina JI et al. Russian Federation Patent application 2006134574). A fragment amplified by PCR using the genomic DNA of ananatis (SC17 (0) strain as a template was used. The strain with enhanced ybjL gene expression was named MG16551P4071-ybjL :: Km.

<13-5> Deletion of gltA gene, sdhABCD gene, and sucAB gene and construction of sucCD gene expression-enhanced strain The entire base sequence of genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3) SucCD gene encoding citrate synthase, sdhCDAB gene encoding succinate dehydrogenase, sucA gene encoding 2-oxoglutarate decarboxylase, sucB gene encoding dihydrolipoamide dehydrogenase, sucCD gene encoding succinyl CoA synthase Base sequences have also been reported. These genes are continuously present on the genome of Escherichia coli MG1655 strain. That is, in the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3, the gltA gene is a complementary sequence of nucleotide numbers 752408 to 753691, the sdhCDAB gene is nucleotide numbers 754400 to 757628, and the sucA gene is nucleotide numbers 757929 to In 760730, the sucB gene corresponds to nucleotide numbers 760745 to 761962, the sucC gene corresponds to nucleotide numbers 762237 to 763403, and the sucD gene corresponds to nucleotide numbers 763403 to 764272, respectively.

The gltA gene and other genes are present in opposite directions, and the gltA gene and the sdhA gene are present with the start codons facing each other. Therefore, an oligonucleotide having a sequence downstream of the gltA gene and an attL sequence (SEQ ID NO: 61), and an oligonucleotide having a sequence downstream from the start codon of the sucCD gene and a sequence corresponding to a part of the tac-like promoter (SEQ ID NO: 62) ) Was used as a template for the genomic DNA of a strain having a tac-like promoter sequence having the −35 region of SEQ ID No. 20 of the above-mentioned document (Katashkina JI et al. Russian Federation Patent Application 2006134574). The DNA sequence from the −35 region of the tac-like promoter sequence to the start codon including the ribosome binding site (RBS) is shown in SEQ ID NO: 74. Using the fragment amplified by PCR, a strain in which the gltA gene, the sdhABCD gene, and the sucAB gene were deleted and the sucCD gene expression was enhanced was constructed by the Red driven integration method described in Example <1-2>. The constructed strain was named MG1655ΔgltA-sucABP4074-sucCD :: Km.

<13-6> Construction of strain lacking ackA gene and pta gene The entire nucleotide sequence of the genomic DNA of Escherichia coli MG1655 strain is known (GenBank accession number NC_000913.3), and the ackA gene encoding phosphate kinase and phosphate The nucleotide sequence of the pta gene encoding acetyltransferase has also been reported. That is, in the genome sequence of Escherichia coli MG1655 strain described in GenBank accession number NC_000913.3, the ackA gene corresponds to base numbers 2414192 to 2412694, and the pta gene corresponds to base numbers 2412769 to 2414913.

AckA gene encoding acetate kinase of Escherichia coli MG1655 strain and pta gene encoding phosphate acetyltransferase are continuously present on the genome. Deletion of the region containing these genes was carried out by the Red driven integration method described in Example <1-1>. Specifically, a fragment amplified by PCR using pMW118-attL-Km-attR as a template using the oligonucleotides of SEQ ID NO: 63 and SEQ ID NO: 64 was used. A strain lacking the region containing the ackA gene and the pta gene was named MG16551ΔackA-pta :: Km.

<13-7> Construction of succinic acid-producing strain derived from Escherichia coli MG1655 strain Using the EMC1.4 strain as a recipient, the strain constructed in Examples <13-1> to <13-6> As described above, an evaluation strain was prepared by sequentially performing transduction. Transduction was performed using P1kc phage as in Example <1-11>, and the helper plasmid pMW-intxis-sacB (Cm) or pMW-intxis-sacB (Spc) was used to remove the drug resistance gene. It was. The resulting stock was named EMSF stock. The genotype of the EMSF strain is MG1655ΔgltA ΔsdhABCD ΔsucAB P4074-sucCD ΔaceB-P4071-aceA ΔaceK ΔglcB Δgcl ΔmaeA ΔmaeB ΔldhA P4071-ybjL ΔadhE ΔackA-pta ΔpoxB.

<Example 14: Desensitization mutation introduction into phosphoenolpyruvate carboxylase gene (ppc)>
The activity of phosphoenolpyruvate carboxylase (PEPC) is inhibited by L-malate, an intermediate product of the succinate biosynthetic pathway (Masato Yano and Katsura Izui, Eur. Biochem. FEBS, 247, 74-81, 1997). ). Inhibition by L-malic acid can be reduced, for example, by introducing a desensitizing mutation by substitution of one amino acid into PEPC. Specific examples of the desensitizing mutation by substitution of one amino acid include a mutation that substitutes the 620th amino acid of PEPC protein derived from Escherichia coli from lysine to serine (the same document).

RSFPPG (WO2008 / 020654) is a plasmid carrying a prpC gene encoding methylcitrate synthase, a ppc gene encoding PEPC, and a gdh gene encoding glutamate dehydrogenase. Among these genes, the desensitizing mutation described above was introduced into the ppc gene encoding PEPC. The specific procedure is shown below.

PCR was performed using RSFPPG as a template and the synthetic oligonucleotides of SEQ ID NO: 65 and SEQ ID NO: 66 as primers, and the entire RSFPPG containing a PEPC gene in which the 620th amino acid of the PEPC protein was substituted from lysine to serine was amplified. The obtained PCR product was treated with DpnI, and JM109 was transformed to obtain tetracycline resistant colonies. It was confirmed that the target mutation was introduced into the plasmid obtained from the strain forming the colony. The RSFPPG obtained by introducing the desensitizing mutation into the PEPC gene thus obtained was named RSFPP ^* G.

<Example 15: Production of glutamic acid from sugar by Escherichia coli evaluated strain introduced with malate thiokinase gene and malyl-CoA lyase gene>
<15-1> Construction of Escherichia coli evaluation strain Competent cells of EMSF strain, which is a succinic acid production strain prepared in Example <13-7>, were prepared according to a conventional method, and transformed with RSFPP ^* G. Plated on LBGM9 agarose plate medium containing 25 mg / L tetracycline hydrochloride at 37 ° C. to select tetracycline resistant strains. Competent cells of the tetracycline resistant strain obtained here were prepared according to a conventional method, further transformed with each of pHSG396 and pHSG-MLO_AK, and at 37 ° C, 25 mg / L tetracycline hydrochloride and 25 mg / L Plated on LBGM9 agarose plate medium containing chloramphenicol, and the grown strain was used as the evaluation strain. Table 11 shows the evaluation strains.

<15-2> Production of glutamic acid from sugar by Escherichia coli evaluated strain into which malate thiokinase gene and malyl CoA lyase gene have been introduced. The evaluated strain prepared in Example <15-1> The cells were cultured at 30 ° C. for 18 hours on an LBGM9 agarose plate containing chloramphenicol / mL. The whole amount of the obtained bacterial cells was inoculated into a 1 L jar fermenter into which 300 mL of a seed culture medium having the composition shown below was injected.

[Seed culture medium composition]
20 g / L Bacto trypton, 10 g / L Bacto Yeast extract, 10 g / L NaCl, 6% glucose, 0.05% magnesium sulfate heptahydrate, 1.72% disodium hydrogenphosphate 12 hydrate, 0.3% phosphoric acid 2 Potassium hydrogen, 0.1% ammonium chloride, 0.4 g / L lysine hydrochloride, 0.4 g / L diaminopimelic acid, 0.4 g / L methionine, 25 mg / L tetracycline hydrochloride, 25 mg / L chloramphenicol, 0.1 mL / L GD-113 (Antifoaming agent)

After controlling the pH to 7.0 with ammonia at a temperature of 30 ° C, aeration 1 VVM, stirring 700 rpm, and culturing for 18 hours, 80% of the aeration was switched to nitrogen gas, stirring was reduced to 500 rpm, The culture was further performed for 5 hours.

The cells grown in the seed medium were collected by centrifugation, washed with 10 μmL of the main culture medium having the composition shown below, and then suspended in 60 μmL of the main culture medium to obtain a seed cell suspension. 20 μL of the seed cell suspension was inoculated into a 100 μmL jar fermenter into which 80 μmL of the main culture medium was injected.

[Main culture medium composition]
50 g / L glucose, 0.4 g / L MgSO ₄ , 0.1 mL / L GD-113 (antifoam), 4 g / L (NH ₄ ) ₂ S0 ₄ , 2 g / L KH ₂ PO ₄ , 4 g / L yeast extract, 10 mg / L FeSO ₄ · 7H ₂ 0, 10 mg / L MnSO ₄ · 4 to 5H ₂ 0, O.4 g / L L-lysine, 0.4 g / L DL-methionine, 0.4 g / L Diaminopimelic acid, 25 mg / L tetracycline, 25 mg / L chloramphenicol

The main culture was performed at a temperature of 30 ° C., stirring at 700 rpm, and controlling the pH to 7.0 with ammonia. In addition, aeration was performed under conditions of nitrogen gas 10 L / min, air 40 L / min, and carbon dioxide gas 50 L / min.

The glucose concentration in the medium and the concentration of accumulated L-glutamic acid were analyzed using Biotech Analyzer AS-310 (Sakura S Eye Co., Ltd.).

