JP5599319B2 - Method for producing menaquinone-4 - Google Patents

Description

The present invention relates to an efficient method for producing menaquinone-4, a microorganism used in the production method, and a method for breeding the microorganism.

  Menaquinone, a kind of vitamin K, is a generic name for 2-methyl-3-polyprenyl 1,4-naphthalenedione, is a component of the bacterial plasma membrane, and is a redox agent in electron transport and oxidative phosphorylation systems. (Non-Patent Document 1). Among these, menaquinone-4 is considered to be an active menaquinone in the human body and has been shown to have a higher blood coagulation activity than a longer-chain menaquinone (Non-patent Document 2).

As a method for producing menaquinone-4, an extraction method from a microorganism and a chemical synthesis method are known.
Although microorganisms that preferentially produce menaquinone-4 are not known in nature, microorganisms belonging to the genus Flavobacterium that have preferentially produced menaquinone-4 by a breeding method using a mutation agent ( Patent Document 1) and microorganisms belonging to the genus Arthrobacter (Patent Document 2) are known.

However, in the production of menaquinone-4 by these strains, there is a problem that a considerable amount of long-chain menaquinone, which is a byproduct, accumulates. There is also a need for further improvement in productivity.
In the case of ubiquinone, which is a quinone to which a prenyl group is added in the same manner as menaquinone, 1-deoxy-D-xylulose 5-phosphate synthase, 2-C-methyl-D-erythritol 4-phosphate synthase, decaprenyl diphosphate synthase and The production of ubiquinone-10 is increased by enhancing the activity of p-hydroxybenzoate-decaprenyl transferase (Patent Documents 3 and 4), and the ubiquinone produced by introducing a mutation into polyprenyl transferase. It is known that the chain length of the isoprene side chain changes (Non-patent Document 3 and Patent Document 5).

  The biosynthetic pathway of menaquinone in cells of Escherichia coli and Bacillus subtilis has been clarified, and the production amount of menaquinone-8 may be increased by enhancing the expression of 1-deoxy-D-xylulose 5-phosphate synthase in Escherichia coli. Known (Patent Document 6). However, it has not been known that menaquinone-4 is produced by enhancing expression of a specific gene or that productivity is improved specifically for menaquinone-4.

  Moreover, although the function of dehydroxynaphthoic acid prenyltransferase encoded by the menA gene has been clarified, a method for producing menaquinone by a microorganism in which the expression level of the menA gene is increased is not known.

JP 61-216696 JP 61-265097 JP2000-300256 WO01 / 027286 JP 09-173076 JP2000-300256

J Infect Dis. 1984 Aug; 150 (2): 213-8. Y. Akiyama et al, (1995) Biochem. Phermacol., 49: 1801-1807 K. Wang et al., 1999, Trends Biochem. Sci., 24: 445-451

  Provided are an efficient method for producing menaquinone-4, a microorganism used in the production method, and a method for breeding the microorganism.

The present invention relates to the following [1] to [8].
[1] A microorganism whose dehydroxynaphthoic acid prenyltransferase activity is enhanced compared to a wild type strain is cultured in a medium, menaquinone-4 is produced and accumulated in the culture, and the menaquinone-4 is collected from the culture A process for producing menaquinone-4, characterized in that
[2] The process according to [1] above, wherein the enhancement of dehydroxynaphthoic acid prenyltransferase activity increases the copy number of a gene containing a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity.
[3] The production method of the above-mentioned [1], wherein the enhancement of dehydroxynaphthoic acid prenyltransferase activity modifies an expression regulatory sequence of a gene containing a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity.
[4] The process according to [2] or [3] above, wherein the DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity is a DNA encoding a dehydroxynaphthoic acid prenyltransferase derived from Arthrobacter nicotianae. .
[5] Any of [2] to [4] above, wherein the DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity is a DNA encoding a polypeptide selected from the following (a) to (c): The manufacturing method.
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and dehydroxyl Polypeptide having naphthoic acid prenyltransferase activity (c) Polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2 and having dehydroxynaphthoic acid prenyltransferase activity [6] The method according to any one of [2] to [4] above, wherein the DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity is the following DNA (a) or (b):
(A) DNA comprising the base sequence represented by SEQ ID NO: 1
(B) a DNA that hybridizes with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 under a stringent condition and encodes a protein having dehydroxynaphthoic acid prenyltransferase activity
[7] The method according to any one of [1] to [6] above, wherein the microorganism is a microorganism belonging to the genus Arthrobacter or Corynebacterium.
[8] The method according to any one of [1] to [7] above, wherein the microorganism belongs to Arthrobacter nicotianae or Corynebacterium glutamicum.

