WO2001002547A1

WO2001002547A1 - Process for producing l-lysine

Info

Publication number: WO2001002547A1
Application number: PCT/JP2000/004347
Authority: WO
Inventors: Masakazu Sugimoto; Hisao Ito; Osamu Kurahashi
Original assignee: Ajinomoto Co., Inc.
Priority date: 1999-07-02
Filing date: 2000-06-30
Publication date: 2001-01-11
Also published as: AU5571200A; JP2003169674A

Abstract

A process for producing L-lysine by a further improved fermentation method compared with the existing methods which comprises transferring a gene encoding ribose phosphate isomerase into a coryneform bacterium capable of producing L-lysine and thus enhancing the ribose phosphate isomerase activity to thereby elevate the L-lysine productivity; and a strain to be used therein.

Description

Statement

TECHNICAL FIELD The present invention relates to a method for producing L-lysine by a fermentation method. L-Lysine is widely used as a feed additive. BACKGROUND ART Conventionally, L-lysine has been industrially produced by a fermentation method using a coryneform bacterium belonging to the genus Brevipacterium and Corynebacterium having the ability to produce L-lysine. For these coryneform bacteria, strains isolated from the natural world or artificial mutants of the strains are used in order to improve productivity.

In addition, various techniques for increasing L-amino acid producing ability by enhancing L-lysine biosynthetic enzyme activity by recombinant DNA technology have been disclosed. For example, in a coryneform bacterium capable of producing L-lysine, a gene (mutant lysC) encoding aspartokinase from which feedback inhibition by L-lysine and L-threonine has been released, a dihydrodipicolinate reductase gene (DapB), dihydrodipicolinate synthase gene (dapA), diaminopimelate decarboxylase gene (lysA), and diaminopimelate dehydrogenase gene (ddh) (W096 / 40934), lysA and ddh (Japanese Patent Laid-Open No. 9-322774). No.), lysC, lysA and phosphoenolpyruvate carboxylase gene (ppc) (JP-A-10-165180), variant lysC, dapB, dapA, lysA and aspartate aminotransferase gene (aspC) (JP-A-10-215883) Is known to improve the production of L-lysine in the bacterium. .

In the bacterium belonging to the genus Escherichia, dapA, mutant lys dapB, diaminobimeric acid dehydrogenase gene (ddh) (or tetrahydrodipicolinate succinylase gene (dapD), and succinyldiaminopimelate deacylase gene It is known that the sequential amplification or introduction of (dapE)) enhances L-lysine production ability (W095 / 16042). In WO95 / 16042, tetrahydrodipicolinate succinylase is erroneously described as succinyldiaminopimelate transaminase. However, the structure of the gene encoding ribosephosphate isomerase has not been reported in coryneform bacteria, and the gene encoding ribosephosphate isomerase can be used for breeding coryneform bacteria. Not even known. DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for producing L-lysine by a fermentation method which has been further improved than before, and a bacterial strain used therefor.

The present inventors have conducted intensive studies to solve the above problems, and as a result, introduced a gene encoding ribose phosphate isomerase into a coryneform bacterium, and demonstrated the activity of ribose phosphate isomerase. It has been found that the production of L-lysine can be increased by increasing the amount of L-lysine, and the present invention has been completed. That is, the present invention is as follows.

(1) Coryneform bacterium having enhanced ribose phosphate isomerase activity in cells and capable of producing lysine.

(2) The coryneform according to (1), wherein the enhancement of the ribosephosphate isomerase activity is caused by increasing the copy number of a gene encoding ribosephosphate isomerase in the bacterial cell. Type bacteria.

(3) The coryneform bacterium according to (2), wherein the gene encoding the ribosephosphate isomerase is derived from a bacterium belonging to the genus Escherichia.