Table 12 shows the glutamic acid concentration accumulated in the culture solution after 27 hours of main culture. In addition, glucose in the culture solution after performing the main culture for 27 hours was completely consumed. The strain (EC / mtk mclA) introduced with malate thiokinase gene and malyl-CoA lyase gene accumulates more glutamate than the target strain (EC / Vec), and produces glutamate with a high sugar yield. It was done.

<Example 16: Glutamic acid production from sugar by Escherichia coli evaluated strain introduced with malyl CoA lyase gene and mutant succinyl CoA synthase gene or succinyl CoA: malate CoA transferase gene>
<16-1> Introduction of malyl-CoA lyase gene into the genome of EMSF strain Using the P1kc phage obtained from MG1655ΔglcB :: pnlp8F10-MLO_A :: Km by the method described in Example <11-3>, MLO_mclA The gene was introduced on the genome of EMSF strain. Although the glcB gene of the EMSF strain has already been destroyed, this operation replaces the glcB gene region with a sequence containing the Km resistance gene that can be removed by the λ phage-derived excision system and the MLO_mclA gene expressed by pnlp8F10. Got a stock. From this strain, a strain from which the kanamycin resistance gene was removed was prepared by the excision system derived from λ phage by the method described in Example <1-1>, and named strain EMSFA.

<16-2> Construction of Escherichia coli Evaluated Strains Competent cells of EMSF strain were prepared according to a conventional method, transformed with RSFPP ^* G, and LBGM9 agarose plate containing 25 mg / L tetracycline hydrochloride at 37 ° C And a tetracycline resistant strain was selected. The tetracycline resistant competent cells obtained here were prepared according to a conventional method, transformed with pSTV29, and LBGM9 containing 25 mg / L tetracycline hydrochloride and 25 mg / L chloramphenicol at 37 ° C. The plate grown on an agarose plate and grown was used as an evaluation strain. Similarly, RSFPP * G was introduced into the EMSFA strain, and each of pSTV29, pSTVSTK ^** , and pSTV-Mm_T was introduced as an evaluation strain. Table 13 shows the evaluation strains.

<16-3> Glutamic acid production from sugar by Escherichia coli evaluated strain introduced with malyl-CoA lyase gene and mutant succinyl-CoA synthase gene or succinyl-CoA: malate-CoA transferase gene

The evaluation strain prepared in Example <16-2> was cultured at 30 ° C. for 18 hours on an LBGM9 agarose plate containing 25 μg / mL tetracycline and 25 μg / mL chloramphenicol. The total amount of the obtained bacterial cells was inoculated into a 1 L jar fermenter into which 300 mL of seed culture medium having the following composition was injected.

The cells grown in the seed medium were collected by centrifugation, washed with 50 μmL of the main culture medium having the composition shown below, and suspended in the main culture medium to obtain a seed cell suspension stock solution. Absorbance at a wavelength of 600 nm of a liquid obtained by diluting this seed cell suspension stock solution 100 times was measured with a spectrophotometer U-2900 (Hitachi). From the obtained results, each cell suspension stock solution was measured at a wavelength of 600 、 nm. Dilution was performed so that the absorbance was 106 to obtain a seed cell suspension. 30 μL of the seed cell suspension was inoculated into a 100 μmL jar fermenter into which 70 μmL of the main culture medium was injected.

Table 14 shows the concentration of glutamic acid accumulated in the culture after 42 hours of main culture. In addition, glucose in the culture solution after performing main culture for 42 hours was completely consumed. It was shown that the strain into which the malyl CoA lyase gene was introduced (EC + mclA / pSTV) accumulated more glutamic acid than the target strain (EC / pSTV), and produced glutamic acid at a high sugar yield. Furthermore, by introducing a mutant succinyl CoA synthase gene or a succinyl CoA: malate CoA transferase gene into a strain into which a malyl CoA lyase gene was introduced, more glutamate was accumulated (EC + mclA / stk ^** and EC + mclA / smt). From these results, it was shown that glutamic acid was produced at a high sugar yield by introducing a mutant succinyl CoA synthase gene or a succinyl CoA: malate CoA transferase gene into a strain into which a malyl CoA lyase gene was introduced. .

Example 17: Production of glutamic acid from sugar via an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase, and isocitrate lyase in an Escherichia coli evaluated strain <17-1> Succinyl CoA: malate CoA transferase Introduction of the gene into the genome PCR was performed using pTWV-Km-pnlp8F10-MLO_A prepared in Example <11-1> as a template and the oligonucleotides of SEQ ID NO: 209 and SEQ ID NO: 210, and the amplified DNA fragment was Purified by conventional methods. This DNA fragment contains an attL-Km-attR sequence and a pnlp8F10 sequence, and further has a sequence complementary to a part of pSTV29 upstream of the attL-Km-attR sequence. On the other hand, PCR was performed using pSTV-Mm_T prepared in Example <5-2> as a template and the oligonucleotides of SEQ ID NO: 211 and SEQ ID NO: 212, and the amplified DNA fragment was purified by a conventional method. This DNA fragment contains the entire length of pSTV-Mm_T excluding the tac promoter sequence (SEQ ID NO: 72), and further pnlp8F10 sequence upstream of the Mm_smt gene sequence (SEQ ID NO: 213) modified according to the codon usage frequency of Escherichia coli. It has a complementary sequence in the part. These two DNA fragments were mixed and ligated using In-Fusion HD Cloning Kit (Clontech). JM109 was transformed with the ligated product to obtain kanamycin resistant colonies. The plasmid obtained from the colony-forming strain was confirmed to have a Km resistance gene that can be removed by the λ phage-derived excision system and the Mm_smt gene (SEQ ID NO: 213) expressed by pnlp8F10. This plasmid was named pSTV-Km-pnlp8F10-Mm_T.

Subsequently, using the primers shown in SEQ ID NO: 214 and SEQ ID NO: 215, PCR was performed using the plasmid pSTV-Km-pnlp8F10-Mm_T as a template, and a Km resistance gene that can be removed by the excision system derived from λ phage, and pnlp8F10 A DNA fragment containing the Mm_smt gene expressed by was obtained. It should be noted that homologous sequences are included at both ends of this DNA fragment upstream and downstream of the ptsG gene. Using this DNA fragment, a Km resistance gene capable of removing the ptsG gene region on the genome of Escherichia coli MG1655 strain by the excision system derived from λ phage by the Red driven integration method described in Example <4-1> The sequence was replaced with a sequence containing the Mm_smt gene expressed by pnlp8F10. The obtained strain was named MG1655ΔptsG :: pnlp8F10-Mm_T :: Km.

On the other hand, using the primers shown in SEQ ID NO: 216 and SEQ ID NO: 217, PCR was performed using plasmid pSTV-Km-pnlp8F10-Mm_T as a template, and a Km resistance gene that can be removed by the excision system derived from λ phage, and pnlp8F10 A DNA fragment containing the Mm_smt gene expressed by was obtained. Note that both ends of this DNA fragment contain homologous sequences upstream and downstream of the mqo gene. Using this DNA fragment, the mqo gene region on the genome of Escherichia coli MG1655 strain, which can be removed by the excision system derived from λ phage, by the Red driven integration method described in Example <4-1>, The sequence was replaced with a sequence containing the Mm_smt gene expressed by pnlp8F10. The obtained strain was named MG1655Δmqo :: pnlp8F10-Mm_T :: Km.

<17-2> Preparation of Escherichia coli Evaluated Strains According to the following procedure, Escherichia coli AJ111087 (NITE BP-01708) encodes a gene encoding an enzyme that synthesizes malyl CoA from L-malic acid and malyl CoA lyase. Each of the genes to be introduced was introduced to prepare an evaluation strain. AJ111087 is a derivative of the EMSF strain described in Example <13-7>. That is, AJ111087 has enhanced expression of the aceA gene encoding isocitrate lyase, but lacks the gltA gene encoding citrate synthase. On the other hand, AJ111087, unlike the EMSF strain, is deficient in the ptsG gene encoding PTS glucose enzyme II BC and the mqo gene encoding malate: quinone oxidoreductase. AJ111087 was deposited on September 18, 2013 at the Patent Microorganism Depositary Center (Postal Code: 292-0818, Address: 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture) on September 18, 2013 And the accession number NITE BP-01708 is assigned.

First, the malyl-CoA lyase gene was introduced into the genome of AJ111087. The gene was introduced by transduction using P1kc phage as in Example <11-3>. Specifically, by using the P1kc phage obtained from MG1655ΔglcB :: pnlp8F10-MLO_A :: Km by the method described in Example <1-11>, the region where the glcB gene is deleted on the genome of AJ111087. The Km resistance gene that can be removed by the excision system derived from λ phage was replaced with a sequence containing the MLO_mclA gene expressed by pnlp8F10. From the obtained strain, a strain from which the kanamycin resistance gene was removed by the excision system derived from λ phage was constructed by the method described in Example <1-11>, and named AJ111087A.

Next, two copies of a gene encoding succinyl CoA: malate CoA transferase were introduced onto the genome of AJ111087A. The gene was introduced by transduction using P1kc phage as in Example <1-11>. Specifically, using the method described in Example <1-11>, a region lacking the ptsG gene on the genome of AJ111087A was obtained using a P1kc phage prepared using MG1655ΔptsG :: pnlp8F10-Mm_T :: Km. The Km resistance gene that can be removed by the excision system derived from λ phage and the sequence containing the Mm_smt gene expressed by pnlp8F10 were substituted. From the obtained strain, a strain from which the kanamycin resistance gene of this strain was removed by the excision system derived from λ phage was constructed by the method described in Example <1-11> and named AJ111087APT. Further, by using the P1kc phage prepared using MG1655Δmqo :: pnlp8F10-Mm_T :: Km by the method described in Example <1-11>, a region lacking the mqo gene on the genome of AJ111087APT was obtained. The sequence was replaced with a sequence containing the Km resistance gene that can be removed by the excision system and the Mm_smt gene expressed by pnlp8F10. From the obtained strain, a strain from which the kanamycin resistance gene was removed was constructed by the excision system derived from λ phage by the method described in Example <1-11> and named AJ111087APTMT.

