JP2004173685A - Method for production of target substance by fermentation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a useful substance such as an L-amino acid by a fermentation process using Escherichia genus bacteria, capable of increasing production efficiency or production speed. <P>SOLUTION: This method for production of the target substance by utilizing microorganisms comprises cultivation of the Escherichia genus bacteria in a medium, accumulating the target substance in the medium or the bacterial cells and collecting the target substance, wherein a strain in which FIS (factor for inversion stimulation) protein does not normally function in the cell due to disruption of fis gene on the chromosome, is used for the Escherichia genus bacteria. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates to the fermentation industry, and more particularly, to a method for efficiently producing a target substance such as L-amino acid by a fermentation method using a microorganism.

  The chromosomal DNA of Escherichia coli is folded by a plurality of DNA binding proteins into a nucleosome-like structure called a nucleoid. Such a protein is called a nucleoid constituent protein.

  FIS (factor for inversion stimulation), which is one of the major nucleoid constituent proteins, is encoded by the fis gene located at 73.4 minutes on the Escherichia coli chromosome. FIS expression is induced during the growth phase and is suppressed during the stationary phase. Disruption of the fis gene of a wild strain of Escherichia coli results in a phenotypically slow growth rate in a very nutrient-rich medium, i.e. under conditions of high growth rate, but not in the case of a low growth rate. It has been reported that the growth rate does not change (Non-Patent Document 1). The growth rate of the fungus under the conditions for producing the substance corresponds to the latter. FIS is a global transcriptional regulator that positively or negatively regulates the expression of a plurality of genes. In addition to transfer RNA and ribosomal RNA (Non-Patent Document 2), FIS has been used to regulate the expression of genes involved in metabolism. It is reported that it is controlled (Non-Patent Document 3).

  H-NS is known as a nucleoid-constituting protein that is induced in the growth phase similarly to FIS and is known to positively or negatively regulate the expression of a plurality of genes (Non-Patent Document 4). H-NS is encoded by the hns gene located at 27.8 minutes on the Escherichia coli chromosome.

  Other major nucleoid-constituting proteins include, for example, HU and DPS. HU is composed of HUα and HUβ heterodimers, and is encoded by the hupA and hupB genes located at 90.5 minutes and 9.7 minutes, respectively, on the Escherichia coli chromosome (Non-patent Document 5). It is seen both in the stationary phase. DPS is encoded by the dps gene located at 18.3 minutes on the Escherichia coli chromosome, and its expression is suppressed during the growth phase and is induced during the stationary phase (Non-patent Document 6).

Until now, there has been no report on the improvement of substance production by controlling the expression of genes encoding nucleoid constituent proteins such as the fis gene, the hns gene, the hapAB gene, and the dps gene.
Nilsson et al., The Journal of Biological Chemistry, Vol. 269, 9460-9465, 1994 Nilsson et al., The EMBO Journal, Vol. 9, 727-734, 1990 Xu et al., Journal of Bacteriology, Vol.177, 938-947, 1995 Hulton et al., Cell, Vol. 63, 631-642, 1990. Wada et al., Journal of Molecular Biology, Vol. 204, 581-591, 1988 Almion et al., Genes and Development, Vol. 6, 2646-2654, 1992

  An object of the present invention is to improve production efficiency or production rate when producing a useful substance by a fermentation method using a bacterium belonging to the genus Escherichia.

The present inventors have conducted intensive studies to solve the above problems, and by modifying a gene encoding a nucleoid constituent protein that is universally present in Escherichia bacteria, it is possible to improve substance production by Escherichia bacteria. Was found. Specifically, they found that disrupting the fis gene in a bacterium belonging to the genus Escherichia improves the ability to produce the target substance, and completed the present invention.
That is, the present invention is as follows.

(1) A method for producing a target substance using a bacterium belonging to the genus Escherichia, wherein the bacterium belonging to the genus Escherichia is cultured in a medium, the target substance is produced and accumulated in the medium or the cells, and the target substance is collected. Is a method for producing a target substance, which has the ability to produce the target substance, and wherein the FIS protein does not function normally in cells.
(2) The method according to (1), wherein the Escherichia bacterium has a FIS protein that does not function normally due to disruption of the fis gene on the chromosome.
(3) The method according to (1) or (2), wherein the Escherichia bacterium is Escherichia coli.
(4) The method according to any one of (1) to (3), wherein the target substance is an L-amino acid or a protein.
(5) The method according to (4), wherein the L-amino acid is an amino acid belonging to the aspartic acid family.
(6) The method according to (5), wherein the aspartic acid amino acid is L-lysine, L-threonine, or L-methionine.
(7) The method according to (6), wherein the amino acid is L-lysine.

  According to the present invention, the production rate or production efficiency can be improved when producing useful substances such as L-amino acids using Escherichia bacteria.

