L-Amino Acid-Producing Bacterium and Method for Producing L-Amino Acid

- AJINOMOTO CO., INC.

An L-amino acid is produced by culturing a Methylophilus bacterium which can grow by using methanol as the main carbon source and has L-amino acid-producing ability, for example, a Methylophilus bacterium in which dihydrodipicolinate synthase activity and aspartokinase activity are enhanced by transformation of cells with a DNA coding for dihydrodipicolinate synthase that is desensitized to feedback inhibition by L-lysine and a DNA coding for aspartokinase that is desensitized to feedback inhibition by L-lysine, or a Methylophilus bacterium which is casamino acid auxotrophic, in a medium containing methanol as a main carbon source, to produce and accumulate an L-amino acid in culture, and collecting the L-amino acid from the culture.

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Description

This application is a Continuation of, and claims priority under 35 U.S.C. §120 to, U.S. patent application Ser. No. 11/738,617, filed Apr. 23, 2007, which was a Divisional of, and claims priority under U.S.C. §120 to, U.S. patent application Ser. No. 09/926,299, filed Oct. 9, 2001, now U.S. Pat. No. 7,223,572, issued May 29, 2007, which was a U.S. national phase filing under 35 U.S.C. §371 of PCT Patent Application No. PCT/JP00/02295, filed Apr. 7, 2000, which in turn claimed priority under 35 U.S.C. §119 to Japanese Patent Application 11-103143, filed Apr. 9, 1999, Japanese Patent Application 11-169447, filed Jun. 16, 1999, and Japanese Patent Application 11-368097, filed Dec. 24, 1999. These applications are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to techniques useful in the microbial industry. In particular, the present invention relates to a method for producing an L-amino acid by fermentation, and a microorganism which can be used in the method.

2. Brief Description of the Related Art

Amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine, L-isoleucine, L-valine, and L-phenylalanine are typically produced in industry by fermentation using microorganisms that belong to the genus Brevibacterium, Corynebacterium, Bacillus, Escherichia, Streptomyces, Pseudomonas, Arthrobacter, Serratia, Penicillium, Candida, or the like. In order to improve productivity, microorganism strains isolated from nature or artificial mutants thereof have typically been used. To increase the ability to produce L-glutamic acid, various techniques have been disclosed to enhance or increase the activities of L-glutamic acid biosynthetic enzymes using recombinant DNA techniques.

The production of L-amino acids has been considerably increased by breeding microorganisms such as those mentioned above. However, in order to meet increased demand in the future, development of more efficient production methods at lower cost are desirable.

Methanol is a raw material useful in fermentation since it is readily available and inexpensive. Known methods using methanol in fermentation typically use microorganisms that belong to the genus Achromobacter or Pseudomonas (Japanese Patent Publication (Kokoku) No. 45-25273/1970), Protaminobacter (Japanese Patent Application Laid-open (Kokai) No. 49-125590/1974), Protaminobacter or Methanomonas (Japanese Patent Application Laid-open (Kokai) No. 50-25790/1975), Microcyclus (Japanese Patent Application Laid-open (Kokai) No. 52-18886/1977), Methylobacillus (Japanese Patent Application Laid-open (Kokai) No. 4-91793/1992), Bacillus (Japanese Patent Application Laid-open (Kokai) No. 3-505284/1991), and so forth.

To date, however, the use of Methylophilus bacteria in production of L-amino acids has not been reported. Although the methods described in EP 0 035 831 A, EP 0 037 273 A, and EP 0 066 994 A are methods for transforming Methylophilus bacteria using recombinant DNA, the use of such techniques to improve the amino acid productivity of Methylophilus bacteria has not been reported.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel bacterium which is able to produce L-amino acids, and a method for producing an L-amino acid by using the L-amino acid-producing bacterium.

As a result of the present inventors' efforts to achieve the aforementioned objectives, is was found that Methylophilus bacteria were suitable for producing L-amino acids. Furthermore, although it has conventionally been considered difficult to obtain auxotrophic mutants of Methylophilus bacteria (FEMS Microbiology Rev. 39, 235-258 (1986) and Antonie van Leeuwenhoek 53, 47-53 (1987)), the present inventors have succeeded in obtaining auxotrophic mutants of said bacteria. Thus, the present invention has been accomplished.

That is, the present invention provides the following.

It is an object of the present invention to provide a Methylophilus bacterium having L-amino acid-producing ability.

It is an object of the present invention to provide the Methylophilus bacterium as described above, wherein the L-amino acid is L-lysine, L-valine, L-leucine, L-isoleucine, or L-threonine.

It is an object of the present invention to provide the Methylophilus bacterium as described above, which has resistance to an L-amino acid analogue or L-amino acid auxotrophy.

It is an object of the present invention to provide the Methylophilus bacterium as described above, wherein L-amino acid biosynthetic enzyme activity is enhanced.

It is an object of the present invention to provide the Methylophilus bacterium as described above, wherein dihydrodipicolinate synthase activity and aspartokinase activity are enhanced, and the bacterium has L-lysine-producing ability.

It is an object of the present invention to provide the Methylophilus bacterium as described above, wherein dihydrodipicolinate synthase activity is enhanced, and the bacterium has L-lysine-producing ability.

It is an object of the present invention to provide the Methylophilus bacterium as described above, wherein aspartokinase activity is enhanced, and the bacterium has L-lysine-producing ability.

It is an object of the present invention to provide the Methylophilus bacterium as described above, wherein an activity or activities of one, two, or three enzymes selected from aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate reductase, and diaminopimelate decarboxylase is/are enhanced.

It is an object of the present invention to provide the Methylophilus bacterium as described above, wherein the dihydrodipicolinate synthase activity and the aspartokinase activity are enhanced by transformation through introduction into cells, of a DNA coding for dihydrodipicolinate synthase that is not subject to feedback inhibition by L-lysine and a DNA coding for aspartokinase that is not subject to feedback inhibition by L-lysine.

It is an object of the present invention to provide the bacterium as described above, wherein activities of aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase, is/are enhanced, and the bacterium has L-threonine-producing ability.

It is an object of the present invention to provide the bacterium as described above, wherein the Methylophilus bacterium is Methylophilus methylotrophus.

It is an object of the present invention to provide a method for producing an L-amino acid, which comprises culturing a Methylophilus bacterium as described above in a medium to produce and accumulate an L-amino acid in culture and collecting the L-amino acid from the culture.

It is an object of the present invention to provide the method as described above, wherein the medium contains methanol as a main carbon source.

It is an object of the present invention to provide a method for producing bacterial cells of a Methylophilus bacterium with an increased content of an L-amino acid, which comprises culturing a Methylophilus bacterium as described above in a medium to produce and accumulate an L-amino acid in bacterial cells of the bacterium.

It is an object of the present invention to provide the method for producing bacterial cells of the Methylophilus bacterium as described above, wherein the L-amino acid is L-lysine, L-valine, L-leucine, L-isoleucine or L-threonine.

It is an object of the present invention to provide a DNA which codes for a protein defined in the following (A) or (B):

(A) a protein which has the amino acid sequence of SEQ ID NO: 6, or

(B) a protein which has an amino acid sequences of SEQ ID NO: 6 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has aspartokinase activity.

It is an object of the present invention to provide the DNA as described above, which is a DNA defined in the following (a) or (b):

(a) a DNA which has a nucleotide sequence comprising the nucleotide sequence of the nucleotide numbers 510 to 1736 of SEQ ID NO: 5; or

(b) a DNA which is hybridizable with a probe having the nucleotide sequence of the nucleotide numbers 510 to 1736 of SEQ ID NO: 5 or a part thereof under a stringent condition, and codes for a protein having aspartokinase activity.

It is an object of the present invention to provide a DNA which codes for a protein defined in the following (C) or (D):

(C) a protein which has the amino acid sequence of SEQ ID NO: 8, or

(D) a protein which has an amino acid sequences of SEQ ID NO: 8 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has aspartic acid semialdehyde dehydrogenase activity.

It is an object of the present invention to provide the DNA as described above, which is a DNA defined in the following (c) or (d):

(c) a DNA which has a nucleotide sequence comprising the nucleotide sequence of the nucleotide numbers 98 to 1207 of SEQ ID NO: 7; or

(d) a DNA which is hybridizable with a probe having the nucleotide sequence of the nucleotide numbers 98 to 1207 of SEQ ID NO: 7 or a part thereof under a stringent condition, and codes for a protein having aspartic acid semialdehyde dehydrogenase activity.

It is an object of the present invention to provide a DNA which codes for a protein defined in the following (E) or (F):

(E) a protein which has the amino acid sequence of SEQ ID NO: 10, or

(F) a protein which has an amino acid sequences of SEQ ID NO: 10 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate synthase activity.

It is an object of the present invention to provide the DNA as described above, which is a DNA defined in the following (e) or (f):

(e) a DNA which has a nucleotide sequence comprising the nucleotide sequence of the nucleotide numbers 1268 to 2155 of SEQ ID NO: 9; or

(f) a DNA which is hybridizable with a probe having the nucleotide sequence of the nucleotide numbers 1268 to 2155 of SEQ ID NO: 9 or a part thereof under a stringent condition, and codes for a protein having dihydrodipicolinate synthase activity.

It is an object of the present invention to provide a DNA which codes for a protein defined in the following (G) or (H):

(G) a protein which has the amino acid sequence of SEQ ID NO: 12, or

(H) a protein which has an amino acid sequences of SEQ ID NO: 12 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate reductase activity.

It is an object of the present invention to provide the DNA as described above, which is a DNA defined in the following (g) or (h):

(g) a DNA which has a nucleotide sequence comprising the nucleotide sequence of the nucleotide numbers 2080 to 2883 of SEQ ID NO: 11; or

(h) a DNA which is hybridizable with a probe having the nucleotide sequence of the nucleotide numbers 2080 to 2883 of SEQ ID NO: 11 or a part thereof under a stringent condition, and codes for a protein having dihydrodipicolinate reductase activity.

It is an object of the present invention to provide a DNA which codes for a protein defined in the following (I) or (J):

(I) a protein which has the amino acid sequence of SEQ ID NO: 14, or

(J) a protein which has an amino acid sequences of SEQ ID NO: 14 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has diaminopimelate decarboxylase activity.

It is an object of the present invention to provide the DNAas described above, which is a DNA defined in the following (i) or (j):

(i) a DNA which has a nucleotide sequence comprising the nucleotide sequence of the nucleotide numbers 751 to 1995 of SEQ ID NO: 13; or

(j) a DNA which is hybridizable with a probe having the nucleotide sequence of the nucleotide numbers 751 to 1995 of SEQ ID NO: 13 or a part thereof under a stringent condition, and codes for a protein having diaminopimelate decarboxylase activity.