  By using the microorganism of the present invention, a microorganism that efficiently produces menaquinone-4 can be obtained, and menaquinone-4 can be efficiently produced using the microorganism.

1. Preparation of microorganism used in the production method of the present invention The microorganism having enhanced dehydroxynaphthoic acid prenyltransferase activity used in the production method of the present invention specifically includes (i) a protein having dehydroxynaphthoic acid prenyltransferase activity. By increasing the copy number of a gene containing DNA encoding DNA, or (ii) a gene containing DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity. This refers to a microorganism in which dehydroxynaphthoic acid prenyltransferase activity is enhanced as compared to the wild type strain by modifying the expression regulatory sequence.

In this specification, the gene includes a region having a control function related to expression of a gene such as a promoter and an operator, in addition to a region (ORF) encoding the amino acid sequence of a protein encoded by the gene.
A wild strain refers to a strain having the phenotype most frequently observed in the wild population.
Such wild strain, for example, Arthrobacter Nikotianae (Arthrobacter nicotianae) Arthrobacter nicotianae ATCC15236 as the wild-type strain, as wild-type strain of Corynebacterium glutamicum (Corynebacterium glutamicum) be mentioned Corynebacterium glutamicum ATCC13032 it can.

The protein having dehydroxynaphthoic acid prenyltransferase activity is not particularly limited as long as it is a protein having dehydroxynaphthoic acid prenyltransferase activity.
As a protein having dehydroxynaphthoic acid prenyltransferase activity, specifically,
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having dehydroxynaphthoic acid prenyltransferase activity, or (c) SEQ ID NO: And amino acid sequence having a homology of 80% or more, preferably 90%, more preferably 95%, more preferably 98%, most preferably 99% with the amino acid sequence represented by 2 and prenyl dehydroxynaphthoate A polypeptide having transferase activity,
Can give.

In the above, a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added, and having dehydroxynaphthoic acid prenyltransferase activity, is Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press ( 1989) (hereinafter abbreviated as Molecular Cloning 2nd edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research, 10 , 6487 (1982), Proc. Natl. Acad. Sci. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Using site-directed mutagenesis described in Acad. Sci. USA, 82 , 488 (1985) etc., for example, site-directed mutagenesis is introduced into DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2. By doing Can be acquired.

The number of amino acids to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a known method such as the above-described site-directed mutagenesis method, 1 to several tens, Preferably they are 1-20 pieces, More preferably, they are 1-10 pieces, More preferably, they are 1-5 pieces.
In the amino acid sequence represented by SEQ ID NO: 2, one or more amino acids are deleted, substituted or added when one or more amino acids are deleted, substituted or added at any position in the same sequence. Good.

Examples of amino acid positions at which amino acid deletion or addition can be performed include one to several amino acids on the N-terminal side and C-terminal side of the amino acid sequence represented by SEQ ID NO: 2.
Deletions, substitutions or additions may occur simultaneously, and the amino acids substituted or added may be natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

Examples of amino acids that can be substituted with each other are shown below. Amino acids included in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3 -Hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine In addition, in order for the protein of the microorganism used in the present invention to have dehydroxynaphthoic acid prenyltransferase activity, SEQ ID NO: 2 Amino represented by It is desirable that the homology with the acid sequence is 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 98% or more, particularly preferably 99% or more.

The homology of amino acid sequences and base sequences is based on the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (1993)] and FASTA [Methods Enzymol., 183 , 63 (1990)] by Karlin and Altschul. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215 , 403 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. Further, when an amino acid sequence is analyzed by BLASTX based on BLAST, parameters are set to score = 50 and wordlength = 3, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known (http://www.ncbi.nlm.nih.gov).

Further, specific examples of the protein having dehydroxynaphthoic acid prenyltransferase activity include the protein having the amino acid sequence represented by SEQ ID NO: 2 and the Corynebacterium glutamicum ATCC13032 strain having the amino acid sequence represented by SEQ ID NO: 4. protein, Bacillus subtilis subsp. subtilis having an amino acid sequence represented by SEQ ID NO: 6 (Bacillus subtilis subsp. subtilis) str. 168 derived proteins, Arthrobacter having an amino acid sequence represented by SEQ ID NO: 8, A protein derived from an auresense ( Arthrobacter aurescens ) TC-1 strain, a protein derived from Flavobacterium psychrophilim JIP02 / 86 having the amino acid sequence represented by SEQ ID NO: 10, and an amino acid represented by SEQ ID NO: 12 Derived from Escherichia coli K12 strain having sequence Can be mentioned.

The DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity is not particularly limited as long as it is a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity.
As a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity, specifically,
Hybridizes under stringent conditions with (a) DNA consisting of the base sequence represented by SEQ ID NO: 1 or (b) DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1; DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity,
Can give.

  The term “hybridize” as used herein refers to a step in which DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Accordingly, the DNA having the specific base sequence or a part of the base sequence of the DNA is useful as a probe for Northern or Southern blot analysis, or is a length of DNA that can be used as an oligonucleotide primer for PCR analysis. May be. Examples of DNA used as a probe include DNA of at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more, but may be DNA of at least 10 bases, preferably 15 bases or more. .

  Methods for DNA hybridization experiments are well known, such as Molecular Cloning 2nd Edition, 3rd Edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press ( In addition to those described in Molecular), hybridization conditions can be determined and experiments can be performed according to a number of other standard textbooks.

  The above stringent conditions include, for example, a filter on which DNA is immobilized and a probe DNA, 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6). ), Incubate overnight at 42 ° C. in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA, for example in a 0.2 × SSC solution at about 65 ° C. Conditions for washing the filter can be raised, but lower stringent conditions can also be used. Stringent conditions can be changed by adjusting the formamide concentration (lower stringent concentration reduces the stringency), and changing the salt concentration and temperature conditions. As low stringent conditions, for example, 6 × SSCE (20 × SSCE is 3 mol / l sodium chloride, 0.2 mol / l sodium dihydrogen phosphate, 0.02 mol / l EDTA, pH 7.4), 0.5% Incubate overnight at 37 ° C in a solution containing SDS, 30% formamide, 100 μg / l denatured salmon sperm DNA, then wash with 50 ° C 1x SSC, 0.1% SDS solution. I can give you. Further, examples of lower stringent conditions include conditions in which hybridization is performed using a solution having a high salt concentration (for example, 5 × SSC) under the above-described low stringency conditions and then washed.

The various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of hybridization experiments. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions in order to adapt the conditions.
The DNA that can hybridize under the above stringent conditions includes, for example, at least the base sequence represented by SEQ ID NO: 1 when calculated based on the above parameters using a program such as BLAST and FASTA described above. Examples thereof include DNA having a base sequence having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 98% or more, and particularly preferably 99% or more.

The fact that the DNA that hybridizes with the above DNA under stringent conditions is a DNA that encodes a protein having dehydroxynaphthoic acid transferase activity. This means that a recombinant DNA that expresses the DNA is prepared, Culturing a microorganism obtained by introducing body DNA into a host cell deficient in dehydroxynaphthoic acid transferase activity, adjusting a cell extract or membrane fraction containing the protein from the obtained culture, The method of Suvarna et al. (Suvarna, K et.al., Journal of Bacteriology, 180 , 2782-2787 (1998) detects the resulting demethylmenaquinone mixed with the substrates dehydroxynaphthoic acid and isoprenyl diphosphate. )).

As a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity, in addition to the DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, the nucleotide sequence represented by SEQ ID NO: 3 derived from Corynebacterium glutamicum ATCC13032 strain A DNA comprising the nucleotide sequence represented by SEQ ID NO: 5 derived from Bacillus subtilis subspecies subtilis str.168, a base represented by SEQ ID NO: 7 derived from Arthrobacter aurecens TC-1 strain DNA consisting of the sequence, DNA consisting of the base sequence represented by SEQ ID NO: 9 derived from Flavobacterium cyclophilum JIP02 / 86 strain, and DNA consisting of the base sequence represented by SEQ ID NO: 11 derived from Escherichia coli K12 strain be able to. Furthermore, the DNA encoding the de-hydroxynaphthoic acid prenyl transferase, based on homology to the above DNA, Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Brevibacterium lactofermentum (Brevibacterium lactofermentum) such Examples include DNA that can be cloned from bacteria belonging to the genus Mycobacterium such as coryneform bacteria and Mycobacterium tuberuculosis .

  By increasing the copy number of DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity, a microorganism with enhanced dehydroxynaphthoic acid prenyltransferase activity can have one or more dehydroxynaphthoic acid prenyltransferases on its chromosomal DNA. It can be obtained by introducing DNA encoding a protein having activity or by introducing an autonomously replicating recombinant DNA containing DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity into a microorganism. .