Hereinafter, the present invention will be described in detail.
The bacterium belonging to the genus Escherichia used in the present invention is not particularly limited as long as it is a microorganism belonging to the genus Escherichia and has the ability to produce the target substance. Specifically, for example, those described in the book by Neidhardt et al. (Neidhardt, FC et al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington DC, 1208, table 1) can be used. More specifically, Escherichia coli is mentioned.

  The “ability to produce a target substance” refers to the ability to produce a target substance to the extent that it can be recovered from cells or medium when the bacterium belonging to the genus Escherichia used in the present invention is cultured in the medium. Preferably, it refers to the ability to produce a larger amount of a target substance than a wild-type or unmodified strain of a bacterium belonging to the genus Escherichia.

The target substance is not particularly limited as long as it can be produced by a bacterium belonging to the genus Escherichia. For example, various L-amino acids such as L-lysine, L-threonine, L-homoserine, L-glutamic acid, L-leucine, L-isoleucine, L-valine and L-phenylalanine, proteins (including peptides), guanine, Examples thereof include those conventionally produced by bacteria belonging to the genus Escherichia, such as nucleic acids such as inosine, guanylic acid, and inosine acid, vitamins, antibiotics, growth factors, and physiologically active substances. In addition, the present invention can be applied to those that have not been produced by Escherichia bacteria.
The target substance may be an amino acid of the aspartic acid family. Homologs include L-lysine, L-threonine, L-methionine.

  Examples of L-lysine-producing Escherichia bacteria include mutants having resistance to L-lysine analogs. This L-lysine analog is such that it inhibits the growth of Escherichia bacteria, but its inhibition is such that when L-lysine coexists in the medium, it is totally or partially released. . For example, there are oxalidine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam and the like. Mutants having resistance to these lysine analogs can be obtained by subjecting a microorganism of the genus Escherichia to ordinary artificial mutation procedures. Specific examples of strains used for producing L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see JP-A-56-18596 and US Pat. No. 4,346,170) and Escherichia coli VL611. No. Aspartokinase of these microorganisms is released from feedback inhibition by L-lysine.

  Other examples include L-threonine-producing bacteria described below. This is because the inhibition of aspartokinase by L-lysine is generally released also from L-threonine-producing bacteria.

  In Examples described later, strain WC196 was used as an L-lysine-producing bacterium of Escherichia coli. This strain was bred by imparting AEC resistance to the W3110 strain derived from Escherichia coli K-12. The strain was named Escherichia coli AJ13069 strain, and dated December 6, 1994, National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 305-8566 Japan). Deposit No. FERM P-14690 at 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki Pref., Japan, deposited under accession number FERM P-14690, transferred to an international deposit under the Budapest Treaty on September 29, 1995, and deposited under accession number FERM BP-5252. (See WO 96/17930, International Publication Pamphlet).

Examples of L-threonine-producing Escherichia bacteria include Escherichia coli VKPM B-3996 (RIA 1867) (see US Pat. No. 5,175,107) and MG442 strain (Gusyatiner et al.).
al., Genetika (in Russian), 14, 947-956 (1978)).

Examples of L-homoserine-producing Escherichia bacteria include the NZ10 strain, which is a Leu ⁺ revertant strain of the C600 strain (see Appleyard RK, Genetics, 39, 440-452 (1954)).

  Examples of L-glutamic acid-producing bacteria belonging to the genus Escherichia include AJ12624 strain (FERM BP-3853) (see French Patent Application Publication No. 2,680,178) and L-valine-resistant strains such as Escherichia coli B11 and Escherichia coli K-12. (ATCC10798), Escherichia coli B (ATCC11303) and Escherichia coli W (ATCC9637).

  Examples of L-leucine-producing Escherichia bacteria include β-2-thienylalanine-resistant strains, β-2-thienylalanine-resistant and β-hydroxyleucine-resistant strains (Japanese Patent Publication No. 62-34397). , 4-azaleucine resistance or 5,5,5-trifluoroleucine resistance (JP-A-8-70879). A specific example is AJ11478 strain (FERM P-5274) (see Japanese Patent Publication No. 62-34397).

Examples of L-isoleucine-producing Escherichia bacteria include Escherichia coli KX141 (VKPM B-4781) (see European Patent Application Publication No. 519,113).
Examples of L-valine-producing Escherichia bacteria include Escherichia coli VL1970 (VKPM B-4411) (see European Patent Application Publication No. 519,113).
Examples of L-phenylalanine producing bacteria include Escherichia coli AJ12604 (FERM BP-3579) (see European Patent Application Publication No. 488,424).