(4) A method comprising culturing the coryneform bacterium according to any of (1) to (3) in a medium, producing and accumulating L-lysine in the culture, and collecting L-lysine from the culture. The production method of L-lysine. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

<1> Coryneform bacterium of the present invention

The coryneform bacterium of the present invention is a coryneform bacterium having L-lysine-producing ability and enhanced ribosephosphate isomerase activity in cells.

The coryneform bacterium referred to in the present invention is a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, p. 599 (1974). Yes, aerobic, Gram-positive, non-acid-fast, non-spore-forming bacilli, including bacteria that were previously classified as Brevipacterium but are now integrated as Corynebacterium. (Int. J. Syst. Bacterid., 41, 255 (1981)), and also include bacteria of the genus Brevipacterium and Microbatterium, which are very closely related to the genus Corynepacterium. The strains of coryneform bacteria suitably used for the production of L-lysine include, for example, those shown below.

Corynebacterium acetoacid film ATCC13870

Corynebacterium acetoglu mikum ATCC15806

Corynebacterium carnaet ATCC15991

Corynepaterum · Guru Yu Mikum ATCC13032

(Brevipactivium dibaricatam) ATCC14020

(Brevipactium · Lactofamentum) ATCC13869

(Corynebacterium. Lilium) ATCC15990

(Brevipactium / Flavum) ATCC14067

Coli non-bacterium Melase cola ATCC17965

Brevibacterium Saccharolyticum ATCC1 wound 6

Brevibacterium in Mario film ATCC14068

Brevibacterium roseum ATCC13825

Brevibacterium Choge Niyu Squirrel ATCC 19240

Microbacterium ammonia Filum ATCC15354

Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) To obtain these, for example, American Type Culture Collection (American Type Culture Collection ^ Address 12301 Park lawn Drive, Rockville, Maryland 20852, United States of America). That is, a registration number corresponding to each microorganism is assigned, and the microorganism can be ordered by referring to this registration number. The registration number corresponding to each microorganism can be found in the American Type Culture Collection catalog. Also,

The AJ12340 strain has been deposited with the Ministry of International Trade and Industry at the Institute of Biotechnology and Industrial Technology (ZIP 1-3-1, Tsukuba, Ibaraki, Japan) under the Budapest Treaty.

In addition to the above strains, mutants having L-lysine-producing ability derived from these strains can also be used in the present invention. Such artificial mutants include the following. S— (2-aminoethyl) one cysteine (hereinafter abbreviated as “AEC”) resistant mutant (for example, Brevipacterium lactofarmentum AJ11082 (NRR LB-11470), Japanese Patent Publication No. 56-1914, JP-B-56-1915, JP-B-57-14157, JP-B-57-14158, JP-B-57-30474, JP-B-58-10075, JP-B-59-4993, JP-B-61-35840, (See Japanese Patent Publication No. 62-24074, Japanese Patent Publication No. 62-36673, Japanese Patent Publication No. 5-11958, Japanese Patent Publication No. 7-112437, Japanese Patent Publication No. 7-112438), and their growth requires amino acids such as L-homoserine. (JP-B-48-28078, JP-B-56-6499), which are resistant to AEC, and furthermore, L-leucine, L-homoserine, L-proline, L-serine, L-arginine, L-alanine, Mutants that require amino acids such as L-parin (US Pat. Nos. 3,708,395 and 3,825,472), DL-amino-ε L-lysine-producing mutants resistant to caprolactam, hyaminoaminolauryl lactam, aspartic acid monoanalog, sulfa drugs, quinoids, Ν-lau mouth ylleucine, oxaza mouth acetate decarboxylase (decarboxylase) or respiration L-lysine-producing mutants showing resistance to an enzyme inhibitor (Japanese Patent Application Laid-Open Nos. 50-53588, 50-31093, 52-102498, 53-9394, and 53-9394, 53-86089, JP-A-55-9783, JP-A-55-9759, JP-A-56-32995, JP-B-56-39778, JP-B-53-43591, JP-B-53-1833 ), L-lysine-producing mutants that require inositol or acetic acid (JP-A-55-9784, JP-A-56-8692), L-lysine that is sensitive to fluoropyruvic acid or a temperature of 34 ° C or higher. —Lysine-producing variants (JP-A-55-9783, JP-A-53-86090), ethylene glycol Le to show resistance, A mutant strain of Brevipacterium or Corynepacterium that produces L-lysine (US Patent No. 4411997).

