JP2002306191A - Method for producing object material by fermentation process - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an object material by a fermentation process, capable of increasing production efficiency or production speed, when the useful material, such as an L-amino acid, a protein, and nucleic acids, is produced by the fermentation process for which Escherichia bacteria are used. SOLUTION: This method for producing the object material by the fermentation process comprises culturing the Escherichia bacteria in a culture medium, making the object material formed and accumulated in the culture medium or the bacteria cell bodies, and collecting the object material, wherein a strain of which the RMF protein does not normally function in the cell due to disruption of rmf gene on the chromosome is, for example, used as the Escherichia bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the fermentation industry,
Specifically, the present invention relates to a method for efficiently producing a target substance such as an L-amino acid by a fermentation method utilizing a microorganism.

[0002]

2. Description of the Related Art Escherichia coli has a reduced protein translation activity during a transition from a growth phase to a stationary phase. This phenomenon is thought to be due to intracellular ribosome dimerization. As a protein involved in ribosome dimerization, RMF protein, a small protein consisting of 55 amino acid residues and having a molecular weight of 6.5 kDa, is considered. Is found (W
ada et al., Proc. Natl. Acad. Sci. USA, Vol. 87, 26
57-2661, 1990).

[0003] The rmf gene encoding the RMF protein is
It was confirmed that the gene was located at 21.8 minutes on the Escherichia coli chromosome. The expression of the rmf gene is suppressed during the logarithmic growth phase in which cells are growing at a high rate, and its expression is induced at the time of transition to the stationary phase or at a low growth rate. It has been revealed that the regulation of gene expression is not due to the σS factor, which is known to control stationary phase-specific gene expression (Yamagishi et.a.
l., The EMBO Journal., Vol. 12, 625-630, 1993). It has also been reported that disruption of the rmf gene results in the absence of ribosome dimers as a phenotype even in the stationary phase and a decrease in the survival rate in the stationary phase (Yamagishi et al.). al., The EMBO Jou
rnal, Vol. 12, 625-630, 1993). Furthermore, it has been reported that a strain overexpressing this gene becomes nonviable (Wada et al., Biochem. Biophys. Res. Comm., V
ol. 214, 410-417, 1995).
It has been strongly suggested that it is a factor involved in E. coli growth and stationary phase survival.

[0004] However, there has been no report on improvement of the rmf gene under low growth rate conditions or improvement of substance productivity.

[0005]

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

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found a universal factor expressed in the stationary phase, and by modifying its gene, Escherichia It has been found that substance production by genus bacteria can be improved. That is, the DNA array method (H. Tao, C. B.
ausch, C. Richmond, FR Blattner, T. Conway (199
9) The rmf gene was identified as a gene in Escherichia coli which showed a marked increase in expression in the stationary phase by effectively using the gene expression analysis technology of the Journal of Bacteriology, 181, 6425-6440). Then, it was found that by disrupting the rmf gene, growth in the stationary phase was improved, and the ability to produce the target substance was improved, thereby completing the present invention. That is, the present invention is as follows.

(1) A method for producing a target substance using a microorganism, comprising culturing a bacterium belonging to the genus Escherichia in a medium, producing and accumulating the target substance in the medium or the cells, and collecting the target substance. A method for producing a target substance, wherein the RMF protein does not function normally in cells of bacteria. (2) The method according to (1), wherein in the bacterium belonging to the genus Escherichia, the RMF protein does not function normally due to disruption of the rmf 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 L-lysine.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION 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, a book by Neidhardt, FC et al.
al., Escherichia coli and Salmonella Typhimurium,
American Society for Microbiology, Washington D.
C., 1208, table 1) can be used.
More specifically, Escherichia coli is mentioned.

The target substance produced by the present invention 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 Escherichia bacteria, such as nucleic acids such as inosine, guanylic acid, and inosinic 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.

[0010] 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 if L-lysine coexists in the medium, it is totally or partially released. . For example, 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. L
-As a strain used for lysine production, specifically, Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185;
JP-A-56-18596 and U.S. Pat. No. 4,346,170), and Escherichia coli VL611. Aspartokinase of these microorganisms is released from feedback inhibition by L-lysine.