  In addition, a bacterium belonging to the genus Escherichia having an L-amino acid-producing ability can also be bred by introducing and enhancing DNA carrying genetic information involved in L-amino acid biosynthesis by genetic recombination technology. For example, in an L-lysine producing bacterium, the introduced gene is phosphoenolpyruvate carboxylase, aspartokinase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, succinyldiaminopimelate transaminase, succinyldiaminopimelate deacylase. And other genes encoding enzymes on the biosynthetic pathway of L-lysine. In the case of an enzyme gene that is subject to feedback inhibition by L-aspartic acid or L-lysine, such as phosphoenolpyruvate carboxylase or aspartokinase and dihydrodipicolinate synthase, encodes an enzyme in which such inhibition has been released It is desirable to use a mutant gene.

  In the L-glutamic acid producing bacteria, the introduced genes include glutamate dehydrogenase, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, pyruvate dehydrogenase, and pyruvate. Kinases, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase, etc. is there.

  In the L-valine-producing bacterium, examples of the gene to be introduced include, for example, the ilvGMEDA operon, preferably the ilvGMEDA operon in which the threonine deaminase activity is not expressed and the attenuation is released (see JP-A-8-47397). ).

  Furthermore, the activity of an enzyme that catalyzes a reaction that produces another compound by branching off from the target L-amino acid biosynthetic pathway may be reduced or lacking. For example, homoserine dehydrogenase is an enzyme that catalyzes a reaction that produces a compound other than L-lysine by branching off from the L-lysine biosynthetic pathway (see WO95 / 23864). Examples of enzymes that catalyze a reaction that produces a compound other than L-glutamic acid by branching off from the biosynthetic pathway of L-glutamic acid include α-ketoglutarate dehydrogenase, isocitrate lyase, phosphate acetyltransferase, acetate kinase, and acetohydroxy acid. Synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase and the like.

In addition, bacteria belonging to the genus Escherichia having the ability to produce nucleic acids are described in detail, for example, in WO99 / 03988 International Publication Pamphlet. More specifically, the Escherichia coli FADRaddG-8-3 :: KQ strain (purFKQ, purA-, deoD-, purR-, add-, gsk-) described in the pamphlet can be mentioned. The strain has the ability to produce inosine and guanosine. This strain has a mutant purF encoding a PRPP amide transferase in which the lysine residue at position 326 has been replaced with a glutamine residue and the feedback inhibition by AMP and GMP has been released, and the succinyl-AMP synthase gene (purA); The purine nucleoside phosphorylase gene (deoD), the purine repressor gene (purR), the adenosine deaminase gene (add), and the inosine-guanosine kinase gene (gsk) have been disrupted. The shares were granted a private number, AJ13334, and dated June 24, 1997, the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute (now the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 305-8566 It has been internationally deposited in accordance with the Budapest Treaty at 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki, Japan, under the Budapest Treaty, and has been assigned a deposit number of FERM BP-5993.

  The protein to which the present invention is applied is not particularly limited as long as it can be produced by a genetic engineering technique, and specific examples include acid phosphatase, GFP (Green Fluorescent Protein), and the like.

  In addition, Escherichia bacteria having the ability to produce other useful substances such as L-amino acids, proteins (including peptides), nucleic acids, or vitamins, antibiotics, growth factors, bioactive substances, etc. It can be used for the invention.

  In breeding a bacterium belonging to the genus Escherichia having the ability to produce the target substance as described above, a gene is introduced into the bacterium belonging to the genus Escherichia. And transforming Escherichia coli therewith. In addition, transduction, transposon (Berg, DE and Berg, CM, Bio / Technol. 1, 417, (1983)), Mu phage, (Japanese Patent Laid-Open No. 2-109985) or homologous recombination ( Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)) can integrate the target gene into the host chromosome.

  The bacterium belonging to the genus Escherichia used in the present invention is a bacterium having the ability to produce the target substance as described above and in which the FIS protein does not function normally in cells. "The FIS protein does not function normally" means that the transcription or translation of the fis gene is prevented, and the FIS protein, which is the product of the gene, may not be produced, or may be in a state where production is reduced, or it may be produced. The FIS protein may be mutated and the original function of the FIS protein reduced or lost. As a bacterium belonging to the genus Escherichia in which the FIS protein does not function normally, typically, a gene-disrupted strain in which the fis gene on the chromosome is disrupted by genetic recombination technology, and an expression regulatory sequence or coding region of the fis gene on the chromosome A mutant strain in which a functional FIS protein is not produced due to the mutation is included.

  SEQ ID NOS: 21 and 22 show an example of the nucleotide sequence of the fis gene of Escherichia coli (the sequence of nucleotide numbers 1067-1363 of the nucleotide sequence of GenBank accession AE000405) and the amino acid sequence of the FIS protein.