In the present specification, “L-amino acid-producing ability” refers to the ability to accumulate a significant amount of an L-amino acid in a medium or to increase the amino acid content in the microbial cells when a microorganism of the present invention is cultured in the medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the production process for the plasmid RSF24P, which includes the mutant dapA. The term “dapA*24” refers to the mutant dapA that codes for the mutant DDPS, wherein the 118-histidine residue is replaced with a tyrosine residue.

FIG. 2 shows the production process for the plasmid RSFD80 which includes the mutant dapA and mutant lysC. The term “lysC*80” refers to the mutant lysC that codes for the mutant AKIII, wherein the 352-threonine residue is replaced with an isoleucine residue.

FIG. 3 shows the aspartokinase activity of E. coli strains transformed with the ask gene.

FIG. 4 shows the aspartic acid semialdehyde dehydrogenase activity of E. coli strains transformed with the asd gene.

FIG. 5 shows the dihydrodipicolinate synthase activity of E. coli strains transformed with the dapA gene.

FIG. 6 shows the dihydrodipicolinate reductase activity of an E. coli strain transformed with the dapB gene.

FIG. 7 shows the diaminopimelate decarboxylase activity of E. coli strains transformed with the lysA gene.

 DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

<1> Microorganism of the Present Invention

The microorganism of the present invention is a bacterium belonging to the genus Methylophilus which is able to produce L-amino acids. The Methylophilus bacterium of the present invention includes, for example, Methylophilus methylotrophus AS1 strain (NCIMB10515) and so forth. This strain is available from the National Collections of Industrial and Marine Bacteria (Address: NCIMB Lts., Torry Research Station 135, Abbey Road, Aberdeen AB9 8DG, United Kingdom).

L-amino acids which can be produced according to the present invention include L-lysine, L-glutamic acid, L-threonine, L-valine, L-leucine, L-isoleucine, L-tryptophan, L-phenylalanine, L-tyrosine, and so forth. One or more types of such amino acids may be produced.

Methylophilus bacteria which are able to produce L-amino acids can be obtained by imparting this L-amino acid-producing ability to wild-type strains of the Methylophilus bacteria. In order to impart L-amino acid-producing ability, methods conventionally used for breeding coryneform bacteria, Escherichia bacteria, or the like, may be used. These methods include breeding auxotrophic mutant strains, strains resistant to L-amino acid analogues or metabolic control mutant strains, and methods for producing recombinant strains wherein L-amino acid biosynthetic enzyme activities are enhanced (see “Amino Acid Fermentation”, the Japan Scientific Societies Press [Gakkai Shuppan Center], 1st Edition, published on May 30, 1986, pp. 77 to 100). When breeding amino acid-producing bacteria, characteristics such as auxotrophy, L-amino acid analogue resistance, and metabolic control mutations may be imparted alone or in combination. One or more L-amino acid biosynthetic enzymes may be enhanced, and/or one or more of the methods mentioned above may be combined with enhancing one or more of the biosynthetic enzymes. For example, bacteria which produce L-lysine are bred to be auxotrophic for L-homoserine or L-threonine, and L-methionine (Japanese Patent Publication (Kokoku) Nos. 48-28078/1973 and 56-6499/1981), inositol or acetic acid (Japanese Patent Application Laid-open (Kokai) Nos. 55-9784/1980 and 56-8692/1981). These bacteria have also been bred to be resistant to oxalysine, lysine hydroxamate, S-(2-aminoethyl)-cysteine, β-methyllysine, α-chlorocaprolactam, DL-α-amino-ε-caprolactam, α-amino-lauryllactam, aspartic acid analogues, sulfa drugs, quinoid, or N-lauroylleucine.

Furthermore, L-glutamic acid-producing bacteria can be bred as mutants which are auxotrophic for oleic acid or the like. Bacteria which produce L-threonine can be bred to be resistant to α-amino-β-hydroxyvaleric acid. Bacteria which produce L-homoserine can be bred to be auxotrophic for L-threonine, or to be resistant to L-phenylalanine analogues. Bacteria which produce L-phenylalanine can be bred to be auxotrophic for L-tyrosine. Bacteria which produce L-isoleucine can be bred to be auxotrophic for L-leucine. Bacteria which produce L-proline can be bred to be auxotrophic for L-isoleucine.

Furthermore, strains that produce one or more kinds of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine) can be bred to be auxotrophic for casamino acid.

In order to obtain mutants of Methylophilus bacteria, the optimal mutagenesis conditions were examined using emergence frequency of streptomycin-resistant strains as an index. As a result, the maximum emergence frequency of streptomycin resistant strains was obtained when the survival rate after mutagenesis was about 0.5%. Therefore, auxotrophic strains were obtained under these conditions. Auxotrophic strains were also obtained by screening mutants on a large scale, which had been previously reported to be difficult, as compared with that previously reported for E. coli and so forth.

As described above, since mutants can be obtained by mutagenizing Methylophilus bacteria under suitable conditions, it is possible to readily obtain desirable mutants by suitably setting conditions that result in a survival rate after mutagenesis of about 0.5%, depending on the mutagenesis method.

Mutagenesis methods for obtaining mutants of Methylophilus bacteria include UV irradiation and treatments with typical mutagenesis agents, such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid. Methylophilus bacteria which are able to produce L-amino acids can also be obtained by selecting naturally occurring mutants of Methylophilus bacteria.

Mutants resistant to L-amino acid analogues can be obtained by, for example, inoculating mutagenized Methylophilus bacteria into agar medium containing an L-amino acid analogue at a variety of concentrations, and selecting for bacterial colonies.

Auxotrophic mutants can be obtained by allowing Methylophilus bacteria to form colonies on an agar medium containing a target nutrient (for example, an L-amino acid), replicating the colonies on an agar medium which does not contain said nutrient, and selecting strains that cannot grow on this agar medium.

Methods for imparting, enhancing, and/or increasing the ability to produce L-amino acids by enhancing L-amino acid biosynthetic enzyme activity is exemplified below.

L-lysine

The ability to produce L-lysine can be imparted by, for example, enhancing dihydrodipicolinate synthase activity and/or aspartokinase activity.

The dihydrodipicolinate synthase and/or the aspartokinase activity in Methylophilus bacteria can be enhanced by ligating a gene fragment coding for dihydrodipicolinate synthase and/or a gene fragment coding for aspartokinase with a vector that functions in Methylophilus bacteria, preferably a multiple-copy type vector, to create a recombinant DNA. This recombinant DNA is then used to transform a Methylophilus bacterium. As a result of the increase in the copy number of the gene coding for dihydrodipicolinate synthase and/or the gene coding for aspartokinase in the transformant strain, the activity or activities thereof is/are enhanced. Hereinafter, dihydrodipicolinate synthase, aspartokinase, and aspartokinase III are also referred to as DDPS, AK, and AKIII, respectively.

Any microorganism can be used to provide the genes encoding for DDPS and/or AK, so long as the chosen microorganism has genes enabling expression of DDPS activity and AK activity in Methylophilus microorganisms. Such microorganisms may be wild-type strains or mutant strains derived therefrom. Specifically, examples of such microorganisms include E. coli (Escherichia coli) K-12 strain, Methylophilus methylotrophus AS1 strain (NCIMB10515), and so forth. Since the nucleotide sequences of the gene coding for DDPS (dapA, Richaud, F. et al., J. Bacteriol., 297, (1986)) and the gene coding for AKIII (lysC, Cassan, M., Parsot, C., Cohen, G. N. and Patte, J. C., J. Biol. Chem., 261, 1052 (1986)) native to and derived from Escherichia bacteria have both been reported, these genes can be obtained by PCR using primers synthesized based on the nucleotide sequences of these genes and chromosomal DNA of a microorganism such as E. coli K-12, or the like, as the template. As specific examples, dapA and lysC derived from E. coli will be explained below. However, genes used in the present invention are not limited to these.

It is preferred that DDPS and AK are not inhibited by L-lysine, i.e. feedback inhibition. It has been reported that wild-type DDPS derived from E. coli is subject to such feedback inhibition by L-lysine, and that wild-type AKIII derived from E. coli is also subject to suppression and feedback inhibition by L-lysine. Therefore, the dapA and lysC which are used to transform Methylophilus bacteria preferably code for DDPS and AKIII which have a mutation that desensitizes this feedback inhibition. Hereinafter, the DDPS which has a mutation that desensitizes feedback inhibition by L-lysine is also referred to as “mutant DDPS”, and the DNA coding for this mutant DDPS is also referred to as “mutant dapA”. AKIII derived from E. coli which has a mutation that desensitizes feedback inhibition by L-lysine is also referred to as “mutant AKIII”, and the DNA coding for this mutant AKIII is also referred to as “mutant lysC”.

According to the present invention, DDPS and AK are not necessarily required to be a mutated as such. It is known that, for example, DDPS native to Corynebacterium bacteria does not suffer feedback inhibition by L-lysine.

The nucleotide sequence of wild-type dapA native to E. coli is shown in SEQ ID NO: 1. The amino acid sequence of wild-type DDPS coded by this nucleotide sequence is shown in SEQ ID NO: 2. The nucleotide sequence of wild-type lysC native to E. coli is shown in SEQ ID NO: 3. The amino acid sequence of wild-type ATIII coded by this nucleotide sequence is shown in SEQ ID NO: 4.

The DNA coding for mutant DDPS that is not subject to feedback inhibition by L-lysine includes the DNA coding for DDPS having the amino acid sequence shown in SEQ ID NO: 2, but wherein the 118-histidine residue is replaced with a tyrosine residue. The DNA coding for mutant AKIII that is not subject to feedback inhibition by L-lysine includes a DNA coding for AKIII having the amino sequence shown in SEQ ID NO: 4, but wherein the 352-threonine residue is replaced with an isoleucine residue.

Any plasmid may be used for gene cloning, so long as it can replicate in microorganisms such as Escherichia bacteria or the like. Specifically, useful plasmids may include pBR322, pTWV228, pMW119, pUC19, and so forth.

Vectors that function in Methylophilus bacteria include, for example, plasmids that can autonomously replicate in Methylophilus bacteria. Specifically, RSF1010, which is a broad host spectrum vector, and derivatives thereof, for example, pAYC32 (Chistorerdov, A. Y., Tsygankov, Y. D. Plasmid, 16, 161-167, (1986)), pMFY42 (Gene, 44, 53, (1990)), pRP301, pTB70 (Nature, 287, 396, (1980)), and so forth, may be used.

The chosen vector may be digested with a restriction enzyme that corresponds to the terminus of the DNA fragments containing dapA and lysC. The vector is then ligated to the gene fragments, and ligation is usually performed with a ligase such as T4 DNA ligase. dapA and lysC may be individually ligated into separate vectors or into a single vector.

