JP4582573B2 - Method for producing pyruvic acid and method for producing L-valine - Google Patents

Description

  The present invention relates to a method for producing L-valine or pyruvic acid by a fermentation method.

  Examples of microorganisms that produce pyruvic acid by fermentation include the genus Corynebacterium (Non-patent document 1), the genus Acinetobacter (non-patent document 2), the genus Candida (non-patent document 3), or the genus Tolropsis (non-patent document 4). Etc. are known. It also belongs to the genus Proteus, Xanthomonas, Chryseobacterium, Cytophaga, Acinetobacter, Saccharomyces, Ogatea, Sporoboromyces, Sporidiobaras, Candida, Waltmyces, Mycobacterium, or Nocardia A method for producing pyruvic acid using a microorganism is also known (Patent Document 1). As a method for producing pyruvic acid using an Escherichia bacterium, a method using a lipoic acid-requiring strain is known (Patent Document 2).

  L-valine-producing bacteria belonging to the genus Brevibacterium, Corynebacterium, Serratia (Non-patent Document 5) or Escherichia (Patent Document 3) are known as microorganisms that produce L-valine by fermentation.

  In general, in order to efficiently produce primary metabolites such as L-valine and pyruvic acid, weakening of enzyme activity catalyzing a reaction branched from its biosynthetic pathway, introduction of metabolic regulatory mutation, degradation pathway of product It is effective to block the expression and enhance the expression of the biosynthetic gene of the target substance. In order to enhance the expression of a biosynthetic gene, a method of amplifying the gene using a plasmid or the like or modifying an expression regulatory region of the gene is generally performed (Non-patent Document 6).

  Further, not only the target gene itself but also the mutation of the DNA binding protein that regulates the expression of the gene can achieve the enhanced expression of the target gene. For example, it has been reported that the expression level of a gene encoding pyruvate kinase increases in a strain lacking the cra (fruR) gene encoding cra, which is a DNA binding protein (Non-patent Document 7). It has also been reported that the L-amino acid-producing ability is improved although the amount is small due to the deletion of the cra gene (Patent Document 4).

  By the way, mRNA transcribed from DNA in vivo is degraded by ribonuclease (RNase). For example, in bacteria belonging to the genus Escherichia, RNase III, RNase E (Non-Patent Document 8), RNase G (Non-Patent Document 9), and the like are known as RNases that degrade mRNA. In order to stably express a target gene product, it is preferable to increase the transcription amount of mRNA and suppress degradation of mRNA. Therefore, it is also conceivable to suppress the degradation of the target enzyme mRNA by suppressing the RNase activity.

Known strains with reduced RNase activity include strains in which mutations have been introduced into the RNase G gene (Non-patent Document 10) and strains in which the RNase G gene has been disrupted (Non-patent Document 11). However, since the substrate specificity of RNase has not been completely clarified, it has been difficult to suppress degradation of specific mRNA by regulating the activity of RNase. Therefore, strains with reduced RNase activity were not used for the production of metabolites.
Japanese Patent Laid-Open No. 11-2431979 JP-A-5-137568 International Publication No. 96/06926 Pamphlet US Patent Application Publication No. 20030049803A1 Ikuo Shimamura, Toshikazu Yoshitake: Journal of Agricultural Chemistry, 44, 195-201, (1970) Agric. Biol. Chem., 46, p2673-2679, (1982) Abstracts of Annual Meeting of the Japanese Society for Agricultural Chemistry, 129, (1975) J. Ferment. Bioeng. 78, 155, (1994) Amino Acid Fermentation, Academic Publishing Center, pp. 397-422, 1986 Amino Acid Fermentation, Academic Publishing Center, 31-100, 1986 Journal of Bacteriol. 1996, vol. 178 (1), p280-283 Escherichia coli and Salmonella Vol.1 2nd edition, ASM press, p855 Genes Cells, 6, 2001, p403-410 Biochem. Biophys. Res. Commun., 289 (5), 1301-1306, 2001 Genes Cells. 2001 May; 6 (5): 403-10.

  An object of the present invention is to provide a method capable of efficiently producing pyruvic acid or L-valine using Escherichia bacteria.

  As a result of intensive studies to solve the above problems, the present inventors have found that pyruvic acid and L-valine accumulate by reducing RNaseG activity. Furthermore, it has been found that the accumulation of pyruvate and L-valine is further improved by combining mutation introduction into the cra gene and conferring L-valine resistance, and the present invention has been completed.

