JP2009232840A - Method for producing useful substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein active for complementing the alkalinity-susceptibility of coryneform bacteria, a DNA encoding the protein, a recombinant DNA containing the DNA, and a coryneform bacterium genetically transformed by the recombinant DNA, and to provide a method for producing a useful substance. <P>SOLUTION: The protein has an amino acid sequence expressed by SEQ ID NO.1 or 3. The DNA encodes the protein. The recombinant DNA contains the DNA. The coryneform bacterium is genetically transformed by the recombinant DNA. The method for producing a useful substance comprises culturing the genetically transformed coryneform bacterium in a medium, generating and accumulating the useful substance in the medium, and collecting the useful substance from the cultured product. The useful substance includes amino acids and nucleic acids such as glutamine, arginine, etc. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a protein having an activity to restore the alkalinity of an alkaline-sensitive coryneform bacterium, a DNA encoding the protein, a recombinant DNA containing the DNA, and a coryneform transformed with the recombinant DNA The present invention relates to a method for producing bacteria and useful substances.

  Fermentative production of useful substances such as amino acids and nucleic acids at an industrial level is performed using a large-scale fermenter having a volume of several hundred kiloliters. However, in such a large-scale manufacturing site, the ability of the production strain is not exhibited to the maximum, and the result of preliminary examination using a small facility is often not reproduced.

  It is considered that the decrease in productivity of useful substances due to such scale-up is mainly due to a decrease in stirring efficiency proportional to the amount of culture solution. For example, the distribution of pH caused by a decrease in stirring efficiency gives considerable stress to cell physiology, and is a major factor in poor performance and unstable performance.

  As a method for dealing with this problem, it is possible to increase the mixing efficiency of the liquid in the fermenter, but there is a problem in that it requires improvement in terms of equipment and costs. If it becomes possible to deal with strains without relying on equipment, it will be a new technology that improves fermentation productivity.

In Escherichia coli and the like, genes encoding Na ⁺ / H ⁺ antiporter involved in pH homeostasis have been identified, and a method using Escherichia coli (see Patent Document 1) in which those genes are enhanced is known. However, in coryneform bacteria useful for the production of useful substances, such studies have not been conducted, and there is no report on what functions govern pH homeostasis.

JP2007-185184A

  An object of the present invention is to provide a protein having an activity complementary to the alkaline sensitivity of coryneform bacteria, a DNA encoding the protein, a recombinant DNA containing the DNA, and a coryneform bacterium transformed with the recombinant DNA Another object is to provide a method for producing a useful substance.

The present invention relates to the following (1) to (6).
(1) A transformant obtained by incorporating a DNA encoding a protein having an activity to restore the alkalinity of an alkaline-sensitive coryneform bacterium into the coryneform bacterium, and having the ability to produce useful substances.
(2) The transformation according to (1) above, wherein the DNA encoding a protein having an activity to restore the alkalinity of an alkaline-sensitive coryneform bacterium is any one of the following [1] to [3]: body.
[1] DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3
[2] DNA having the base sequence represented by SEQ ID NO: 2 or 4
[3] It hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 or 4, and has an activity of restoring the alkalinity of an alkali-sensitive coryneform bacterium DNA encoding protein
(3) A useful substance characterized by culturing the transformant of (1) or (2) above in a medium, producing and accumulating a useful substance in the culture, and collecting the useful substance from the culture Manufacturing method.
(4) The protein according to any one of [1] to [3] below.
[1] a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3 [2] consisting of 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: 1 or 3; And a protein having an activity to restore the alkalinity of coryneform bacteria sensitive to alkalinity [3] consisting of an amino acid sequence having 80% or more homology or identity with the amino acid sequence represented by SEQ ID NO: 1 or 3, and being alkaline A protein having an activity of restoring the alkaline sensitivity of a sensitive coryneform bacterium (5) DNA of any one of [1] to [3] below.
[1] DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3
[2] DNA having the base sequence represented by SEQ ID NO: 2 or 4
[3] It hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 or 4, and has an activity of restoring the alkalinity of an alkali-sensitive coryneform bacterium DNA encoding protein
(6) A recombinant DNA containing the DNA of (5) above.

  According to the present invention, a protein having an activity to restore the alkalinity of an alkaline-sensitive coryneform bacterium, a DNA encoding the protein, a recombinant DNA containing the DNA, and transformed with the recombinant DNA A method for producing a coryneform bacterium or a useful substance can be provided.

  When the transformant of the present invention is used, productivity and yield of useful substances are improved.

  As a transformant of coryneform bacterium used in the present invention, a DNA encoding a protein (hereinafter also referred to as the protein of the present invention) having an activity of restoring the alkaline sensitivity of an alkali-sensitive coryneform bacterium (hereinafter referred to as the present invention). And a transformant (hereinafter also referred to as a transformant of the present invention) obtained by incorporating a DNA into a coryneform bacterium and capable of producing useful substances such as amino acids and nucleic acids.