Other examples include the 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 the examples described below, strain WC196 was used as an L-lysine-producing bacterium of Escherichia coli. This strain is an AEC strain of Escherichia coli K-12 derived from W3110.
It was bred by imparting resistance. The strain was named Escherichia coli AJ13069 strain, and on December 6, 1994, the Institute of Biotechnology and Industrial Technology (now the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Japan), 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 WO96 / 17930, International Publication Pamphlet).

Examples of L-threonine-producing Escherichia bacteria include Escherichia coli VKPM B-3996 (RIA 186).
7) (see US Pat. No. 5,175,107), MG442 strain (Gusyatine
r etal., Genetika (in Russian), 14, 947-956 (1978)
Reference).

As the L-homoserine-producing Escherichia bacterium, strain C600 (Appleyard RK, Genetics, 39,
440-452 (1954)) and the NZ10 strain which is a Leu ⁺ revertant.

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.
Coli K-12 (ATCC10798), Escherichia coli B (ATCC11
303) and Escherichia coli W (ATCC 9637).

Examples of L-leucine-producing bacteria belonging to the genus Escherichia include strains having β-2-thienylalanine resistance, strains having β-2-thienylalanine resistance and β-hydroxyleucine resistance (these are JP-B-62-34397). JP-A-8-70879) and strains having 4-azaleucine resistance or 5,5,5-trifluoroleucine resistance. Specifically, AJ11478 strain (FERM P-52
74) (see JP-B-62-34397).

Examples of L-isoleucine-producing Escherichia bacteria include Escherichia coli KX141 (VKPM B-478).
1) (see EP-A-519,113). Examples of L-valine-producing Escherichia bacteria include:
Escherichia coli VL1970 (VKPM B-4411) (see EP-A-519,113). L-phenylalanine producing bacteria include Escherichia coli AJ126.
04 (FERM BP-3579) (see EP-A-488,424).

The bacterium belonging to the genus Escherichia having the ability to produce L-amino acids can also be bred by introducing and enhancing DNA carrying genetic information relating to the biosynthesis of L-amino acids by genetic recombination techniques. . For example,
In L-lysine-producing bacteria, the introduced gene is phosphoenolpyruvate carboxylase, aspartokinase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, succinyldiaminopimelate transaminase, succinyldiaminopimelate deacylase, and the like. It is a gene encoding an enzyme 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, it encodes the enzyme whose inhibition has been released. It is desirable to use a mutant gene.

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

In the L-valine-producing bacterium, examples of the gene to be introduced include, for example, the ilvGMEDA operon, preferably the ilvGMEDA operon which does not express threonine deaminase activity and has been attenuated (Japanese Patent Laid-Open No. 8-47397). Reference).

Further, the activity of an enzyme that catalyzes a reaction that diverges from the target L-amino acid biosynthetic pathway to produce another compound may be reduced or lacking. For example,
As an enzyme that catalyzes a reaction that produces a compound other than L-lysine by branching off from the L-lysine biosynthetic pathway, there is homoserine dehydrogenase (see WO95 / 23864). Examples of enzymes that catalyze a reaction that produces a compound other than L-glutamic acid by branching from the L-glutamic acid biosynthetic pathway 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.

Further, the bacterium belonging to the genus Escherichia having the ability to produce nucleic acids is described in detail, for example, in WO99 / 03988 International Publication Pamphlet. More specifically, the Escherichia coli FADRaddG-8-3 :: KQ strain (purFKQ, p
urA-, deoD-, purR-, add-, gsk-). 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); Purine nucleoside phosphorylase gene (deoD), purine repressor gene (pu
rR), the adenosine deaminase gene (add) and the inosine-guanosine kinase gene (gsk) have been disrupted. The stock was granted a private number, AJ13334, and dated June 24, 1997, 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, Higashi 1-1, Tsukuba, Ibaraki, Japan, based on the Budapest Treaty.
P-5993 has been granted.