That is, the present invention is as follows.
(1) culturing an Escherichia bacterium having an ability to produce pyruvic acid and modified so that the activity of ribonuclease (RNase) G is reduced in the medium, and generating and accumulating pyruvic acid in the medium or the cell; A method for producing pyruvic acid, which comprises collecting pyruvic acid from a medium or cells.
(2) The method for producing pyruvic acid according to (1), wherein the bacterium belonging to the genus Escherichia is a bacterium belonging to the genus Escherichia whose activity of RNaseG is reduced by disrupting a gene encoding RNaseG.
(3) The method for producing pyruvic acid according to (1) or (2), wherein the Escherichia bacterium is an Escherichia bacterium further modified so that expression of the cra gene is lowered.
(4) The method for producing pyruvic acid according to (3), wherein the Escherichia bacterium is an Escherichia bacterium having L-valine resistance.
(5) A bacterium belonging to the genus Escherichia having L-valine-producing ability, wherein the bacterium belonging to the genus Escherichia modified so as to reduce the activity of ribonuclease G is cultured in a medium, and L-valine is produced in the medium or the cells. A method for producing L-valine, which comprises accumulating and collecting L-valine from the medium or cells.
(6) A bacterium belonging to the genus Escherichia having an ability to produce L-valine, wherein the RNaseG activity is reduced and the expression of the cra gene is reduced, and further, L-valine resistance is imparted. A method for producing L-valine, which comprises culturing bacteria in a medium, producing and accumulating L-valine in the medium or cells, and collecting L-valine from the medium or cells.
(7) The method for producing L-valine according to (5) or (6), wherein the Escherichia bacterium is an Escherichia bacterium further attenuated in expression of a gene encoding acetolactate synthase III.
(8) The L-valine according to any one of (5) to (7), wherein the Escherichia bacterium is an Escherichia bacterium further enhanced in expression of a gene encoding acetolactate synthase I or acetolactate synthase II. Manufacturing method.
(9) A bacterium belonging to the genus Escherichia that has been modified so that the activity of RNaseG is reduced and the expression of the cra gene is reduced, and further L-valine resistance is imparted.

  According to the method of the present invention, the fermentation yield of pyruvic acid or L-valine can be improved.

Hereinafter, the present invention will be described in detail. <1> Method for producing pyruvic acid The method for producing pyruvic acid according to the first invention of the present application has an ability to produce pyruvic acid and has been modified so that the activity of RNaseG is reduced. It is a method characterized by culturing a genus bacterium in a medium, producing and accumulating pyruvic acid in the medium or cells, and collecting pyruvic acid from the medium or cells.

  Here, “pyruvic acid producing ability” refers to the ability to accumulate pyruvic acid in the medium or the cells when the bacterium of the present invention is cultured in the medium. “Pyruvate-producing ability” refers to the expression of pyruvate biosynthetic enzymes by gene recombination, or by mutating the parent strain with a normal mutagen, It can be acquired by making a selection. It may be modified to have pyruvic acid-producing ability by modifying so that the activity of RNaseG is reduced.

Examples of the strain having pyruvic acid-producing ability include a lipoic acid-requiring strain (Japanese Patent Laid-Open No. 5-137568). Specific examples include Escherichia coli AJ12631 (FERM P-12381) and Escherichia coli W1485 lip2 (ATCC25645). Lipoic acid-requiring mutant strains can be obtained by normal mutagenesis procedures such as UV, X-ray irradiation, or chemical treatment with N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, etc. It is obtained by culturing the treated cells on an agar plate medium and isolating colonies that have become lipoic acid-requiring (J.
Gen. Microbiol., 53, 363-381, (1968)).

  The Escherichia bacterium used in the present invention (also referred to as the Escherichia bacterium of the present invention) can be obtained by modifying the strain having the ability to produce pyruvate as described above so that the RNaseG activity is reduced. In the breeding of the genus Escherichia of the present invention, either of giving the ability to produce pyruvate and modifying to reduce the RNase G activity may be performed first.

  In the present invention, “RNaseG activity” refers to the activity of degrading RNA serving as a substrate for RNaseG. Examples of RNA serving as a substrate for RNaseG include a gene eno (EnBank Accession No. X82400) encoding enolase, a gene adhE (GenBank Accession No. M33504) encoding alcohol dehydrogenase, and the like. For example, RNA is extracted from a strain in which RNA synthesis is suppressed by rifampicin, and the activity is measured by measuring the degradation half-life of mRNA such as enol-encoding gene eno or alcohol dehydrogenase-encoding gene adhE. Can be known indirectly. In addition, the activity can be determined by isolating and purifying RNaseG and measuring the cleavage reaction of an artificial substrate such as an oligoribonucleotide containing an RNase G cleavage site. Such an activity measurement method has already been disclosed (J. Biol. Chem., 275, 8726-8732, 2000).

Examples of the protein having the above activity include a protein having the amino acid sequence of SEQ ID NO: 6. Further, as long as it has the above activity, it may be a protein having a sequence in which one or several amino acids are substituted, deleted, or added in the amino acid sequence of SEQ ID NO: 6. Here, the number is preferably 2 to 20, more preferably 2 to 2.
10 means, particularly preferably 2-5.

  `` Modified to reduce RNaseG activity '' means that the specific activity of RNaseG contained in the bacterial cell extract is lower than that of an unmodified strain, such as a wild-type Escherichia bacterium. Say. For example, the case where the number of RNaseG molecules per cell decreases, the case where the RNaseG activity per molecule decreases, and the like are applicable. The “decrease” includes the case where the activity is completely lost. Examples of wild-type bacteria belonging to the genus Escherichia that serve as controls include Escherichia coli MG1655 strain.