As coryneform bacteria, Corynebacterium (Corynebacterium) genus Brevibacterium (Brevibacterium) genus, although a microorganism belonging to the micro Brevibacterium (Microbacterium) genus can be mentioned microorganisms belonging to the genus Corynebacterium are preferably used.

Corynebacterium as the microorganisms belonging to the genus Corynebacterium aceto A Sid Fi Lamb (Corynebacterium acetoacidophilum) ATCC13870 such as Corynebacterium aceto A Sid Fi lamb, Corynebacterium aceto glutamicum (Corynebacteriumacetoglutamicum) ATCC15806 such as Corynebacterium - aceto glutamicum, Corynebacterium Karunae (Corynebacteriumcallunae) ATCC15991 like Corynebacterium Karunae, Corynebacterium glutamicum (Corynebacteriumglutamicum) ATCC13032, Corynebacterium glutamicum ATCC13060, Corynebacterium glutamicum ATCC 13826, Corynebacterium Glutamicum ATCC14020, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum AHP-3 strain (FERM BP-7382), Corynebacterium glutamicum RB26 I strain (WO2006 / 035831), Corynebacterium glutamicum ATCC31833 strain, Corynebacterium · ATCC13032 strain, etc. glutamicum Corynebacterium glutamicum, Corynebacterium Hakyurisu (Corynebacteriumherculis) ATCC13868 such as Corynebacterium Hakyurisu, Korinebakuteri um Ririamu (Corynebacteriumlilium) ATCC15990 like Corynebacterium Ririamu, Corynebacterium Merasekora (Corynebacteriummelassecola) ATCC17965 like Corynebacterium Merasekora, Corynebacterium thermo amino monocytogenes (Corynebacteriumthermoaminogenes) ATCC9244 and the like of Corynebacterium thermo Examples include microorganisms belonging to aminogenes and the like.

As a microorganism belonging to the genus Brevibacterium, Brevibacterium Sacca Lori tee cam (Brevibacterium saccharolyticum) ATCC14066 such as Brevibacterium Sacca Lori tee cam, Brevibacterium such as Brevibacterium Imari office lamb (Brevibacteriumimmariophilum) ATCC14068 - Imari office lamb, Brevibacterium Rozeamu (Brevibacteriumroseum) ATCC13825 Brevibacterium Rozeamu such, is a microorganism belonging to the Brevibacterium Chiogenitarisu (Brevibacteriumthiogenitalis) Brevibacterium Chiogenitarisu such as ATCC19240 and the like.

Examples of microorganisms belonging to the genus Microbacterium include microbacterium ammoniaphilum such as Microbacterium ammoniaphilum ATCC 15354.

  The useful substance that can be produced according to the present invention may be any industrially useful substance that can be produced by microorganisms belonging to coryneform bacteria, such as amino acids, nucleic acids, vitamins, and various enzymes. Examples include proteins, peptides such as glutathione, sugars such as xylose, sugar alcohols such as xylitol, alcohols such as ethanol, organic acids such as lactic acid and succinic acid, lipids, and the like, preferably amino acids, nucleic acids and vitamins, particularly preferably Is an amino acid.

  As amino acids, L-alanine, glycine, L-glutamine, L-glutamic acid, L-asparagine, L-aspartic acid, L-lysine, L-methionine, L-threonine, L-leucine, L-valine, L-isoleucine , L-proline, L-histidine, L-arginine, L-tyrosine, L-tryptophan, L-phenylalanine, L-serine, L-cysteine, L-3-hydroxyproline, L-4-hydroxyproline, etc. Examples of nucleic acids include inosine, guanosine, inosinic acid, guanylic acid, and examples of vitamins include riboflavin, thiamine, and ascorbic acid.

  In the present invention, the coryneform bacterium is “alkaline-sensitive” means that a wild-type coryneform bacterium, preferably Corynebacterium glutamicum ATCC31833 or Corynebacterium glutamicum ATCC13032, more preferably Corynebacterium glutamicum ATCC31833. The strain grows even if the coryneform bacterium cannot grow or can grow in an alkaline medium that can grow (pH 8 to 10, for example, pH 8, 9, 9.25, 9.5, 9.75, 10 etc.). The rate is usually 50% or less, preferably 20% or less, more preferably 10% or less of the wild type strain.

  In a medium for determining whether or not coryneform bacteria are sensitive to alkalinity, the carbon source includes glucose, fructose, sucrose, maltose, starch hydrolysates, etc., alcohols such as ethanol, acetic acid, lactic acid, sucrose. Examples thereof include organic acids such as acids.

  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate and other inorganic and organic ammonium salts, urea, other nitrogen-containing compounds, meat extract, yeast extract, corn steep liquor, soybean hydrolysate And nitrogen-containing organic substances such as

  Examples of inorganic salts include potassium hydrogen phosphate, potassium hydrogen dihydrogen phosphate, ammonium sulfate, sodium chloride, magnesium sulfate, calcium carbonate and the like.

  In addition, you may contain trace nutrient sources, such as biotin and a thiamine, as needed. These micronutrient sources can be substituted with medium additives such as meat extract, yeast extract, corn steep liquor, soybean hydrolysate, and casamino acid.