The plasmid containing mutant dapA coding for mutant DDPS and mutant lysC coding for mutant AKIII has been reported. This plasmid is the broad host spectrum plasmid RSFD80 (WO95/16042). E. coli JM109 strain transformed with this plasmid was designated AJ12396, and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Oct. 28, 1993 and received an accession number of FERM P-13936, and it was converted to an international deposit under the provisions of the Budapest Treaty on Nov. 1, 1994, and received an accession number of FERM BP-4859. RSFD80 can be obtained from the AJ12396 strain using known techniques.

The mutant dapA from RSFD80 has a substitution of the C at nucleotide 597 in the wild-type dapA (SEQ ID NO: 1), with a T. As a result, the encoded mutant DDPS has a tyrosine at position 118 in SEQ ID NO: 2 instead of the native histidine. The mutant lysC from RSFD80 has a substitution of the C at nucleotide 1638 in the wild-type lysC (SEQ ID NO: 3), with a T. As a result, the encoded mutant AKIII has an isoleucine at position 352 instead of the native threonine.

Any method can be used to introduce the recombinant DNA prepared as described above into the Methylophilus bacteria, so long as sufficient transformation efficiency is acheived. For example, electroporation can be used (Canadian Journal of Microbiology, 43, 197 (1997)).

The DDPS activity and/or the AK activity can also be enhanced by the presence of multiple copies of dapA and/or lysC on the chromosomal DNA of Methylophilus bacteria. Homologous recombination can be used to introduce multiple copies of dapA and/or lysC into chromosomal DNA of Methylophilus bacteria. As a target, a sequence that is present in multiple copies on the chromosomal DNA of Methylophilus bacteria can be used, such as repetitive DNA, inverted repeats present at the end of a transposable element, or the like. Alternatively, as disclosed in Japanese Patent Application Laid-open (Kokai) No. 2-109985/1990, multiple copies of dapA and/or lysC can be transfered to the chromosomal DNA using a transposon. In both of these methods, the DDPS and AK activities should be increased by increasing the copy numbers of dapA and/or lysC.

Other than increasing the gene copy numbers, the DDPS activity and/or the AK activity can be amplified by replacing an expression control sequence such as the promoters of dapA and/or lysC with stronger ones (Japanese Patent Application Laid-open (Kokai) No. 1-215280/1989). Strong promoters are known in the art and include, for example, the lac promoter, trp promoter, trc promoter, tac promoter, PR promoter and PL promoter of lambda phage, tet promoter, amyE promoter, spac promoter, and so forth. Substitution of native promoters with these stronger promoters enhances the expression of dapA and/or lysC, and thus the DDPS activity and the AK activity are amplified. Enhancing the expression control sequences can be combined with increasing the copy numbers of dapA and/or lysC.

In order to prepare a recombinant DNA of a gene fragment and a vector, the vector is digested with a restriction enzyme corresponding to the terminus of the gene fragment, and ligation is performed using a ligase such as T4 ligase. The methods used for digestion, ligation, preparation of chromosomal DNA, PCR, preparation of plasmid DNA, transformation, design of oligonucleotides used as primers and so forth, can be typical methods well known to those skilled in the art. Such methods are described in Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning: A Laboratory Manual, 2nd Edition”, Cold Spring Harbor Laboratory Press, (1989) and so forth.

In addition to enhancing the DDPS activity and/or the AK activity, the activities of other enzymes involved in the L-lysine biosynthesis may also be enhanced. Such enzymes include diaminopimelate pathway enzymes such as dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase (WO96/40934 for all of the foregoing enzymes), phosphoenolpyruvate carboxylase (Japanese Patent Application Laid-open (Kokai) No. 60-87788/1985), aspartate aminotransferase (Japanese Patent Publication (Kokoku) No. 6-102028/1994), diaminopimelate epimerase, aspartic acid semialdehyde dehydrogenase and so forth, or aminoadipate pathway enzymes such as homoaconitate hydratase and so forth. Preferably, the activity of at least aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate reductase, and/or diaminopimelate decarboxylase is/are enhanced.

Aspartokinase, aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, and diaminopimelate decarboxylase derived from Methylophilus methylotrophus will be described later.

Furthermore, the activity of an enzyme that catalyzes a reaction that generates a compound other than L-lysine via a branch of the L-lysine biosynthetic pathway may be decreased in the chosen host microorganism. An example of such an enzyme is homoserine dehydrogenase (see WO95/23864).

The aforementioned techniques for enhancing enzyme activity can be similarly used for other amino acids, as mentioned below.

L-glutamic acid

The ability to produce L-glutamic acid can be imparted to Methylophilus bacteria by, for example, introducing a DNA that codes for any one of the following enzymes: glutamate dehydrogenase (Japanese Patent Application Laid-open (Kokai) 61-268185/1986), glutamine synthetase, glutamate synthase, isocitrate dehydrogenase (Japanese Patent Application Laid-open (Kokai) Nos. 62-166890/1987 and 63-214189/1988), aconitate hydratase (Japanese Patent Application Laid-open (Kokai) No. 62-294086/1987), citrate synthase (Japanese Patent Application Laid-open (Kokai) Nos. 62-201585/1987 and 63-119688/1988), phosphoenolpyruvate carboxylase (Japanese Patent Application Laid-open (Kokai) Nos. 60-87788/1985 and 62-55089/1987), pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase (Japanese Patent Application Laid-open (Kokai) No. 63-102692/1988), glucose phosphate isomerase, glutamine-oxoglutarate aminotransferase (WO99/07853), and so forth.

Furthermore, the activity of an enzyme that catalyzes a reaction that generates a compound other than L-glutamic acid via a branch of the L-glutamic acid biosynthetic pathwaymay be decreased in the chosen host. Examples of such enzymes are α-ketoglutarate dehydrogenase (αKGDH), isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and so forth.

L-threonine

The ability to produce L-threonine can be imparted or increased by, for example, enhancing the activities of aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase. The activities of these enzymes can be enhanced by, for example, transforming Methylophilus bacteria with a recombinant plasmid containing the threonine operon (Japanese Patent Application Laid-open (Kokai) Nos. 55-131397/1980, 59-31691/1984 and 56-15696/1981 and Japanese Patent Application Laid-open (Kohyo) No. 3-501682/1991).

Production of L-threonine can also be imparted or enhanced by amplifying or introducing a threonine operon which includes the gene coding for aspartokinase which has been desensitized to feedback inhibition by L-threonine (Japanese Patent Publication (Kokoku) No. 1-29559/1989), the gene coding for homoserine dehydrogenase (Japanese Patent Application Laid-open (Kokai) No. 60-012995/1985), or the genes coding for homoserine kinase and homoserine dehydrogenase (Japanese Patent Application Laid-open (Kokai) No. 61-195695/1986).

Furthermore, the production of L-threonine can be improved by introducing a DNA coding for a mutant phosphoenolpyruvate carboxylase which has been desensitized to feedback inhibition by aspartic acid.

L-valine

The ability to produce L-valine can be imparted by, for example, introducing into Methylophilus bacteria an L-valine biosynthesis gene with a substantially desensitized control mechanism. A mutation that substantially desensitizes the control mechanism of an L-valine biosynthesis gene in Methylophilus bacteria may also be introduced.

Examples of the L-valine biosynthesis gene include, for example, the ilvGMEDA operon of E. coli. Threonine deaminase encoded by the ilvA gene catalyzes the deamination reaction which results in conversion of L-threonine to 2-ketobutyric acid, which is the rate-determining step of L-isoleucine biosynthesis. Therefore, in order for efficient progression of the L-valine synthesis reactions, it is preferable to use an operon that does not express threonine deaminase activity. Examples of such an ilvGMEDA operon include the ilvGMEDA operon with an inactive ilvA gene, whether the gene is mutated, disrupted, or deleted. Since L-valine, L-isoleucine, and/or L-leucine attenuate expression of the ilvGMEDA operon, the region causing the attenuation is preferably removed or mutated so to desensitize this attenuation by L-valine.

An ilvGMEDA operon which does not express threonine deaminase activity and and with desensitized attenuation as described above can be obtained by subjecting the wild-type ilvGMEDA operon to mutagenesis or modifying it using gene recombination techniques (see WO96/06926).

L-leucine

The ability to produce L-leucine is imparted or enhanced by, for example, introducing into a Methylophilus bacteria an L-leucine biosynthesis gene with a substantially desensitized control mechanism, in addition to the above-described manipulations required for the production of L-valine. It is also possible to eliminate the control mechanism of an L-leucine biosynthesis gene in Methylophilus via mutation. A mutation in the leuA gene which substantially eliminates inhibition by L-leucine is one example.

L-isoleucine

The ability to produce L-isoleucine can be imparted by, for example, substantially desensitizing the host microorganism to inhibition by L-threonine by introducing the thrABC operon with the E. coli thrA gene coding for aspartokinase I/homoserine dehydrogenase I. Also, inhibition by L-isoleucine can be substantially desensitized in the host by removing the region of the ilvA gene required for attenuation in the ilvGMEDA operon (Japanese Patent Application Laid-open (Kokai) No. 8-47397/1996).

Other Amino Acids:

Biosyntheses of L-tryptophan, L-phenylalanine, L-tyrosine, L-threonine, and L-isoleucine can be enhanced by increasing the ability of the Methylophilus bacteria to produce phosphoenolpyruvate (WO97/08333).

The ability to produce L-phenylalanine and L-tyrosine can be improved by amplifying or introducing a desensitized chorismate mutase-prephenate dehydratase (CM-PDT) gene (Japanese Patent Application Laid-open (Kokai) Nos. 5-236947/1993 and 62-130693/1987) and a desensitized 3-deoxy-D-arabinoheptulonate-7-phosphate synthase (DS) gene (Japanese Patent Application Laid-open (Kokai) Nos. 5-236947/1993 and 61-124375/1986).

The ability to produce L-tryptophan can be improved by amplifying or introducing a tryptophan operon with a gene coding for desensitized anthranilate synthase (Japanese Patent Application Laid-open (Kokai) Nos. 57-71397/1982, 62-244382/1987 and US Pat. No. 4,371,614).

In the present specification, the expression that the “enzyme activity is enhanced” usually means that the intracellular activity of the enzyme is higher than that in a wild-type strain. Furthermore, when the activity of the enzyme is enhanced by modification using gene recombinant techniques or the like, the intracellular activity of the enzyme is higher than that in the strain before the modification. The expression that “enzyme activity is decreased” usually means that the intracellular activity of the enzyme is lower than that in a wild-type strain. Similarly, when the activity of the enzyme is decreased by modification using gene recombinant techniques or the like, the intracellular activity of the enzyme is lower than that in the strain before the modification.