  Reduction of RNaseG activity is achieved by replacing the RNaseG-encoding gene (rng) with a defective or mutated form, or by modifying expression regulatory sequences such as the rng gene promoter or Shine-Dalgarno (SD) sequence. . More specifically, by substituting or deleting a partial sequence of the rng gene and transforming an Escherichia bacterium with a DNA containing an rng gene (mutant) modified so as not to produce a normally functioning RNaseG, By causing homologous recombination with a gene on the chromosome, the rng gene on the chromosome can be mutated.

rng gene, rng gene of Escherichia coli registered in GenBank (GenBank Accession No. NC _- 000913 in the nucleotide numbers complement of 3,393,963 to 3,395,450: SEQ ID NO: 5) based on the sequence of the synthetic oligonucleotides were synthesized, Cloning can be performed by performing PCR reaction using Escherichia coli chromosome as a template. When the rng gene is deleted by homologous recombination, it has a certain degree of homology with the rng gene on the chromosome, for example, 80% or more, preferably 90% or more, more preferably 95% or more. Genes can also be used. A gene that hybridizes with a rng gene on a chromosome under stringent conditions can also be used. The stringent conditions include, for example, a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, and more preferably 2 times. The condition of washing three times is mentioned.

Gene disruption by gene replacement using homologous recombination has already been established, and there are a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin.
In order to replace the deletion type rng with the rng on the host chromosome, for example, the following may be performed. A recombinant DNA containing a temperature-sensitive replication origin, a mutant rng and a marker gene resistant to drugs such as ampicillin is prepared, and Escherichia bacteria are transformed with this recombinant DNA, and the temperature at which the temperature-sensitive replication origin does not function By culturing the transformed strain in the above, and subsequently culturing it in a medium containing a drug, a transformed strain in which the recombinant DNA is incorporated into the chromosomal DNA can be obtained.

  In this way, the strain in which the recombinant DNA is integrated into the chromosome undergoes recombination with the rng sequence originally present on the chromosome, and two fusion genes of the chromosome rng and the deletion type rng are in other parts of the recombinant DNA ( The vector portion, the temperature sensitive replication origin and the drug resistance marker) are inserted in the chromosome. Therefore, since normal rng is dominant in this state, the transformed strain expresses a normal repressor.

Next, in order to leave only the deletion-type rng on the chromosomal DNA, one copy of rng is removed from the chromosomal DNA together with the vector portion (including the temperature-sensitive replication origin and drug resistance marker) by recombination of two rngs. Let At that time, normal rng is left on the chromosomal DNA and the deletion-type rng is cut out, and conversely, the deletion-type rng is left on the chromosomal DNA and normal rng is cut out. In either case, if the cells are cultured at a temperature at which the temperature-sensitive replication origin functions, the excised DNA is retained in the cell in the form of a plasmid. Next, when cultured at a temperature at which the temperature-sensitive replication origin does not function, rng on the plasmid falls off the cell together with the plasmid. Then, deletion type rng on the chromosome by PCR or Southern hybridization etc.
By selecting a strain in which rng remains, a strain in which rng is destroyed can be obtained.

  Methods such as chromosomal DNA preparation, hybridization, PCR, plasmid DNA preparation, DNA cleavage and ligation, transformation, and setting of oligonucleotides used as primers are described in Sambrook, J., Fritsch, EF, and Maniatis, T. , "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1.2 (1989).

  Alternatively, the rng gene can be mutated to obtain a gene encoding RNaseG having low activity. For example, since the expression of the gene adhE encoding alcohol dehydrogenase depends on the function of the rng gene (Biochemical and Biophysical Research Communications 295 (2002) 92-97), a plasmid in which a promoter of adhE is combined with a reporter gene such as β-galactosidase Is allowed to coexist with the mutant rng gene in the cell, and β-galactosidase activity is measured, so that the activity-reduced rng gene can be screened.

  In order to reduce the activity of RNaseG, in addition to the above-described genetic manipulation methods, for example, Escherichia bacteria are irradiated with ultraviolet light, or normal mutations such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrite Examples include a method of selecting a strain that has been treated with the mutagen used in the treatment and has reduced RNaseG activity. Examples of mutant strains with reduced RNaseG activity include strains in which the maturation activity at the 5 'end of 16S rRNA remains but only mRNA degradation activity has decreased, such as mutant strains DC430 and GM1430 (Biochem. Biophys. Res. Commun., 289 (5), 1301-1306, 2001).

  The bacterium used in the first invention may be further modified so that the expression of the cra gene is reduced. The cra gene is also called a fruR gene and is a repressor of a gene encoding PEP: fructose phosphotransferase system (Mol Gen Genet. 1991 Apr; 226 (1-2): 332-6). Specific examples of the cra gene include a gene having a sequence (SEQ ID NO: 7) registered in GenBank Accession No. X55457. In order to modify so that the expression of the cra gene is reduced, for example, by performing homologous recombination using the cra gene as described above, the cra gene on the chromosome is changed to a mutant or deletion type cra gene. What is necessary is just to select the strain which substituted or performed the mutation process and the expression of cra gene fell. Homologous recombination and the like can be performed in the same manner as the above rng gene. In addition, “decrease” includes the case where expression completely disappears. Examples of the strain lacking the cra gene include mutant strain MC1061 described later.