Subsequently, an expression plasmid for a mutant succinyl-CoA synthase gene derived from Escherichia coli was introduced into AJ111087APTMT. Specifically, pSTV-STK (V161A, G271A: β) prepared in Example <4-2> was introduced into AJ111087APTMT by a conventional method, and colonies were collected on an LBGM9 agarose plate containing 25 mg / L chloramphenicol. The strain that formed was acquired. The strain purified with LBGM9 agarose plate containing 25 mg / L chloramphenicol was named AJ111087APTMT / pSTV-STK (V161A, G271A: β).

On the other hand, as a control strain of AJ111087APTMT / pSTV-STK (V161A, G271A: β), a strain in which pSTV29 was introduced into AJ111087 was prepared. Specifically, pSTV29 was introduced into AJ111087 by a conventional method, and a chloramphenicol resistant strain that formed colonies on an LBGM9 agarose plate containing 25 mg / L chloramphenicol was obtained. A strain purified with an LBGM9 agarose plate containing 25 mg / L chloramphenicol was named AJ111087 / pSTV29.

<17-3> Production of glutamic acid from sugar via an enzyme that synthesizes malyl-CoA from L-malic acid, malyl-CoA lyase, and isocitrate-lyase in an Escherichia coli evaluated strain. Prepared in Example <17-2> Using AJ111087APTMT / pSTV-STK (V161A, G271A: β) and AJ111087 / pSTV29 as evaluation strains, glutamic acid was produced from sugar. Hereinafter, AJ111087APTMT / pSTV-STK (V161A, G271A: β) is referred to as “Malyl-CoA strain”, and AJ111087 / pSTV29 is referred to as “control strain”.

The Malyl-CoA strain and the control strain were each applied to one LBGM9 agarose plate containing 25 μg / mL chloramphenicol and cultured at 30 ° C. for 18 hours. LBGM9 agarose plate containing 25 μg / mL chloramphenicol on which Malyl-CoA and control strains were grown was placed at 30 ° C in a 2.5 L square jar containing one Aneropac Kenki (Mitsubishi Gas Chemical Co., Ltd.) package. For 6 hours. Thereafter, the cells were suspended in ice-cooled 1 mL of 1.72% disodium hydrogen phosphate 12 hydrate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride solution, and collected by centrifugation. Further, suspension and centrifugation with this solution were repeated twice to wash the bacteria. The washed cells are suspended in the above solution so that OD600 becomes 100, and 50 μl is added to a 12-well plate (Nihon Becton Dickinson Co., Ltd., catalog number 351143) into which 2 μmL of glutamic acid production medium having the following composition is injected. Inoculated.

[Glutamic acid production medium composition]
1.5% Glucose, 200 mM MOPS-KOH (pH 7.0), 10 mg / L L-Met, 10 mg / L L-Cys hydrochloride, 10 mg / L L-Trp, 10 mg / L L-His, 10 mg / L L-Phe, 10 mg / L L-Tyr, 10 mg / L L-Asn monohydrate, 10 mg / L L-Ser, 10 mg / L L-Thr, 10 mg / L L-Ile , 10 mg / L L-Ala, 10 mg / L L-Asp, 10 mg / L Gly, 10 mg / L L-Lys hydrochloride, 10 mg / L L-Leu, 10 mg / L L-Arg hydrochloride , 10 mg / L L-Val, 1.72% disodium hydrogen phosphate 12 hydrate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.5 g / L MgSO ₄ 7H ₂ O, 15 g / L ammonium sulfate , 10 μg / L thiamine hydrochloride, 25 mg / L chloramphenicol, 50 g / L calcium carbonate, 250 mM sodium nitrate

After attaching Breeze Easy (Toho Co., Ltd.) to the top of the 12-well plate containing the inoculated culture solution, in a 2.5 mm L square jar containing 1 pack of Anero Pack Kenki (Mitsubishi Gas Chemical Co., Ltd.) At 30 ° C., the medium was shaken using a medium shaker double shaker NR-30 (Tytec Corp.) at 140 rpm. After culturing for 50 hours, the concentration of L-glutamic acid accumulated in the medium was analyzed by Biotech Analyzer AS-310 (Sakura Seye Co., Ltd.).

The results are shown in Table 15. The control strain produced no L-glutamic acid, whereas the Malyl-CoA strain produced L-glutamic acid. This indicates that glutamic acid is produced from sugar via enzymes that synthesize malyl CoA from L-malic acid (mutant succinyl CoA synthase and succinyl CoA: malate CoA transferase), malyl CoA lyase, and isocitrate lyase. Indicated.

<Example 18: Production of glutamic acid from sugar by Pantoea ananatis-evaluated strain>
In this example, an isocitrate lyase gene, malyl-CoA lyase gene, and a mutant succinyl-CoA synthase gene or a succinyl-CoA: malate-CoA transferase gene were introduced to construct an evaluation strain derived from Pantoea ananatis SC17 strain, and glutamate production from sugar Went.

<18-1> Construction of SC17sucA / Ptac84-yhfK strain By replacing the promoter of the yhfK gene on the genome of P. ananatis SC17sucA strain (FERM BP-08646) with the modified Ptac promoter (Ptac84) by the following procedure. SC17sucA / Ptac84-yhfK with enhanced expression of the yhfK gene was constructed. The SC17sucA strain is a deletion strain of the α-ketoglutarate dehydrogenase-E1o subunit gene (sucA gene) of the P. ananatis SC17 strain (FERM BP-11091) (US Pat. No. 6,596,517). The yhfK gene is a gene encoding a glutamate excretion factor. The entire nucleotide sequence of the genomic DNA of P. ananatis AJ13355, which is an ancestor of the SC17 strain, is known, and the yhfK gene corresponds to nucleotide numbers 3448100-3450193 of the AJ13355 strain genomic sequence described in GenBank accession number NC_017531.1. . The Ptac84 promoter is obtained by replacing the -35 region of the Ptac promoter from TTGACA to TTTGCA. From the strength of LacZ activity, the transcriptional activity of the Ptac84 promoter is suppressed to about 1/3 compared to the transcriptional activity of the Ptac promoter. (Katashkina JI et al. Russian Federation Patent application 2006134574). The Ptac84 promoter is a P. ananatis SC17 (0) λattL-Kmr-λattR-Ptac84-lacZ strain (Katashkina JI et al. Russian Federation Patent application 2006134574). )

<18-1-1> Construction of SC17 (0) / RSF-Rsd-TER strain The RSF-Red-TER plasmid (WO2008 / 090770A1) was transformed into P. ananatis SC17 (0) strain (VKPM B-9246) by the electric pulse method. And cultured overnight at 34 ° C. on an L agarose plate containing 25 μg / mL of chloramphenicol. The grown strain was purified on the same plate, and the resulting strain was designated SC17 (0) / RSF-Rsd-TER. The RSF-Red-TER plasmid is a helper plasmid for recombination by the λ-Red system that expresses the gam, bet, and exo genes of the λ phage (hereinafter, “λRed gene”). The SC17 (0) strain is a P. ananatis strain resistant to the λRed gene product. The SC17 (0) stock was held on 21 September 2005 in the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika (address: Russia, 117545 Moscow, 1 Dorozhny 1) is deposited under accession number VKPM B-9246.

<18-1-2> Construction of SC17 (0) / Km-Ptac84-yhfK strain An SC17 (0) / RSF-Red-TER electric pulse competent cell was prepared by the following procedure. First, SC17 (0) / RSF-Red-TER was grown overnight at 34 ° C. in L medium containing 25 μg / mL chloramphenicol. Thereafter, the culture was diluted 100-fold with a fresh L medium containing 25 μg / mL chloramphenicol, and cultured under the condition of adding 1 mM IPTG. The cells were collected from the culture solution, washed 3 times with ice-cooled 10% glycerin, resuspended in 70 μl of 10% cold glycerin to obtain competent cells.

The synthetic DNA primers shown in SEQ ID NOs: 218 and 219 were synthesized by a conventional method. The primer shown in SEQ ID NO: 218 has a structure in which the homologous sequence at the 5 ′ end of λattL-Kmr-λattR-Ptac84 is connected to the homologous sequence upstream of the yhfK gene of P. ananatis. The primer of SEQ ID NO: 219 has a configuration in which the complementary sequence at the 3 ′ end of λattL-Kmr-λattR-Ptac84 is connected to the complementary sequence at the 5 ′ end including the start codon of the yhfK gene of P. ananatis. Using these primers, PCR was performed using the genomic DNA of P. ananatis SC17 (0) λattL-Kmr-λattR-Ptac84-lacZ as a template, so that the yhfK at the 5 ′ end of the sequence of λattL-Kmr-λattR-Ptac84 was obtained. A fragment of about 1.6 kbp was amplified in which the upstream sequence of the gene was added with the 5 'end sequence containing the start codon of the yhfK gene at the 3' end. The amplified PCR fragment was purified by a conventional method, introduced into SC17 (0) / RSF-Red-TER by an electric pulse method, and a transformant was selected on an L agarose plate containing 40 mg / L kanamycin. About the grown kanamycin resistant strain, it was confirmed by PCR using the synthetic DNA primers shown in SEQ ID NO: 220 and SEQ ID NO: 221 that the above fragment was inserted upstream of the yhfK gene. (0) / Km-Ptac84-yhfK.