L-amino acids can be produced by culturing the Methylophilus bacteria obtained as described above in a medium under conditions which allow for production and accumulation of the L-amino acids in the medium, and collecting the L-amino acids from the medium.

Methylophilus bacteria with an increased amount of L-amino acid as compared with wild-type strains of Methylophilus bacteria can be produced by culturing Methylophilus bacteria with an ability to produce L-amino acids in a medium under conditions which allow for production and accumulation of the L-amino acids.

Microorganisms used in the present invention can be cultured by methods which are typically used for culturing methanol-assimilating microorganisms. The medium for the culture may be a natural or synthetic medium so long as it contains a carbon source, a nitrogen source, inorganic ions, and other trace organic components as required.

By using methanol as the main source of carbon, L-amino acids can be inexpensively produced. 0.001 to 30% of methanol is typically necessary in the culture medium. Ammonium sulfate or the like can be used as the nitrogen source. Otherwise, small amounts of the trace components such as potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate, and manganese sulfate may be added to the medium.

The culture is usually performed under aerobic conditions, for example, shaking or stirring for aeration, at pH 5 to 9 and a temperature of 20 to 45° C., and it is usually completed within 24 to 120 hours.

Collection of the L-amino acids from the culture can be attained by a combination of known methods, such as by using ion exchange resin, precipitation, and others.

Furthermore, the Methylophilus bacterial cells can be separated from the medium by typical methods which are know in the art for separating microbial cells.

<2> Gene of the Present Invention

The DNA of the present invention is a gene which codes for one of the following enzymes: aspartokinase (henceforth also abbreviated as “AK”), aspartic acid semialdehyde dehydrogenase (henceforth also abbreviated as “ASD”), dihydrodipicolinate synthase (henceforth also abbreviated as “DDPS”), dihydrodipicolinate reductase (henceforth also abbreviated as “DDPR”), and diaminopimelate decarboxylase (henceforth also abbreviated as “DPDC”) derived from Methylophilus methylotrophus.

The DNA of the present invention can be obtained by, for example, transforming a mutant strain of a microorganism which is deficient in AK, ASD, DDPS, DDPR, or DPDC using a gene library from Methylophilus methylotrophus, and selecting a clone in which the auxotrophy is recovered.

A gene library of Methylophilus methylotrophus can be produced as follows, for example. First, total chromosomal DNA is prepared from a Methylophilus methylotrophus wild-type strain, for example, the Methylophilus methylotrophus AS1 strain (NCIMB 10515), by the method of Saito et al. (Saito, H. and Miura, K., Biochem. Biophys. Acta 72, 619-629, (1963)) or the like, and partially digested with a suitable restriction enzyme, for example, Sau3AI or AluI, to obtain a mixture of various fragments. By controlling the degree of the digestion by adjusting the digestion reaction time and so forth, a wide range of restriction enzymes can be used.

Subsequently, the digested chromosomal DNA fragments are ligated to vector DNA which is able to autonomously replicate in Escherichia coli cells to produce recombinant DNA. Specifically, a restriction enzyme producing the same terminal nucleotide sequence as that produced by the restriction enzyme used for the digestion of chromosomal DNA is allowed to act on the vector DNA to fully digest and cleave the vector. Then, the mixture of chromosome DNA fragments and the digested and cleaved vector DNA are ligated with a ligase, preferably T4 DNA ligase, to obtain recombinant DNA.

A gene library solution can be obtained by transforming Escherichia coli, for example, the Escherichia coli JM109 strain or the like, with the recombinant DNA, and preparing recombinant DNA from the culture broth of the transformant. This transformation can be performed by the method of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the method of treating recipient cells with calcium chloride so as to increase the permeability of DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), and so forth. In the examples mentioned hereinafter, electroporation was used.

Vectors which can be used as described above include pUC19, pUC18, pUC118, pUC119, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218, pSTV28, pSTV29, and so forth. Phage vectors can also be used. Since pUC118 and pUC119 contain an ampicillin resistance gene, and pSTV28 and pSTV29 contain a chloramphenicol resistance gene, for example, only transformants with the vector or the recombinant DNA can be grown in a medium containing ampicillin or chloramphenicol.

The alkali SDS method and the like can be used to culture the transformants and collect the recombinant DNA from the bacterial cells.

A mutant microbial strain which does not contain AK, ASD, DDPS, DDPR, or DPDC is transformed by using the gene library solution of Methylophilus methylotrophus as described above, and clones whose auxotrophy is recovered are selected.

Examples of a mutant microbial strain deficient in AK include E. coli GT3 deficient in three different genes coding for AK (thrA, metLM, lysC). Examples of a mutant microbial strain deficient in ASD include E. coli Hfr3000 U482 (CGSC 5081 strain). Examples of a mutant microbial strain deficient in DDPS include E. coli AT997 (CGSC 4547 strain). Examples of a mutant microbial strain deficient in DDPR include E. coli AT999 (CGSC 4549 strain). Examples of a mutant microbial strain deficient in DPDC include E. coli AT2453 (CGSC 4505 strain). These mutant strains can be obtained from the E. coli Genetic Stock Center (the Yale University, Department of Biology, Osborn Memorial Labs., P.O. Box 6666, New Haven 06511-7444, Connecticut, U.S.).

Although all of the aforementioned mutant strains cannot grow in M9 minimal medium, transformant strains which contain a gene coding for AK, ASD, DDPS, DDPR, or DPDC can grow in M9 minimal medium because these genes are able to function in the transformants. Therefore, by selecting transformant strains that can grow in the minimal medium and collecting recombinant DNA from the strains, DNA fragments containing a gene that codes for each enzyme can be obtained. E. coli AT999 (CGSC 4549 strain) shows an extremely slow growth rate even in a complete medium such as L medium when diaminopimelic acid is not added to the medium. However, normal growth is observed for the transformant strains which contain the gene coding for DDPR derived from Methylophilus methylotrophus, because of the function of the gene. Therefore, a transformant strain that contains the gene coding for DDPR can also be obtained by selecting a transformant strain which can normally grow in L medium.

By extracting an insert DNA fragment from the recombinant DNA and determining its nucleotide sequence, the amino acid sequence of each enzyme and nucleotide sequence coding for it can be determined.

The gene coding for AK of the present invention (henceforth also referred to “ask”) codes for AK which has the amino acid sequence of SEQ ID NO: 6. As a specific example of the ask gene, the DNA having the nucleotide sequence of SEQ ID NO: 5 can be used. The ask gene of the present invention may have a sequence in which the codon corresponding to each of the amino acids is replaced with an equivalent codon so long as it codes for the same amino acid sequence as shown in SEQ ID NO: 6.

The gene which codes for ASD of the present invention (henceforth also referred to as “asd”) codes for ASD which has the amino acid sequence of SEQ ID NO: 8. As a specific example of the asd gene, the DNA which contains the nucleotide sequence of nucleotide 98-1207 shown in SEQ ID NO: 7 can be used. The asd gene of the present invention may have a sequence in which the codon corresponding to each of the amino acids is replaced with an equivalent codon so long as it codes for the same amino acid sequence as shown in SEQ ID NO: 8.

The gene which codes for DDPS of the present invention (henceforth also referred to as “dapA”) codes for DDPS which has the amino acid sequence of SEQ ID NO: 10. As a specific example of the dapA gene, the DNA which has the nucleotide sequence of nucleotide 1268-2155 in SEQ ID NO: 9 can be used. The dapA gene of the present invention may have a sequence in which the codon corresponding to each of the amino acids is replaced with an equivalent codon so long as it codes for the same amino acid sequence as shown in SEQ ID NO: 10.

The gene which codes for DDBR of the present invention (henceforth also referred to as “dapB”) codes for DDBR which has the amino acid sequence of SEQ ID NO: 12. As a specific example of the dapB gene, the DNA which has the nucleotide sequence of numbers 2080-2883 in SEQ ID NO: 11 can be used. The dapB gene of the present invention may have a sequence in which the codon corresponding to each of the amino acids is replaced with an equivalent codon so long as it codes for the same amino acid sequence as shown in SEQ ID NO: 12.

The gene which codes for DPDC of the present invention (henceforth also referred to as “lysA”) codes for DPDC which has the amino acid sequence of SEQ ID NO: 14. As a specific example of the lysA gene, the DNA which has the nucleotide sequence of numbers 751-1995 in SEQ ID NO: 13 can be used. The lysA gene of the present invention may have a sequence in which the codon corresponding to each of the amino acids is replaced with an equivalent codon so long as it codes for the same amino acid sequence as shown in SEQ ID NO: 14.

The enzymes of the present invention may have the amino acid sequences of SEQ ID NO: 6, 8, 10, 12, or 14, and these sequences may include substitutions, deletions, insertions, additions, or inversions of one or several amino acids, as long as the activity of the enzyme is maintained. The expression “one or several” used herein preferably means 1 to 10, more preferably 1 to 5, and more preferably 1 to 2.

The DNA which codes for the substantially same protein as AK, ASD, DDPS, DDPR, or DPDC such as those described above can be obtained by modifying each nucleotide sequence so that the encoded amino acid sequence contains substitutions, deletions, insertions, additions, or inversions of an amino acid(s) at a particular site by, for example, site-specific mutagenesis. This modified DNA may also be obtained using conventional mutagenesis treatments. Examples of mutagenesis treatments include in vitro treatment of DNA coding for AK, ASD, DDPS, DDPR or DPDC with hydroxylamine or the like, treatment of a microorganism such as Escherichia bacteria containing a gene coding for AK, ASD, DDPS, DDPR or DPDC with UV irradiation or with typical mutagenesis agents such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.

The aforementioned substitution, deletion, insertion, addition, or inversion of nucleotides and/or amino acids includes naturally occurring mutations (mutant or variant) such as those between species or strains of microorganisms containing AK, ASD, DDPS, DDPR, or DPDC, and so forth.

The DNA which codes for substantially the same protein as AK, ASD, DDPS, DDPR or DPDC can be obtained by expressing a DNA having such a mutation as described above in a suitable cell, and examining the AK, ASD, DDPS, DDPR, or DPDC activity of the expression product. The DNA which codes for substantially the same protein as AK, ASD, DDPS, DDPR or DPDC can also be obtained by isolating, a DNA which is able to hybridize to a probe containing a nucleotide sequence of numbers 510-1736 of SEQ ID NO: 5, a nucleotide sequence of numbers 98-1207 of SEQ ID NO: 7, a nucleotide sequence of numbers 1268-2155 of SEQ ID NO: 9, a nucleotide sequence of numbers 2080-2883 of SEQ ID NO: 11, or a nucleotide sequence of numbers 751-1995 of SEQ ID NO: 13, or portions of these nucleotide sequences under stringent conditions, and coding for a protein having AK, ASD, DDPS, DDPR or DPDC activity. In the present specification, to have a nucleotide sequence or a portion thereof means to have the nucleotide sequence or the portion thereof, or a nucleotide complementary thereto.