  When the cra gene is deleted by homologous recombination, it has a certain degree of homology with the cra gene on the chromosome, for example, 80% or more, preferably 90% or more, more preferably 95% or more. Genes can also be used. A gene that hybridizes with a cra gene on a chromosome under stringent conditions can also be used. The stringent conditions include, for example, a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, and more preferably 2 times. The condition of washing three times is mentioned.

The bacterium used in the first invention may further have L-valine resistance. “L-valine resistance” means that it can grow in a medium supplemented with a concentration of L-valine at which a parent strain such as Escherichia coli K-12 cannot grow. The concentration at which the parent strain cannot grow is preferably 25 μg / ml or more. For example, a strain that can grow on M9 minimal medium containing 25 μg / ml or more of L-valine can be said to have L-valine resistance. A strain having L-valine resistance can be obtained by mutating the strain and selecting a strain that grows in a medium containing a high concentration of L-valine from the mutant strains. The L-valine resistant strain may be isolated as an L-valine analog resistant strain. Examples of analogs of L-valine include α-aminobutyric acid and 2-thiazolealanine. Moreover, L-valine resistance can also be conferred by transducing a normal type ilvGM gene (GenBank Accession No. X04890) (see Proc Natl Acad Sci USA Vol 78, No. 2, p922-925). Examples of pyruvic acid-producing bacteria that have been modified so that the activity of ribonuclease G is reduced and the expression of the cra gene is reduced and L-valine resistance is imparted include, for example, Escherichia coli as shown in the Examples. B16 strain can be mentioned.

  Pyruvate can be produced by culturing the bacterium as described above in a medium, producing and accumulating pyruvic acid in the medium or cells, and collecting pyruvic acid from the medium or cells.

  The cultivation of bacteria and the collection and purification of pyruvic acid from the culture solution in the present invention may be carried out in the same manner as the conventional method for producing pyruvic acid by fermentation using microorganisms. As a medium used for producing pyruvic acid, a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids, vitamins and nucleic acids as necessary is used. Any carbon source may be used as long as the mutant strain to be used is available. For example, saccharides such as glucose, fructose, and starch-decomposed molasses are used. Organic acids such as normal paraffin, etc. are used alone or in combination with other carbon sources. As the nitrogen source, for example, ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, and inorganic or organic nitrogen sources such as nitrates, urea, ammonia, and meat extracts are used. Organic micronutrients include, for example, amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acids, yeast extracts, proteolysates containing these, and auxotrophic mutations that require amino acids for growth. When using strains, it is necessary to supplement the required nutrients.

  Cultivation is preferably carried out under aerobic conditions. During the culture period, the culture is shaken or aerated and stirred for 1 to 4 days while controlling the pH of the medium at 5 to 9 and the temperature at 20 to 40 ° C. It is preferable to culture by. The pH of the medium can be adjusted with ammonia, calcium carbonate, various acids, various bases, buffer solutions, and the like. The method for collecting pyruvic acid from the culture solution may be carried out according to a known method, and it is collected by separating and removing cells from the culture solution and then concentrating and crystallizing as pyruvate or using an ion exchange resin. .

<2> Method for Producing L-Valine The method for producing L-valine of the second invention of the present application has an ability to produce L-valine and has been modified so that the activity of RNaseG is reduced, or A bacterium belonging to the genus Escherichia that has L-valine-producing ability, is modified so that the activity of RNaseG is decreased and the expression of the cra gene is decreased, and is further imparted with L-valine resistance, is cultured in a medium, It is a method characterized by producing and accumulating L-valine in a medium or cells and collecting L-valine from the medium or cells.

  “L-valine-producing ability” refers to the ability to accumulate L-valine in a medium or in a microbial cell when the bacterium is cultured in the medium. The L-valine production ability can be obtained by enhancing the expression of the L-valine biosynthesis gene or selecting a mutant strain that has acquired L-valine production ability by performing a mutation treatment. In addition, a strain that originally has L-valine-producing ability, or a strain that has become capable of producing L-valine by modifying the RNaseG gene or cra gene can also be used.

Examples of bacteria having L-valine-producing ability include Escherichia coli VL1970 strain (US Pat. No. 5,658,766). Further, as described in WO96 / 06926, an L-valine-producing bacterium having a mutation requiring lipoic acid for growth or / and a mutation deficient in H + -ATPase, or at least ilvG, ilvM, ilvE In addition, Escherichia bacteria that express each gene of ilvD and ilvD and into which a DNA fragment containing the ilvGMEDA operon has been introduced into cells can also be suitably used in the present invention. In addition, since the ilvGMEDA operon is subjected to expression regulation (attenuation) of the operon by L-valine and / or L-isoleucine and / or L-leucine, in order to release the expression suppression by the produced L-valine, It is preferable that a region necessary for nuation is removed or mutated (Japanese Patent Laid-Open No. 8-47397).