  As a method for introducing DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity onto the chromosomal DNA of a microorganism, a method utilizing homologous recombination can be mentioned. As a general method utilizing homologous recombination, a plasmid DNA having a drug resistance gene that cannot autonomously replicate in a host cell into which a DNA fragment encoding a protein having dehydroxynaphthoic acid prenyltransferase activity is to be introduced. And a method of using a plasmid for homologous recombination that can be prepared by ligating with a DNA.

  As a method utilizing homologous recombination, (i) after introducing the plasmid for homologous recombination into a microbial cell by a conventional method, the plasmid for homologous recombination is chromosomally DNAd by homologous recombination using drug resistance as an index. (Ii) After culturing the obtained transformant for several hours to one day in a medium not containing the drug, the drug-containing agar medium and the drug-free agar medium are selected. Applying (iii) selecting a strain that does not grow on the former medium and that can grow on the latter medium, can give a method of obtaining a strain that has undergone the second homologous recombination on the chromosomal DNA. .

As a plasmid having a drug resistance gene that cannot autonomously replicate in cells of a microorganism, a plasmid having a drug resistance gene and a Bacillus subtilis levanshu clease gene sacB (Mol. Microbiol., 6 , 1195 (1992)) is used. It is also possible to obtain a strain in which the second homologous recombination has occurred using a selection method (J. Bacteriol., 174 , 5462 (1992)) that utilizes the production of a substance that is harmful to host cells. it can.

As a method for introducing a plasmid for homologous recombination into a microorganism cell, any method can be used as long as it can introduce DNA into the microorganism cell. For example, electroporation (Appl. Microbiol. Biotech., 52 , 541 (1999)) and the protoplast method (J. Bacteriol., 159 , 306 (1984)).
Introduction of a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity into chromosomal DNA of a microorganism can be performed, for example, using a sequence present in multiple copies on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used.

  The microorganism may be any microorganism as long as it is a microorganism that expresses dehydroxynaphthoic acid prenyltransferase, but is preferably a prokaryote, more preferably an Arthrobacter, Corynebacterium, Bacillus, Staphylococcus, A microorganism belonging to the genus Escherichia or Salmonella, more preferably a microorganism belonging to the genus Arthrobacter or Corynebacterium, most preferably an Arthrobacter nicotianae or Corynebacterium glutamicum can be mentioned.

  In addition, inserting a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity into the chromosomal DNA of a microorganism means ligating the DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity to a transposon. It can also be obtained by transferring it and inserting it into chromosomal DNA (Japanese Patent Laid-Open No. 2-109985).

By determining the base sequence of the peripheral region into which a DNA fragment encoding a protein having dehydroxynaphthoic acid prenyltransferase activity on chromosomal DNA has been introduced, or by Southern hybridization using a part of the DNA fragment as a probe, etc. Then, it can be confirmed that the DNA fragment has been introduced at the target position on the chromosomal DNA.
For example, a microorganism into which a recombinant DNA containing a DNA encoding a protein having dehydroxynaphthoic acid prenyltransferase activity has been introduced. For example, a DNA cell encoding a protein having dehydroxynaphthoic acid prenyltransferase activity can be used as a host cell. A recombinant DNA produced by ligation to a vector having a promoter at a position capable of transcribing a DNA encoding a protein having dehydroxynaphthoic acid prenyl transferase activity that is capable of autonomous replication in a host microbial cell It can be obtained by.

  Examples of the vector include coryneforms such as pCG1, pCG2, pCG4, pCG11, pCE52, pCE53, pCE54 (Japanese Patent Laid-Open Nos. 57-134500, 57-183799, 58-105999). A plasmid capable of autonomous replication in bacteria can be exemplified, and a plasmid having a pCG1 replication origin can also be used as it is in a microorganism belonging to the genus Arthrobacter. An example of such a plasmid is pCS299P (WO 00/63388 pamphlet).

  Introduction of the recombinant DNA prepared as described above into a microorganism can be performed according to transformation methods reported so far. Such methods include, for example, a method in which recipient cells are treated with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and methods for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson GAand Young, FE, Gene, 1, 153 (1977)). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, can be placed in a protoplast or spheroplast state that easily incorporates recombinant DNA and the recombinant DNA is introduced into the DNA-receptor. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nature, 274, 398 (1978); Hinnen, A Hicks, JB and Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). Further, transformation of coryneform bacteria can also be performed by the electric pulse method (Sugimoto et al., Japanese Patent Laid-Open No. 2-207791). The transformation of Arthrobacter bacteria is also known by the electric pulse method (PC Shaw et al. (1988) J. Gen. Microbiol. 134: 903-911, M. Morikawa et al. (1994) Appl. Microbiol. Biotechnol. 42: 300-303).