  In the above alkaline medium, coryneform bacteria are cultured at 20 to 42 ° C., preferably 30 ° C. to 40 ° C. for 1 to 6 days, and the growth rate is examined to determine whether the coryneform bacteria are alkaline or not. Can do.

  As the DNA of the present invention, when introduced into an alkaline-sensitive coryneform bacterium, the activity of recovering the obtained transformant until the growth rate in the alkaline medium is 80% or more, preferably 90% or more of the wild-type strain. DNA encoding a protein having The growth rate can be determined according to a conventional method from the relationship between the culture time and the number of growing bacteria. In addition, when the activity of the protein is reduced or eliminated by, for example, destroying the DNA in the coryneform bacterium having the DNA of the present invention according to a conventional method, the coryneform bacterium is imparted with alkaline sensitivity.

Examples of the protein of the present invention having such activity include the following proteins.
[1] a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3 [2] consisting of 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: 1 or 3; And a protein having the activity to restore the alkaline sensitivity of coryneform bacteria [3] comprising an amino acid sequence having 80% or more homology or identity with the amino acid sequence represented by SEQ ID NO: 1 or 3, and Protein with activity to restore alkaline sensitivity

In the above, a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having an activity of restoring the alkaline sensitivity of coryneform bacteria, 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. Acad. Sci. USA, 82 , 488 (1985) and the like, using a site-directed mutagenesis method, for example, a DNA encoding a protein comprising an amino acid sequence represented by SEQ ID NO: 1 or 3 (for example, In SEQ ID NO: 2 or 4 By introducing site-specific mutations in DNA) having a nucleotide sequence, it 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 it is 1-20, More preferably, it is 1-10, More preferably, it is 1-5.

  In the amino acid sequence represented by SEQ ID NO: 1 or 3, one or more amino acids are deleted, substituted or added. One or more amino acids are deleted, substituted or added at any position in the same sequence. May be.

  Examples of the amino acid position at which amino acid can be deleted or added include 1 to several amino acids on the N-terminal side and C-terminal side of the amino acid sequence represented by SEQ ID NO: 1 or 3.

  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 present invention to have the activity of restoring the alkaline sensitivity of coryneform bacteria, the homology or identity with the amino acid sequence represented by SEQ ID NO: 1 or 3 is 80% or more, preferably 90%. As described above, it is desirable to have homology or identity of 95% or more, more preferably 98% or more, particularly preferably 99% or more.

The homology or identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (1993)] and FASTA [Methods Enzymol., 183 , 63 (1990) by Karlin and Altschul. ] Can be used. 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. In addition, when an amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. To obtain gapped alignment, Gapped BLAST can be used as described in Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects the positional relationship (Id.) Between molecules and the relationship between molecules that share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs can be used. See http://www.ncbi.nlm.nih.gov.

  It has 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, particularly preferably 99% or more of homology or identity with the amino acid sequence represented by SEQ ID NO: 1 or 3. A protein having an amino acid sequence and having an activity of restoring the alkaline sensitivity of coryneform bacteria is also a protein of the present invention.

  The protein of the present invention is a protein having the activity of restoring the alkaline sensitivity of coryneform bacteria, indicating that the growth rate of the alkaline sensitive coryneform bacteria expressing the protein in the alkaline medium is 80% of that of the wild type strain. As described above, it can be confirmed by preferably recovering to 90% or more.

Examples of the DNA of the present invention to be introduced into a coryneform bacterium include the following DNAs [4] to [6].
[4] DNA encoding the protein of the present invention of [1] to [3] above,
[5] Hybridization under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 2 or 4 and [6] DNA having a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2 or 4 DNA encoding a protein that has the activity to restore the alkaline susceptibility of soy and coryneform bacteria

  The DNAs having the base sequences described in SEQ ID NOs: 2 and 4 are DNAs registered as Cgl1281 (NCgl1232) and Cgl1434 (NCgl1379) in DDBJ / GenBank / EMBL, respectively. It encodes a protein having the amino acid sequence represented by SEQ ID NO: 1 described as pertaining to the zinc / cadmium excretion system and a protein having the amino acid sequence represented by SEQ ID NO: 3 described as pertaining to the zinc transporter ing.

  Here, “hybridize” means that the DNA hybridizes to DNA having a specific base sequence or a part of the DNA under specific conditions. 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 has a length 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, for example, Molecular Cloning 2nd edition, 3rd edition (2001), Methods for General and Molecular Bacteriolgy, 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 DNA-immobilized filter and probe DNA, 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6). ), Incubated in a solution containing 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA overnight at 42 ° C., 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 concentration of formamide (the lower the formamide concentration, the lower 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 under which hybridization is performed using a solution having a high salt concentration (for example, 5 × SSC) under the low stringent conditions described above 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, the base sequence represented by SEQ ID NO: 2 or 4 when calculated based on the above parameters using the above-described programs such as BLAST and FASTA. And at least 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more of DNA having a base sequence having homology or identity. .