As used herein, the term "L_lysine-producing ability" refers to the ability to accumulate a significant amount of L-lysine in a medium when a coryneform bacterium is cultured in the medium, or the L-lysine in the cells. The ability to increase lysine.

<2> Enhancement of ribose phosphate isomerase activity

In order to enhance ribosephosphate isomerase activity in coryneform bacterial cells, a gene fragment encoding ribosephosphate isomerase is ligated to a vector, preferably a multicopy vector, which functions in the bacterium. Then, a recombinant DNA may be prepared and introduced into a coryneform bacterium capable of producing L-lysine for transformation. As a result of an increase in the copy number of the gene encoding ribosephosphate isomerase in the cells of the transformed strain, ribosephosphate isomerase activity is enhanced. Ribose phosphate isomerase is encoded in the rpi gene in Escherichia coli.

As the ribosephosphate isomerase gene, either a gene of a coryneform bacterium or a gene derived from another organism such as a bacterium belonging to the genus Escherichia can be used.

The nucleotide sequence of the rpi gene of Escherichia coli has already been elucidated (Sorensen, K.I. et al., J. Bacteriol., 178 (4), 1003-1011 (1996), Genbank / EMBL / DDBJ accetion No. X82203), using a primer prepared based on the nucleotide sequence, for example, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing, and using the chromosomal DNA of Escherichia coli as a type II (PCR: polymerase chain reaction) White, TJ et al; see Trends Genet. 5, 185 (1989)) to obtain the rpi gene. Genes encoding ribosephosphate isomerase of other microorganisms such as coryneform bacteria can be obtained in a similar manner.

Chromosomal DNA is obtained from bacteria that are DNA donors, for example, by the method of Saito and K. Miura (H. Saito and K. Miura Biochem. Biophys. Acta, 72, 619 (1963)), Biological Engineering Experiment, Japan Society for Biotechnology Pp. 97-98, Baifukan, 1992). The gene encoding ribosephosphate isomerase amplified by the PCR method is connected to a vector DNA capable of autonomous replication in cells of Escherichia coli and / or coryneform bacteria to prepare recombinant DNA, If this is introduced into Escherichia coli cells, subsequent operations will be slow. As a vector capable of autonomous replication in Escherichia coli cells, a plasmid vector is preferable, and a vector capable of autonomous replication in a host cell is preferable.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSF1010 and the like are preferable. No.

Examples of vectors capable of autonomous replication in coryneform bacterium cells include PAM330 (see Japanese Patent Application Laid-Open No. 58-67699), pHM1519 (see Japanese Patent Application Laid-Open No. 58-77895), and the like. In addition, a DNA fragment capable of autonomously replicating plasmid in coryneform bacteria is extracted from these vectors and inserted into the Escherichia coli vector, whereby autonomous replication in both Escherichia coli and coryneform bacteria occurs. It can be used as a shuttle vector that is not possible. The following are examples of such shuttle vectors. Microorganisms carrying the respective vectors and the accession numbers of the international depository organizations are shown in parentheses.

PAJ655 Escherichia 'Kori AJ11882 (FERM BP-136)

Coryneha ,, Kteridum ,, Rutamicum SR8201 (ATCC39135)

PAJ1844; [Seriheria coli] AJ11883 (FERM BP-137)

Coryneha ,, Kteridum, Rutamikumu SR8202 (ATCC39136)

PAJ611 Escherichia Cori AJ11884 (FERM BP-138)

PAJ3148 Coryneha, Cterizum, Rutamicum SR8203 (ATCC39137)

PAJ440; r, f 'r7 WAJ11901 (FERM BP-140)

pHC4 I colihori 'coli AJ12617 (FERM BP-3532)

To prepare recombinant DNA by ligating the gene encoding ribosephosphate isomerase and the vector that functions in coryneform bacterium, a restriction enzyme that matches the end of the gene encoding ribosephosphate isomerase is used. Cut the vector. Ligation is usually performed using a ligase such as T4DNA ligase.

