WO2001002547A1 - Procede de production de l-lysine - Google Patents

Procede de production de l-lysine Download PDF

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Publication number
WO2001002547A1
WO2001002547A1 PCT/JP2000/004347 JP0004347W WO0102547A1 WO 2001002547 A1 WO2001002547 A1 WO 2001002547A1 JP 0004347 W JP0004347 W JP 0004347W WO 0102547 A1 WO0102547 A1 WO 0102547A1
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WIPO (PCT)
Prior art keywords
lysine
producing
gene
isomerase
ribosephosphate
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PCT/JP2000/004347
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English (en)
Japanese (ja)
Inventor
Masakazu Sugimoto
Hisao Ito
Osamu Kurahashi
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Ajinomoto Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ajinomoto Co., Inc. filed Critical Ajinomoto Co., Inc.
Priority to AU55712/00A priority Critical patent/AU5571200A/en
Publication of WO2001002547A1 publication Critical patent/WO2001002547A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • 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.
  • 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.
  • Coryneform bacterium having enhanced ribose phosphate isomerase activity in cells and capable of producing lysine.
  • 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 thermoaminogenes AJ12340 (FERM BP-1539)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a plasmid vector 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.
  • 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.
  • 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.
  • PAJ611 Escherichia Cori AJ11884 (FERM BP-138)
  • 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.
  • the transformation may be carried out according to the transformation method reported in (1).
  • 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)
  • 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)).
  • 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.
  • a DNA-accepting bacterium such as those known for Bacillus subtilis, actinomycetes and yeast
  • 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.
  • a sequence present in multiple copies on chromosomal DNA is used as a target. Perform by homologous recombination.
  • repetitive DNA and inverted repeats present at the end of a transposable element can be used.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • reduced activity 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.
  • 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.
  • 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.
  • inorganic ions small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added.
  • 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.
  • 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. .
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Protein hydrolyzate (bean concentrate) 30 ml
  • Ribose phosphate isomerase is an enzyme in the pentose phosphate cycle.
  • 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.

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  • Chemical & Material Sciences (AREA)
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Abstract

Cette invention concerne un procédé de production de L-lysine qui fait appel à une technique de fermentation améliorée par rapport aux techniques traditionnelles. Cette technique consiste à transférer un gène codant l'isomérase de phosphate de ribose dans une bactérie corynéforme capable de produire de la L-lysine, ce qui augmente l'activité de l'isomérase de phosphate de ribose et accroît la production de L-lysine. Cette invention concerne également une souche utilisée dans cette technique.
PCT/JP2000/004347 1999-07-02 2000-06-30 Procede de production de l-lysine WO2001002547A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU55712/00A AU5571200A (en) 1999-07-02 2000-06-30 Process for producing l-lysine

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JP11/189514 1999-07-02
JP18951499A JP2003169674A (ja) 1999-07-02 1999-07-02 Ｌ−リジンの製造法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2348107A2 (fr) 2003-02-18 2011-07-27 Metabolic Explorer Procédé de préparation de microorganismes évolués permettant la création ou la modification de voies métaboliques

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040934A1 (fr) * 1995-06-07 1996-12-19 Ajinomoto Co., Inc. Procede de production de l-lysine
EP0854189A2 (fr) * 1996-12-05 1998-07-22 Ajinomoto Co., Inc. Procédé de préparation de L-Lysine
EP0857784A2 (fr) * 1996-12-05 1998-08-12 Ajinomoto Co., Inc. Procédé de production de L-Lysine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040934A1 (fr) * 1995-06-07 1996-12-19 Ajinomoto Co., Inc. Procede de production de l-lysine
EP0854189A2 (fr) * 1996-12-05 1998-07-22 Ajinomoto Co., Inc. Procédé de préparation de L-Lysine
EP0857784A2 (fr) * 1996-12-05 1998-08-12 Ajinomoto Co., Inc. Procédé de production de L-Lysine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM I. SORENSEN ET AL.: "Ribose catabolism of Escherichia coli: Characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene, which is involved in regulation of rpiB expression", JOURNAL OF BACTERIOLOGY,, vol. 178, no. 4, 1996, pages 1003 - 1011, XP002927745 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2348107A2 (fr) 2003-02-18 2011-07-27 Metabolic Explorer Procédé de préparation de microorganismes évolués permettant la création ou la modification de voies métaboliques

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AU5571200A (en) 2001-01-22
JP2003169674A (ja) 2003-06-17

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