The term “stringent conditions” used herein means conditions that allow for formation of a so-called specific hybrid and does not allow for formation of a non-specific hybrid. These conditions may vary depending on the nucleotide sequence and length of the probe. However, for example, conditions that allow for hybridization of highly homologous DNA such as DNA having homology of 40% or higher, but does not allow for hybridization of DNA of lower homology than defined above, or conditions that allow for hybridization using washing conditions typical in Southern hybridization, of a temperature of 60° C. and salt concentrations corresponding to 1×SSC and 0.1% SDS, preferably 0.1 x SSC and 0.1% SDS.

A partial sequence of each gene can also be used as the probe. Such a probe can be produced by PCR (polymerase chain reaction) using oligonucleotides produced based on the nucleotide sequence of each gene as primers and a DNA fragment containing each gene as the template. When a DNA fragment having a length of about 300 bp is used as the probe, washing conditions for hybridization may be, for example, 50° C., 2×SSC and 0.1% SDS.

Genes that hybridize under conditions as described above also include those having a stop codon in its sequence and those encoding an enzyme which has lost its activity due to a mutation in the active center. However, such genes can readily be eliminated by ligating the genes to a commercially available activity expression vector, and measuring AK, ASD, DDPS, DDPR or DPDC activity.

Since the nucleotide sequences of the genes that code for AK, ASD, DDPS, DDPR and DPDC derived from Methylophilus methylotrophus are first described herein, DNA sequences which code for AK, ASD, DDPS, DDPR, and DPDC can be obtained from a Methylophilus methylotrophus gene library by hybridization using oligonucleotide probes produced based on the reported sequences. Moreover, DNA sequences which code for these enzymes can also be obtained by amplifying them from Methylophilus methylotrophus chromosomal DNA by PCR using oligonucleotide primers produced based on the aforementioned nucleotide sequences.

The aforementioned genes can suitably be utilized to enhance the ability of Methylophilus bacteria to produce L-lysine.

EXAMPLES

The present invention will further specifically be explained with reference to the following non-limiting examples.

The reagents used were obtained from Wako Pure Chemicals or Nakarai Tesque unless otherwise indicated. The compositions of the media used in each example are shown below. pH was adjusted with NaOH or HCl for all media.

L Medium:

Bacto trypton (DIFCO) 10 g/L  Yeast extract (DIFCO) 5 g/L NaCl 5 g/L

steam-sterilized at 120° C. for 20 minutes

L Agar Medium:

L medium Bacto agar (DIFCO) 15 g/L

steam-sterilized at 120° C. for 20 minutes

SOC Medium:

Bacto trypton (DIFCO) 20 g/L Yeast extract (DIFCO)  5 g/L  10 mM NaCl 2.5 mM KCl  10 mM MgSO4  10 mM MgCl2  20 mM Glucose

The constituents except for the magnesium solution and glucose were steam-sterilized (120° C., 20 minutes), then 2 M magnesium stock solution (1 M MgSO4, 1 M MgCl2) and 2 M glucose solution, which had been passed through a 0.22-μm filter, were added thereto, and the mixture was passed through a 0.22-μm filter again.

121M1 Medium:

K2HPO4 1.2 g/L KH2PO4 0.62 g/L NaCl 0.1 g/L (NH4)2SO4 0.5 g/L MgSO4•7H2O 0.2 g/L CaCl2•6H2O 0.05 g/L FeCl3•6H2O 1.0 mg/L H3BO3 10 μg/L CuSO4•5H2O 5 μg/L MnSO4•5H2O 10 μg/L ZnSO4•7H2O 70 μg/L NaMoO4•2H2O 10 μg/L CoCl2•6H2O 5 μg/L Methanol 1% (vol/vol), pH 7.0

The constituents except for methanol were steam-sterilized at 121° C. for 15 minutes. After the constituents sufficiently cooled, methanol was added.

Composition of 121 production medium:

Methanol   2% Dipotassium phosphate 0.12% Potassium phosphate 0.062%  Calcium chloride hexahydrate 0.005%  Magnesium sulfate heptahydrate 0.02% Sodium chloride 0.01% Ferric chloride hexahydrate  1.0 mg/L  Ammonium sulfate  0.3% Cupric sulfate pentahydrate  5 μg/L Manganous sulfate pentahydrate 10 μg/L Sodium molybdate dihydrate 10 μg/L Boric acid 10 μg/L Zinc sulfate heptahydrate 70 μg/L Cobaltous chloride hexahydrate  5 μg/L Calcium carbonate (Kanto Kagaku)   3% pH 7.0

121M1 Agar Medium:

121M1 medium 15 g/L Bacto agar (DIFCO)

The constituents except for methanol were steam-sterilized at 121° C. for 15 minutes. After the constituents sufficiently cooled, methanol was added.

M9 Minimal Medium:

Na2HPO4•12H2O 16 g/L KH2PO4 3 g/L NaCl 0.5 g/L NH4Cl 1 g/L MgSO4•7H20 246.48 mg/L Glucose 2 g/L pH 7.0

MgSO4 and glucose were separately sterilized (120° C., 20 minutes) and added. A suitable amount of amino acids and vitamins were added as required.

M9 Minimal Agar Medium:

M9 minimal medium 15 g/L Bacto agar (DIFCO)

Example 1

Creation of L-lysine-Producing Bacterium: (I)

(1) Introduction of Mutant lysC and Mutant dapA into Methylophilus Bacterium

A Methylophilus bacterium was transformed with plasmid RSFD80 (see WO95/16042) which contained a mutant lysC and a mutant dapA. RSFD80 is plasmid pVIC40 (International Publication WO90/04636, Japanese Patent Application Laid-open (Kohyo) No. 3-501682/1991) derived from the broad host spectrum vector plasmid pAYC32 (Chistorerdov, A. Y., Tsygankov, Y. D., Plasmid, 16, 161-167, (1986)), which is a derivative of RSF1010. RSF1010 contains a mutant dapA and a mutant lysC derived from E. coli located downstream of the promoter (tetP) of the tetracycline resistance gene of pVIC40 in this order, so that the transcription directions of the genes are ordinary with respect to tetP. The mutant dapA codes for a mutant DDPS with a tyrosine in place of the histidine at position 118. The mutant lysC codes for a mutant AKIII with an isoleucine in place of the threonine at position 352.

RSFD80 was constructed as follows. The mutant dapA on plasmid pdapAS24 was ligated to pVIC40 downstream of the promoter of the tetracycline resistance gene to obtain RSF24P as shown in FIG. 1. Then, the plasmid RSFD80 which had the mutant dapA and a mutant lysC was prepared from RSF24P and pLLC*80 containing the mutant lysC as shown in FIG. 2. That is, while pVIC40 contains a threonine operon, this threonine operon is replaced with a DNA fragment containing the mutant dapA and a DNA fragment containing the mutant lysC in RSFD80.

The E. coli JM109 strain transformed with the RSFD80 plasmid was designated AJ12396, and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Oct. 28, 1993 and received an accession number of FERM P-13936, and it was converted to an international deposit under the provisions of the Budapest Treaty on Nov. 1, 1994, and received an accession number of FERM BP-4859.

The E. coli AJ1239 strain was cultured in 30 ml of LB medium containing 20 mg/L of streptomycin at 30° C. for 12 hours, then the RSFD80 plasmid was purified from the cells using Wizard® Plus Midipreps DNA Purification System (sold by Promega).

The RSFD80 plasmid as described above was introduced into the Methylophilus methylotrophus AS1 strain (NCIMB 10515) by electroporation (Canadian Journal of Microbiology, 43, 197 (1997)). As a control, the DNA region coding for the threonine operon was deleted from the pVIC40 plasmid from which the RSFD80 plasmid was derived, to produce a pRS plasmid having only the vector region (see Japanese Patent Application Laid-open (Kohyo) No. 3-501682/1991). The pRS plasmid was introduced into the AS1 strain in the same manner as that used for RSFD80.

(2) AKIII Activity of Methylophilus Bacterium Containing mutant lysC and Mutant dapA Derived from E. coli

Cell-free extracts were prepared from the Methylophilus methylotrophus AS1 strain containing the RSFD80 plasmid (also referred to as “AS1/RSFD80” hereinafter) and the Methylophilus methylotrophus AS1 strain containing the pRS plasmid (also referred to as “AS1/pRS” hereinafter), and AK activity was measured. The cell-free extracts (crude enzyme solutions) were prepared as follows. The AS1/RSFD80 strain and AS1/pRS strain were each inoculated into the above-described 121 production medium containing 20 mg/L of streptomycin, cultured at 37° C. for 34 hours with shaking, and then calcium carbonate was removed and cells were harvested.

The bacterial cells obtained as described above were washed with 0.2% KCl at 0° C., suspended in 20 mM potassium phosphate buffer (pH 7) containing 10 mM MgSO4, 0.8 M (NH4)2SO4 and 0.03 M β-mercaptoethanol, and disrupted by sonication (0° C., 200 W, 10 minutes). The sonicated cell suspension was centrifuged at 33,000 rpm for 30 minutes at 0° C., and the supernatant was removed. To the supernatant, ammonium sulfate was added to 80% saturation, and the mixture was left at 0° C. for 1 hour, and centrifuged. The pellet was dissolved in 20 mM potassium phosphate buffer (pH 7) containing 10 mM MgSO4, 0.8 M (NH4)2SO4 and 0.03 M β-mercaptoethanol.

AK activity was measured in accordance with the method of Stadtman (Stadtman, E. R., Cohen, G. N., LeBras, G., and Robichon-Szulmajster, H., J. Biol. Chem., 236, 2033 (1961)). That is, a following reaction solution was incubated at 30° C. for 45 minutes, resulting in color development (2.8 N HCl: 0.4 ml, 12% TCA: 0.4 ml, 5% FeCl3.6H2O/0.1 N HCl: 0.7 ml). The reaction solution was centrifuged, and absorbance of the supernatant was measured at 540 nm. The activity was expressed in terms of the amount of hydroxamic acid produced in 1 minute (1 U=1 μmol/minute). The molar extinction coefficient was set at 600. A reaction solution without potassium aspartate was used as a blank. When the enzymatic activity was measured, L-lysine was added to the enzymatic reaction solution at various concentrations to examine the degree of inhibition by L-lysine. The results are shown in Table 1.