  In the present invention, the “FIS protein” may be an Escherichia coli FIS protein having the amino acid sequence shown in SEQ ID NO: 22, or a homolog of the protein. The disrupted fis gene may be a homolog of the Escherichia coli fis gene having the nucleotide sequence shown in SEQ ID NO: 21, in addition to the fis gene. For example, examples of the homolog of the fis gene include a gene that hybridizes with a probe having the nucleotide sequence of SEQ ID NO: 21 or a part thereof under stringent conditions and encodes a protein having a function of the FIS protein. Can be The term "stringent conditions" used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, as an example, DNAs with high homology, for example, DNAs having homology of 50% or more, preferably 70% or more, more preferably 90% or more DNA hybridizes with each other, and DNAs with lower homology do not hybridize with each other, or at 60 ° C., 1 × SSC, 0.1% SDS, which is the usual washing condition for Southern hybridization. Conditions for hybridization at a salt concentration corresponding to 1 × SSC and 0.1% SDS are exemplified.

A part of the base sequence of SEQ ID NO: 21 can also be used as a probe. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the nucleotide sequence of SEQ ID NO: 21 as a primer and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 21 as a template. When a DNA fragment having a length of about 300 bp is used as a probe, the washing conditions for hybridization include 50 ° C., 2 × SSC, and 0.1% SDS.

  As the fis gene used for the gene disruption described below, it is preferable to use the same gene as the fis gene on the chromosome of the target bacterium of the genus Escherichia. Can also be used.

  Hereinafter, an example of a method for destroying a fis gene on a chromosome by a genetic recombination technique will be described. A Escherichia bacterium is transformed with a DNA containing a fis gene (deleted fis gene) modified so as not to produce a normally functioning FIS by deleting a part of the fis gene. By causing recombination with the above fis gene, the fis gene on the chromosome can be disrupted. Such gene disruption by homologous recombination has already been established, and there are a method using linear DNA, a method using a plasmid containing a temperature-sensitive replication control region, and the like, and a method using a plasmid containing a temperature-sensitive replication control region. Is preferred from the viewpoint of certainty.

  The replacement of the deleted fis gene with the fis gene on the host chromosome may be performed as follows. That is, a recombinant DNA is prepared by inserting a temperature-sensitive replication control region, a mutant fis gene and a marker gene exhibiting resistance to drugs such as ampicillin, and the recombinant DNA is used to transform Escherichia bacteria. By culturing the transformant at a temperature at which the replication control region does not function, and subsequently culturing the transformant in a medium containing a drug, a transformant in which the recombinant DNA has been integrated into the chromosomal DNA can be obtained.

  In this way, the strain in which the recombinant DNA has been integrated into the chromosome undergoes recombination with the fis gene sequence originally present on the chromosome, and two fusion genes of the chromosomal fis gene and the deleted fis gene are combined with the recombinant DNA. (Vector portion, temperature-sensitive replication control region and drug resistance marker) are inserted into the chromosome. Therefore, in this state, the transformed strain expresses a normal FIS protein because the normal fis gene is dominant.

  Next, in order to leave only the deleted fis gene on the chromosomal DNA, one copy of the fis gene was recombined with the vector part (including the temperature-sensitive replication control region and drug resistance marker) by recombination of the two fis genes. Drop off chromosomal DNA. At that time, the normal fis gene is left on the chromosomal DNA and the deleted fis gene is cut out, and conversely, the deleted fis gene is left on the chromosomal DNA and the normal fis gene is cut out. is there. In any case, if the cells are cultured at a temperature at which the temperature-sensitive replication control region functions, the cut-out DNA is retained in the cells in the form of a plasmid. On the other hand, when cultured at a temperature at which the temperature-sensitive replication control region does not function, when the deleted fis gene is left on the chromosomal DNA, the plasmid containing the normal fis gene is dropped from the cells. Therefore, by confirming the structure of the fis gene in the cell by colony PCR or the like, a strain in which the deleted fis gene is retained on the chromosomal DNA and the normal fis gene has dropped out of the cell can be obtained.

  Examples of a plasmid having a temperature-sensitive replication control region that functions in a bacterium belonging to the genus Escherichia include pMAN997 (see WO99 / 03988) used in Examples described later.

Techniques used for general gene recombination such as DNA cleavage and ligation, transformation, extraction of recombinant DNA from a transformed strain, and PCR are well known to those skilled in the art, for example, molecular cloning ( Sambrook, J., Fritsch, EF, Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)) and the like.

  In addition, a mutant strain in which a functional FIS protein is not produced can be subjected to ultraviolet irradiation or a normal mutation treatment such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrous acid to a bacterium belonging to the genus Escherichia. It can be obtained by treating with the mutagen used.