<18-1-3> Construction of SC17sucA / Ptac84-yhfK strain Genomic DNA was extracted from SC17 (0) / Km-Ptac84-yhfK and introduced into the SC17sucA strain by the electric pulse method. Transformants were selected on LBGM9 agarose plates containing 40 mg / L kanamycin. About the grown kanamycin resistant strain, it was confirmed by PCR using the synthetic DNA primer shown in SEQ ID NO: 220 and SEQ ID NO: 221 that the above fragment was inserted upstream of the yhfK gene. It was named SC17sucA / Km-Ptac84-yhfK.

PMW-intxis-sacB (Cm) prepared in Example <1-1> was introduced into SC17sucA / Km-Ptac84-yhfK strain by the electric pulse method. Transformants were selected on LBGM9 agarose plates containing 25 mg / L chloramphenicol. The emerged colonies were streaked on LBGM9 agarose plates containing 25 mg / L chloramphenicol, and strains sensitive to kanamycin were selected from the single colonies. This strain was transferred on a LBGM9 agarose plate containing no drugs (antibiotics), and LBSM9 (L medium was supplemented with minimal medium components (2 mM magnesium sulfate, monopotassium phosphate 3 g / L, sodium chloride 0.5 g / L). , Ammonium chloride 1g / L, disodium phosphate 6g / L), 1mM IPTG, sucrose 100g / L added) streak culture on agarose plate, pMW-intxis-sacB (Cm) A strain from which the plasmid was lost was obtained. The strain thus obtained was named SC17sucA / Ptac84-yhfK strain.

<18-2> Construction of SC17sucA / Ptac84-yhfKΔaceB :: tet strain (FKGP1 strain) By the following procedure, the aceB gene on the genome of SC17sucA / Ptac84-yhfK strain is deleted, and SC17sucA / Ptac84-yhfKΔaceB :: tet strain (FKGP1 strain) was constructed. The aceB gene is a gene encoding malate synthase A. The aceB gene corresponds to nucleotide numbers 4066662 to 4068260 in the genome sequence of P. ananatis AJ13355 described in GenBank accession number NC — 017531.1.

<18-2-1> Construction of SC17 (0) ΔaceB :: tet Strain First, the aceB gene of P. ananatis SC17 (0) strain was deleted as follows. The DNA fragment for deletion of the aceB gene was amplified by PCR using pMW118-attL-Tc-attR (WO2005 / 010175, JP2005-58227) as a template and the oligonucleotides of SEQ ID NO: 222 and SEQ ID NO: 223. The amplified PCR fragment was purified using Wizard PCR Prep DNA Purification System (Promega). 500-700 ng of the purified PCR fragment was introduced into the SC17 (0) / RSF-Rsd-TER strain by the same method as in <18-1-2>, and transformed on an L agarose plate containing tetracycline 12.5 mg / L. A transformant was selected. About the grown tetracycline resistant strain, it was confirmed by PCR using synthetic DNA primers shown in SEQ ID NO: 224 and SEQ ID NO: 225 that the aceB gene was deleted, and the strain in which the aceB gene deletion was confirmed was SC17 (0) It was named ΔaceB :: tet.

<18-2-2> Construction of SC17sucA, P4073-yhfKΔaceB :: tet strain (FKGP1 strain) The genome of SC17 (0) ΔaceB :: tet was extracted by a conventional method, and 700 μg of SC17sucA / Ptac84- Introduced into yhfK. The cells were spread on an LBGM9 agarose plate containing tetracycline 12.5 μg / mL and cultured at 34 ° C. for about 20 hours. About the grown tetracycline resistant strain, the aceB gene deficiency (introduction of the ΔaceB :: tet trait) was confirmed by PCR using the synthetic DNA primers shown in SEQ ID NO: 224 and SEQ ID NO: 225, and the ΔaceB :: tet trait was introduced. The confirmed strain was named FKGP1 strain. The genotype of the FKGP1 strain is SC17sucA, P4073-yhfKΔaceB :: tet.

<18-3> Construction of SC17sucA / Ptac84-yhfKΔaceB :: tet, RyliG :: P4071-aceA :: Km strain (FKGP4 strain) The aceA gene was introduced into the yliG gene region on the genome of FKGP1 strain by the following procedure. SC17sucA / Ptac84-yhfKΔaceB :: tet, RyliG :: P4071-aceA :: Km strain (FKGP4 strain) with enhanced expression of aceA gene was constructed. The aceA gene is a gene encoding isocitrate lyase. The aceB gene and the aceA gene are continuously present on the genome of the P. ananatis AJ13355 strain. That is, the aceB gene corresponds to base numbers 4066662 to 4068260 of the genome sequence of P. ananatis AJ13355 strain described in GenBank accession number NC_017531.1, and the aceA gene is the genome sequence of P. ananatis AJ13355 strain described in GenBank accession number NC_017531.1. Correspond to base numbers 4068278 to 4069579. The yliG gene is a gene encoding 2-methylthioadenine synthetase. The yliG gene corresponds to the complementary sequence of base numbers 745519 to 746847 of the genome sequence of P. ananatis AJ13355 described in GenBank accession number NC — 017531.1.

<18-3-1> Construction of SC17 (0), RyliG :: P4071-aceA :: Km strain First, as shown below, the expression enhancement of aceA gene and the deficiency of aceB gene of P. ananatis SC17 (0) went. Using the oligonucleotides of SEQ ID NO: 226 and SEQ ID NO: 227, PCR with the genomic DNA of P. ananatis SC17 (0) strain as a template, the highest expression intensity in literature (Katashkina JI et al. Russian Federation Patent application 2006134574) A DNA fragment containing a high tac-like promoter sequence (SEQ ID No. 16 in the same document) was amplified. The DNA sequence from the −35 region of the tac-like promoter sequence to the initiation codon including the ribosome binding site (RBS) is shown in SEQ ID NO: 73. The amplified DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega). The purified DNA fragment was introduced into the SC17 (0) / RSF-Rsd-TER strain by the same method as in <18-1-2>. The cells were spread on an L agarose plate containing 40 μg / mL kanamycin and incubated overnight at 34 ° C. to select transformants. It was confirmed by PCR that the strain selected using kanamycin resistance as an index was a strain in which the tac-like promoter sequence (SEQ ID NO: 73) was inserted immediately before the aceA gene of P. ananatis SC17 (0) and the aceB gene was deleted. This strain was named SC17 (0) ΔaceB P4071-aceA :: Km.

Subsequently, as described below, the aceA gene expressed by the tac-like promoter sequence (SEQ ID NO: 73) was inserted into the yliG gene region of the P. ananatis SC17 (0) strain. SC17 (0) ΔaceB P4071-aceA :: Km genomic DNA was purified by a conventional method, and the DNA fragment was amplified by PCR using the present genomic DNA as a template and the oligonucleotides of SEQ ID NO: 228 and SEQ ID NO: 229. The amplified DNA fragment includes a Km resistance gene that can be removed by the excision system derived from λ phage, and a tac-like promoter sequence (SEQ ID NO: 73) that has the highest expression intensity in the literature (Katashkina JI et al. The aceA gene sequence expressed by) is contained, and 50 bases homologous to the upstream and downstream of the yliG gene of P. ananatis AJ13355 strain are present at both ends thereof. The amplified DNA fragment was purified using a Wizard® PCR® Prep® DNA® Purification System (Promega). The purified DNA fragment was introduced into the SC17 (0) / RSF-Rsd-TER strain by the same method as in <18-1-2>. The cells were spread on an L agarose plate containing 40 μg / mL kanamycin and incubated overnight at 34 ° C. to select transformants. The strain selected using kanamycin resistance as an index is a strain in which the yliG gene region of the P. ananatis SC17 (0) strain is replaced with the aceA gene sequence expressed by the Km resistance gene and the tac-like promoter sequence (SEQ ID NO: 73). Was confirmed by PCR using the synthetic DNAs shown in SEQ ID NO: 230 and SEQ ID NO: 231 as primers, and this strain was named SC17 (0), RyliG :: P4071-aceA :: Km.

<18-3-2> Construction of SC17sucA / Ptac84-yhfKΔaceB :: tet, RyliG :: P4071-aceA :: Km strain (FKGP4 strain) SC17 (0), RyliG :: P4071-aceA :: Km genomic DNA Extraction was performed by a conventional method, and 700 μg was introduced into the FKGP1 strain by an electric pulse method. The cells were spread on an LBGM9 agarose plate containing tetracycline 12.5 μg / mL and kanamycin 50 μg / mL, and cultured at 34 ° C. for about 20 hours. About the grown kanamycin resistant strain, introduction of aceA gene (introduction of RyliG :: P4071-aceA :: Km trait) was confirmed by PCR using the synthetic DNAs shown in SEQ ID NO: 230 and SEQ ID NO: 231 as primers, and RyliG :: A strain in which the P4071-aceA :: Km trait was confirmed to be introduced was named FKGP4 strain. The genotype of the FKGP4 strain is SC17sucA / Ptac84-yhfKΔaceB :: tet, RyliG :: P4071-aceA :: Km.