Composition of Reaction Solution:

Reaction mixture *1 0.3 ml Hydroxylamine solution *2 0.2 ml 0.1 M Potassium aspartate (pH 7.0) 0.2 ml Enzyme solution 0.1 ml Water (balance) Total 1 ml *1 M Tris-HCl (pH 8.1): 9 ml, 0.3 M MgSO4: 0.5 ml and 0.2 M ATP (pH 7.0): 5 ml *2 8 M Hydroxylamine solution neutralized with KOH immediately before use

TABLE 1 AK activity Specific Desensitization (Specific activity with degree of Strain activity*1) 5 mM L-lysine inhibition*2 (%) AS1/pRS 7.93 9.07 114 AS1/RSFD80 13.36 15.33 115 *1nmol/minute/mg protein *2Activity retention ratio in the presence of 5 mM L-lysine

As shown in Table 1, AK activity was increased by about 1.7 times by the introduction of the RSFD80 plasmid. Furthermore, it was confirmed that the inhibition by L-lysine was completely desensitized in E. coli AK encoded by the RSFD80 plasmid. Moreover, it was found that AK that was originally retained by the AS1 strain was not inhibited by L-lysine alone. The inventors of the present invention have discovered that the AK derived from the AS1 strain was inhibited by 100% when 2 mM each of L-lysine and L-threonine were present in the reaction solution (concerted inhibition).

(3) Production of L-lysine by Methylophilus Bacterium Containing Mutant lysC and Mutant dapA Derived from E. coli

Then, the AS1/RSFD80 strain and the AS1/pRS strain were inoculated into 121 production medium containing 20 mg/L of streptomycin, and cultured at 37° C. for 34 hours with shaking. After the culture was completed, the bacterial cells and calcium carbonate were removed by centrifugation, and L-lysine concentration in the culture supernatant was measured by an amino acid analyzer (JASCO Corporation [Nihon Bunko], high performance liquid chromatography). The results are shown in Table 2.

TABLE 2 Production amount of L-lysine Strain hydrochloride (g/L) AS1/pRS 0 AS1/RSFD80 0.3

Example 2

Creation of L-lysine-Producing Bacterium (II)

(1) Introduction of the Tac Promoter Region into a Broad Host Spectrum Vector

In order to produce a large amount of a L-lysine biosynthetic enzyme in Methylophilus methylotrophus, the tac promoter was used for gene expression of this target enzyme. This promoter is frequently used in E. coli.

The tac promoter region was obtained by amplification through PCR using pKK233-3 (Pharmacia) as the template, DNA fragments having the nucleotide sequences of SEQ ID NOS: 15 and 16 as primers, and a heat-resistant DNA polymerase. The PCR was performed with cycles of 94° C. for 20 seconds, 60° C. for 30 seconds, and 72° C. for 60 seconds, repeated 30 times. Then, the amplified DNA fragment was collected and treated with restriction enzymes EcoRI and PstI. The broad host spectrum vector pRS (see Japanese Patent Application Laid-open (Kohyo) No. 3-501682/1991) was also digested with the same restriction enzymes, and the aforementioned DNA fragment containing the tac promoter region was introduced into the restriction enzyme digestion termini to construct pRS-tac.

(2) Preparation of dapA Gene (Dihydrodipicolinate Synthase Gene) Expression Plasmid pRS-dapA24 and lysC Gene (Aspartokinase Gene) Expression Plasmid pRS-lysC80

A mutant gene (dapA*24) coding for dihydrodipicolinate synthase with partially desensitized feedback inhibition by Lys was was introduced into the plasmid pRS-tac, which was prepared as described above (1).

First, the dapA*24 gene region was obtained by amplification through PCR using RSFD80 (see Example 1) as the template, and DNA fragments having the nucleotide sequences of SEQ ID NOS: 17 and 18 as primers. The PCR was performed with cycles of 94° C. for 20 seconds, 60° C. for 30 seconds, and 72° C. for 90 seconds, repeated 30 times. Then, the fragment was treated with restriction enzymes Sse8387I and XbaI. pRS-tac was also treated with Sse8387I and partially digested with XbaI in the same manner as described above. To this digested plasmid, the aforementioned dapA*24 gene fragment was ligated with T4 ligase to obtain pRS-dapA24.

Similarly, the gene (lysC*80) coding for aspartokinase with partially desensitized feedback inhibition by Lys was obtained by PCR using RSFD80 as the template, and DNA fragments having the nucleotide sequences of SEQ ID NOS: 19 and 20 as primers. The PCR was performed with cycles of 94° C. for 20 seconds, 60° C. for 30 seconds, and 72° C. for 90 seconds, repeated 30 times. Then, the DNA fragment was treated with restriction enzymes Sse8387I and SapI. The vector pRS-tac was also treated with Sse8387I and SapI. To this digested plasmid, the aforementioned lysC*80 gene fragment was ligated with T4 ligase to obtain pRS-lysC80.

(3) Introduction of pRS-dapA24 or pRS-lysC80 into Methylophilus methylotrophus and Evaluation of the Culture

pRS-dapA24 and pRS-lysC80 obtained as described above were each separately introduced into the Methylophilus methylotrophus AS1 strain (NCIMB 10515) by electroporation to obtain AS1/pRS-dapA24 and AS1/pRS-lysC80, respectively. Each strain was inoculated into 121 production medium containing 20 mg/L of streptomycin, and cultured at 37° C. for 48 hours with shaking. As a control strain, AS1 strain harboring pRS was also cultured in a similar manner. After the culture was completed, the cells and calcium carbonate were removed by centrifugation, and the L-lysine concentration in the culture supernatant was measured by an amino acid analyzer (JASCO Corporation [Nihon Bunko], high performance liquid chromatography). The results are shown in Table 3.

TABLE 3 Production amount of L-lysine Strain hydrochloride (g/L) AS1/pRS <0.01 AS1/pRS-lysC80 0.06 AS1/pRS-dapA24 0.13

Example 3

Creation of L-lysine-Producing Bacterium (III)

The Methylophilus methylotrophus AS1 strain (NCIMB10515) was inoculated into 121M1 medium and cultured at 37° C. for 15 hours. The obtained bacterial cells were treated with NTG in a conventional manner (NTG concentration: 100 mg/L, 37° C., 5 minutes), and spread onto 121M1 agar medium containing 7 g/L of S-(2-aminoethyl)-cysteine (AEC) and 3 g/L of L-threonine. The cells were cultured at 37° C. for 2 to 8 days, and the colonies which formed were picked up to obtain AEC-resistant strains.

The aforementioned AEC-resistant strains were inoculated into 121 production medium, and cultured at 37° C. for 38 hours under aerobic conditions. After the culture was completed, the cells and calcium carbonate were removed from the medium by centrifugation, and the L-lysine concentration in the culture supernatant was measured by an amino acid analyzer (JASCO Corporation [Nihon Bunko], high performance liquid chromatography). The strain with improved L-lysine-producing ability as compared with the parent strain was selected, and designated Methylophilus methylotrophus AR-166 strain. The L-lysine production amounts of the parent strain (AS1 strain) and the AR-166 strain are shown in Table 4.

TABLE 4 Production amount of L-lysine Strain hydrochloride (mg/L) AS1 5.8 AR-166 80

The Methylophilus methylotrophus AR-166 strain was given a private number of AJ13608, and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Jun. 10, 1999 and received an accession number of FERM P-17416, and it was converted to an international deposit under the provisions of the Budapest Treaty on Mar. 31, 2000, and received an accession number of FERM BP-7112.

Example 4

Creation of L-threonine-Producing Bacterium

(1) Introduction of Threonine Operon Plasmid into a Methylophilus Bacterium

Plasmid pVIC40 (International Publication WO90/04636, Japanese Patent Application Laid-open (Kohyo) No. 3-501682/1991) containing a threonine operon derived from E. coli was introduced into the Methylophilus methylotrophus AS1 strain (NCIMB 10515) by electroporation (Canadian Journal of Microbiology, 43, 197 (1997)) to obtain AS1/pVIC40 strain. As a control, pRS (Japanese Patent Application Laid-open (Kohyo) No. 3-501682/1991) with only the vector region was obtained by deleting the DNA region coding for the threonine operon from the pVIC40 plasmid, and it was introduced into the AS1 strain in the same manner as for pVIC40 to obtain AS1/pRS strain.

(2) Production of L-threonine by Methylophilus Bacterium Containing the Threonine Operon Derived from E. coli

The AS1/pVIC40 and AS1/pRS strains were each inoculated into 121 production medium containing 20 mg/L of streptomycin, 1 g/l of L-valine and 1 g/l of L-leucine, and cultured at 37° C. for 50 hours with shaking. After the culture was completed, the cells and calcium carbonate were removed by centrifugation, and the L-threonine concentration in the culture supernatant was measured by an amino acid analyzer (JASCO Corporation [Nihon Bunko], high performance liquid chromatography). The results are shown in Table 5.

TABLE 5 Production amount of Strain L-threonine (mg/L) AS1/pRS 15 AS1/pVIC40 30

Example 5

Creation of Branched Chain Amino Acid-Producing Bacterium

The Methylophilus methylotrophus AS1 strain (NCIMB 10515) was inoculated into 121M1 medium and cultured at 37° C. for 15 hours. The obtained bacterial cells were treated with NTG in a conventional manner (NTG concentration: 100 mg/L, 37° C., 5 minutes), and spread onto 121M1 agar medium containing 0.5% of casamino acid (DIFCO). The cells were cultured at 37° C. for 2 to 8 days, and allowed to form colonies. The colonies were picked up, and inoculated into 121M1 agar medium and 121M1 agar medium containing 0.5% casamino acid. Strains exhibiting better growth on the latter medium compared with on the former medium were selected as casamino acid auxotrophic strains. In this way, 9 leaky casamino acid auxotrophic strains were obtained from the NTG-treated 500 strains. From these casamino acid auxotrophic strains, one strain produced more L-valine, L-leucine, and L-isoleucine in the medium as compared with its parent strain. This strain was designated Methylophilus methylotrophus C138 strain.

The Methylophilus methylotrophus C138 strain was given a private number of AJ13609, and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Jun. 10, 1999 and received an accession number of FERM P-17417, and it was converted to an international deposit under the provisions of the Budapest Treaty on Mar. 31, 2000, and received an accession number of FERM BP-7113.

The parent strain (AS1 strain) and the C138 strain were inoculated into 121 production medium, and cultured at 37° C. for 34 hours under aerobic conditions. After the culture was completed, the cells and calcium carbonate were removed from the medium by centrifugation, and the concentrations of L-valine, L-leucine, and L-isoleucine in the culture supernatant were measured by an amino acid analyzer (JASCO Corporation [Nihon Bunko], high performance liquid chromatography). The results are shown in Table 6.