  The Escherichia bacterium having the target substance-producing ability and having the FIS protein not functioning normally in the cells obtained as described above is cultured in a medium, and the target substance is produced and accumulated in the medium or the cells. By collecting the target substance, the target substance can be produced. In the present invention, by using a bacterium belonging to the genus Escherichia having the above properties, the production rate or production efficiency of the target substance can be improved. This is because the wild-type strain of the genus Escherichia concerning the fis gene expresses the fis gene during culture and promotes or suppresses the expression of a group of genes (including known and unknown genes) whose transcription is regulated by the FIS protein. It is presumed that in a strain in which the FIS protein does not function normally, the expression of the genes is not regulated.

  In the present invention, the medium used for culturing the genus Escherichia may be a known medium conventionally used depending on the type of the strain or the target substance to be used. That is, it is a normal medium containing a carbon source, a nitrogen source, inorganic ions and other organic components as required. A special medium for carrying out the present invention is not particularly required.

  As the carbon source, sugars such as glucose, lactose, galactose, hydrolysates of fructose and starch, alcohols such as glycerol and sorbitol, and organic acids such as fumaric acid, citric acid, and succinic acid can be used.

  Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as soybean hydrolysate; ammonia gas; and aqueous ammonia.

As organic trace nutrients, it is desirable to add required substances such as vitamin B ₁ , L-homoserine, L-tyrosine, yeast extract and the like in an appropriate amount depending on the properties of the bacteria belonging to the genus Escherichia. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, and the like are added as needed.

  The cultivation may be performed under well-known conditions conventionally used depending on the strain to be used. For example, culturing is preferably performed under aerobic conditions for 12 to 120 hours, and the culturing temperature is controlled at 25 ° C to 45 ° C, and the pH is controlled at 5 to 8 during the culturing. For pH adjustment, an inorganic or organic acidic or alkaline substance, furthermore, ammonia gas or the like can be used.

  No special method is required in the present invention for collecting the target substance from the medium or the cells after the completion of the culture. That is, the target substance can be collected by combining conventionally known ion exchange resin methods, precipitation methods and other methods according to the type of the target substance. The target substance accumulated in the cells can be collected by physically or enzymatically destroying the cells and collecting from the cell extract or the membrane fraction depending on the target substance. It should be noted that, depending on the target substance, the target substance can be used as a microbial catalyst or the like in a state where it is present in the cells.

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

<Example 1> Disruption of the gene encoding the nucleoid-constituting protein of Escherichia coli MG1655 and its effect on growth Destruction of the gene encoding the nucleoid-constituting protein of Escherichia coli was carried out by crossover PCR (Link, AJ, Phillips, D , Church, GM, J. Bacteriol., Vol. 179. 6228-6237, 1997).

(1) Destruction of fis gene Oligonucleotides (primers 1 and 2) shown in SEQ ID NOs: 1 and 2 as primers to amplify a coding region of about 20 bp at the N-terminus of the fis gene and a region of about 300 bp including the upstream region, The oligonucleotides (primers 3 and 4) shown in SEQ ID NOs: 3 and 4 were synthesized as primers for amplifying a C-terminal coding region of about 20 bp and a region of about 300 bp including the downstream region. Primers 2 and 3 partially have a common sequence complementary to each other, and are designed such that when the respective amplification products are ligated at this portion, a part of the fis gene ORF is deleted. I have.

  The first PCR reaction was performed using a combination of the primers 1 and 2 and the primers 3 and 4 and using the genomic DNA of the wild-type MG1655 strain prepared by a conventional method as a template. At this time, the molar ratio of primers 1 and 2 and primers 4 and 3 was 10: 1. A second PCR was performed using the obtained first PCR product as a template and primers 1 and 4. A DNA fragment having a deleted fis gene constructed by the second PCR was cloned into a cloning vector kit pGEMT-easy manufactured by Promega according to the protocol to obtain a recombinant vector pGEM-fis.

  The pGEM-fis was digested with EcoRI to obtain a DNA fragment containing the deleted fis gene. This cleaved fragment was ligated with a temperature-sensitive plasmid pMAN997 (see WO99 / 03988, International Publication Pamphlet) cut and purified by the same enzyme using DNA ligation Kit Ver.2 (Takara Shuzo Co., Ltd.). The above-mentioned pMAN997 is obtained by reconnecting the respective VspI-HindIII fragments of pMAN031 (J. Bacteriol., 162, 1196 (1985)) and pUC19 (Takara Shuzo).

Escherichia coli JM109 competent cells (Takara Shuzo Co., Ltd.) were transformed with the above ligation reaction solution, and spread on an LB agar plate (LB + ampicillin) containing 25 μg / ml of ampicillin (Meiji Seika Co., Ltd.). C., and ampicillin-resistant colonies were selected. The colony was cultured in an LB medium containing 25 μg / ml of ampicillin at 30 ° C. in a test tube, and the plasmid was extracted from the cells using Wizard Plus Miniprep (Promega). These plasmids were digested with EcoRI, and the plasmid containing the desired length fragment was designated as fis disruption plasmid pMANΔfis.
Escherichia coli MG1655 was transformed using pMANΔfis.