The protein to which the present invention is applied is not particularly limited as long as it can be produced by genetic engineering techniques, and specific examples include acid phosphatase. Examples of the bacterium belonging to the genus Escherichia producing acid phosphatase include a plasmid pMPI501 or pMPI505 containing an acid phosphatase gene derived from Morganella morgani NCIMB 10466 described later (Japanese Patent Application Laid-Open No.
No. 6,010,851), or a mutant acid phosphatase in which the glycine residue at position 92 is replaced by an aspartic acid residue and the isoleucine residue at position 171 is replaced by a threonine residue in the gene. Plasmid pMPI700 (Applied and Envir
onmental Microbiology, July 2000, p.2811-2816 Vo
l. 66, No. 7), and the like. Also, Escherichia Brattae JCM1650, Providencia Stuarty ATCC29851, Enterobacter aerogenes IFO12010, Klebsiella Planticola IFO14939 and Serratia Ficaria IAM135
Bacteria belonging to the genus Escherichia carrying a gene encoding acid phosphatase derived from microorganisms such as 40 (Japanese Patent Laid-Open No. 9-37)
No. 785, US Pat. No. 6,010,851) can also be suitably used.

In addition, a bacterium belonging to the genus Escherichia having the ability to produce other L-amino acids, proteins (including peptides), nucleic acids, or useful substances such as vitamins, antibiotics, growth factors, and physiologically active substances. Can also be used in the present invention.

In the breeding of a bacterium belonging to the genus Escherichia having the ability to produce a target substance as described above, a gene can be introduced into the bacterium belonging to the genus Escherichia by linking the gene to a vector capable of autonomous replication in the bacterium belonging to the genus Escherichia. There is a method of producing a recombinant DNA and transforming Escherichia coli therewith. In addition, transduction, transposon (Berg, DE and Berg, CM, Bio / Techno)
l. 1,417, (1983)), Mu phage, (JP-A-2-10998)
No. 5) or homologous recombination (Experiments in Molecular Ge
netics, Cold Spring Harbor Lab. (1972)), the target gene can also be integrated 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 above-mentioned target substance and in which the RMF protein does not function normally in cells.
"RMF protein does not function normally" means that rmf
The transcription or translation of the gene may be hindered, and the RMF protein, the product of the gene, may not be produced or may be in a reduced production state, or the produced RMF protein may be mutated, and the original function of the RMF protein May be reduced or eliminated. As a bacterium belonging to the genus Escherichia in which the RMF protein does not function normally, typically, a gene-disrupted strain in which the rmf gene on the chromosome is disrupted by genetic recombination technology, and an rmf on the chromosome
Mutants in which a functional RMF protein is not produced due to a mutation in a gene expression regulatory sequence or a coding region are included.

Hereinafter, an example of a method for destroying the rmf gene on a chromosome by a gene recombination technique will be described. rm
transforming a bacterium belonging to the genus Escherichia with a DNA containing an rmf gene (deleted rmf gene) modified so as not to produce a normally functioning RMF by deleting a part of the f gene;
By causing recombination between the deletion type rmf gene and the rmf gene on the chromosome, the rmf gene on the chromosome can be destroyed. 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 deleted rmf gene is inserted into the host chromosome by r
The following procedure may be used to replace the mf gene. That is, a recombinant DNA is prepared by inserting a temperature-sensitive replication control region, a mutant rmf gene, and a marker gene exhibiting resistance to a drug such as ampilin, 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 it in a medium containing a drug, the recombinant DNA
Is obtained in the chromosomal DNA.

The strain in which the recombinant DNA has been integrated into the chromosome thus undergoes recombination with the rmf gene sequence originally present on the chromosome, and the chromosome rmf gene and the deletion r
Two fusion genes with the mf gene are inserted into the chromosome with the other part of the recombinant DNA (vector part, temperature-sensitive replication control region and drug resistance marker) interposed therebetween.
Therefore, in this state, the normal rmf gene is dominant and the transformed strain expresses normal RMF protein.