<18-4> SC17sucA / Ptac84-yhfKΔaceB , RyliG :: P4071-aceA, the following construction procedure ycaJ :: Pnlp8φ10-MLO_mclA strain (FKGP10Km ^s Ltd.), a MLO_mclA gene YcaJ gene region on the genome of FKGP4 strain introduced, it was constructed SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA strain (FKGP10Km ^s Co., Ltd.). The ycaJ gene is a gene encoding AAA family ATPase. The ycaJ gene corresponds to base numbers 790234 to 791601 of the genome sequence of Pantoea ananatis AJ13355 described in GenBank accession number NC — 017531.1.

<18-4-1> Construction of plasmid RSFRedIX Using pKD46 (Datsenko, KA, Wanner, BL, Proc. Nat1. Acad. Sci. USA, 97, 6640-6645, (2000)) as a template, SEQ ID NO: 232 and the sequence PCR was performed using the primers shown in No. 233, to amplify a DNA fragment containing a promoter region and orf region of araC and P _araBAD promoter. This was digested with PvuI and BsaI and inserted into RSF-int-xis (Japanese Patent Application No. 2008-110275) previously treated with PvuI and BsaI to obtain plasmid RSFParaIX. RSFParaIX carries the int-xis gene downstream of the ParaB promoter, and the expression of the int-xis gene is induced by arabinose. Then, this RSFParaIX as a template, PCR was performed using the primers shown in SEQ ID NO: 235 and SEQ ID NO: 234, the int-xis gene linked promoter region and orf region of araC, and a P _araBAD promoter downstream thereof The containing DNA fragment was amplified. This was digested with NotI and inserted into RSFredTER (WO2008 / 090770A1) previously treated with NotI to obtain plasmid RSFRedIX. RSFRedIX has gam, bet, and exo λ-Red genes downstream of the Plac promoter, and an int-xis gene downstream of the ParaB promoter. According to RSFRedIX, integration by the λ-Red system is induced by IPTG induction and arabinose induction. Thus, the drug resistance gene can be removed without using another helper plasmid.

<18-4-2> Construction of SC17 (0), ycaJ :: pnlp8F10-MLO_A :: Km strain The ycaJ gene region on the SC17 (0) genome was replaced with a sequence containing the MLO_mclA gene as follows. Specifically, a DNA fragment containing the MLO_mclA gene was amplified by PCR using the oligonucleotides of SEQ ID NO: 236 and SEQ ID NO: 237 using pTWV-Km-pnlp8F10-MLO_A constructed in Example <11-1> as a template. did. The amplified DNA fragment was purified by a conventional method and introduced into the SC17 (0) / RSF-Rsd-TER strain by a method similar to <18-1-2>. A transformant is selected using kanamycin resistance as an index, and a strain in which the ycaJ gene is replaced with a sequence containing the Km resistance gene and the MLO_mclA gene expressed by pnlp8F10 is SC17 (0), ycaJ :: pnlp8F10-MLO_A: : Named Km strain.

<18-4-3> Construction of SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA strain RSFRedIX plasmid was introduced into FKGP4 strain by electric pulse method, and chloramphenicol 25 μg / mL Was applied onto an LBGM9 agarose plate and cultured at 34 ° C. for about 20 hours. In order to remove the drug resistance gene of the grown strain, the strain was purified twice on an LB agarose plate containing 10 mM arabinose and 25 μg / mL chloramphenicol. Among the obtained colonies, a strain showing sensitivity to kanamycin and tetracycline was designated as FKGP8 strain.

SC17 (0), ycaJ :: Pnlp8φ10-MLO_mclA :: Km genome was extracted by a conventional method, and 700 μg was introduced into the FKGP8 strain by an electric pulse method. The cells were spread on an LBGM9 agarose plate containing kanamycin 50 μg / mL and cultured at 34 ° C. for about 20 hours. About the grown kanamycin resistant strain, it was confirmed that the trait of ycaJ :: Pnlp8φ10-MLO_mclA :: Km was introduced by PCR using the synthetic DNAs shown in SEQ ID NO: 238 and SEQ ID NO: 239 as primers, and the strain was designated as SC17sucA / Ptac84-yhfKΔaceB :: tet, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA :: Km / RSFRedIX strain. In order to remove the kanamycin resistance gene of the same strain, it was purified twice on an L agarose plate containing 10 μm arabinose and 25 μg / mL chloramphenicol. Among the obtained colonies, a strain showing sensitivity to kanamycin and tetracycline was named SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA / RSFRedIX strain.

SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclARS / RSFRedIX strain is removed by agarose containing IPTG 1 mM and sucrose 100 g / L And purified. Among the obtained colonies, the strain showing chloramphenicol sensitivity due to the loss of the RSFRedIX plasmid was named SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA strain.

<18-5> Construction of SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA, ldh :: P4071-pyc :: Km strain (FKGP40 strain) SC17sucA / Ptac84- pyc gene is introduced into the ldh gene region of yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA, and SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8cl10-MLO: A P4071-pyc :: Km strain (FKGP40 strain) was constructed. The pyc gene is a gene encoding pyruvate carboxylase. As the pyc gene, the pyc gene of Brevibacterium lactofermentum (Corynebacterium glutamicum) 2256 strain (ATCC 13869) was used. The nucleotide sequence of the same strain pyc gene and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs: 248 and 249, respectively. The ldh gene is a gene encoding lactate dehydrogenase. The ldh gene corresponds to nucleotide numbers 1621607 to 1622599 of the genome sequence of Pantoea ananatis AJ13355 described in GenBank accession number NC — 017531.1.

<18-5-1> Construction of SC17 (0), ppc :: P4071-pyc :: Km strain First, the ldh gene region on the SC17 (0) genome was replaced with a sequence containing the pyc gene as follows. . PCR reaction was performed using the chromosomal DNA of B. lactofermentum 2256 strain as a template and the synthetic DNAs described in SEQ ID NO: 240 and SEQ ID NO: 241 as primers to amplify a DNA fragment containing the orf region of the pyc gene. Further, a DNA fragment containing the ^{λattL-Km r -λattR-Ptac (} WO2008090770A1) as a template, PCR was performed using the synthetic DNA described in SEQ ID NO: 242 and SEQ ID NO: 243 as primers, λattL-Km ^r -λattR- A DNA fragment containing Ptac was amplified. Next, a DNA fragment containing the DNA fragment and λattL-Km ^r -λattR-Ptac containing ORF region of pyc gene as a template, a PCR reaction using synthetic DNA shown in SEQ ID NO: 242 and SEQ ID NO: 241 as a primer conducted to give the ^{λattL-Km r -λattR-P4071-} pyc gene fragment having a sequence homologous to the upstream and downstream of the ppc gene at both ends. The ^{λattL-Km r -λattR-Ptac-} pyc gene fragment was purified using the Promega Corp. Wizard PCR Prep, in the same manner as <18-1-2>, SC17 (0) / the RSF-Rsd-TER strain Introduced. SC17 (0), ppc :: P4071-pyc :: Km / RSF in which the desired trait has been introduced into the ppc gene region by selecting transformants on L agarose plates containing 40 mg / L kanamycin -Obtained Red-TER stock. The obtained strain was purified with an L agarose plate containing 10% sucrose and 1 mM IPTG to obtain a strain from which the RSF-Red-TER plasmid was dropped. The obtained strain was named SC17 (0), ppc :: P4071-pyc :: Km strain.

<18-5-2> Construction of SC17 (0), ldh :: P4071-pyc :: Km strain In the SC17 (0), ppc :: P4071-pyc :: Km strain, the pyc gene is inserted into the ppc gene region. As a result, the ppc gene is deficient. Therefore, in order to change the insertion position of the pyc gene from the ppc gene region to the ldh gene region, the following operation was performed. The genome of SC17 (0), ppc :: P4071-pyc :: Km strain is extracted, using this as a template, PCR reaction is performed using the primers shown in SEQ ID NOs: 244 and 245, and both ends of the ldh of P. ananatis It was obtained ^{λattL-Km r -λattR-Ptac-} pyc gene fragment having a sequence homologous to the upstream and downstream of the gene. This was introduced into the SC17 (0) / RSF-Rsd-TER strain by the same method as in <18-1-2>. Transformants were selected on an L agarose plate containing 40 mg / L kanamycin, the genome structure was confirmed by PCR using the synthetic DNAs shown in SEQ ID NO: 246 and SEQ ID NO: 247 as primers, and the target of ldh gene region The SC17 (0), ldh :: P4071-pyc :: Km / RSF-Red-TER strain into which the traits were introduced was obtained. As a result, the ldh gene is missing. The obtained strain was purified on an L agarose plate containing 10% sucrose and 1 mM IPTG to obtain a strain from which the RSFRedTER plasmid was removed. The obtained strain was named SC17 (0), ldh :: P4071-pyc :: Km strain.

<18-5-3> Construction of SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA, ldh :: P4071-pyc :: Km strain (FKGP40 strain) SC17 (0), ldh: The genome of: P4071-pyc :: Km was extracted by a conventional method, and 700 μg was introduced into SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA strain by an electric pulse method. The cells were spread on an L agarose plate containing kanamycin 50 μg / mL and cultured at 34 ° C. for about 20 hours. About the grown kanamycin resistant strain, it was confirmed that the trait of P4071-pyc :: Km was introduced into the ldh gene region by PCR using the synthetic DNAs shown in SEQ ID NO: 246 and SEQ ID NO: 247 as primers. FKGP40 strain was designated. The genotype of the FKGP40 strain is SC17sucA / Ptac84-yhfKΔaceB, RyliG :: P4071-aceA, ycaJ :: Pnlp8φ10-MLO_mclA, ldh :: P4071-pyc :: Km.