  The fact that the DNA that hybridizes with the above DNA under stringent conditions encodes a protein having the activity of restoring the alkaline sensitivity of coryneform bacteria means that the DNA is an alkaline sensitive coryneform bacterium and the DNA It can be confirmed by confirming that the growth rate of the transformant obtained by introducing the above in the alkaline medium is 80% or more, preferably 90% or more of the wild type strain.

The DNA of the present invention is represented by SEQ ID NO: 2 or 4 using, for example, a chromosomal DNA prepared from a microorganism belonging to coryneform bacteria according to the method of Saito et al. [Biochim. Biophys. Acta, 72 , 619 (1963)]. It can be obtained by PCR using a primer DNA designed and synthesized based on the base sequence.

  For example, chromosomal DNA is prepared from Corynebacterium glutamicum ATCC13032 strain or ATCC31833 strain, and the 5 ′ end and 3 ′ end region sequences of the respective base sequences represented by SEQ ID NO: 2 or 4 are prepared using the DNA as a template. The DNA of the present invention can be obtained by PCR using the contained DNA as a primer set.

  Specific examples of the DNA that can be obtained include Cgl1281 (NCgl1232) having the base sequence represented by SEQ ID NO: 2, Cgl1434 (NCgl1379) having the base sequence represented by SEQ ID NO: 4, and the like.

  Moreover, based on a hybridization method using a part or all of the DNA consisting of the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 as a probe, or based on the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, This method can also be obtained by a method of chemically synthesizing DNA having the base sequence.

  Further, the DNA sequence encoding the amino acid sequence represented by SEQ ID NO: 1 or 3 with respect to various gene sequence databases and 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98%. % Or more, particularly preferably 99% or more of the sequence having homology or identity, and based on the base sequence obtained by the search, the above-mentioned from the chromosomal DNA, cDNA library, etc. of the organism having the base sequence The DNA of the present invention or the DNA used in the production method of the present invention can also be obtained by the method.

The base sequence of DNA is determined by a commonly used base sequence analysis method such as dideoxy method [Proc. Natl. Acad. Sci., USA, 74 , 5463 (1977)], 373A DNA sequencer (manufactured by Perkin Elmer), etc. It can be determined by analyzing using a base sequence analyzer.

  If the obtained DNA has a partial length as a result of determining the base sequence, the full-length DNA can be obtained by Southern hybridization or the like for a chromosomal DNA library using the partial length DNA as a probe. .

  Prepare the DNA obtained by replacing the base as it is, or, if necessary, so as to be an optimal codon for the expression of coryneform bacteria, and if necessary, an appropriate length including a portion encoding the protein. And is inserted downstream of the promoter of an expression vector suitable for coryneform bacteria to prepare recombinant DNA.

  As the vector, a vector which can autonomously replicate in coryneform bacteria or can be integrated into a chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed is preferably used.

For example, pCG1 (JP 57-134500), pCG2 (JP 58-35197), pCG4 (JP 57-183799), pCG11 (JP 57-134500), pCG116, pCE54, pCB101 (all JP-A-58-105999), pCE51, pCE52, pCE53 [all of which are Molecular and General Genetics, 196 , 175 (1984)] and the like are preferably used.

  The recombinant DNA obtained by incorporating the DNA of the present invention into a vector is preferably composed of a promoter, a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence. A gene that controls the promoter may also be included.

Any promoter can be used as long as it functions in a host cell (coryneform bacterium). For example, trp promoter (P _trp), lac promoter, P _L promoter, P _R promoter, can be mentioned promoters derived from such T7 promoter, E. coli or phage, or the like. Further, artificially designed and modified promoters such as a promoter in which two _Ptrps are connected in series (P _trp × 2), a tac promoter, a lacT7 promoter, and a let I promoter can be used.

P54-6 promoter [Appl. Microbiol. Biotechnol., 53 , 674-679 (2000)] for expression in microorganisms belonging to the genus Corynebacterium can be preferably used as such a promoter. .

  It is preferable that an appropriate distance (for example, 6 to 18 bases) is adjusted between the Shine-Dalgarno sequence that is a ribosome binding sequence and the initiation codon. A transcription termination sequence is not necessarily required, but it is preferable that the transcription termination sequence is located immediately below the structural gene.

  The obtained recombinant DNA is introduced into a coryneform bacterium serving as a host.

As a method for introducing recombinant DNA, any method that can introduce DNA into coryneform bacteria can be used. For example, a method using calcium ion [Proc. Natl. Acad. Sci., USA, 69 , 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electroporation method [Nucleic Acids Res., 16 , 6127 (1988)] and the like.

  The ability to produce a useful substance in the microorganism of the present invention may be an ability to produce one or more useful substances. In the process of breeding, the ability to produce a desired useful substance by a known method as necessary. It may be given or augmented artificially.

As the known method,
(A) a method for mitigating or releasing at least one of the mechanisms controlling biosynthesis of useful substances;
(B) a method for enhancing expression of at least one enzyme involved in biosynthesis of useful substances,
(C) a method of increasing at least one copy number of an enzyme gene involved in biosynthesis of a useful substance,
(D) a method for weakening or blocking at least one metabolic pathway that branches from a biosynthetic pathway of a useful substance to a metabolite other than the useful substance, and (e) a degree of resistance to an analog of the useful substance compared to a wild-type strain To select high cell lines,
The above known methods can be used alone or in combination.