Next, in order to leave only the deleted rmf gene on the chromosomal DNA, one copy of the rmf gene was recombined by recombination of the two rmf genes, and the vector portion (the temperature-sensitive replication control region and the drug resistance marker were replaced with each other). Along with chromosomal DNA. At that time, the normal rmf gene is left on the chromosomal DNA and the deleted rmf gene is cut out, and conversely, the deleted rmf gene is left on the chromosomal DNA and the normal rmf 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 excised 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, the deletion type r
If the mf gene remains on the chromosomal DNA, the plasmid containing the normal rmf gene will fall out of the cell. Therefore, by confirming the structure of the rmf gene in the cell by colony PCR or the like, a strain in which the deleted rmf gene is retained on the chromosomal DNA and the normal rmf 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, International Publication Pamphlet) used in Examples described later.

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

In addition, mutants in which a functional RMF protein is no longer produced can be obtained by irradiating a bacterium belonging to the genus Escherichia with ultraviolet light or by using a normal cell such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrite. It can be obtained by treating with a mutagen used in the mutation treatment.

The bacterium belonging to the genus Escherichia, which has the ability to produce the target substance and in which the RMF protein does not function normally in the cells, obtained as described above, is cultured in a medium, and the target substance is added to the medium or the cells. The target substance can be produced by producing and accumulating the target substance and collecting the target substance. In the present invention, the production rate or production efficiency of the target substance can be improved by using a bacterium belonging to the genus Escherichia having the above properties. This indicates that the wild type strain of the genus Escherichia for the rmf gene
This is presumed to be because the mf gene is expressed and the protein translation activity is reduced, whereas the strain in which the normal RMF protein does not function normally prevents or reduces the protein translation activity.

In the present invention, the medium used for culturing the bacterium belonging to the genus Escherichia may be a conventionally known medium, depending on the strain used or the kind of the target substance. 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. it can.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

[0038] Organic trace nutrients include vitamin B ₁ ,
Desirable substances such as L-homoserine and L-tyrosine or yeast extract are desirably contained in appropriate amounts. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, and the like are added as necessary.

The cultivation may be performed under well-known conditions conventionally used depending on the strain to be used. For example,
The cultivation is preferably performed under aerobic conditions for 16 to 120 hours, the culturing temperature is 25 ° C to 45 ° C, and the pH during culturing is 5 to 8 hours.
To control. For pH adjustment, an inorganic or organic acidic or alkaline substance, and ammonia gas or the like can be used.

The collection of the target substance from the medium or the cells after the completion of the culture does not require any special method in the present invention. 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. In addition, collection of the target substance accumulated in the cells
The cells can be physically or enzymatically destroyed and collected from a cell extract or a 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.

[0041]

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

[0042]

Example 1 Extraction of Total RNA from Escherichia coli Cells and Analysis by DNA Macroarray Escherichia coli wild-type strain W3110 was isolated from MS culture medium (glucose 20 g / g) using a 1 L small jar fermenter.
L, KH ₂ PO ₄ 1 g / L, MgSO ₄ 1 g / L, yeast extract 2 g / L, (NH ₃ ) ₂
(SO ₄ ) The cells were cultured at 37 ° C. in 300 ml of a medium containing 16 g / L. The culture is aerated at a flow rate of 150 ml / min, and the dissolved oxygen concentration is 5%.
The stirring was performed while controlling the number of stirring as described above. Further, the pH of the medium was adjusted by feeding ammonia water so as to be constant at 6.8, and after the glucose in the medium was depleted, the glucose concentration in the culture solution was adjusted to 0.1 to 10 g / L with a glucose 500 g / L solution. Feed was made to be in the range of L.