To introduce the recombinant DNA prepared as described above into coryneform bacteria, The transformation may be carried out according to the transformation method reported in (1). For example, a method for increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for E. coli K_12 (Mandel, M. and Higa, A., J. Mol. Biol. , 53, 159 (1970)), and a method for preparing DNA from transgenic cells and introducing DNA as described in Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)). Alternatively, cells of a DNA-accepting bacterium, such as those known for Bacillus subtilis, actinomycetes and yeast, are transformed into protoplasts or spheroplasts that readily incorporate the recombinant DNA, and the recombinant DNA is transferred to the DNA recipient. (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979) Bibb, MJ, Ward, JMand Hopwood, 0.A., Nature, 274, 398 (1978) Hinnen, A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). The transformation method used in the examples of the present invention is the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791).

Enhancement of the activity encoding ribosephosphate isomerase can also be achieved by the presence of multiple copies of the gene encoding reporter phosphate isomerase on the chromosomal DNA of the host. In order to introduce multiple copies of a gene encoding ribosephosphate isomerase into the chromosomal DNA of a microorganism belonging to the coryneform bacterium, a sequence present in multiple copies on chromosomal DNA is used as a target. Perform by homologous recombination. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-1099985, a gene encoding ribose phosphate isomerase is mounted on a transposon and transferred, and multiple copies are introduced into chromosomal DNA. Is also possible. Either method increases the copy number of the gene encoding ribosephosphate isomerase in the transformant, resulting in enhanced ribosephosphate isomerase activity.

Enhancement of ribosephosphate isomerase activity is not only due to the above gene amplification, but also as ribosephosphate isomerase on chromosome DNA or plasmid. This can also be achieved by replacing the expression control sequence such as the promoter of the gene encoding zeo with a strong one (see Japanese Patent Application Laid-Open No. 1-215280). For example, lac promoter, trp promoter, trc promoter, tac bromo overnight, lambda phage PR promoter, PL promoter, etc. are known as strong promoters. Substitution of these promoters enhances ribose phosphate isomerase activity by enhancing expression of the gene encoding reporter phosphate isomerase.

Further, the coryneform bacterium of the present invention can enhance the enzymatic genes of other L-lysine biosynthetic pathways or glycolytic pathways in addition to the ribose phosphate isomerase activity, thereby enhancing their enzymatic activities. Good. Examples of such a gene include a gene coding for Aspartokinase subunit protein or /? Subunit protein in which synergistic feedback inhibition by L-lysine and L-threonine has been substantially eliminated (W094 / 25605 international publication pamphlet), a wild-type phosphoenolpyruvate carboxylase gene derived from a coryneform bacterium (Japanese Patent Application Laid-Open No. 60-87788), a gene encoding a wild-type dihydrodipicolinate synthase derived from a coryneform bacterium (Japanese Patent Publication No. No. 55149).

In addition, the activity of an enzyme that catalyzes a reaction that produces a compound other than L-lysine by branching off from the L-lysine biosynthetic pathway may be reduced or lacking. One such enzyme is homoserine dehydrogenase (see WO 95/23864).

As used herein, the phrase “enhanced activity” of an enzyme generally means that the enzyme activity in a cell is higher than that of a wild-type strain, and is modified by a gene recombination technique or the like. When a strain with enhanced enzyme activity is obtained, it means that the enzyme activity in the cell is higher than that of the strain before modification. The term “reduced activity” of an enzyme usually means that the enzyme activity in a cell is lower than that of a wild-type strain, and the enzyme activity has been reduced by modification by genetic recombination technology or the like. When a strain is obtained, it means that the enzyme activity in the cell is lower than that of the strain before modification.