  As another approach, it may be effective to introduce a mutation (ileS or valS) of aminoacyl tRNA synthase having a reduced affinity for each corresponding amino acid (Km increased). The operon preferably does not express threonine deaminase activity. Escherichia coli VL1970 with the ileS17 mutation, which is detained as described above, is the Lucian National Collection of Industrial Microorganisms (VKPM) Depositary GNIIgenetika (Russian National Collection of Industrial Microorganisms (VKPM) Depositary, GNIIgenetika) (address: 1, Dorozhny Proezd., 1, 113545, Moscow, Russia), with a registration number of VKPM B-4411.

  A strain capable of efficiently producing L-valine can be obtained by modifying the strain having the ability to produce L-valine as described above so that the RNaseG activity is reduced. Either L-valine-producing ability or modification for reducing RNaseG activity may be performed first.

  In addition, the strain having the ability to produce L-valine as described above is modified so that the RNaseG activity is decreased and the expression of the cra gene is decreased, and further, L-valine resistance is imparted. -A strain capable of efficiently producing valine can be obtained. The L-valine production ability, the modification for reducing the RNaseG activity, the modification for reducing the expression of the cra gene, and the provision of L-valine resistance may be performed in any order.

  The modification for reducing the RNaseG activity, the modification for reducing the expression of the cra gene, and imparting L-valine resistance can be carried out in the same manner as described in the first invention. Examples of L-valine-producing bacteria that have been modified so that the activity of ribonuclease G is reduced and the expression of the cra gene is reduced, and L-valine resistance is conferred include, for example, Escherichia Cori B16 strain can be mentioned.

  The microorganism used for the production of L-valine may further have a weakened expression of a gene encoding acetolactate synthase III (AHASIII). Examples of the gene encoding acetolactate synthase III include the ilvIH gene (GenBank Accession No. X01609).

In order to reduce the expression of the gene encoding acetolactate synthase III, for example, the ilvIH gene is replaced with a deletion type or a mutant type, or the expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence of the ilvIH gene May be modified. More specifically, by substituting or deleting a partial sequence of the ilvIH gene and transforming an Escherichia bacterium with a DNA containing the ilvIH gene (mutant) modified so as not to produce normally functioning AHASIII, By causing recombination with a gene on the chromosome, the ilvIH gene on the chromosome can be made into a mutant type. Moreover, the expression of the ilvIH gene was reduced by treating the Escherichia coli with ultraviolet rays or a mutagen that is usually used for mutagenesis such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid. Also by a method of selecting a strain, a strain in which expression of a gene encoding acetolactate synthase III is reduced can be obtained. Furthermore, the strain used in the second invention is one in which the expression of the ilvIH gene is reduced by modifying the cra gene and / or the rng gene, or the ilvIH gene is inherently deleted like the MC1061 strain. There may be.

The microorganism used for the production of L-valine may also have an enhanced expression of a gene encoding acetolactate synthase I (AHASI) or acetolactate synthase I (AHASII). Examples of the gene encoding acetolactate synthase I include the ilvBN gene (GenBank Accession No. X02541). In addition, as long as it encodes a protein having AHAS activity, a gene that hybridizes with the gene under stringent conditions can also be used. The stringent conditions include, for example, a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, and more preferably 2 times. The condition of washing three times is mentioned.
Examples of the gene encoding acetolactate synthase II include ilvGM gene (GenBank Accession No. X04890). In addition, as long as it encodes a protein having AHAS activity, a gene that hybridizes with the gene under stringent conditions can also be used. The stringent conditions include, for example, a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, and more preferably 2 times. The condition of washing three times is mentioned.

  In order to enhance the expression of the AHASI gene, for example, a DNA fragment containing the AHASI gene is ligated with a vector functioning in bacteria belonging to the genus Escherichia, preferably a multicopy type vector, to produce a recombinant DNA, which is then transformed into Escherichia. What is necessary is just to introduce | transduce into a genus bacteria. Examples of vectors that can function in bacteria belonging to the genus Escherichia include vectors that can autonomously replicate in the cells of the host microorganism. Vectors that can replicate autonomously in Escherichia bacteria include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184 (available from Takara Bio Inc.), RSF1010, pBR322, pMW219 (pMW available from Nippon Gene) ) And the like.

  In order to introduce the recombinant DNA into the microorganism, transformation methods that have been reported so far may be used. For example, a method for increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) There is a method (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)) that prepares competent cells from DNA and introduces DNA.

  Furthermore, increasing the expression level of the AHASI gene can also be achieved by making multiple copies of the AHASI gene present on the chromosomal DNA of the genus Escherichia. In order to introduce the AHASI gene in multiple copies on the chromosomal DNA of a microorganism, homologous recombination is performed using a sequence present on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is possible to mount the AHASI gene on a transposon, transfer it, and introduce multiple copies onto the chromosomal DNA.