On the other hand, the W3110 strain was transformed into an E-100 medium (20 mM NH ₄ C
_{l, 2 mM MgSO 4, 40} mM NaHPO 4, 30mM KH 2 PO 4, 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 (manufactured by Difco), 250 mM MES-NaOH (pH6.8)
Was reciprocally shaken at 37 ° C. and 120 rpm using a 500 ml Sakaguchi flask containing 50 ml of a medium containing

In order to extract total RNA from Escherichia coli cells, the growth curves were about 1 ml from the jar fermenter and about 10 ml from the flask during the logarithmic and stationary phases.
Were sampled, respectively. The sampled culture solution was immediately cooled on ice, and then centrifuged at 10,000 g for 2 minutes using a cooling centrifuge to remove the culture supernatant. Total RNA was recovered from the collected cells using the RNeasy kit of QIAGEN according to the attached protocol. The obtained total RNA was confirmed to be recovered without degradation by agarose gel electrophoresis, and the RNA concentration was quantified by measuring the absorbance at 260 nm. The obtained total RNA was sealed and stored at -80 ° C, and subjected to gene expression analysis using a DNA macroarray.

Using 20 μg of the obtained total RNA as a template, dATP,
Using 1 mM each of dGTP and dTTP and 9.25 MBq of [α- ³² P] dCTP as a substrate, Reverse Transcription K of Promega was used.
Reverse transcription reaction was performed using it to obtain labeled cDNAs in logarithmic growth phase and stationary phase.

Using the obtained cDNA as a probe, Sigma-Ge
Hybridization was performed using a Nosys Panorama E. coli Gene Arrays macroarray membrane according to the protocol. After completion of the hybridization, the membrane was washed. The membrane after washing is sealed and placed on an imaging plate manufactured by Fuji Film Co., for 48 hours.
It was brought into close contact and exposed. The exposed imaging plate was read by a fluoro imaging analyzer FLA-3000G manufactured by Fuji Film Co., Ltd. The obtained read image was quantified using a DNA array image analysis system AIS (manufactured by Imaging Research), and the concentration of each spot was quantified and converted as an expression ratio. Data obtained.

From the obtained DNA array data, a group of genes whose expression level was low in the logarithmic growth phase but increased in the stationary phase was selected from all Escherichia coli genes. The ratio of the expression ratio in the stationary phase to the expression ratio in the logarithmic growth phase was calculated for each obtained from the jar fermenter and the flask culture. The top 20 were extracted from those with higher values obtained by multiplying the increase ratio of the expression ratio in each culture. Among them, the rmf gene was extracted as the one with the highest numerical value among genes with known functions. Table 1 shows changes in the growth rate and the expression ratio of the rmf gene. In both the jar fermenter and the flask culture, it was revealed that the gene showed a marked increase in expression in the stationary phase.

[0048]

Table 1 Relationship between growth rate and rmf gene expression level in each culture condition and culture phase. Medium Incubator Incubation time Specific growth rate rmf expression ratio ^* hr 1 / hr ──────────────────────────── MS jar 1.5 0.32 1.000 MS Jar 9 0.02 11.068 E-100 flask 4 0.75 1.063 E-100 flask 24 -0.019 47.711 ──────────────────────────── *: Jar Relative value when the value of 1.5 hours of culture is 1

[0049]

[Example 2] Disruption of the rmf gene of Escherichia coli and its effect on L-lysine production Disruption of the rmf gene of Escherichia coli was carried out by crossover PCR (Link, AJ, Phillips, D., Church, GM, J. Am.
Bacteriol., Vol. 179. 6228-6237, 1997).

Oligonucleotides (primers 1 and 2) shown in SEQ ID NOs: 1 and 2 as primers for amplifying a coding region of about 600 bp at the N-terminal of the rmf gene and a region of about 1 kbp including the upstream region thereof, Code area about 60
Oligonucleotides (primers 3, 4) shown in SEQ ID NOs: 3 and 4 were synthesized as primers for amplifying a region of about 1 kbp including 0 bp and a downstream region thereof. Primers 2 and 3
Is designed so that a part of the rmf gene ORF is deleted when an amplification product is ligated at this part.

Primers 1 and 2 and primers 3 and 4
The first PCR reaction was performed using the combination of genomic DNA of wild-type strain W3110 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. The DNA fragment having the defective rmf 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-R.