<18-6> Construction of FKGP40 / pSTV strain, FKGP40 / stk ** strain, FKGP40 / smt strain, FKGP40 / RSFPPG + pSTV strain, FKGP40 / RSFPPG + stk ** strain, and FKGP40 / RSFPPG + smt strain pSTV28 (TAKARA ), PSTV :: STK ** constructed in Example <4-2>, and pSTV-Mm_T constructed in Example <5-2> were introduced into the FKGP40 strain by the electric pulse method, respectively. The cells were spread on an LBGM9 agarose plate containing 25 μg / mL of chloramphenicol and cultured at 34 ° C. The grown strains were purified on the same plate and named FKGP40 / pSTV strain, FKGP40 / stk ** strain, and FKGP40 / smt strain, respectively. Furthermore, RSFPPG (WO2008 / 020654) was introduced into FKGP40 / pSTV strain, FKGP40 / stk ** strain, and FKGP40 / smt strain by the electric pulse method. The cells were spread on an LBGM9 agarose plate containing 25 μg / mL chloramphenicol and 12.5 μg / mL tetracycline, and cultured at 34 ° C. The grown strains were purified on the same plate and named FKGP40 / RSFPPG + pSTV strain, FKGP40 / RSFPPG + stk ** strain, and FKGP40 / RSFPPG + smt strain, respectively.

<18-7> Production of glutamic acid from sugar by Pantoea ananatis-evaluated strains FKGP40 / pSTV strain, FKGP40 / stk ** strain, and FKGP40 / smt strain, each containing LBGM9 agarose plate medium containing 25 μg / mL chloramphenicol Incubated overnight at 34 ° C. FKGP40 / RSFPPG + pSTV strain, FKGP40 / RSFPPG + stk ** strain, and FKGP40 / RSFPPG + smt strain were each 34 ° C. on an LBGM9 agarose plate containing 25 μg / mL of chloramphenicol and 12.5 μg / mL of tetracycline, Cultured overnight. The cells were collected, washed with physiological saline, and suspended in physiological saline so that OD620 was 100. 1.3 mL glutamic acid production medium (glucose 20 g / L, MgSO ₄ · 7H ₂ O 1 g / L, yeast extract 2 g / L, ammonium sulfate 2 g / L L, KH ₂ SO ₄ 1 g / L, MnSO ₄ · 5H ₂ O 10 mg / L, FeSO ₄ 10 mg / L, biotin 1 mg / L, calcium carbonate 50 g / L) Incubation was performed at 34 ° C. for 48 hours at 1400 rpm using a shaker (eppendorf).

After completion of the culture, the concentration of L-glutamic acid accumulated in the medium and the concentration of residual sugar in the medium were analyzed with Biotech Analyzer AS-310 (Sakura Seye Co., Ltd.). The cell turbidity (OD) was measured using a spectrophotometer U-2900 (HITACHI).

The results are shown in Table 16. The L-glutamic acid yield (vs. sugar yield) of the FKGP40 / stk ** and FKGP40 / smt strains was improved by 1.2% and 1.4%, respectively, compared with the control FKGP40 / pSTV strain. On the other hand, with respect to the FKGP40 / RSFPPG + stk ** strain and the FKGP40 / RSFPPG + smt strain, the L-glutamic acid yield (sugar yield) was 3.5%, respectively, compared with the control FKGP40 / RSFPPG + pSTV strain. 1.4% improvement. The results of both experiments revealed that the ability to produce L-glutamic acid was improved by introducing a mutant succinyl CoA synthase gene or a succinyl CoA: malate CoA transferase gene.

According to the present invention, the ability of microorganisms to produce target substances can be improved, and target substances can be produced efficiently.