TABLE 6 Strain L-valine (mg/L) L-leucine (mg/L) L-isoleucine (mg/L) AS1 7.5 5.0 2.7 C138 330 166 249

Example 6

Preparation of Chromosomal DNA Library of Methylophilus methylotrophus AS1 Strain

(1) Preparation of Chromosome DNA of Methylophilus methylotrophus AS1 Strain

One platinum loop of the Methylophilus methylotrophus AS1 strain (NCIMB 10515) was inoculated into 5 ml of 121M1 medium in a test tube, and cultured at 37° C. overnight with shaking. Then, the culture broth was inoculated into 50 ml of 121M1 medium in a 500 ml-volume Sakaguchi flask to 1%, and cultured at 37° C. overnight with shaking. Then, the cells were harvested by centrifugation, and suspended in 50 ml of TEN solution (solution containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA and 20 mM NaCl (pH 8.0)). The cells were collected by centrifugation, and suspended again in 5 ml of the TEN solution containing 5 mg/ml of lysozyme and 10 μg/ml of RNase A. The suspension was maintained at 37° C. for 30 minutes, and then proteinase K and sodium laurylsulfate were added thereto to final concentrations of 10 μg/ml and 0.5% (wt/vol), respectively.

The suspension was maintained at 70° C. for 2 hours, and then an equal amount of a saturated solution of phenol (phenol solution saturated with 10 mM Tris-HCl (pH 8.0)) was added and mixed. The suspension was centrifuged, and the supernatant was collected. An equal amount of phenol/chloroform solution (phenol:chloroform:isoamyl alcohol=25:24:1) was added and mixed, and the mixture was centrifuged. The supernatant was collected, and an equal amount of chloroform solution (chloroform:isoamyl alcohol=24:1) was added thereto to repeat the same extraction procedure. To the supernatant, a 1/10 volume of 3 M sodium acetate (pH 4.8) and 2.5-fold volume of ethanol were added to precipitate the chromosomal DNA. The precipitates were collected by centrifugation, washed with 70% ethanol, dried under reduced pressure, and dissolved in a suitable amount of TE solution (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)).

(2) Preparation of the Gene Library

A 50 μl portion of the chromosomal DNA (1 μg/μl) obtained in the above (1), 20 μl of H buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol, 1000 mM NaCl (pH 7.5)) and 8 units of a restriction enzyme Sau3AI (Takara Shuzo) were allowed to react at 37° C. for 10 minutes in a total volume of 200 μl, and then 200 μl of the phenol/chloroform solution was added and mixed to stop the reaction. The reaction mixture was centrifuged, and the upper layer was collected and separated on a 0.8% agarose gel. DNA corresponding to 2 to 5 kilobase pair (henceforth abbreviated as “kbp”) was collected by using Concert™ Rapid Gel Extraction System (DNA collecting kit, GIBCO BRL Co.). In this way, 50 μl of a solution of DNA with fractionated sizes was obtained.

2.5 μg of plasmid pUC118 (Takara Shuzo), 2 μl of K buffer (200 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol, 1000 mM KCl (pH 8.5)) and 10 units of restriction enzyme BamHI (Takara Shuzo) were allowed to react at 37° C. for 2 hours in a total volume of 20 μl, then 20 units of calf small intestine alkaline phosphatase (Takara Shuzo) was added and mixed, and the mixture was allowed to react for an additional 30 minutes. The reaction mixture was mixed with an equal amount of the phenol/chloroform solution, and the mixture was centrifuged. The supernatant was collected, and an equal amount of the chloroform solution was added thereto to repeat a similar extraction procedure. To the supernatant, a 1/10 volume of 3 M sodium acetate (pH 4.8) and 2.5-fold volume of ethanol were added to precipitate DNA. The DNA was collected by centrifugation, washed with 70% ethanol, dried under reduced pressure, and dissolved in a suitable amount of TE solution.

A Sau3AI digestion product of the chromosomal DNA prepared as described above and a BamHI digestion product of pUC118 were ligated by using a Ligation Kit ver. 2 (Takara Shuzo). To the reaction mixture, a 1/10 volume of 3 M sodium acetate (pH 4.8) and 2.5-fold volume of ethanol were added to precipitate DNA. The DNA was collected by centrifugation, washed with 70% ethanol, dried under reduced pressure, and dissolved in TE solution (Ligase solution A).

In the same manner as in the above procedure, fragments obtained by partial digestion of the chromosomal DNA with a restriction enzyme AluI (Takara Shuzo) and a SmaI digestion product of plasmid pSTV29 (Takara Shuzo) were ligated (Ligase solution B).

One platinum loop of E. coli JM109 was inoculated into 5 ml of L medium in a test tube, and cultured at 37° C. overnight with shaking. Then, the culture broth was inoculated into 50 ml of L medium in a 500 ml-volume Sakaguchi flask to 1%, cultured at 37° C. until OD660 of the culture became 0.5 to 0.6, and cooled on ice for 15 minutes. Then, the cells were harvested by centrifugation at 4° C. The cells were suspended in 50 ml of ice-cooled water and centrifuged to wash the cells. This operation was repeated once again, and the cells were suspended in 50 ml of ice-cooled 10% glycerol solution, and centrifuged to wash the cells. The cells were suspended in an equal volume of 10% glycerol solution, and divided into 50 μl aliquots. To the cells in the 50 μl volume, 1 μl of Ligase solution A or Ligase solution B prepared above was added. Then, the mixture was put into a special cuvette (0.1 cm width, preliminarily ice-cooled) for an electroporation apparatus of BioRad.

The setting of the apparatus was 1.8 kV and 25 μF, and the setting of pulse controller was 200 ohms. The cuvette was mounted on the apparatus and pulses were applied thereto. Immediately after the application of pulse, 1 ml of ice-cooled SOC medium was added thereto, and the mixture was transferred to a sterilized test tube, and cultured at 37° C. for 1 hour with shaking. Each cell culture broth was spread onto L agar medium containing an antibiotic (100 μg/ml of ampicillin when Ligase solution A was used, or 20 μg/ml of chloramphenicol when Ligase solution B was used), and incubated at 37° C. overnight. The colonies which grew on each agar medium were scraped, inoculated into 50 ml of L medium containing respective antibiotic in a 500 ml-volume Sakaguchi flask, and cultured at 37° C. for 2 hours with shaking. Plasmid DNA was extracted from each culture broth by the alkali SDS method to form Gene library solution A and Gene library solution B, respectively.

Example 7

Cloning of the Lysine Biosynthesis Gene of Methylophilus methylotrophus AS1 Strain

(1) Cloning of the Gene Coding for Aspartokinase (AK)

E. coli GT3 deficient in the three genes coding for AK (thrA, metLM and lysC) was transformed with Gene library solution B by the same electroporation procedure as mentioned above. SOC medium containing 20 μg/ml of diaminopimelic acid was added to the transformation solution, and cultured at 37° C. with shaking. Then, the culture broth was spread onto L medium containing 20 μg/ml of diaminopimelic acid and 20 μg/ml of chloramphenicol, and colonies grew. This was replicated as a master plate to M9 agar medium containing 20 μg/ml of chloramphenicol, and the replicate was incubated at 37° C. for 2 to 3 days. The host could not grow in M9 minimal medium without diaminopimelic acid since it did not have AK activity. In contrast, it was expected that the transformant strain that contained the gene coding for AK derived from Methylophilus methylotrophus could grow in M9 minimal medium because of the function of the gene.

Two transformants out of about 3000 transformants formed colonies on M9 medium. Plasmids were extracted from the colonies which emerged on M9 medium and analyzed. As a result, the presence of an inserted fragment on the plasmids was confirmed. The plasmids were designated pMMASK-1 and pMMASK-2, respectively. By using these plasmids, E. coli GT3 was transformed again. The obtained transformants grew on M9 minimal medium. Furthermore, the transformant which contained each of these plasmids was cultured overnight in L medium containing 20 μg/ml of chloramphenicol, and the cells were collected by centrifugation of the culture broth. Cell-free extracts were prepared by sonicating the cells, and AK activity was measured according to the method of Miyajima et al. (Journal of Biochemistry (Tokyo), vol. 63, 139-148 (1968)) (FIG. 3: pMMASK-1, pMMASK-2). In addition, a GT3 strain harboring the vector pSTV29 was similarly cultured in L medium containing 20 μg/ml of diaminopimelic acid and 20 μg/ml of chloramphenicol, and AK activity was measured (FIG. 3: Vector). As a result, increase in AK activity was observed in two of the clones containing the inserted fragments compared with the transformant harboring only the vector. Therefore, it was confirmed that the gene that could be cloned on pSTV29 was the gene coding for AK derived from Methylophilus methylotrophus. This gene was designated as ask.

The DNA nucleotide sequence of the ask gene was determined by the dideoxy method. It was found that pMMASK-1 and pMMASK-2 contained a common fragment. The nucleotide sequence of the DNA fragment containing the ask gene derived from Methylophilus methylotrophus is shown in SEQ ID NO: 5. The amino acid sequence that can be encoded by the nucleotide sequence is shown in SEQ ID NOS: 5 and 6.

(2) Cloning of Gene Coding for Aspartic Acid Semialdehyde Dehydrogenase (ASD)

E. coli Hfr3000 U482 (CGSC 5081 strain) deficient in the asd gene was transformed by electroporation using Gene library solution B in the same manner as described above. To the transformation solution, SOC medium containing 20 μg/ml of diaminopimelic acid was added and the mixture was cultured at 37° C. with shaking. The cells were harvested by centrifugation. The cells were washed by suspending them in L medium and centrifuging the suspension. The same washing operation was repeated once again, and the cells were suspended in L medium. Then, the suspension was spread onto L agar medium containing 20 μg/ml of chloramphenicol, and incubated overnight at 37° C. The host grew extremely slowly in L medium without diaminopimelic acid since it was deficient in the asd gene. In contrast, it was expected that normal growth would be observed for a transformant strain which contained the gene coding for ASD derived from Methylophilus methylotrophus even in L medium because of the function of the gene. Furthermore, the host E. coli could not grow in M9 minimal medium, but a transformant strain that contained the gene coding for ASD derived from Methylophilus methylotrophus was expected to be able to grow in M9 minimal medium because of the function of the gene. Therefore, colonies of transformants that normally grew on L medium were picked up, streaked and cultured on M9 agar medium. As a result, growth was observed. Thus, it was confirmed that the gene coding for ASD functioned in these transformant strains as expected.