In addition, the method for producing menaquinone-4 of the present invention includes a microorganism having enhanced dehydroxynaphthoic acid prenyltransferase activity by modifying an expression regulatory sequence of a gene encoding a protein having dehydroxynaphthoic acid prenyltransferase activity. Can also be used.
According to the method described in the pamphlet of International Publication No. 00/18935, such a microorganism can replace expression control sequences such as a promoter of dehydroxynaphthoic acid prenyltransferase gene on a chromosomal DNA or a plasmid with a strong one, It can also be obtained by amplifying a regulator that increases the expression of dehydroxynaphthoic acid prenyltransferase and deleting or weakening a regulator that decreases the expression of dehydroxynaphthoic acid prenyltransferase.

  For example, since the lac promoter, trp promoter, trc promoter and the like derived from bacteria belonging to the genus Escherichia are known as strong promoters, the above promoter and the promoter of the dehydroxynaphthoic acid prenyltransferase gene can be replaced. It is also possible to introduce a base substitution into the promoter region of the dehydroxynaphthoic acid prenyltransferase gene and modify it to be more powerful. Methods for evaluating promoter strength and examples of strong promoters are described in Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128.

  Furthermore, it is known that substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon, has a great influence on the translation efficiency of mRNA, These can be modified. Expression regulatory regions such as a promoter of dehydroxynaphthoic acid prenyl transferase can be determined using a promoter search vector or gene analysis software such as GENETYX (Software Development Co., Ltd.).

These promoter substitutions or modifications can enhance the expression of the dehydroxynaphthoic acid prenyltransferase gene.
Substitution of an expression regulatory sequence such as a promoter can be performed using, for example, a temperature sensitive plasmid. Examples of temperature sensitive plasmids for coryneform bacteria include p48K and pSFKT2 (Japanese Patent Laid-Open No. 2000-262288), pHSC4 (Japanese Patent Laid-Open No. 5-7491), and the like. These plasmids can replicate autonomously in coryneform bacteria at least at 25 ° C, but cannot replicate at 37 ° C.

The modification of the expression regulatory sequence may be combined with increasing the copy number of the dehydroxynaphthoic acid prenyltransferase gene.
The activity of dehydroxynaphthoic acid prenyltransferase is improved compared to host cells. For example, the amount of mRNA of dehydroxynaphthoic acid prenyltransferase is compared with that of host cells that do not enhance dehydroxynaphthoic acid prenyltransferase activity. It can be confirmed by doing. Examples of the method for confirming the mRNA expression level include Northern hybridization and RT-PCR (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). Here, the host cell may be a wild strain or a strain bred from a wild strain.

The degree of improvement in dehydroxynaphthoic acid prenyltransferase activity is not limited as long as it is increased compared to the wild strain, but for example, the expression level of dehydroxynaphthoic acid prenyltransferase is 1.5 times or more that of the wild strain More preferably, it is 2 times or more, more preferably 3 times or more.
2. Process for producing menaquinone-4 of the present invention
According to the method for producing menaquinone-4 of the present invention, the microorganism prepared by the method described in 1 above is cultured in a medium, menaquinone-4 is produced and accumulated in the culture, and menaquinone-4 is collected from the culture. It is a manufacturing method characterized by this.

As a medium to be used, a medium conventionally used in the fermentation production of menaquinone using microorganisms can be used (Japanese Patent Publication No. 7-51070, Japanese Patent Laid-Open No. 61-265097). That is, a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required can be used.
As the carbon source, saccharides such as glucose, sucrose, lactose, galactose, fructose and starch hydrolysate, alcohols such as glycerol and sorbitol, organic acids such as fumaric acid, citric acid and succinic acid can be used. .

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.
As an organic trace nutrient source, it is desirable to contain an appropriate amount of a required substance such as vitamin B1, L-homoserine or a yeast extract. In addition to these, a small amount of potassium phosphate, magnesium sulfate, iron ion, manganese ion or the like is added as necessary.

The medium used in the present invention may be a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic ions, and other organic trace components as required.
Cultivation is preferably carried out under aerobic conditions for 1 to 10 days, the culture temperature is preferably 24 ° C to 37 ° C, and the pH during the culture is preferably 5 to 9.
In addition, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment.