3> L-Lysine production

If coryneform bacteria having enhanced ribose phosphate isomerase activity and L-lysine producing ability are cultured in a suitable medium, L-lysine accumulates in the medium. You.

The medium used for producing L-lysine using the microorganism of the present invention is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, other organic micronutrients. Carbon sources include carbohydrates such as glucose, lactose, galactose, fructose, sucrose, molasses, starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, citric acid, succinic acid, etc. Organic acids can be used.

Nitrogen sources include inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, corn 'steep' liquor, soybean hydrolyzate, etc. Organic nitrogen, ammonia gas, aqueous ammonia, etc. can be used.

As inorganic ions, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added. As organic trace nutrients, it is desirable to include a required substance such as vitamin B1 or a yeast extract in an appropriate amount as necessary.

The culture is preferably carried out for 16 to 72 hours under aerobic conditions, such as shaking culture and aeration / agitation culture.The culture temperature is 30 to 45, and the pH during culture is 5 to 9. Control It is to be noted that inorganic or organic acidic or alkaline substances, ammonia gas and the like can be used for pH adjustment.

The collection of L-lysine from the fermentation broth can usually be carried out by a combination of ion exchange resin method, precipitation method and other known methods. Examples Hereinafter, the present invention will be described more specifically with reference to Examples.

<1> Cloning of rpi gene of Escherichia coli JM109

The nucleotide sequence of the rpi gene of Escherichia coli has already been elucidated (Sorensen, K. I. et al., J. Bacterid., 178 (4), 1003-1011 (1996), Genbank / EMBL / J accetion No. X82203 ). Based on the reported nucleotide sequence, the primers shown in SEQ ID NOs: 1 and 2 were synthesized, and the pyruvate dehydrogenase gene was amplified by PCR using the chromosomal DNA of Escherichia coli JM109 strain as type III. .

Among the synthesized primers, SEQ ID NO: 1 corresponds to the sequence from the 1st to 24th base of the base sequence of the rpi gene described in Genbank / EMBL / DDBJ accetion No. X 82203, and SEQ ID NO: 2 23 1 corresponds to the sequence from the 2nd to the 2289th base.

The chromosomal DNA of Escherichia coli JM109 strain was prepared by a conventional method (Biotechnological Experiments, edited by Biotechnology Society of Japan, pp. 97-98, Baifukan, 19992). For the PCR reaction, standard reaction conditions described on page 185 of the front line of the PCR method (edited by Takeo Sekiya et al., Kyoritsu Shuppan, 1989) were used.

The resulting PCR product was purified by a conventional method, ligated with a smal-cleaved plasmid pHC4 using a ligation kit (Takara Shuzo), and then combined with Escherichia coli KM JM109 (Takara Shuzo). Lactic acid medium containing 30 g / ml of chloram fenicol (Bacto Tribton 10 g / L, Bactoist Extract 5 g / L, NaCl 5 g / L, Agar 15 g / L, After the culture was completed, the white colonies that appeared were picked up and separated into single colonies to obtain a transformed strain. Plasmid was extracted from the obtained transformant to obtain a plasmid pHC4rpi in which the rpi gene was linked to the vector.

Escherichia coli, which retains pH C4, was named private number AJ12617, and on April 24, 1999, the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (ZIP code 305-8566, Ibaraki, Japan) Deposit No. FE RM P—122 1-5 at Higashi 1-3-chome, Tsukuba City, Prefectural Government, and transferred to an international deposit under the Budapest Convention on August 26, 1999, and deposited under accession number FERM BP. — 3532 is granted.

Next, in order to confirm that the cloned DNA fragment encodes a protein having ribose phosphate isomerase activity, the ribose phosphate of the JM109 strain and the JM109 strain carrying pHC4rpi were The fate isomerase activity Juo was transferred to Rack, E. et al., Methods of Enzymatic Analysis (Bergmeyer, H.