Specific methods of the above (a) to (e) are, for example, when the useful substance is an amino acid, and for the method of (a) above, Agric. Biol. Chem., 43 , 105-111 (1979), J. Bacteriol., 110 , 761-763 (1972) and Appl. Microbiol. Biotechnol., 39 , 318-323 (1993). The method (b) is described in Agric. Biol. Chem., 43 , 105-111 (1979) and J. Bacteriol., 110 , 761-763 (1972). The method (c) is described in Appl. Microbiol. Biotechnol., 39 , 318-323 (1993) and Agric. Biol. Chem., 39 , 371-377 (1987). The method (d) is described in Appl. Environ. Micribiol., 38 , 181-190 (1979) and Agric. Biol. Chem., 42 , 1773-1778 (1978). Regarding the method of (e) above, Agric. Biol. Chem., 36 , 1675-1684 (1972), Agric. Biol. Chem., 41 , 109-116 (1977), Agric. Biol. Chem., 37 , 2013-2023 (1973) and Agric. Biol. Chem., 51 , 2089-2094 (1987). Microorganisms having the ability to generate and accumulate various amino acids can be prepared with reference to the above documents and the like.

Furthermore, for a method for preparing a microorganism having the ability to produce and accumulate amino acids by any one or a combination of the above (a) to (e), Biotechnology 2nd ed., Vol. 6, Products of Primary Metabolism (VCH Verlagsgesellschaft mbH , Weinheim, 1996) section 14a, 14b, Advances in Biochemical Engineering / Biotechnology 79 , 1-35 (2003), Amino Acid Fermentation, Academic Publishing Center, Hiroshi Aida et al. (1986). In addition to the above, methods for preparing microorganisms having the ability to produce and accumulate specific amino acids are disclosed in JP 2003-164297, Agric. Biol. Chem., 39 , 153-160 (1975), Agric. Biol. Chem., 39 , 1149-1153 (1975), JP 58-13599, J. Gen. Appl.Microbiol., 4 , 272-283 (1958), JP 63-94985, Agric. Biol. Chem. , 37 , 2013-2023 (1973), WO97 / 15673, JP-A-56-18596, JP-A-56-144092, and JP-T-2003-511086, etc. A microorganism having the ability to produce the above amino acids can be prepared.

  There have also been many reports on methods for imparting microorganisms with the ability to produce useful substances other than amino acids, and all methods known in the art can be used for the preparation of microorganisms used in the production method of the present invention. .

  DNA encoding a protein having an activity to restore alkaline sensitivity may be incorporated into the chromosome of a coryneform bacterium, or may be introduced into a coryneform bacterium as a plasmid to form a transformant. In addition, DNA encoding a protein having an activity to restore alkaline sensitivity is preferably DNA derived from coryneform bacteria.

  In addition, when a coryneform bacterium used as a host is one that has the ability to produce useful substances such as amino acids and nucleic acids, the resulting transformant can be used as it is for the production of useful substances. In the case of using those that do not have the ability to produce useful substances, a method for introducing other genes related to useful substances into the obtained transformants, introduction of auxotrophic mutations by mutation treatment, It can be used as the transformant of the present invention by imparting the ability to produce useful substances by methods such as introduction of amino acid analog resistance mutations.

  The transformant of the present invention is cultured by a normal culture method for coryneform bacteria.

  As the carbon source, nitrogen source, inorganic salt, and other additives for the culture medium for transformant, the same medium as that for determining alkalinity sensitivity can be used.

  The transformant is cultured under aerobic conditions such as shaking culture and deep aeration stirring culture. The culture temperature is usually 20 ° C to 42 ° C, preferably 30 ° C to 40 ° C. The pH in the medium is preferably maintained at 5-9. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

  The culture period is usually 1 to 6 days. Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.

When culturing coryneform bacteria transformed with recombinant DNA using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when culturing a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like with a recombinant vector using the trp promoter. Indoleacrylic acid or the like may be added to the medium.

  The method for producing the protein of the present invention includes a method for producing in a host cell, a method for secreting it outside the host cell, or a method for producing it on the outer membrane of the host cell. The host cell to be used and the structure of the protein to be produced The method can be selected by changing.

When the protein of the present invention is produced in the host cell or on the outer membrane of the host cell, the method of Paulson et al. [J. Biol. Chem., 264 , 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad Sci. USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], or JP-A-5-336963, WO94 / 23021, etc. It can be actively secreted outside. That is, the protein of the present invention is actively secreted outside the host cell by expressing the polypeptide containing the active site of the protein of the present invention with a signal peptide added in front of the polypeptide using a gene recombination technique. Can be made. Further, according to the method described in JP-A-2-27075, the production amount can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.

  Useful substances are produced by collecting useful substances produced and accumulated in a culture obtained by culturing the transformant of the present invention in a medium.