<Explanation of Sequence Listing>
SEQ ID NOs: 1 to 38: Primer SEQ ID NO: 39: Nucleotide sequence of a DNA fragment containing the mclA gene derived from Methylobacterium extruens AM1 strain modified according to the codon usage of Escherichia coli, etc. SEQ ID NO: 40: Escherichia Nucleotide sequence SEQ ID NO: 41 of a DNA fragment containing the mtkAB gene derived from Methylobacterium extorens AM1 strain modified according to the codon usage frequency of E. coli: Mesozobium roti modified according to the codon usage frequency of Escherichia coli Nucleotide sequence of DNA fragment containing mclA gene and mtkAB gene from MAFF303099 strain SEQ ID NO: 42: DNA fragment containing mclA gene and mtkAB gene from Granulibacter bethesdensis CGDNIH1 strain modified according to codon usage of Escherichia coli SEQ ID NO: 43: Codon usage frequency of Escherichia coli SEQ ID NO: 44 of the DNA fragment containing the Ca_smtAB gene modified according to SEQ ID NO: 44: The nucleotide sequence of the DNA fragment containing the Ap_smtAB gene modified according to the codon usage of Escherichia coli SEQ ID NO: 45: Codon usage of the Escherichia coli SEQ ID NO: 46 of the DNA fragment containing the Mm_smt gene modified according to SEQ ID NO: 46: Nucleotide sequence of the DNA fragment containing the Rr_smt gene gene modified according to the codon usage frequency of Escherichia coli, etc. SEQ ID NO: 47 to 66: Primer SEQ ID NO: 67: Nucleotide sequence of a DNA fragment containing a double mutant sucCD gene SEQ ID NO: 68 to 71: Primer SEQ ID NO: 72: tac promoter SEQ ID NO: 73, 74: tac-like promoter SEQ ID NO: 75: mtkA of Methylobacterium Extorens AM1 strain Base sequence of the gene SEQ ID NO: 76: MtkA of Methylobacterium extremens AM1 strain Protein amino acid sequence SEQ ID NO: 77: MtkB gene nucleotide sequence SEQ ID NO: 78: Methylobacterium EXTRUSENCE AM1 strain MtkB protein amino acid sequence SEQ ID NO: 79: Mesozobium The nucleotide sequence of the mtkA gene of the roti MAFF303099 strain SEQ ID NO: 80: the amino acid sequence of the MtkA protein of the mesozobium roti MAFF303099 strain SEQ ID NO: 81: the nucleotide sequence of the mtkB gene of the mesozobium roti MAFF303099 strain SEQ ID NO: 82: the strain of the mesozobium roti MAFF303099 Amino acid sequence of the MtkB protein SEQ ID NO: 83: Nucleotide sequence of the mtkA gene of Granulibacter bethesdensis CGDNIH1 strain SEQ ID NO: 84: Amino acid sequence of the MtkA protein of the Granulobacter bethesdensis strain CGDNIH1 SEQ ID NO: 85: Granulibacter bethesdensi The nucleotide sequence of the mtkB gene of SGD CGDNIH1 SEQ ID NO: 86: The amino acid sequence of the MtkB protein of CGDNIH1 strain SEQ ID NO: 87: The nucleotide sequence of the sucC gene of E. coli MG1655 SucC of E. coli MG1655 Protein amino acid sequence SEQ ID NO: 89: E. coli MG1655 sucD gene base sequence SEQ ID NO: 90: E. coli MG1655 SucD protein amino acid sequence SEQ ID NO: 91: Pantoea ananatis AJ13355 sucC gene base sequence SEQ ID NO: 92: Pantoea amino acid sequence of the SucC protein of ananatis AJ13355 SEQ ID NO: 93: nucleotide sequence of the sucD gene of Pantoea ananatis AJ13355 SEQ ID NO: 94: amino acid sequence of the SucD protein of Pantoea ananatis AJ13355 SEQ ID NO: 95: nucleotide sequence of the sucC gene of Corynebacterium glutamicum ATCC13032 : Amino acid sequence of the SucC protein of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 97: Coryne Nucleotide sequence of sucD gene of bacterium glutamicum ATCC13032 SEQ ID NO: 98: Amino acid sequence of SucD protein of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 99: Nucleotide sequence of Ca_smtA gene of Chloroflexus aurantiax J-10-fl SEQ ID NO: 100: Chloroflexus The amino acid sequence of the Ca_SmtA protein of Aurantax Ax J-10-fl SEQ ID NO: 101: the nucleotide sequence of the Ca_smtB gene of Chloroflexus auslanx Ax J-10-fl SEQ ID NO: 102: Chloroflex auranthax J- Amino acid sequence of Ca_SmtB protein of 10-fl strain SEQ ID NO: 103: Accumulactor phosphatis (candidate strain) clade IIAstr .. Nucleotide sequence of Ap_smtA gene of UW-1 strain SEQ ID NO: 104: Accumulactor phosphatis (candidate strain) clade IIAstr .. Amino acid sequence of Ap_SmtA protein of UW-1 strain SEQ ID NO: 105: Accum Rabacter phosphatis (candidate strain) clade IIAstr .. Nucleotide sequence of Ap_smtB gene of UW-1 strain SEQ ID NO: 106: amino acid sequence number of Ap_SmtB protein of Accumulacter phosphatis (candidate strain) clade IIAstr .. UW-1 strain 107: Rr_smt gene nucleotide sequence of Rhodospirillum rambum ATCC 11170 strain SEQ ID NO: 108: Rr_Smt protein amino acid sequence SEQ ID NO. 109 of Rhodospirylum rumum ATCC strain 11170: Nucleotide sequence of Mm_smt gene of Magnetospirillum magneticum AMB-1 strain No. 110: Amino acid sequence of Mm_Smt protein of Magnetospirillum magneticumum strain AMB-1 SEQ ID NO: 111: Nucleotide sequence of mclA gene of Methylobacterium Extorcense AM1 sequence No. 112: Methylobacterium Extorcense Amino acid sequence of MclA protein of AM1 strain SEQ ID NO: 11 3: base sequence of mclA gene of Mesozobium roti MAFF303099 strain SEQ ID NO: 114: amino acid sequence of MclA protein of Mesozobium roti MAFF303099 sequence SEQ ID NO: 115: base sequence of mclA gene of Cgrunichacter bethesdensis CGDNIH1 strain: 116 Amino acid sequence of the MclA protein of the strain CGDNIH1 of Reactor Bethesdensis SEQ ID NO: 117: Nucleotide sequence of the aceA gene of E. coli MG1655 SEQ ID NO: 118: Amino acid sequence of the AceA protein of E. coli MG1655 SEQ ID NO: 119: The aceA gene of Pantoea ananatis AJ13355 SEQ ID NO: 120: AceA protein amino acid sequence of Pantoea ananatis AJ13355 SEQ ID NO: 121: nucleotide sequence of ICL1 gene of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 122: amino acid sequence of protein encoded by ICL1 of Corynebacterium glutamicum ATCC13032: E c oli MG1655 aceB gene base sequence SEQ ID NO: 124: E. coli MG1655 AceB protein amino acid sequence SEQ ID NO: 125: Pantoea ananatis AJ13355 aceB gene base sequence SEQ ID NO: 126: Pantoea ananatis AJ13355 amino acid sequence sequence number 127: nucleotide sequence of the aceB gene of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 128: amino acid sequence of the AceB protein of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 129: nucleotide sequence of the ptsG gene of E. coli MG1655 SEQ ID NO: 130: of the PtsG protein of E. coli MG1655 Amino acid sequence SEQ ID NO: 131: base sequence of the ptsG gene of Pantoea ananatis AJ13355 SEQ ID NO: 132: amino acid sequence of the PtsG protein of Pantoea ananatis AJ13355 SEQ ID NO: 133: base sequence of the ptsG1 gene of Corynebacterium glutamicum ATCC13032: ptsG1 of Corynebacterium glutamicum ATCC13032 Protein encoded by Amino acid sequence SEQ ID NO: 135: base sequence of ldhA gene of E. coli MG1655 SEQ ID NO: 136: amino acid sequence of LdhA protein of E. coli MG1655 SEQ ID NO: 137: base sequence of ldhA gene of Pantoea ananatis AJ13355 SEQ ID NO: 138: Pantoea ananatis AJ13355 The amino acid sequence of the LdhA protein of SEQ ID NO: 139: The nucleotide sequence of the adhE gene of E. coli MG1655 SEQ ID NO: 140: The amino acid sequence of the AdhE protein of E. coli MG1655 SEQ ID NO: 141: The nucleotide sequence of the adhE gene of Pantoea ananatis AJ13355 : Amino acid sequence of the AdhE protein of Pantoea ananatis AJ13355 SEQ ID NO: 143: base sequence of the pta gene of E. coli MG1655 SEQ ID NO: 144: amino acid sequence of the Pta protein of E. coli MG1655 SEQ ID NO: 145: base of the pta gene of Pantoea ananatis AJ13355 SEQ ID NO: 146: Amino acid sequence of the Pta protein of Pantoea ananatis AJ13355 SEQ ID NO: 147: PflB gene of SEQ ID NO: 148: amino acid sequence of PflB protein of E. coli MG1655 SEQ ID NO: 149: nucleotide sequence of pflD gene of E. coli MG1655 SEQ ID NO: 150: of PflD protein of E. coli MG1655 Amino acid sequence SEQ ID NO: 151: base sequence of tdcE gene of E. coli MG1655 SEQ ID NO: 152: amino acid sequence of TdcE protein of E. coli MG1655 SEQ ID NO: 153: base sequence of pflB gene of Pantoea ananatis AJ13355 SEQ ID NO: 154: Pantoea ananatis AJ13355 Amino acid sequence of the PflB protein of SEQ ID NO: 155: nucleotide sequence of the sucA gene of Pantoea ananatis AJ13355 SEQ ID NO: 156: amino acid sequence of the SucA protein of Pantoea ananatis AJ13355 SEQ ID NO: 157: nucleotide sequence of the sucB gene of Pantoea ananatis AJ13355: SEQ ID NO: 158: Pantoea amino acid sequence of SucB protein of ananatis AJ13355 SEQ ID NO: 159: lpdA gene of Pantoea ananatis AJ13355 SEQ ID NO: 160: amino acid sequence SEQ ID NO: 161 of the LpdA protein of Pantoea ananatis AJ13355: SEQ ID NO: 162 of the odhA gene of Corynebacterium glutamicum ATCC13032: E1o subunit amino acid sequence SEQ ID NO: 163 of Corynebacterium glutamicum ATCC13032: Corynebacterium glutamicum ATCC13032 lpd gene nucleotide sequence SEQ ID NO: 164: Corynebacterium glutamicum ATCC13032 E3 subunit amino acid sequence SEQ ID NO: 165: Corynebacterium glutamicum ATCC13032 NCgl2126 nucleotide sequence SEQ ID NO: 166: Corynebacterium glutamicum ATCC13032 NCgl2126 encoded amino acid sequence SEQ ID NO: 167: Corynebacterium nucleotide sequence of ICL2 gene of glutamicum ATCC13032 SEQ ID NO: 168: amino acid sequence of protein encoded by ICL2 of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 169: of ICL1 gene of Corynebacterium glutamicum 2256 (ATCC13869) Base sequence SEQ ID NO: 170: amino acid sequence SEQ ID NO: 171 of the protein encoded by ICL1 of Corynebacterium glutamicum 2256 (ATCC13869) ICL2 base sequence of SEQ ID NO: 172: ICL2 of Corynebacterium glutamicum 2256 (ATCC13869) Amino acid sequence of encoded protein SEQ ID NO: 173: Base sequence of glcB gene of E. coli MG1655 SEQ ID NO: 174: Amino acid sequence of GlcB protein of E. coli MG1655 SEQ ID NO: 175: Base sequence of aceB gene of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 176: AceB protein amino acid sequence of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 177: Base sequence of ptsG2 gene of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 178: Amino acid sequence of protein encoded by ptsG2 of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 179: Corynebacterium glutamic The nucleotide sequence of the ptsG1 gene of um 2256 (ATCC13869) SEQ ID NO: 180: The amino acid sequence of the protein encoded by ptsG1 of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 181: The nucleotide sequence of the ptsG2 gene of Corynebacterium glutamicum 2256 (ATCC13869): The amino acid sequence of the protein encoded by ptsG2 of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 183: The nucleotide sequence of sucC gene of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 184: The amino acid sequence of SucC protein of Corynebacterium glutamicum 2256 (ATCC13869) : Nucleotide sequence of the sucD gene of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 186: amino acid sequence of the SucD protein of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 187: nucleotide sequence of the ldh gene of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 188: Corynebacterium glutamicum ATCC13032 Ldh protein amino acid sequence SEQ ID NO: 189: Corynebacterium glutamicum 2256 (ATCC13869) ldh gene base sequence SEQ ID NO: 190: Corynebacterium glutamicum 2256 (ATCC13869) Ldh protein amino acid sequence SEQ ID NO: 191: Corynebacterium glutamicum ATCC13032 adhE gene base sequence SEQ ID NO: 192: amino acid sequence of AdhE protein of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 193: nucleotide sequence of ilvB gene of E. coli MG1655 SEQ ID NO: 194: amino acid sequence of IlvB protein of E. coli MG1655 SEQ ID NO: 195: of E. coli MG1655 nucleotide sequence of the ilvI gene SEQ ID NO: 196: amino acid sequence of the IlvI protein of E. coli MG1655 SEQ ID NO: 197: nucleotide sequence of the ilvG gene of Pantoea ananatis AJ13355 SEQ ID NO: 198: amino acid sequence of the IlvG protein of Pantoea ananatis AJ13355 SEQ ID NO: 199: Pantoea base sequence of the ilvI gene of ananatis AJ13355 No. 200: amino acid sequence of the IlvI protein of Pantoea ananatis AJ13355 SEQ ID NO: 201: nucleotide sequence of the ilvB gene of Corynebacterium glutamicum ATCC13032 202: amino acid sequence of the IlvB protein of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 203: base of the budA gene of Pantoea ananatis AJ13355 SEQ ID NO: 204: Pantoea ananatis AJ13355 amino acid sequence of BudA protein SEQ ID NO: 205: Pantoea ananatis AJ13355 budC gene base sequence SEQ ID NO: 206: Pantoea ananatis AJ13355 BudC protein amino acid sequence SEQ ID NO: 207: Corynebacterium glutamicum ATCC13032 butA gene SEQ ID NO: 208: Amino acid sequence of ButA protein of Corynebacterium glutamicum ATCC13032 SEQ ID NO: 209-212: Primer SEQ ID NO: 213: Base sequence of Mm_smt gene modified according to the codon usage of Escherichia coli Column numbers 214 to 247: Primer SEQ ID NO: 248: Base sequence of pyc gene of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 249: Amino acid sequence of Pyc protein of Corynebacterium glutamicum 2256 (ATCC13869) SEQ ID NO: 250: Corynebacterium glutamicum 2256 (ATCC13869) nucleotide sequence of yggB gene SEQ ID NO: 251: amino acid sequence of YggB protein of Corynebacterium glutamicum 2256 (ATCC13869)