The transformant was cultured at 30 ° C. on an LB + ampicillin plate, and ampicillin-resistant colonies were selected. After the selected colonies were liquid-cultured at 30 ° C. overnight, they were diluted 10 ^{−3 and} spread on LB + ampicillin plates, and ampicillin-resistant colonies were selected at 42 ° C. At this stage, pMANΔfis has been integrated into the chromosomal DNA.

Next, the selected colonies were spread on an LB + ampicillin plate, cultured at 30 ° C., and then an appropriate amount of cells were suspended in 2 ml of LB medium and cultured at 42 ° C. for 4 to 5 hours with shaking. The culture solution diluted to 10 ^-5 was spread on an LB plate, and several hundred colonies among the obtained colonies were inoculated on an LB plate and an LB + ampicillin plate, and the growth was confirmed, thereby confirming the ampicillin sensitivity or resistance. In the ampicillin-sensitive strain, the vector portion of pMANΔfis and the normal fis gene or the deleted fis gene originally present on the chromosomal DNA are omitted from the chromosomal DNA. Colony PCR was performed on several ampicillin-sensitive strains, and fis
A strain in which the gene was replaced with the deletion type was selected. Thus, a fis gene-disrupted strain MG1655Δfis was obtained from MG1655 of Escherichia coli.

(2) Disruption of hns gene Using the same method as in (1), an hns gene-disrupted strain was obtained from MG1655. Oligonucleotides shown in SEQ ID NOS: 5 and 6 (primers 5 and 6) as primers for amplifying a coding region of about 40 bp at the N-terminal of the hns gene and a region of about 600 bp including the upstream region thereof, and a coding region of the C-terminal of the hns gene Oligonucleotides (primers 7, 8) shown in SEQ ID NOs: 7 and 8 were synthesized as primers to amplify a region of about 600 bp including 40 bp and a downstream region thereof.

  Using the combination of primers 5, 6 and 7, 8, a first PCR reaction was performed using the genomic DNA of wild-type MG1655 strain prepared by a conventional method as a template. Using the obtained first PCR product as a template, a second PCR was performed using primers 5 and 8. A DNA fragment having the deleted hns gene constructed by the second PCR was obtained. Subsequent operations were performed in the same manner as in (1) to obtain an hns gene-disrupted strain MG1655Δhns.

(3) Disruption of dps gene Using the same method as in (1), a dps gene-disrupted strain was obtained from MG1655. Oligonucleotides shown in SEQ ID NOS: 9 and 10 (primers 9 and 10) as primers for amplifying a region of about 20 bp of the N-terminal coding region of the dps gene and a region of about 400 bp including the upstream region, and a coding region of the C-terminal of the dps gene Oligonucleotides (primers 11, 12) shown in SEQ ID NOs: 11 and 12 were synthesized as primers for amplifying a region of about 300 bp including 20 bp and a downstream region thereof.

  Using the combination of primers 9, 10 and primers 11, 12, a first PCR reaction was performed using genomic DNA of the wild-type MG1655 strain prepared by a conventional method as a template. Using the obtained first PCR product as a template, a second PCR was performed using primers 9 and 12. A DNA fragment having the deleted dps gene constructed by the second PCR was obtained. The subsequent operation was performed in the same manner as in (1) to obtain a dps gene-disrupted strain MG1655Δdps.

(4) Disruption of hupAB gene Since hapA and hopB are not adjacent on the genome, the genes were separately disrupted. First, the hupA gene was disrupted. Oligonucleotides shown in SEQ ID NOs: 13 and 14 (primers 13 and 14) as primers for amplifying the N-terminal coding region of the hapA gene of about 20 bp and the region of about 300 bp including the upstream region, Oligonucleotides (primers 15, 16) shown in SEQ ID NOS: 15 and 16 were synthesized as primers for amplifying a region of about 300 bp including 20 bp and a downstream region thereof.

  Using the combination of the primers 13, 14 and the primers 15, 16, a first PCR reaction was performed using the genomic DNA of the wild-type MG1655 strain prepared as a template as a template. A second PCR was carried out using the obtained first PCR product as a template and primers 13 and 16. A DNA fragment having the deleted hapA gene constructed by the second PCR was obtained. The subsequent operation was performed in the same manner as in (1) to obtain a hupA gene-disrupted strain MG1655ΔhupA.

  Next, the hupB gene was disrupted from the hupA gene disruption strain MG1655ΔhupA. Oligonucleotides (primers 17, 18) shown in SEQ ID NOs: 17 and 18 as primers to amplify a region of about 20 bp including the N-terminal coding region of the hopB gene and a region of about 300 bp including the upstream region, Oligonucleotides (primers 19 and 20) shown in SEQ ID NOs: 19 and 20 were synthesized as primers for amplifying a region of about 300 bp including 20 bp and a downstream region thereof.