The pGEMdR was digested with EcoRI to obtain a defective DNA fragment containing the rmf gene. This fragment and
Temperature-sensitive plasmid pMAN99 cut and purified with the same enzyme
7 (see WO99 / 03988, International Publication Pamphlet)
Ligation Kit Ver.2 (Takara Shuzo Co., Ltd.) was used for ligation. Incidentally, the pMAN997, pMAN031 (J. Bacteriol., 162,
1196 (1985)) and pUC19 (Takara Shuzo Co., Ltd.)
HindIII fragment was reconnected.

In the above ligation reaction solution, Escherichia coli
Transform JM109 competent cells (Takara Shuzo Co., Ltd.)
Spread ampicillin (Meiji Seika Co., Ltd.) on an LB agar plate (LB + ampicillin) containing 25 μg / ml, culture at 30 ° C,
Ampicillin resistant colonies were selected. 25μ colony
Test tubes were cultured in an LB medium containing g / ml ampicillin at 30 ° C., and plasmids were extracted from the cells using Wizard Plus Miniprep (Promega). EcoR
After digestion with I, the plasmid containing the target length fragment was designated as plasmid pMANΔrmf for rmf disruption. Escherichia coli WC196 strain was transformed using pMANΔrmf. WC196 shares AE
A C-resistant Escherichia coli L-lysine-producing bacterium, designated as Private Number AJ13069 by the National Institute of Advanced Industrial Science and Technology Accession number FERM at 1-1, Higashi 1-1, Tsukuba City, Ibaraki Pref.
Deposited as P-14690 and transferred to an international deposit under the Budapest Treaty on September 29, 1995, with accession number FERM B
P-5252 (see WO96 / 17930, International Publication Pamphlet).

Transformants were plated on LB + ampicillin plates
The cells were cultured at 30 ° C., and ampicillin-resistant colonies were selected. After liquid culture of the selected colonies at 30 ° C overnight, 10
^{-3 was} diluted and spread on an LB + ampicillin plate, and ampicillin-resistant colonies were selected at 42 ° C. At this stage, pM
ANΔrmf is integrated into the chromosomal DNA.

Next, the selected colonies were spread on an LB + ampicillin plate and cultured at 30 ° C. After that, an appropriate amount of the cells was suspended in 2 ml of LB medium and cultured at 42 ° C. with shaking for 4 to 5 hours. 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 to confirm the sensitivity or resistance to ampicillin. Ampicillin-sensitive strains are derived from chromosomal DNA by the vector portion of pMANΔrmf and normal
The rmf gene or the deleted rmf gene is missing. Colony PCR was performed on several ampicillin-sensitive strains, and strains in which the rmf gene had been replaced with a deletion type as desired were selected. Thus, the rmf gene-disrupted strain WC196Δrmf was obtained from the L-lysine producing bacterium WC196 of Escherichia coli.

The rmf gene-disrupted strain WC196Δrmf and its parent strain WC196 were subjected to 20 mM NH ₄ Cl, 2 mM MgSO ₄ , 40 mM NaH
PO 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, 50 mM glucose, 2 mM thiamine hydrochloride, 2.5 g / L casamino acid (Difco), 250 mM MES-
The cells were cultured in a medium containing NaOH (pH 6.8) using an Erlenmeyer flask with a 200-ml baffle. The culture volume at the start of culture is
The volume was adjusted to 20 ml, the mixture was shaken at a rotation speed of 144 rpm, and culture was performed at 37 ° C. All media, containers, etc. were provided after autoclaving.

The cell concentration, glucose concentration, and L-lysine accumulation in the culture solution were measured over time. The cell concentration was determined by using a culture solution diluted with water at an appropriate magnification and measuring turbidity at 600 nm with a spectrophotometer (Beckman).
Glucose concentration, and L-lysine concentration, after diluting the culture supernatant removed by centrifugation with water at an appropriate magnification,
It was measured by Biotech Analyzer (Sakura Seiki). The results are shown in FIGS. In addition, the values of L-lysine accumulation and residual sugar after 17 hours of culture were shown.