  As the culture method, the same method as the above-described protein production method of the present invention can be used, but it is preferable to set the optimum conditions depending on the useful substance to be produced.

  Useful substances produced and accumulated in the culture can be recovered by removing precipitates such as bacterial cells and using a known method such as activated carbon treatment or ion exchange resin treatment in combination.

  Examples of the present invention are shown below, but the present invention is not limited to these Examples.

(1) Corynebacterium glutamicum ATCC31833 strain (hereinafter also simply referred to as ATCC31833 strain) was mutated with nitrosoguanidine by a conventional method.

Treated ATCC31833 strain with pH 7.0 MM agar medium (glucose 10 g, magnesium sulfate heptahydrate 0.4 g, ammonium chloride 4 g, urea 2 g, potassium monohydrogen phosphate 3 g, potassium dihydrogen phosphate 1 g, iron sulfate heptahydrate 10 mg, manganese sulfate pentahydrate 1 mg, nicotinic acid 5 mg, biotin 0.1 mg, thiamine hydrochloride 5 mg, CAPS 0.1 mol and bacto agar 20 g in 1 liter of water, hydroxylated It was applied to a medium whose pH was adjusted with an aqueous sodium solution. On the other hand, a MM agar medium at pH 9.5 was prepared, and a strain that deteriorated in growth and a non-growing strain was selected as an alkali-sensitive strain by the replica method. From these selected strains, two mutant strains having different alkalinity sensitivities were selected as representative strains and named AL-43 strain and AL-77 strain, respectively.

  An MM agar medium adjusted to the pH shown in Table 1 was prepared and cultured at 30 ° C. for one day. The growth of these two strains on the agar medium is shown in Table 1.

As shown in Table 1, the AL-43 and AL-77 strains showed alkaline sensitivity.
(2) Chromosomal DNA of ATCC31833 strain was prepared according to the method described in JP-A-6-169785.

On the other hand, plasmids pCSEK20 (republished patent WO01 / 021774) and pCS299P [Appl. Microbiol. Biotech., 63 , 592 (2004)], which can be replicated with Corynebacterium glutamicum, are stored in Corynebacterium glutamicum ATCC31833. It was prepared from the cultured cells of the strain according to the vector preparation method described in JP-A-6-169785.

The plasmid pCSEK20 is the origin of replication of the plasmid pCG2 derived from Corynebacterium glutamicum (JP 58-35197), the spectinomycin of the plasmid pCG4 derived from Corynebacterium glutamicum (JP 57-183799) and This is a plasmid comprising a streptomycin resistance gene and a kanamycin resistance gene of Escherichiacoli 's general vector pCG22 [J. Bacteriol., 140 , 400 (1979)].

The plasmid pCS299P is the origin of replication of the plasmid pCG116 [Bio / Technology, 11 , 921 (1993)] derived from Corynebacterium glutamicum, and the general vector pHSG299 [Gene, 61 , 63 (1987) of Escherichia coli. The plasmid consisting of the kanamycin resistance gene.

  Chromosomal DNA of ATCC31833 strain and plasmid pCSEK20 were each digested with EcoRI, and both digests were ligated with a ligation kit. On the other hand, the chromosomal DNA of ATCC31833 strain and plasmid pCS299P were each digested with BamHI, and both digests were ligated with a ligation kit.

  Using the two types of genomic libraries thus constructed, the alkaline sensitive strains AL-43 and AL-77 obtained in (1) were transformed according to the method described in JP-A-6-169785.

  From the obtained transformants, a strain capable of growing on a MM agar medium at pH 9.5 containing 20 μg / ml of kanamycin was selected as a transformant whose alkaline sensitivity was restored. Plasmid DNA was isolated from the selected transformant according to the vector preparation method described in JP-A-6-169785.

  The obtained plasmid DNA is cleaved with each restriction enzyme and analyzed to obtain a 4.0 Kb EcoRI cleaved fragment as a DNA fragment that restores the alkalinity sensitivity of the AL-43 strain, and the alkalinity sensitivity of the AL-77 strain is restored. A 2.8 Kb BamHI cleavage fragment was obtained as a DNA fragment. The plasmid containing the 4.0 Kb EcoRI fragment thus obtained was named pEco4.0, while the plasmid containing the 2.8 Kb BamHI fragment was named pBam2.8.

  The base sequence of each DNA fragment inserted into pEco4.0 and pBam2.8 was determined by the dideoxynucleotide enzyme method described in the republished patent WO01 / 021774.

  As a result of analyzing the determined nucleotide sequence based on the whole genome information of Corynebacterium glutamicum (accession number BA000036), the 4.0 Kb EcoRI fragment contained in pEco4.0 contains 4 of Cgl1278, Cgl1279, Cgl1280 and Cgl1281. There were two genes. On the other hand, Cgl1434 was present in the 2.8 Kb BamHI fragment contained in pBam2.8.

  As a result of subcloning various DNA fragments from pEco4.0 by a conventional method and examining the ability to recover alkali-sensitivity of the AL-43 strain, the plasmid pCgl1281 containing only the Cgl1281 gene restored the alkali-sensitivity of the AL-43 strain. .