Plasmids were extracted from the three transformant strains which emerged on M9 medium, and the presence of an inserted fragment in the plasmids was confirmed. The plasmids were designated pMMASD-1, pMMASD-2 and pMMASD-3, respectively. When the E. coli Hfr3000 U482 was transformed again with these plasmids, each transformant grew in M9 minimal medium. Furthermore, each transformant was cultured overnight in L medium containing 20 μg/ml of chloramphenicol, and the cells were collected by centrifugation of the culture broth. The cells were sonicated to prepare a crude enzyme solution, and ASD activity was measured according to the method of Boy et al. (Journal of Bacteriology, vol. 112 (1), 84-92 (1972)) (FIG. 4: pMMASD-1, pMMASD-2, pMMASD-3). In addition, the host harboring the vector was similarly cultured in L medium containing 20 μg/ml of diaminopimelic acid and 20 μg/ml of chloramphenicol, and ASD activity was measured as a control experiment (FIG. 4: Vector). As a result, the enzymatic activity could not be detected for the transformant harboring only the vector, whereas the ASD activity could be detected in three of the clones having an insert fragment. Therefore, it was confirmed that the obtained gene was a gene coding for ASD derived from Methylophilus methylotrophus (designated as asd).

The DNA nucleotide sequence of the asd gene was determined by the dideoxy method. It was found that all of the three obtained clones contained a common fragment. The nucleotide sequence of the DNA fragment containing the asd gene derived from Methylophilus methylotrophus is shown in SEQ ID NO: 7. The amino acid sequence that can be encoded by the nucleotide sequence is shown in SEQ ID NOS: 7 and 8.

(3) Cloning of Gene Coding for Dihydrodipicolinate Synthase (DDPS)

E. coli AT997 (CGSC 4547 strain) deficient in the dapA gene was transformed by the same electroporation procedure using Gene library solution A. To the transformation solution, SOC medium containing 20 μg/ml of diaminopimelic acid was added, and the mixture was cultured at 37° C. with shaking. Then, the culture broth was spread onto L medium containing 20 μg/ml of diaminopimelic acid and 100 μg/ml of ampicillin, and colonies grew. This was replicated as a master plate to M9 minimal agar medium containing 100 μg/ml of ampicillin, and the replicate was incubated at 37° C. for 2 to 3 days. The host could not grow in M9 minimal medium that did not contain diaminopimelic acid since it was deficient in dapA gene. In contrast, it was expected that a transformant strain that contained the gene coding for DDPS derived from Methylophilus methylotrophus could grow in M9 minimal medium because of the function of that gene.

Plasmids were extracted from the colonies of two strains emerged on M9 medium, and analyzed. As a result, the presence of the inserted fragment in the plasmids was confirmed. The plasmids were designated pMMDAPA-1 and pMMDAP-2, respectively. When E. coli AT997 was transformed again with these plasmids, each transformant was grown in M9 minimal medium. Furthermore, each transformant containing each plasmid was cultured overnight in L medium containing 100 μg/ml of ampicillin, and the cells were collected by centrifugation of the culture broth. The cells were sonicated to prepare a cell extract, and DDPS activity was measured according to the method of Yugari et al. (Journal of Biological Chemistry, vol. 240, and p. 4710 (1965)) (FIG. 5: pMMDAPA-1, pMMDAPA-2). In addition, the host harboring the vector was similarly cultured in L medium containing 20 μg/ml of diaminopimelic acid and 100 μg/ml of ampicillin, and DDPS activity was measured as a control experiment (FIG. 5: Vector). As a result, the enzymatic activity could not be detected for the transformant harboring only the vector, whereas the DDPS activity could be detected in each of the transformants harboring the plasmids having the insert fragment. Therefore, it was confirmed that this gene was the gene coding for DDPS derived from Methylophilus methylotrophus (designated as dapA).

The DNA nucleotide sequence of the dapA gene was determined by the dideoxy method. It was found that two of the inserted fragments contained a common fragment. The nucleotide sequence of the DNA fragment containing the dapA gene derived from Methylophilus methylotrophus is shown in SEQ ID NO: 9. The amino acid sequence that can be encoded by the nucleotide sequence is shown in SEQ ID NOS: 9 and 10.

(4) Cloning of Gene Coding for Dihydrodipicolinate Reductase (DDPR)

E. coli AT999 (CGSC 4549 strain) deficient in the dapB gene was transformed by the same electroporation procedure as described above using Gene library solution A. To the transformation solution, SOC medium containing 20 μg/ml of diaminopimelic acid was added, and the mixture was cultured at 37° C. with shaking. Then, the cells were harvested by centrifugation. The cells were washed by suspending them in L medium and centrifuging the suspension. The same washing operation was repeated once again, and the cells were suspended in L medium. Then, the suspension was spread onto L agar medium containing 100 μg/ml of ampicillin, and incubated overnight at 37° C. The host grew extremely slowly in L medium not containing diaminopimelic acid since it was deficient in the dapB gene. In contrast, it was expected that normal growth would be observed for the transformant strain that contained the gene coding for DDPR derived from Methylophilus methylotrophus even in L medium because of the function of the gene. Furthermore, the host E. coli could not grow in M9 minimal medium, but it was expected that a transformant strain which contained the gene coding for DDPR derived from Methylophilus methylotrophus would grow in M9 minimal medium because of the function of the gene.

Therefore, a colony of the transformant that grew normally on L medium was streaked and cultured on M9 agar medium. Then, growth was also observed on M9 medium. Thus, it was confirmed that the gene coding for DDPR functioned in the transformant strain. A plasmid was extracted from the colony which grew on M9 medium, and the presence of an inserted fragment in the plasmid was confirmed. When E. coli AT999 was transformed again by using the plasmid (pMMDAPB), the transformant grew in M9 minimal medium. Furthermore, the transformant containing the plasmid was cultured overnight in L medium, and the cells were collected by centrifugation of the culture broth. The cells were sonicated to prepare a cell extract, and DDPR activity was measured according to the method of Tamir et al. (Journal of Biological Chemistry, vol. 249, p. 3034 (1974)) (FIG. 6: pMMDAPB). In addition, the host harboring the vector was similarly cultured in L medium containing 20 μg/ml diaminopimelic acid and 100 μg/ml of ampicillin, and DDPR activity was measured as a control experiment (FIG. 6: Vector). As a result, the enzymatic activity could not be detected for the transformant harboring only the vector, whereas the DDPR activity could be detected for the transformant harboring pMMDAPB. Therefore, it was confirmed that this gene was the gene coding for DDPR derived from Methylophilus methylotrophus (designated as dapB).

The DNA nucleotide sequence of the dapB gene was determined by the dideoxy method. The nucleotide sequence of the DNA fragment containing the dapB gene derived from Methylophilus methylotrophus is shown in SEQ ID NO: 11. The amino acid sequence that can be encoded by the nucleotide sequence is shown in SEQ ID NOS: 11 and 12.

(5) Cloning of Gene Coding for Diaminopimelate Decarboxylase (DPDC)

E. coli AT2453 (CGSC 4505 strain) deficient in the lysA gene was transformed by the same electroporation procedure as described above using Gene library solution A. To the transformation solution, SOC medium was added, and the mixture was cultured at 37° C. with shaking. The cells were harvested by centrifugation. The cells were washed by suspending them in 5 ml of sterilized water and centrifuging the suspension. The same washing operation was repeated once again, and the cells were suspended in 500 μl of sterilized water. Then, the suspension was spread onto M9 minimal agar medium containing 20 μg/ml of chloramphenicol, and incubated at 37° C. for 2 to 3 days. The host did not grow in M9 minimal medium without lysine since it was deficient in the lysA gene. In contrast, it was expected that a transformant strain that contained the gene coding for DPDC derived from Methylophilus methylotrophus would grow in M9 minimal medium because of the function of the gene.

Therefore, plasmids were extracted from the three transformant strains which grew on M9 medium, and analyzed. As a result, the presence of an inserted fragment in the plasmids was confirmed. The plasmids were designated pMMLYSA-1, pMMLYSA-2 and pMMLYSA-3, respectively. When E. coli AT2453 was transformed again by using each of these plasmids, each transformant grew in M9 minimal medium. Furthermore, each transformant containing each plasmid was cultured overnight in L medium containing 20 μg/ml of chloramphenicol, and the cells were collected by centrifugation of the culture broth. The cells were sonicated to prepare a cell extract, and DPDC activity was measured according to the method of Cremer et al. (Journal of General Microbiology, vol. 134, 3221-3229 (1988)) (FIG. 7: pMMLYSA-1, pMMLYSA-2, pMMLYSA-3). In addition, the host harboring the vector was similarly cultured in L medium containing 20 μg/ml of chloramphenicol, and DPDC activity was measured as a control experiment (FIG. 7: Vector). As a result, the enzymatic activity could not be detected for the transformant harboring only the vector, whereas the DPDC activity could be detected in three of the clones having an insert fragment. Therefore, it was confirmed that this gene was the gene coding for DPDC derived from Methylophilus methylotrophus (designated as lysA).

The DNA nucleotide sequence of the lysA gene was determined by the dideoxy method. It was found that all of the three inserted fragments contained a common DNA fragment. The nucleotide sequence of the DNA fragment containing the lysA gene derived from Methylophilus methylotrophus is shown in SEQ ID NO: 13. The amino acid sequence that can be encoded by the nucleotide sequence is shown in SEQ ID NOS: 13 and 14.

INDUSTRIAL APPLICABILITY

According to the present invention, a Methylophilus bacterium having the ability to produce L-amino acids, a method for producing an L-amino acid using the Methylophilus bacterium, and Methylophilus bacterial cells with increased content of an L-amino acid are provided. By the method of the present invention, L-amino acids can be produced using methanol as a raw material. Moreover, novel L-lysine biosynthesis enzyme genes derived from Methylophilus bacteria are provided.

While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.

Claims

1. A method for producing L-lysine comprising culturing a Methylophilus bacterium in a medium containing methanol to produce and accumulate L-lysine in the medium, and collecting L-lysine from the medium, wherein said Methylophilus bacterium has been modified to have enhanced activities of dihydrodipicolinate synthase and aspartokinase by transformation with a DNA from Escherichia coli that encodes dihydrodipicolinate synthase resistant to feedback inhibition by L-lysine and with a DNA from Escherichia coli that encodes aspartokinase resistant to feedback inhibition by L-lysine.

2. The method according to claim 1, wherein said DNA from Escherichia coli that encodes dihydrodipicolinate synthase encodes dihydrodipicolinate synthase comprising replacement of histidine at position 118 in SEQ ID NO: 2 with tyrosine.

3. The method according to claim 1, wherein said DNA from Escherichia coli that encodes aspartokinase encodes aspartokinase comprising replacement of threonine at position 352 in SEQ ID NO: 4 with isoleucine.

Patent History
Publication number: 20130171700
Type: Application
Filed: Feb 21, 2013
Publication Date: Jul 4, 2013
Applicant: AJINOMOTO CO., INC. (Tokyo)
Inventor: Ajinomoto Co., Inc. (Tokyo)
Application Number: 13/772,959
Classifications
Current U.S. Class: Lysine; Diaminopimelic Acid; Threonine; Valine (435/115)
International Classification: C12P 13/08 (20060101);