The collection of menaquinone from the culture can usually be carried out by combining ion exchange resin method, precipitation method and other known methods. In addition, when menaquinone accumulates in the microbial cell, for example, crush the microbial cell with ultrasonic waves and collect menaquinone from the supernatant obtained by removing the microbial cell by centrifugation, for example, by the ion exchange resin method. Can do.
Examples of the present invention are shown below, but the present invention is not limited to these examples.

Search for MK-4 productivity-enhancing gene expression strain (1) Acquisition of Arthrobacter Nicotianae KS-8-18 expressing menaquinone-4 synthesis-related gene from Corynebacterium glutamicum ATCC13032 DNA fragments containing the menA, menB, menC, menD, menE and menF genes derived from the bacterial glutamicum ATCC13032 strain were obtained by PCR.

  For obtaining a DNA fragment containing the menA gene, a synthetic DNA fragment having the nucleotide sequence represented by SEQ ID NOs: 13 and 14 is used. For obtaining a DNA fragment containing the menB gene, the nucleotide sequence represented by SEQ ID NOs: 15 and 16 is used. A synthetic DNA fragment having the nucleotide sequence represented by SEQ ID NOs: 17 and 18 for obtaining a DNA fragment containing the menC gene, and SEQ ID NOs: 19 and 20 for obtaining a DNA fragment containing the menD gene. A synthetic DNA fragment having the nucleotide sequence represented, a synthetic DNA fragment having the nucleotide sequence represented by SEQ ID NOs: 21 and 22 for obtaining a DNA fragment containing the menE gene, and a sequence for obtaining a DNA fragment comprising the menF gene Synthetic DNA fragments having the base sequences represented by Nos. 23 and 24 were used as primer sets, respectively.

PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, using Pyrobest DNA polymerase and the attached buffer. Each obtained amplified DNA fragment was purified using a QIAquick PCR Purification Kit (manufactured by QIAGEN).
Of the purified DNA fragments obtained above, DNA fragments containing menA are KpnI and Sse8387I, DNA fragments containing menB are BglII and Sse8387I, DNA fragments containing menC and DNA fragments containing menE are DNA containing FbaI, Sse8387I and menD The DNA fragment containing SalI and Sse8387I and menF was digested with two types of restriction enzymes KpnI and SalI.

The digested fragments containing menA, menB, and menF were each treated with the same two types of restriction enzymes, E. coli / Corynebacterium shuttle vector pCS299P (Japanese Patent Application No. 11-110437, Mitsuhashi et al. (2004) Appl. Microbiol. Biotechnol., 63, 592-601) and ligated using Ligation kit ver.1 (Takara Bio Inc.).
Also, PCR was performed using plasmid pKK223-3 (Pharmacia) as a template, synthetic DNA fragments having the nucleotide sequences represented by SEQ ID NOs: 25 and 27 as primer sets, and Pyrobest DNA polymerase and the attached buffer. The Tac promoter portion of pKK223-3 was amplified. The amplified DNA fragment was digested with BamHI and KpnI.

A DNA fragment containing the Tac promoter, a DNA fragment containing menC or menE, and pCS299P cut with KpnI and Sse8387I were mixed and ligated with Ligation kit ver.1.
Similarly, using pKK223-3 as a template, a synthetic DNA fragment having the nucleotide sequences represented by SEQ ID NOs: 25 and 26 as a primer set, PCR reaction was performed using Pyrobest DNA polymerase and an attached buffer, and pKK233-3 The Tac promoter part was amplified. The amplified DNA fragment was cleaved with SalI and KpnI.

A DNA fragment containing the Tac promoter, a DNA fragment containing menD, and pCS299P cleaved with KpnI and Sse8387I were mixed and ligated with Ligation kit ver.1.
Using the 6 kinds of ligation products obtained above, E. coli DH5α was transformed according to a conventional method. The transformant was applied to an LB agar medium containing 20 μg / ml kanamycin, and then cultured at 30 ° C. overnight.

The grown transformant was inoculated into an LB liquid medium containing 20 μg / ml kanamycin and cultured overnight at 30 ° C., and menA, menB, menC, menD, menE or menF were obtained from the obtained culture solution by alkaline SDS method. Plasmids containing each gene were extracted.
Each extracted plasmid was transferred to Arthrobacter nicotianae KS-8-18 strain ((FERM BP-8232, Japanese Patent Publication No. Sho 61-265097)) and electroporation method according to the method of Sandu et al. (Appl. Environ Microbiol., 71 , 8920-4 (2005)), respectively, to obtain the Arthrobacter Nicotianae KS-8-18 strain that expresses each gene.