  When introducing the AHASI gene by plasmid or homologous recombination, the gene may be ligated downstream of a strong promoter. For example, lac promoter, trp promoter, trc promoter and the like are known as strong promoters. It is also possible to introduce a base substitution into the promoter region of the full-length AHASI gene and modify it to be more powerful. These promoter substitutions or modifications enhance the expression of normal AHASI gene. The substitution of the expression regulatory sequence can be performed using, for example, a temperature sensitive plasmid. It should be noted that the expression enhancement of the AHASII gene can be performed in the same manner as the AHASI gene.

  L-valine can be produced by culturing Escherichia bacteria as described above in a medium, producing and accumulating L-valine in the medium, and collecting L-valine from the medium.

  The bacterial culture and the collection and purification of L-valine from the culture solution in the present invention may be performed in the same manner as in the conventional amino acid production method by fermentation using microorganisms. The medium used for the culture may be a synthetic medium or a natural medium as long as it contains a carbon source, a nitrogen source, and an inorganic substance, and if necessary, contains an appropriate amount of a nutrient source required for growth by the strain used. Examples of the carbon source include various carbohydrates including glucose and sucrose, and various organic acids. Moreover, alcohols such as ethanol and glycerol can be used depending on the assimilation property of the microorganism to be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, amines and other nitrogen compounds, and natural nitrogen sources such as peptone, soybean hydrolyzate, and fermentation cell decomposition product can be used. As the inorganic substance, monopotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate and the like are used.

  The culture is preferably performed under aerobic conditions such as shaking culture and aeration and agitation culture, and the culture temperature is preferably 20 to 40 ° C, more preferably 30 to 38 ° C. The pH of the medium is usually in the range of 5-9, preferably in the range of 6.5-7.2. The pH of the medium can be adjusted with ammonia, calcium carbonate, various acids, various bases, buffer solutions, and the like. Usually, the target L-valine accumulates in the culture medium after 1 to 3 days of culture.

  After completion of the culture, solids such as bacterial cells can be removed from the culture solution by centrifugation or membrane separation, and the target L-valine can be collected and purified by ion exchange, concentration, crystallization, etc. .

[Example]
Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following.

<Construction of rng gene-deficient strain>
A rng (hereinafter also referred to as cafA) deficient strain was constructed as follows. A 1.3 kb cat (chloramphenicol acetyltransferase) gene cassette obtained by treating pXX557 with BamHI and HindIII was blunted with T4 DNA polymerase, and then rng (= cafA) was used. It was inserted into the unique BglII site in the rng gene on the containing plasmid pMEL1 (Journal of Bacteriology (1987) 169: 4935-4940). PXX557 is a 1.3 kb cat gene fragment obtained by treating pACYC184 (Nippon Gene) with HaeII at the HincII site of pUC9 (registered as DSM No.3421 in the German Collection of Microorganisms and Cell Cultures). Thus obtained plasmid. From the obtained plasmid pMEL1-cafA :: cat, a 7.8 kb SalI fragment containing rng :: cat and its adjacent region was isolated after agarose gel electrophoresis. Escherichia coli K-12 recD mutant strain FS1576 (registered as ME9019 by the National Institute of Genetics) is transformed with the isolated linear DNA described above and the rng :: Chloramphenicol resistant colonies formed by homologous recombination between the linear DNA containing cat and the chromosomal rng region were isolated. In this strain, the wild-type rng gene on the chromosome is replaced with rng :: cat. After infecting this strain with P1 phage and preparing phage lysate, this strain was prepared as MC1061 strain (registered as CGSC #: 6649 in E.coli Genetic Stock Center) or MG1655 strain (registered with National Institute of Genetics). Registered as ME9044). Transductants were selected using chloramphenicol resistance as an index, and the obtained MC1061 rng :: cat strain and MG1655 rng :: cat strain were named GM11 strain and GG11 strain, respectively. The MC1061 strain is a strain lacking the cra gene (Biochimical and Biophysical Research Communications 295 (2002) 92-97).

<Quantification of intracellular pyruvic acid in rng gene-deficient strain>
The GG11 strain, the GM11 strain and the parent strain constructed in Example 1 were added to M9 minimal medium (Na ₂ HPO ₄ · 7H ₂ O 12.8 g, NH ₂ PO ₄ 3g, containing 0.2% glucose and 100 μg / ml L-leucine, NaCl 0.5 g, NH ₄ Cl 1 g, MgSO ₄ 2 mM, CaCl ₂ 0.1 mM in 1 L) were inoculated and cultured at 30 ° C. with shaking. After 4 hours from the start of the culture, the culture solution was collected, and the cells were collected by centrifugation, and then the intracellular pyruvic acid was extracted with 22% perchloric acid. The obtained extract was neutralized with sodium carbonate, and then pyruvic acid was measured using a pyruvate measuring reagent determiner PA manufactured by Kyowa Medix. Specifically, peroxidase 6.5 units / ml and ascorbate oxidase 4.5 units / ml are dissolved in a phosphate buffer containing a surfactant, and an appropriate amount of neutralized extract is added thereto. Incubated at 37 ° C. for 5 minutes. Pyruvate oxidase and bis [3-bis (4-chlorophenyl) methyl-4-dimethyl-aminophenyl] amine were added to the constant temperature reaction solution, reacted at 37 ° C. for 5 minutes, and the absorbance at 750 nm was measured. The pyruvic acid concentration in the culture solution was quantified by comparing this measured value with a calibration curve prepared with a standard solution of pyruvic acid. The extracted pyruvic acid was converted into intracellular pyruvic acid concentration by dividing by OD660 of the culture solution at the time of extraction. As shown in Table 1, it was shown that the intracellular pyruvate concentration was increased in the rng gene-deficient strain.