  The plasmid pCgl1281 comprises a 1.9 kb DNA fragment obtained by PCR using the plasmid pEco4.0 as a template and DNA having the nucleotide sequences represented by SEQ ID NOs: 5 and 6 as primers and the vector pCSEK20. This is a plasmid obtained by treating with EcoRI with restriction enzymes and ligating them together.

  The amino acid sequence deduced from the base sequence of the Cgl1281 gene is shown in SEQ ID NO: 1, and the base sequence is shown in SEQ ID NO: 2.

Further, the amino acid sequence deduced from the base sequence of the Cgl1434 gene complementary to the AL-77 strain is shown in SEQ ID NO: 3, and the base sequence is shown in SEQ ID NO: 4.
(3) Obtained by introducing plasmid pBam2.8 into AL-43 / pCgl1281 strain, AL-77 strain and AL-77 strain obtained by introducing plasmid pCgl1281 into AL-43 strain and AL-43 strain The AL-77 / pBam2.8 strain was applied to an MM agar medium prepared at the pH shown in Table 2 and cultured at 30 ° C. for 1 day, and the growth of each strain was examined.

  The results are shown in Table 2.

  As shown in Table 2, the AL-43 / pCgl1281 strain and the AL-77 / pBam2.8 strain recovered alkali sensitivity compared to the host strain.

  Using the plasmids pCgl1281 and pBam2.8 obtained in Example 1, L-glutamine producing bacteria are transformed according to the method described in JP-A-6-169785. As an L-glutamine producing bacterium, for example, ATCC14752 strain (Japanese Examined Patent Publication No. Sho 62-511112) which is a wild type strain of Corynebacterium glutamicum is used.

  Each transformant thus obtained was treated at 28 ° C., for example, in BYG agar medium (glucose 10 g, meat extract 7 g, peptone 10 g, sodium chloride 3 g, yeast extract 5 g, bacto agar 18 g in 1 L of water and adjusted to pH 7.2). Cultured for 24 hours in a platinum culture medium (glucose 150 g, ammonium chloride 50 g, potassium monohydrogen phosphate 0.7 g, potassium dihydrogen phosphate 0.7 g, magnesium sulfate heptahydrate 0.5 g, iron sulfate 7 Hydrate 20 mg, manganese sulfate pentahydrate 20 mg, zinc sulfate heptahydrate 10 mg, biotin 6 μg, thiamine hydrochloride 1 mg, meat extract 5 g in 1 L of water and adjusted to pH 7.2, 50 g of calcium carbonate was added Medium) Inoculate a 300 ml Erlenmeyer flask containing 20 ml and incubate at 28 ° C for 96 hours.

  The cells are removed from the culture by centrifugation, and L-glutamine in the supernatant is recovered.

  Using the plasmids pCgl1281 and pBam2.8 obtained in Example 1, L-arginine-producing bacteria are transformed according to the method described in JP-A-6-169785. As an L-arginine-producing bacterium, for example, RB26I strain of Corynebacterium glutamicum (WO2006 / 035831) is used.

  The obtained transformant was cultured on BYG agar medium at 30 ° C for 24 hours, and 1 platinum loop was cultured on seed medium (25g sucrose, 20g corn steep liquor, 20g peptone, 10g yeast extract, magnesium sulfate. 7 hydrate 0.5 g, potassium dihydrogen phosphate 2 g, urea 3 g, ammonium sulfate 8 g, sodium chloride 1 g, nicotinic acid 20 mg, iron sulfate heptahydrate 10 mg, calcium pantothenate 10 mg, zinc sulfate heptahydrate 1 mg, sulfuric acid Inoculate into a large test tube containing 6 ml of copper pentahydrate 1 mg, thiamine hydrochloride 1 mg, biotin 100 μg in 1 L of water, adjusted to pH 7.2 with sodium hydroxide aqueous solution, and then added with 10 g of calcium carbonate. Incubate at 32 ° C for 24 hours.

  2 ml of the obtained seed culture solution was added to the main culture medium (glucose 60 g, corn steep liquor 5 g, ammonium sulfate 30 g, potassium chloride 8 g, urea 2 g, potassium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate 7 hydrate 1 g, sodium chloride 1 g, iron sulfate heptahydrate 20 mg, nicotinic acid 20 mg, beta-alanine 20 mg, manganese sulfate pentahydrate 10 mg, thiamine hydrochloride 10 mg, biotin 200 μg in 1 L of water, aqueous sodium hydroxide solution After adjusting the pH to 7.7, inoculate a 300 ml Erlenmeyer flask containing 20 ml of a medium supplemented with 30 g of calcium carbonate and incubate at 32 ° C. for 48 hours. The cells are removed from the culture by centrifugation, and L-arginine in the supernatant is recovered.