As a result, the rmf gene-disrupted strain was found to have improved growth (FIG. 1), sugar consumption rate (FIG. 2), and L-lysine production rate (FIG. 3) as compared to the control strain. Admitted

[0059]

Table 2 L-Lysine production of WC196Δrmf strain ────────────────────────────── Strain Experiment Culture time L-Lysine Accumulated residual sugar hr (converted to hydrochloride) mg / L g / L ────────────────────────────── WC196 1 17 89 3.4 2 17 90 3.7 ────────────────────────────── WC196Δrmf 1 17 170 1.4 2 17 159 1.5 ─────── ───────────────────────

[0060]

Example 3 Disruption of rmf Gene of Escherichia coli and Effect on Protein Production (1) Introduction and Expression of Acid Phosphatase Overexpression Plasmid into rmf Gene-Disrupted Strain

The rmf gene disrupted strain WC196 obtained in Example 2
The Δrmf strain and its parent strain, WC196, were transformed with the plasmid pMPI700 containing the mutant acid phosphatase gene to obtain the WC196 / pMPI700 strain and the WC196Δrmf / pMPI700 strain. The mutant acid phosphatase is a mutant enzyme having reduced 5′-nucleotidase activity (phosphoester hydrolysis activity) while maintaining nucleoside-5′-phosphate ester formation activity (phosphotransferase activity). The glycine residue is substituted with an aspartic acid residue, and the isoleucine residue at position 171 is substituted with a threonine residue. pMPI700 is a plasmid obtained as follows (Applied and Environmental Microbiology, Ju
ly 2000, p.2811-2816 Vol. 66, No. 7).

Plasmid pMPI501 containing acid phosphatase gene from Morganella morgani NCIMB 10466
(JP-A-9-37785, US Pat. No. 6,010,851) as a template, a random mutation was introduced into the gene by error-prone PCR. The acid phosphatase gene fragment (EcoRI-HindIII) into which the mutation was introduced was introduced into pUC18, and E. coli JM109 was transformed with the obtained recombinant plasmid. One transformant was obtained from the transformant, which had the same transphosphorylation activity as E. coli (pMPI501) and had reduced 5'-nucleotidase activity, and was named JM109 (pMPI600). Using the plasmid pMPI600 carried by this strain,
In the same manner, random mutagenesis was performed again, and plasmid pM
I got PI700.

DNA obtained by further shortening the acid phosphatase gene fragment contained in pMPI501 by subcloning
Plasmid pMPI505 containing the fragment was transferred to Escherichia coli JM1.
The strain retained in 09 was named AJ13143, and this strain is an international depositary organization based on the Budapest Treaty, and is located in Ibaraki, Japan. National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 Japan
It has already been deposited at Address No. 1 Central No. 6) on February 23, 1996, and is assigned the accession number FERM BP-5422.
A plasmid similar to pMPI700 can be obtained by using pMPI505 instead of pMPI501.

WC196 / pMPI700 strain and WC196Δrmf / pMPI7
00 strain, 500 mL in LB medium containing 100 μg / ml ampicillin
Culture was performed using a Nosakaguchi flask. The culture volume at the start of the culture was 50 mL, and the culture was performed at 37 ° C. by reciprocal shaking at a rotation speed of 120 rpm. All media, containers, etc. were provided after autoclaving. At this time, the cell concentration in the culture solution was measured. The bacterial cell concentration was measured using a culture solution diluted with water at an appropriate magnification, and the turbidity at 600 nm was measured with a spectrophotometer (Beckman). In addition, a culture solution containing bacterial cells is collected over time, collected by centrifugation, suspended in 100 mM phosphate buffer (pH 7.0), sonicated for 20 minutes, and centrifuged again. The supernatant was used as a crude enzyme solution and subjected to measurement of protein concentration and acid phosphatase enzyme activity. The protein concentration in the crude enzyme solution was measured by the Bradford method.