<Separation of growth improving strain from GM11>
When the GM11 strain constructed in Example 1 was cultured in a medium in which L-leucine was added at 100 μg / ml in an M9 minimal medium containing 0.2% glucose, a significant growth delay was observed. Therefore, as a result of investigations, the present inventors have found that growth is recovered by adding L-threonine or L-isoleucine. From this result, in the GM11 strain, in addition to the cra mutation, the rng mutation was introduced to increase the intracellular L-valine concentration, and as a result, the biosynthesis of L-isoleucine was suppressed, resulting in the minimal medium. It was estimated that growth delay was observed. For example, ilvBN encoding acetolactate synthase I and ilvIH encoding acetolactate synthase III are known to undergo feedback inhibition with L-valine. Therefore, the inventors next can isolate the growth-improved strain of GM11 strain in a minimal medium, and isolate the strain in which the feedback inhibition by L-valine in the biosynthesis pathway of L-isoleucine has been released. We thought that the delay could be eliminated and tried to isolate a growth-improving strain. Specifically, on a M9 plate containing 0.2% glucose and L-leucine 100 μg / ml, GM11 strain was diluted to about 2 × 10 ⁷ cells, applied, and cultured at 30 ° C. for 3 days. Three well-growing colonies that appeared due to the mutation were isolated. One of these growth improving strains was named B6 strain.

<Confirmation of valine sensitivity of B6 strain>
The sensitivity of the B6 strain obtained in Example 3 to L-valine is indicated by growth on a plate containing 0.2% glycerol and 100 μg / ml L-leucine and each M9 minimal medium added with each predetermined amount of L-valine. Confirmed. As a result, the parent strain MC1061 and its rng mutant GM11 were unable to grow on a medium containing 6.25 μg / ml L-valine, whereas the B6 strain was not able to grow on a medium containing 100 μg / ml L-valine. It was able to grow (FIG. 1). Therefore, it was revealed that the B6 strain acquired as a growth-improved strain of the GM11 strain was a strain to which L-valine resistance was imparted.
In addition, MC1061 strain was diluted and applied to an M9 plate containing 0.2% glucose, Leu 100 μg / ml, Val 10 μg / ml to 2 × 10 ⁷ cells, cultured at 30 ° C. for 4 days, and then spontaneously mutated. Four growing clones were isolated. Of these, 3 clones grew even in the presence of 25 μg / ml or more of L-valine. When rng mutations were introduced into these strains, it was confirmed that they could grow on a medium in which only leucine was added to the M9 minimal medium as in the case of the B6 strain. Thus, even if an L-valine resistant strain is isolated according to a conventional method and an rng mutation is introduced into it, a growth-improved strain similar to the B6 strain can be obtained.

<Accumulation of pyruvate by rng gene-deficient strain>
The B6 strain obtained in Example 3 and its parent strain MC1061 were inoculated in M9 minimal medium containing 0.2% glucose and 100 μg / ml L-leucine, and cultured at 30 ° C. A culture solution at 16 hours after the start of the culture was collected, and pyruvic acid in the culture solution was measured using a pyruvate measuring reagent determiner PA manufactured by Kyowa Medix. Peroxidase 6.5 units / ml and ascorbate oxidase 4.5 units / ml are dissolved in a phosphate buffer containing a surfactant, and an appropriate amount of a centrifugal supernatant obtained by centrifuging the culture solution is added thereto. Incubated at 37 ° C. for 5 minutes. Pyruvate oxidase and bis [3-bis (4-chlorophenyl) methyl-4-dimethyl-aminophenyl] amine were added to the constant temperature reaction solution, reacted at 37 ° C. for 5 minutes, and the absorbance at 750 nm was measured. The pyruvic acid concentration in the culture solution was quantified by comparing this measured value with a calibration curve prepared with a standard solution of pyruvic acid. As shown in Table 2, it was shown that the amount of pyruvic acid accumulation was improved about 11 times in the B6 strain compared to the MC1061 strain. Considering this data in combination with the data in Table 1, it was found that pyruvic acid accumulation was further improved by imparting L-valine resistance.

Confirmation of L-valine productivity of B6 strain
The MC1061 strain and the B6 strain were cultured at 30 ° C. in a medium in which L-leucine and L-isoleucine were added to M9 medium containing 0.2% glucose at 100 μg / ml, respectively. The culture solution at 24 hours after inoculation with the fungus was collected and diluted appropriately, and the amount of L-valine produced was analyzed using Hitachi Amino Acid Analyzer L-8500. As shown in Table 3, the MC1061 strain accumulated about 40 mg / L L-valine, whereas the B6 strain accumulated about 90 mg / L L-valine. From this, it was shown that the L6 valine production ability was remarkably improved in the B6 strain.