  Using the combination of primers 17, 18 and primers 19, 20, a first PCR reaction was performed using genomic DNA of the wild-type MG1655 strain prepared by a conventional method as a template. A second PCR was performed using the obtained first PCR product as a template and primers 17 and 20. A DNA fragment having the deleted hupB gene constructed by the second PCR was obtained. Subsequent operations were performed in the same manner as in (1) to obtain a hupA gene and a hupB gene disruption strain MG1655ΔhupAB.

(5) Culture of gene-disrupted strain The fis gene-disrupted strain MG1655Δfis, the hns gene-disrupted strain MG1655Δhns, the hupAB gene-disrupted strain MG1655ΔhupAB, the dps gene-disrupted strain MG1655Δdps, and the parent MG1655 strain were 20 mM NH ₄ Cl and 2 mM. MgSO ₄ , 40 mM NaHPO ₄ , 30 mM KH ₂ PO ₄ , 0.01 mM CaCl ₂ , 0.01 mM FeSO ₄ , 0.01 mM MnSO ₄ , 5 mM citric acid, 10 mM glucose, 2 mM thiamine hydrochloride, 2.5 g / L casamino acid (Difco) And 50 mM MES-NaOH (pH 6.8) in a medium containing 10 ml L-shaped test tubes. The volume of the culture solution at the start of the culture was 5 ml, the culture was shaken at a rotation speed of 70 rpm and cultured at 37 ° C. All media, containers, etc. were provided after autoclaving.

  The bacterial cell concentration in the culture was measured over time. The cell concentration was determined by measuring the turbidity at 660 nm using a biophotodetector (Advantech). The results are shown in FIG.

  As a result, the growth rate of the dps gene-disrupted strain was comparable to that of the control strain, and it was confirmed that the growth of the hns gene-disrupted strain and the hupAB gene-disrupted strain deteriorated. On the other hand, it was recognized that the growth of the fis gene-disrupted strain was improved as compared with the control strain. Thus, the disruption effect of the fis gene on fermentation production was verified.

<Example 2> Disruption of fis gene in Escherichia coli and effect on L-lysine production (1) Disruption of fis gene In the same manner as in Example 1, a fis gene-disrupted strain WC196Δfis was obtained from Escherichia coli WC196. I got The WC196 strain is an AEC-resistant Escherichia coli L-lysine-producing bacterium, and has a private number AJ13069 as the National Institute of Advanced Industrial Science and Technology (now the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, -8566 Deposited with accession number FERM P-14690 at 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki, Japan, transferred to an international deposit under the Budapest Treaty on September 29, 1995, and received accession number FERM BP -5252 (see WO96 / 17930, International Publication Pamphlet). The fis disruption plasmid pMANΔfis obtained in Example 1 was used to transform L-lysine-producing WC196 strain of Escherichia coli. The subsequent operation was performed in the same manner as in Example 1, and a fis gene-disrupted strain WC196Δfis strain was obtained from L-lysine-producing strain WC196 of Escherichia coli.

(2) Culture of fis gene-disrupted strain The fis gene-disrupted strain WC196Δfis strain and its parent strain, WC196 strain, were transformed into 20 mM NH ₄ Cl, 2 mM MgSO ₄ , 40 mM NaHPO ₄ , 30 mM KH ₂ PO ₄ , 0.01 mM CaCl ₂ , 0.01 mM. 200 ml Erlenmeyer flask containing a medium containing FeSO ₄ , 0.01 mM MnSO ₄ , 5 mM citric acid, 50 mM glucose, 2 mM thiamine hydrochloride, 2.5 g / L casamino acid (Difco), 50 mM MES-NaOH (pH 6.8) Was used for culturing. The culture volume at the start of the culture was 20 ml, and the culture was carried out at 37 ° C. by rotating and shaking at a rotational speed of 144 rpm. All media, containers, etc. were provided after autoclaving.

The bacterial cell concentration, glucose concentration, and L-lysine accumulation in the culture solution were measured over time. The cell concentration was determined by measuring the turbidity at 562 nm with a spectrophotometer (Beckman) using a culture solution diluted with water at an appropriate magnification. Glucose concentration and L-lysine concentration were measured using a Biotech Analyzer (Sakura Seiki) after diluting the culture supernatant, which had been sterilized by centrifugation, with water at an appropriate magnification. The results are shown in FIGS. In addition, the values of L-lysine accumulation and residual glucose concentration after 8 hours of culture were shown.

(Table 1)
Table 1 L-Resin accumulation and residual glucose concentration 8 hours after fis disruption
Strain L-lysine accumulation (mg / L) Glucose (g / L)
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WC196Δfis 205 6.20
WC1996 95 2.00
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  As a result, it was confirmed that the fis gene-disrupted strain improved in growth (FIG. 2), sugar consumption rate (FIG. 3), and L-lysine production rate (FIG. 4) as compared to the control strain. .