(2) Acquisition of DXP synthase-expressing strain derived from Arthrobacter nicotianae KS-8-18 Using the chromosomal DNA of Arthrobacter nicotianae ATCC14929 extracted by a conventional method as a template, it encodes DXP synthase of Arthrobacter nicotianae A DNA fragment was obtained. In PCR, a DNA fragment encoding DXP synthase was synthesized using TAKARA LA PCR cloning kit (manufactured by Takara Bio Inc.) using a synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 28 and 29 as a primer set, according to the attached protocol. Obtained.

Using the DNA fragment encoding DXP synthase obtained above as a template, using the synthetic DNA represented by SEQ ID NO: 30 or 31 as a primer, the DNA fragment encoding DXP synthase was amplified by PCR, and the restriction enzymes BamHI and KpnI were used. Digested.
Similarly, the shuttle vector pCS299P digested with BamHI and KpnI and the DNA fragment amplified above were ligated with Ligation kit ver.2. The plasmid obtained by ligation was transformed into Arthrobacter nicotianae KS-8-18 strain, a strain having kanamycin resistance was selected, and a DXP synthase expression strain was obtained.

(3) Acquisition of menA expression strain derived from Arthrobacter nicotianae KS-8-18 Strain Using the chromosomal DNA of Arthrobacter nicotianae ATCC14929 as a template and synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 32 and 33 as a primer set Using a TAKARA LA PCR cloning kit, a DNA fragment containing the full length of the menA gene was obtained.
The DNA fragment containing the menA gene was amplified by PCR using the DNA fragment containing the menA gene obtained above as a template and the synthetic DNA having the base sequences represented by SEQ ID NOs: 34 and 35 as a primer set. The amplified fragment was digested with restriction enzymes BamHI and Sse8387I, and ligated with the shuttle vector pCS299P digested with BamHI and Sse8387I using Ligation kit ver.2.

  The plasmid obtained by the ligation was transformed into Arthrobacter nicotianae KS-8-18 strain, and a kanamycin resistant strain was selected to obtain a menA expression strain.

(1) Menaquinone-4 productivity evaluation 1
Arthrobacter Nicotianae KS-8-18 expressing a gene related to menaquinone-4 synthesis derived from Corynebacterium glutamicum ATCC13032 obtained in (1) of Example 1 and DXP synthase obtained from (2) Was evaluated for the ability to produce menaquinone-4 for the S. aerobacter Nicotianae KS-8-18 strain. As a control, a strain into which the expression vector pCS299P was introduced was used.

Arthrobacter Nicotianae glycerol stock with seed medium plate [agar 15g / L, peptone (manufactured by Kyokuto) 10g / L, meat extract (manufactured by Kyokuto) 10g / L, NaCl 3g / L, pH 7.2] And then incubated at 30 ° C. for 24 hours.
Part of the bacteria grown on the plate is scraped with ase and 5 ml of seed medium containing 150 mg / L kanamycin [Peptone (manufactured by Kyokuto) 10 g / L, meat extract (manufactured by Kyokuto) 10 g / L, NaCl 3 g / L, pH 7.2] were inoculated into a test tube and cultured at 30 ° C. for 24 hours with shaking. 3 ml of the culture solution was collected, inoculated into a 300 ml baffled flask filled with 30 ml of production medium, and cultured with shaking at 30 ° C. for 96 hours.

To the collected culture broth, equal amounts of 2-butanol and glass beads were added and shaken to disrupt the cells and extract menaquinone. Using the resulting extract, menaquinone-4, menaquinone-7, menaquinone-8 and menaquinone-9 produced in the culture were quantified by high performance liquid chromatography (HPLC).
For HPLC, a development ODS-8 column manufactured by Nomura Chemical Co., Ltd. was used, and a solution of hexane: methanol = 2: 8 was used as a moving bed. Menaquinone was detected by absorption at a wavelength of 270 nm.

  The results of quantification are shown in Table 1.

As shown in Table 1, in the strain expressing menA gene, it was found that the amount of menaquinone-4 produced and the ratio of menaquinone-4 to the total amount of menaquinone were improved compared to the strains expressing other genes.
(2) Menaquinone productivity evaluation 2
KS-8-18 / pAnmenA obtained in (3) of Example 1 was cultured, and menaquinone productivity was evaluated.