The enzyme activity was measured as follows. Ten
10 mM as substrate in 0 mM MES / KOH buffer (pH 6.0)
An enzymatic reaction was performed at 30 ° C. using p-nitrophenyl phosphate. One minute after the addition of the crude enzyme solution, the reaction was stopped by adding 2N KOH to 1/5 of the reaction solution, and after centrifugation, p-nitrophenol phosphate produced in the supernatant was quantified. . The quantification of the generated p-nitrophenol phosphate was performed by measuring the absorbance at 410 nm with a spectrophotometer (Beckman). The enzymatic activity was calculated on the assumption that the molar extinction coefficient of p-nitrophenol phosphate was 17.52 mM ^-1 · cm ^-1 . Table 3 shows the results. FIG. 4 shows the growth (OD ₆₀₀ ) of each strain during culture. As a result, it was confirmed that the rmf gene-disrupted strain had improved growth, specific activity, and total activity per culture solution as compared to the control strain (Table 3).

[0066]

[Table 3] Table 3 Acid phosphatase activity of crude enzyme solution ────────────────────────────── Bacteria culture time Specific activity Total activity hr / mg protein U / ml────────────────────────────── WC196 6 0.071 0.019 WC196Δrmf 6 0.045 0.054 WC196 / pMPI700 6 2.02 0.569 WC196Δrmf / pMPI700 6 4.08 1.190 ──────────────────────────────

Further, the crude enzyme solution was subjected to SDS gel electrophoresis on a 15% polyacrylamide gel and stained with Cypro Orange (Bio-Rad), and the stained gel image was analyzed using a fluoro-imaging analyzer FLA manufactured by Fuji Film Co., Ltd. -3000G was read, and the obtained read image was quantified for the concentration of each spot using image analysis software ImageGauge to confirm the expression of the target enzyme protein and to compare the expression levels. As a result, the target band was detected, and its concentration reflected the specific activity well (FIG. 5).

[0068]

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

[0069]

[Sequence list]

 SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> Production of the target substance by fermentation <130> P-9421 <140> <141> 2001-12-21 <150> JP 2000-390758 <151> 2000 -12-22 <160> 4 <170> PatentIn version 3.0

<210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: primer f
or PCR <400> 1 gaacaggcaa ccagtacgct tt

<210> 2 <211> 42 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: primer f
or PCR <400> 2 cccatccact aaacaccgtc agttgatgtg cccgttccag gc

<210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: primer f
or PCR <400> 3 tgacggtgtt tagtggatgg gcaaaggtca caatggctg

<210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: primer f
or PCR <400> 4 tacagtgaag ttgatggaga tagt

[Brief description of the drawings]

FIG. 1 is a view showing a growth pattern of a WC196Δrmf strain. The results obtained by performing 2 colonies for each experimental group at n = 3 are shown. Error bars indicate standard error. (The same applies to the following figures)

FIG. 2 shows the sugar consumption pattern of the WC196Δrmf strain.

FIG. 3 shows the lysine accumulation pattern of the WC196Δrmf strain.

FIG. 4 is a diagram showing the growth of a WC196Δrmf / pMPI700 strain and a control strain in which an acid phosphatase gene has been introduced into the WC196Δrmf strain.

FIG. 5 is a diagram showing the staining intensity of the acid phosphatase band observed by SDS polyacrylamide gel electrophoresis of the crude enzyme solutions of the WC196Δrmf / pMPI700 strain and the control strain.

Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) C12R 1:19) (72) Inventor Shinichi Sugimoto 1-1 Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Ltd. F term (reference) 4B024 AA03 BA72 CA04 CA06 DA06 EA04 GA11 HA01 4B050 CC03 DD02 LL05 4B064 AE25 CA02 CA19 CC24 DA16

Claims (5)

[Claims] 1. A method for producing a target substance using a microorganism, 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, wherein the RMF protein does not function normally in cells. 2. The bacterium belonging to the genus Escherichia has an RMF in a cell due to disruption of an rmf gene on a chromosome.
2. The method according to claim 1, wherein the protein does not function normally. 3. The method according to claim 1, wherein the Escherichia bacterium is Escherichia coli. 4. The method according to claim 1, wherein the target substance is an L-amino acid or a protein. 5. The method according to claim 4, wherein the L-amino acid is L-lysine.