Claims

A method for producing a target substance, comprising:
Culturing a microorganism having the ability to produce a target substance in a medium to produce and accumulate the target substance in the medium or in the cells of the microorganism, and collecting the target substance from the medium or the cells,
The microorganism has been modified to increase the activity of an enzyme that synthesizes malyl CoA from L-malic acid, malyl CoA lyase activity, and isocitrate lyase activity;
The method, wherein the target substance is a substance biosynthesized with isocitrate as a precursor.
The enzyme that synthesizes malyl CoA from L-malate is one or more enzymes selected from the group consisting of malate thiokinase, succinyl CoA synthase, and succinyl CoA: malate CoA transferase. the method of.
The method according to claim 1 or 2, which has one or more characteristics selected from the group consisting of the following (A) to (C):
(A) by increasing the expression of a gene encoding an enzyme that synthesizes malyl-CoA from L-malate, the activity of the enzyme that synthesizes malyl-CoA from L-malate was increased;
(B) Increased expression of the gene encoding malyl-CoA lyase increased malyl-CoA lyase activity;
(C) Isocitrate lyase activity was increased by increasing the expression of the gene encoding isocitrate lyase.
The method according to claim 3, wherein the expression of the gene is increased by increasing the copy number of the gene and / or modifying an expression regulatory sequence of the gene.
The expression of the gene encoding malate thiokinase has increased,
The gene encoding malate thiokinase is one or more genes selected from the group consisting of the mtkA gene and the mtkB gene;
The mtkA gene is DNA selected from the group consisting of (A) to (D) below:
The method according to claim 3 or 4, wherein the mtkB gene is DNA selected from the group consisting of the following (E) to (H):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 76, 80, or 84;
(B) an amino acid sequence represented by SEQ ID NO: 76, 80, or 84, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and a complex with other subunits DNA encoding a subunit that forms and exhibits malate thiokinase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 75, 79, or 83;
(D) A base sequence complementary to the base sequence shown in SEQ ID NO: 75, 79, or 83 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and complexed with other subunits A DNA encoding a subunit that forms and exhibits malate thiokinase activity;
(E) a DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 78, 82, or 86;
(F) an amino acid sequence represented by SEQ ID NO: 78, 82, or 86, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and a complex with other subunits DNA encoding a subunit that forms and exhibits malate thiokinase activity;
(G) a DNA comprising the base sequence represented by SEQ ID NO: 77, 81, or 85;
(H) a base sequence complementary to the base sequence shown in SEQ ID NO: 77, 81, or 85 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and complexed with other subunits A DNA encoding a subunit that forms and exhibits malate thiokinase activity;
The expression of the gene encoding succinyl-CoA synthase has increased,
The gene encoding succinyl CoA synthase is one or more genes selected from the group consisting of sucC gene and sucD gene;
The sucC gene is a DNA selected from the group consisting of the following (A) to (D):
The method according to any one of claims 3 to 5, wherein the sucD gene is DNA selected from the group consisting of the following (E) to (H):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 88, 92, 96, or 184;
(B) an amino acid sequence represented by SEQ ID NO: 88, 92, 96, or 184, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and combined with other subunits DNA encoding a subunit that forms a body and exhibits succinyl-CoA synthase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 87, 91, 95, or 183;
(D) a base sequence complementary to the base sequence shown in SEQ ID NO: 87, 91, 95, or 183 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and with other subunits DNA encoding a subunit that forms a complex and exhibits succinyl-CoA synthase activity;
(E) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 90, 94, 98, or 186;
(F) an amino acid sequence represented by SEQ ID NO: 90, 94, 98, or 186, comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and combined with other subunits DNA encoding a subunit that forms a body and exhibits succinyl-CoA synthase activity;
(G) a DNA comprising the base sequence represented by SEQ ID NO: 89, 93, 97, or 185;
(H) a base sequence complementary to the base sequence shown in SEQ ID NO: 89, 93, 97, or 185 or a probe that can be prepared from the complementary sequence, hybridized under stringent conditions, and with other subunits DNA encoding a subunit that forms a complex and exhibits succinyl-CoA synthase activity.
The expression of the gene encoding succinyl CoA: malate CoA transferase has increased,
The gene encoding the succinyl CoA: malate CoA transferase is one or more genes selected from the group consisting of smtA gene, smtB gene, and smt gene;
The smtA gene is a DNA selected from the group consisting of (A) to (D) below:
The smtB gene is DNA selected from the group consisting of (E) to (H) below:
The method according to any one of claims 3 to 6, wherein the smt gene is DNA selected from the group consisting of the following (I) to (L):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 100 or 104;
(B) in the amino acid sequence shown in SEQ ID NO: 100 or 104, comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids, and forming a complex with other subunits Succinyl CoA: a DNA encoding a subunit exhibiting malate CoA transferase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 99 or 103;
(D) It hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 99 or 103 or a probe that can be prepared from the complementary sequence, and forms a complex with other subunits. A DNA encoding a subunit exhibiting succinyl CoA: malate CoA transferase activity;
(E) a DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 102 or 106;
(F) an amino acid sequence represented by SEQ ID NO: 102 or 106, which comprises an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions, and forms a complex with other subunits Succinyl CoA: a DNA encoding a subunit exhibiting malate CoA transferase activity;
(G) a DNA comprising the base sequence represented by SEQ ID NO: 101 or 105;
(H) It hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 101 or 105 or a probe that can be prepared from the complementary sequence, and forms a complex with other subunits. A DNA encoding a subunit exhibiting succinyl CoA: malate CoA transferase activity;
(I) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 108 or 110;
(J) a protein comprising an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in SEQ ID NO: 108 or 110, and having succinyl CoA: malate CoA transferase activity The encoding DNA;
(K) a DNA comprising the base sequence represented by SEQ ID NO: 107 or 109;
(L) a protein that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 107 or 109 or a probe that can be prepared from the complementary sequence and that has succinyl CoA: malate CoA transferase activity DNA encoding
The method according to any one of claims 3 to 7, wherein the gene encoding the malyl CoA lyase is DNA selected from the group consisting of the following (A) to (D):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 112, 114, or 116;
(B) a protein having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence shown in SEQ ID NO: 112, 114, or 116, and having malyl-CoA lyase activity The encoding DNA;
(C) DNA comprising the base sequence shown in SEQ ID NO: 111, 113, or 115;
(D) a protein that hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 111, 113, or 115, or a probe that can be prepared from the base sequence, and that has malyl-CoA lyase activity DNA encoding
The method according to any one of claims 3 to 8, wherein the gene encoding the isocitrate lyase is DNA selected from the group consisting of the following (A) to (D):
(A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 118, 120, 122, 168, 170, or 172;
(B) the amino acid sequence shown in SEQ ID NO: 118, 120, 122, 168, 170, or 172, comprising an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions or additions, and isocitrate DNA encoding a protein having lyase activity;
(C) a DNA comprising the base sequence represented by SEQ ID NO: 117, 119, 121, 167, 169, or 171;
(D) hybridizes under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 117, 119, 121, 167, 169, or 171 or a probe that can be prepared from the complementary sequence, and DNA encoding a protein having acid lyase activity.
The method according to any one of claims 2 to 9, wherein succinyl CoA synthase activity and / or malate thiokinase activity is increased by retaining a gene encoding a mutant succinyl CoA synthase.
The gene encoding the mutant succinyl CoA synthase is a succinyl CoA synthase having a mutation corresponding to one or more mutations selected from the group consisting of the following (A) to (E) in the wild type succinyl CoA synthase: 11. The method of claim 10, which is a gene that encodes:
(A) a mutation in which proline at position 124 in SEQ ID NO: 90 is substituted with alanine;
(B) a mutation in which tyrosine at position 157 in SEQ ID NO: 90 is substituted with glycine;
(C) a mutation in which valine at position 161 in SEQ ID NO: 90 is substituted with alanine;
(D) a mutation in which glutamic acid at position 97 in SEQ ID NO: 90 is substituted with aspartic acid;
(E) A mutation in which glycine at position 271 in SEQ ID NO: 88 is substituted with alanine.
The method according to any one of claims 1 to 11, wherein the microorganism is further modified so that malate synthase activity is lowered.
The method according to any one of claims 1 to 12, wherein the microorganism is further modified so that a supplementary pathway of the TCA cycle is enhanced.
The target substance biosynthesized using isocitrate as a precursor is a group consisting of L-glutamic acid, L-glutamine, L-proline, L-arginine, L-ornithine, L-citrulline, itaconic acid, and γ-aminobutyric acid The method according to any one of claims 1 to 13, which is one or more substances selected from the group consisting of more than one substance.
The method according to claim 14, wherein the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.
The method according to any one of claims 1 to 15, wherein the microorganism is a coryneform bacterium or a bacterium belonging to the family Enterobacteriaceae.
The method according to claim 16, wherein the coryneform bacterium is Corynebacterium glutamicum.
The method according to claim 16, wherein the bacterium belonging to the family Enterobacteriaceae is Pantoea ananatis or Escherichia coli.