CA2319716A1 - New nucleotide sequences which code for the eno gene - Google Patents

Abstract

The invention relates to an isolated polynucleotide comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70 % to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70 % to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b) and d) polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of a), b) or c).
and a process for the fermentative preparation of L-amino acids with amplification of the eno gene.

Description

New nucleotide sequences which code for the eno gene The invention provides nucleotide sequences which code for the eno gene and processes for the fermentative preparation of amino acids, in particular L-lysine, using coryneform bacteria in which the eno gene is amplified.
Prior art Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but in particular in animal nutrition.
It is known that amino acids are prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of its great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form by e.g.
ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these -microorganisms. Strains which are resistant to antimetabolites, such as e.g. the lysine analogue S-(2-aminoethyl)-cysteine, or are auxotrophic for amino acids of regulatory importance and produce L-lysine are obtained in this manner.

., Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce amino acids, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
Review articles in this context are to be found, inter alia, in Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).

Object of the invention The inventors had the object of providing new measures for improved fermentative preparation of amino acids, in particular L-lysine.
Description of the invention Amino acids, in particular L-Lysine, are used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.
When L-lysine or lysine are mentioned in the following, not only the base but also the salts, such as e. g. lysine monohydrochloride or lysine sulfate, are also meant.
The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70 % to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence.of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70o to the amino acid sequence of SEQ ID No. 2, ' ~ 990152 BT

c) polynucleotide which is complementary to the polynucleotides of a) or b) and d) polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of a), b) or c).
The invention also provides the polynucleotide according to claim 1, this preferably being a DNA which is capable of replication, comprising:
(i) the nucleotide sequence shown in SEQ ID no. 1, or (ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and optionally (iv) sense mutations of neutral function in (i).
The invention also provides a polynucleotide according to claim 4, comprising the nucleotide sequence as shown in SEQ ID no. 1, a polynucleotide according to claim 6, which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2, a vector containing the polypeptide according to claim 1, in particular a shuttle vector or plasmid vector S
and coryneform bacteria serving as the host cell, which contain the vector.
The invention also provides polynucleot:ides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which contains the complete gene with the polynucleotide sequence corresponding to SEQ
ID no. 1, with a probe which contains the sequence of the polynucleotide mentioned, according to SEQ ID no. 1, or a fragment thereof, and isolation of the DNA sequence mentioned.
Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, cDNA which code for enolase and to isolate those cDNA or genes which have a high similarity of sequence with that of the enolase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the preparation of DNA
of genes which code for enolase by the polymerase chain reaction (PCR).
Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, especially preferably at least 15 successive bases. Oligonucleotides which have a length of at least 40 or 50 base pairs are also suitable.
"Isolated" means separated out of its natural environment.

"Polynucleotide" in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
"Polypeptides" is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
The polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those with the biological activity of enolase, and also those which are identical to the extent of at least 70 % to the polypeptide according to SEQ ID No. 2, and preferably are identical to the extent of 80o and in particular to the extent of at least 90 % to 95 % to the polypeptide according to SEQ ID no. 2, and have the activity mentioned.
The invention also provides a process for the fermentative preparation of amino acids, in particular L-lysine, using coryneform bacteria which in particular already produce an amino acid, and in which the nucleotide sequences which code for the eno gene are amplified, in particular over-expressed.
The term "amplification" in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene which codes for a corresponding enzyme having a high activity, and optionally combining these measures.

The microorganisms which the present invention provides can prepare L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be S representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among specialists for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC17965 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and L-lysine-producing mutants or strains prepared therefrom, such as, for example Corynebacterium glutamicum FERM-P 170 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum. FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSMS71S.