Complementation of GM11 strain with L-isoleucine-requiring cra gene As mentioned above, since the cra gene is deficient in MC1061 strain, the present inventors have found that the cra mutation affects L-valine biosynthesis I thought about the possibility. If so, introduction of the cra gene should complement the growth delay in the minimal medium observed in the GM11 strain. To confirm this, we first cloned the cra gene. Chromosomal DNA is extracted from GW10 strain (Molecular and General Genetics 253 (1997) 515-519) by a conventional method, and PCR reaction is performed using the chromosomal DNA as a template and the synthetic DNAs shown in SEQ ID NOs: 1 and 2 as primers. A 1.1 kb amplified fragment containing the cra gene was obtained. Since the restriction enzyme BamHI recognition site was added to this primer, this amplified product was completely digested with BamHI and inserted into the BamHI site of pMW218 (Nippon Gene), and the plasmid of the desired configuration was designated as pCRA1. Next, GM11 strain was transformed with this plasmid, and a transformant was obtained using Km resistance as an index. When the obtained transformant GM11 / pCRA1 was spotted on an M9 plate, the auxotrophy of L-threonine or L-isoleucine disappeared as shown in FIG. The requirement for L-threonine or L-isoleucine observed in GM11 was shown to be a phenotype caused by the combination of rng mutation and cra deficiency.

Confirmation of overexpression of ilvBN gene by cra gene deletion
When RNA was extracted from the GM11 strain and GM11 / pCRA1 strain and Northern hybridization was performed using the ilvBN gene fragment as a probe, the expression level of ilvBN mRNA was remarkably suppressed in the GM11 / pCRA1 strain (FIG. 3). The probe used was an ilvBN gene fragment prepared by PCR using the chromosomal DNA of MG1655 as a template and the synthetic DNAs shown in SEQ ID NOs: 3 and 4 as primers. Therefore, it can be said that the effect of the cra mutation is the enhancement of L-valine synthesis by overexpression of the ilvBN gene (acetolactic acid synthase I).

Confirmation of the defect around ilvIH in MC1061 strain
As described in Biochimical and Biophysical Research Communications 295 (2002) 92-97, a part of cra gene is deleted in MC1061 strain, but cra gene and ilvIH gene are located near the chromosome (Fig. 4). On the other hand, MC1061 was said to lack the araD-leuC region (Non-patent document: Journal of Molecular Biology 138 (1980) 179-207). I guessed it was correct. Therefore, the chromosome extracted from MC1061 was completely decomposed with EcoRI or EcoRI and SpeI, and Southern hybridization was performed (FIG. 5). PCR was performed using the MG1655 chromosome as a template and the synthetic DNAs shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers, and a DNA fragment containing a partial sequence of the cra gene was used as the probe. As a result, in the MC1061 strain, it was shown that there was no SpeI site that should be present in the leuO gene (FIG. 5). From this result and the partial deletion of the cra gene observed in MC1061, araD-cra was continuously deleted in the MC1061 strain, and the ilvIH gene was also deleted.

  By using the bacterium of the present invention, pyruvic acid or L-valine can be efficiently produced.

The figure (photograph) which shows the valine tolerance degree of a suppressor mutant. Gly means glycerol. The figure (photograph) which shows the growth recovery of GM11 stock | strain by Ile and Thr addition or cra introduction | transduction. Glc means glucose. The figure which shows suppression of the ilvBN gene expression by cra (photograph). The figure which shows the ilvIH peripheral region on the chromosome of MC1061 strain. The figure (photograph) which shows the analysis by Southern hybridization of the area | region around ilvIH of MC1061 strain | stump | stock.

Claims (4)

The bacterium belonging to the genus Escherichia have a pyruvic acid-producing ability were cultured in the medium to produce and accumulate pyruvate to the medium or bacterial cells, and collecting the pyruvate from the medium or cells, the pyruvate A manufacturing method comprising :
A method wherein the bacterium belonging to the genus Escherichia is modified so that the activity of ribonuclease G is reduced by deleting the rng gene or replacing it with a mutant type, or by modifying the expression regulatory sequence of the rng gene .   2. The method of claim 1, wherein the rng gene encodes a protein selected from the group consisting of (A) and (B).
(A) A protein having the amino acid sequence of SEQ ID NO: 6.
(B) A protein having an amino acid sequence in which 1 to 5 amino acids are substituted, deleted, inserted or added in SEQ ID NO: 6 and having ribonuclease G activity.   The method for producing pyruvic acid according to claim 1 or 2, wherein the Escherichia bacterium is a bacterium belonging to the genus Escherichia further modified so that expression of the cra gene is reduced. The Escherichia bacterium further refers to L- valine resistance Ri bacterium der having, as having L- valine resistance can be grown in conditions containing 25 [mu] g / ml or more L- valine, wherein Item 4. A method for producing pyruvic acid according to Item 3.

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