Using the plasmids pCgl1281 and pBam2.8 obtained in Example 1, the L-lysine producing bacterium Corynebacterium glutamicum AHP-3 strain (FERM BP-7382) was transformed according to the method described in JP-A-6-169785. The obtained transformants were named AHP-3 / pCgl1281 and AHP-3 / pBam2.8, respectively. The L-lysine production ability of each of these transformants and the parent strain was evaluated by conducting the following test tube culture. did. 1 platinum ear of cultured cells cultured at 30 ° C for 24 hours in BY agar medium (medium extract 7g, peptone 10g, sodium chloride 3g, yeast extract 5g, bacto agar 15g in 1L water, adjusted to pH 7.2) , Planted in a thick test tube containing 5 ml of seed medium (20 g of glucose, 7 g of meat extract, 10 g of peptone, 3 g of sodium chloride, 5 g of yeast extract in 1 L of water, adjusted to pH 7.2 and then added with 10 g of calcium carbonate) Bacteria were cultured at 30 ° C for 13 hours. 0.5 ml of this seed culture solution is added to the main culture medium (glucose 50 g, corn steep liquor 10 g, ammonium sulfate 45 g, urea 4 g, potassium dihydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.5 g, biotin 0.3 mg in 1 L of water. After adjusting to pH 7.0, the medium was inoculated into a thick test tube containing 5 ml of a medium supplemented with 30 g of calcium carbonate, and cultured with shaking at 30 ° C. for 72 hours. The cells were removed from the culture by centrifugation, and the amount of L-lysine hydrochloride accumulated in the supernatant was quantified by high performance liquid chromatography (HPLC). The results are shown in Table 3.

  As is apparent from Table 3, the AHP-3 strain into which the plasmid containing the gene of the present invention was introduced had an improved production amount of L-lysine per cell compared to the parent strain AHP-3.

  Using the plasmids pCgl1281 and pBam2.8 obtained in Example 1, L-arginine-producing bacteria were transformed according to the method described in JP-A-6-169785. As an L-arginine-producing bacterium, Corynebacterium glutamicum RB26I strain (WO2006 / 035831) was used.

  The L-arginine production test of each of these transformed strains and the parent strain was performed by conical flask culture as follows. 1 platinum ear of cultured cells cultured on BY agar medium at 30 ° C for 24 hours, seed medium (sucrose 25g, corn steep liquor 20g, peptone 20g, yeast extract 10g, magnesium sulfate heptahydrate 0.5g, phosphoric acid Potassium dihydrogen 2g, urea 3g, ammonium sulfate 8g, sodium chloride 1g, nicotinic acid 20mg, iron sulfate heptahydrate 10mg, calcium pantothenate 10mg, zinc sulfate heptahydrate 1mg, copper sulfate pentahydrate 1mg, thiamine hydrochloride The medium was inoculated into a large test tube containing 6 ml of a salt containing 1 mg of salt and 100 μg of biotin in 1 L of water and adjusted to pH 7.2, and then added with 10 g of calcium carbonate, and cultured at 32 ° C. for 24 hours. 2 ml of this seed culture solution was added to the main culture medium (glucose 60 g, corn steep liquor 5 g, ammonium sulfate 30 g, potassium chloride 8 g, urea 4 g, potassium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate. 1 g, sodium chloride 1 g, iron sulfate heptahydrate 20 mg, nicotinic acid 20 mg, beta-alanine 20 mg, manganese sulfate pentahydrate 10 mg, thiamine hydrochloride 10 mg, biotin 200 μg in 1 L of water and adjusted to pH 7.7, then carbonated Medium in which 30 g of calcium was added) A 300 ml Erlenmeyer flask containing 20 ml was inoculated and cultured at 32 ° C. for 48 hours. The cells were removed from the culture by centrifugation, and the amount of L-arginine accumulated in the supernatant was quantified by high performance liquid chromatography (HPLC). The results are shown in Table 4.

  As is apparent from Table 4, in the RB26I strain into which the plasmid containing the gene of the present invention was introduced, the production amount of L-arginine per cell was improved compared to the parent strain RB26I.

  According to the present invention, a protein having an activity to restore the alkalinity of an alkaline-sensitive coryneform bacterium, a DNA encoding the protein, a recombinant DNA containing the DNA, and transformed with the recombinant DNA A method for producing a coryneform bacterium or a useful substance can be provided.

  The transformant of the coryneform bacterium of the present invention improves the productivity of useful substances.

SEQ ID NO: 5-description of artificial sequence: synthetic DNA
SEQ ID NO: 6 Description of Artificial Sequence: Synthetic DNA

Claims (2)

A transformant obtained by incorporating a DNA encoding a protein having an activity to restore the alkalinity of an alkaline sensitive coryneform bacterium into the coryneform bacterium and capable of producing useful substances is cultured in a medium, A useful substance is produced and accumulated, and the useful substance is collected from the culture. The production of a useful substance according to claim 1, wherein the DNA encoding a protein having an activity to restore the alkalinity of coryneform bacteria sensitive to alkalinity is the DNA according to any one of [1] to [3] below. Law.
[1] DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3
[2] DNA having the base sequence represented by SEQ ID NO: 2 or 4
[3] It hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 or 4, and has an activity of restoring the alkalinity of an alkali-sensitive coryneform bacterium DNA encoding protein