Cultivation and quantification were performed in the same manner as in (1) above.
The results are shown in Table 2.

  As shown in Table 2, in the strain expressing the menA gene, it was found that the amount of menaquinone-4 produced and the ratio of menaquinone-4 to the total amount of menaquinone were improved.

Menaquinone productivity evaluation 3
PCS299P and the plasmid pCgmenA prepared in (1) of Example 1 were introduced into Corynebacterium glutamicum ATCC13032, respectively, to obtain Corynebacterium glutamicum ATCC13032 / pCS299P and Corynebacterium glutamicum ATCC13032 / pCgmenA.

Corynebacterium glutamicum ATCC13032 / pCS299P strain and Corynebacterium glutamicum ATCC13032 / pCgmenA strain were cultured in the same medium and method as in Example 2, and menaquinone productivity was evaluated. Menaquinone was also quantified in the same manner as in Example 2.
The results are shown in Table 3.

  As shown in Table 3, it was found that also in Corynebacterium glutamicum, the amount of MK-4 produced and the production ratio thereof were specifically increased by the expression of menA gene.

  By using the microorganism of the present invention, a microorganism that efficiently produces menaquinone-4 can be obtained, and menaquinone-4 can be efficiently produced using the microorganism.

SEQ ID NO: 13—Description of artificial sequence: synthetic DNA
SEQ ID NO: 14—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 15—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 16—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 17—Description of artificial sequence: synthetic DNA
SEQ ID NO: 18—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 19—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 20—Description of artificial sequence: synthetic DNA
SEQ ID NO: 21—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 22—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 23—Description of artificial sequence: synthetic DNA
SEQ ID NO: 24—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 25—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 26—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 27—Description of artificial sequence: synthetic DNA
SEQ ID NO: 28—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 29—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 30—Description of artificial sequence: synthetic DNA
SEQ ID NO: 31—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 32—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 33—Description of artificial sequence: synthetic DNA
SEQ ID NO: 34—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 35—Description of artificial sequence: synthetic DNA

Claims (5)

Dihydroxy-naphthoic acid prenyl transferase activity, Ri by the increasing copy number of a gene comprising a DNA encoding a protein having a di hydroxynaphthoic acid prenyl transferase activity, Arthrobacter enhanced as compared with the wild strain genus or Korinebakuteri A method for producing menaquinone-4, comprising culturing a microorganism belonging to the genus Umum in a medium, producing and accumulating menaquinone-4 in the culture, and collecting the menaquinone-4 from the culture . Dihydroxy-naphthoic acid prenyl transferase activity, Ri by the modifying an expression regulatory sequence of a gene containing DNA encoding a protein having a di hydroxynaphthoic acid prenyl transferase activity, Arthrobacter enhanced as compared with the wild strain genus or A method for producing menaquinone-4, comprising culturing a microorganism belonging to the genus Corynebacterium in a medium, producing and accumulating menaquinone-4 in the culture, and collecting the menaquinone-4 from the culture . DNA encoding a protein having a di hydroxynaphthoic acid prenyl transferase activity is a DNA encoding a polypeptide selected from the following (a) ~ (c), Process according to claim 1 or 2.
(A) SEQ ID NO: 2, 4, 6, 8, 10, or a polypeptide (b) SEQ ID NO: 2 comprising the amino acid sequence represented by the 12 amino acid sequence represented by 4,6,8,10 or 12, 1-10 amino acids are deleted, substituted or added in the amino acid sequence, and polypeptides (c) SEQ ID NO: 2 with a di-hydroxy-naphthoic acid prenyl transferase activity in, 4,6,8,10 or 12, in consists of the amino acid sequence and amino acid sequence having 95% or more identity, and a polypeptide having a di hydroxynaphthoic acid prenyl transferase activity DNA encoding a protein having a di hydroxynaphthoic acid prenyl transferase activity is a DNA of the following (a) or (b), Process according to any one of claims 1 to 3.
(A) DNA comprising a base sequence represented by SEQ ID NO: 1 , 3, 5, 7, 9, or 11
(B) SEQ ID NO: 1, 3, 5, 7, 9 or consists of the nucleotide sequence and nucleotide sequence having 95% identity with 11, and encoding a protein having a di hydroxynaphthoic acid prenyl transferase activity, DNA to do The production method according to any one of claims 1 to 4 , wherein the microorganism belonging to the genus Arthrobacter or Corynebacterium is a microorganism belonging to Arthrobacter nicotianae or Corynebacterium glutamicum.