<Example 3> Introduction of L-lysine-producing plasmid into fis gene-disrupted strain of Escherichia coli and effect on L-lysine production (1) Introduction of L-lysine-producing plasmid into fis gene-disrupted strain The fis gene-disrupted strain WC196Δfis strain obtained in step 2 and its parent strain WC196 were transformed with a mutant dihydrodipicolinate synthase gene, a mutant aspartokinase III gene, a dihydrodipicolinate reductase gene and a Brevibacterium derived from a bacterium belonging to the genus Escherichia. Transformation was carried out with a plasmid pCABD2 (WO95 / 16042) containing a diaminopimelate dehydrogenase gene derived from U. lactofermentum to obtain strains WC196 / pCABD2 and WC196Δfis / pCABD2. Both the mutant dihydrodipicolinate synthase gene and the mutant aspartokinase III gene have mutations in which feedback inhibition by L-lysine has been released.

(2) Culture of fis gene-disrupted strain The fis gene-disrupted strain WC196Δfis / pCABD2 strain and the wild-type fis gene-carrying strain WC196 / pCABD2 were transformed into 20 mM NH ₄ Cl, 2 mM MgSO ₄ , 40 mM NaHPO ₄ , 30 mM KH ₂ PO ₄ , Medium containing 0.01 mM CaCl ₂ , 0.01 mM FeSO ₄ , 0.01 mM MnSO ₄ , 5 mM citric acid, 50 mM glucose, 2 mM thiamine hydrochloride, 2.5 g / L casamino acid (Difco), 50 mM MES-NaOH (pH 6.8) And cultured in a 200 ml Erlenmeyer flask. The culture volume at the start of the culture was 20 ml, and the culture was carried out at 37 ° C. by rotating and shaking at a rotational speed of 144 rpm. All media, containers, etc. were provided after autoclaving.

  The bacterial cell concentration, glucose concentration, and L-lysine accumulation in the culture solution were measured over time. The cell concentration was determined by measuring the turbidity at 562 nm with a spectrophotometer (Beckman) using a culture solution diluted with water at an appropriate magnification. Glucose concentration and L-lysine concentration were measured using a Biotech Analyzer (Sakura Seiki) after diluting the culture supernatant, which had been sterilized by centrifugation, with water at an appropriate magnification. The results are shown in FIGS. In addition, the values of L-lysine accumulation and residual glucose concentration after 8 hours of culture were shown.

(Table 2)
Table 2 L-Resin Accumulation and Residual Coolose Concentration 8 Hours After Fis Disruption
Strain L-lysine accumulation (g / L) Glucose (g / L)
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WC196 / pCABD2 0.80 7.35
WC196Δfis / pCABD2 1.60 4.40
────────────────────────────────

  As a result, the growth (FIG. 5), the sugar consumption rate (FIG. 6), and the L-lysine production rate (FIG. 7) of the fis gene-disrupted strain into which the L-lysine-producing plasmid was introduced were also higher than those of the control strain. It was found to improve.

The figure which shows the growth pattern of MG1655, MG1655Δfis, MG1655Δhns, MGΔdps, MGΔhupAB strain. The figure which shows the growth pattern of WC196 and the WC196Δfis strain. The figure which shows the sugar consumption pattern of WC196 and the WC196Δfis strain. The figure which shows the lysine accumulation pattern of WC196 and WC196Δfis strain. The figure which shows the growth pattern of WC196 / pCABD2 and WC196Δfis / pCABD2 strain. The figure which shows the sugar consumption pattern of WC196 / pCABD2 and WC196Δfis / pCABD2 strain. The figure which shows the lysine accumulation pattern of WC196 / pCABD2 and WC196Δfis / pCABD2 strain.

Claims (7)

Escherichia bacterium is cultured in a medium, the target substance is produced and accumulated in the medium or the cells, and the target substance is collected.In the method for producing a target substance using the genus Escherichia, the bacterium belonging to the genus Escherichia is the target bacterium. A method for producing a target substance, which has the ability to produce the substance, and wherein the FIS protein does not function normally in cells. The method according to claim 1, wherein in the Escherichia bacterium, the FIS protein does not function normally in the cell due to disruption of the fis gene on the chromosome. 3. The method according to claim 1, wherein the Escherichia bacterium is Escherichia coli. The method according to any one of claims 1 to 3, wherein the target substance is an L-amino acid or a protein. The method according to claim 4, wherein the L-amino acid is an amino acid belonging to the aspartic acid family. The method according to claim 5, wherein the amino acid of the aspartic acid family is L-lysine, L-threonine, or L-methionine. The method according to claim 6, wherein the amino acid is L-lysine.

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