Corrected form (Rule 91) G. ed.) Pp. 186-187, Academic Press (1965). As a result, the JM109 strain carrying pHC4 rpi showed about 18 times the ribose phosphate isomerase activity of the JM109 strain not carrying pHC4 rpi, indicating that the rpi gene was expressed. Confirmed that.

<3> Coryneform bacterium of L- lysine introduction pHC4rpi on production lines and L -. Lysine-producing Brevibacterium Park Teri © beam plasmid _P HC4rpi by lactofermentum AJ11082 an electric pulse method (see Japanese Patent Laid-Open No. 2 -207791) And the obtained transformant was obtained. Using the obtained transformant AJ11082 / pHC4rpi, culture for producing L-lysine was performed as follows. AJ11082 / pHC4rpi strain obtained by culturing in a CM2B plate medium containing 5〃g / ml chloramphenicol was mixed with the following composition containing 5〃g / m1 chloramphenicol. L-lysine production medium was inoculated and cultured at 31.5 ° C with shaking until the sugar in the medium was consumed. As a control, a strain obtained by transforming a plasmid pHC4 capable of autonomously replicating with a previously obtained corynebacterium bacterium by the electric pulse method was cultured in the corynebacterium bacterium AJ11082 strain in the same manner as described above.

Brevipacterium. Lactofermentum AJ11082 was established on January 31, 1981 at the Agricultural Research Culture Collection, Illinois, United States, 6 1 604 Peoria Northuniversity Street 1 815 ( 1815 N. University Street, Peoria, Illinois 61604 USA)) and deposited under accession number NRRL B-11470.

(L-lysine production medium)

Dissolve the following components (in 1 L) other than calcium carbonate, adjust to pH 8.0 with K0H, sterilize at 115 ° C for 15 minutes, and add 50 g of dry-heat sterilized calcium carbonate.

100 g glucose

(NH ₄ ) ₂ S 0 ₄ 55 S

KH ₂ P 0 ₄ 1 g

_{_{Mg S 0 4 · 7 Η 2}} 0 1 g

Piotin 500 S

Thiamine 2000 g

Nicotinamide 5 mg

Protein hydrolyzate (bean concentrate) 30 ml

50 g calcium carbonate

After completion of the culture, the amount of L-lysine accumulated in the culture solution was measured using a Biotech Analyzer-AS-210 manufactured by Asahi Kasei Corporation. Table 1 shows the results. Table 1 L-lysine production (g / L)

AJ11082 / pHC4 29.2

AJ11082 / pHC4rpi 34.4

The results in Table 1 show that L-lysine-producing ability was improved by enhancing ribose phosphate isomerase activity.

Ribose phosphate isomerase is an enzyme in the pentose phosphate cycle. On the other hand, L-lysine biosynthesis requires NADPH, and NAD P + is produced with the production of L-lysine. Therefore, it is considered that the pentose phosphate cycle progresses by enhancing the report phosphate isomerase activity, and the LAD-lysine biosynthesis reaction proceeds smoothly by the generated NAD PH. Possibility According to the present invention, the ability of coryneform bacteria to produce L-lysine can be improved.

Claims

The scope of the claims

1. Coryneform bacterium having enhanced ribose phosphate isomerase activity in cells and capable of producing lysine.

2. The coryneform bacterium according to claim 1, wherein the enhancement of ribosephosphate isomerase activity is caused by increasing the copy number of a gene encoding ribosephosphate isomerase in the bacterial cell.

3. The coryneform bacterium according to claim 2, wherein the gene encoding the ribose phosphate isomerase is derived from a bacterium belonging to the genus Escherichia.

4. A method comprising culturing the coryneform bacterium according to any one of claims 1 to 3 in a medium, producing and accumulating L-lysine in the culture, and collecting L-lysine from the culture. Characteristic method for producing L-lysine.