The inventors have succeeded in isolating the eno gene of C. glutamicum which codes for the enzyme enolase (EC
4.2.1.11).
y To isolate the eno gene or also other genes of C.
glutamicum, a gene library of this microorganism is first set up in E. coli. The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene and Klone, Eine Einfuhrung in die Gentechnologie [Genes and Clones, An Introduction to l0 Genetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known gene library is that of the E. coli K-12 strain W3110 set up in ~ vectors by Kohara et al. (Cell 50, 495 - 508 (1987)). Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the E.coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bormann et al. (Molecular Microbiology 6(3), 317-326)) in turn describe a gene library of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). To prepare a gene library of C. glutamicum in E. coli it is also possible to use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Viera et al., 1982, Gene, 19:259-268). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination-defective. An example of these is the strain DHSamcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned with the aid of cosmids can then in turn be subcloned and subsequently sequenced in the usual vectors which are suitable for sequencing, such as is described e.g. by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).
The new DNA sequence of C. glutamicum which codes for the eno gene and which is a constituent of the present invention as SEQ ID NO l.was obtained in this manner. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the eno gene product is shown in SEQ ID NO 2.
Coding DNA sequences which result from SEQ ID NO 1 by the degeneracy of the genetic code are also a constituent of the invention. In the same way, DNA sequences which -hybridize with SEQ ID NO 1 or parts of SEQ ID NO 1 are a constituent of the invention. Conservative amino acid exchanges, such as e.g. exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, are furthermore known among experts as "sense mutations" which do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. It is furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bass<~t et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and 5 molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID NO 2 are also a constituent of the invention.
In the same way, DNA sequences which hybridize with SEQ ID
NO 1 or parts of SEQ ID NO 1 are a constituent of the 10 invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID NO 1 are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 base pairs.
Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook "The DIG System Users Guide for Filter Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260).
Instructions for amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) can be found by the expert, inter alia, in the handbook by Gait:
Oligonukleotide [sic] synthesis: a practical approach (IRL
Press, Oxford, UK, 1984) and in Newton and Graham: PCR
(Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
The inventors have found that coryneform bacteria produce amino acids, in particular L-lysine in an improved manner after over-expression of the eno gene.

To achieve an over-expression, the number of copies of the corresponding genes can be increased, or the promoter and regulation region or the ribosome binding site upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same way. By inducible promoters, it is additionally possible to increase the expression in the course of fermentative L-lysine production. The expression is likewise improved by measures to prolong the life of the m-l0 RNA. Furthermore, the enzyme activity is also increased by preventing the degradation of the enzyme protein. The genes or gene constructions can either be present in plasmids with a varying number of-copies, or can be integrated and amplified in the chromosome. Alternatively, an over-expression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.
Instructions in this context can be found by the expert, inter alia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994))r Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European Patent Specification EPS 0 472 869, in US Patent 4,601,893, in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in Patent Application 6, in Malumbres et al. (Gene 134, 15 - 24 (1993)), in Japanese Laid-Open Specification JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.
By way of example, the eno gene according to the invention was over-expressed with the aid of plasmids. Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as e.g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102:
93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLl or pGAl. Other plasmid vectors, such as e. g. those based on pCG4~(US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS
Microbiology Letters 66, 119-124 (1990)), or pAGl (US-A
5,158,891) can be used in the same manner.
In addition, it may be advantageous for the production of amino acids, in particular L-lysine, to over-express one or more enzymes of the particular biosynthesis route, of glycolysis, of anaplerosis, of the citric acid cycle or of amino acid export, in addition to the eno gene.
Thus, for example, for the preparation of L-lysine ~ at the same time the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0 197 335),-or ~ at the same time the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992). Journal of Bacteriology 174:6076-6086), or ~ at the same time the tpi gene which codes for triose phosphate isomerase (Eikmanns (1992). Journal of Bacteriology 174:6076-6086), or ~ at the same time the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns (1992). Journal of Bacteriology 174:6076-6086), or ~ at the same time the pyc gene which codes for pyruvate carboxylase (Eikmanns (1992). Journal. of Bacteriology 174:6076-6086), or ~ at the same time the lysE gene which codes for lysine export (DE-A-195 48 222) can be over-expressed.
In addition to over-expression of the eno gene it may furthermore be advantageous, for the production of amino acids, in particular L-lysine, to eliminate undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982). , The microorganisms prepared according to the invention can be cultured continuously or discontinuously in t-he batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of amino acids, in particular L-lysine. A summary of known culture methods are [sic]
described in the textbook by Chmiel (Bioprozesstechnik 1.

Einfuhrung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen S [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 181). Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e. g. Soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e. g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e. g. glycerol and ethanol, and organic acids, such as e. g. acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture. Organic nitrogen- _ containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e. g.
magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the 5 abovementioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.
10 Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to 15 control the development of foam. Suitable substances having a selective action, e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g, air, are introduced into the culture. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C. Culturing is continued until a maximum of lysine has formed. This target is usually reached within 10 hours to 160 hours.
The analysis of L-lysine takes place can be carried out [sic) by anion exchange chromatography with subsequent ninhydrin derivatization, as described by Spackman et al.
(Analytical Chemistry, 30, (1958), 1190).

The process according to the invention is used for the fermentative preparation of amino acids, in particular L-lysine.

Examples The present invention is explained in more detail in the following with the aid of embodiment examples.
Example 1 Preparation of a genomic cosmid gene library from Corynebacterium glutamicum ATCC 13032 Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250). The DNA of the cosmid vector SuperCosl (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164), obtained from the company Stratagene (La Jolla, USA, Product Description SuperCosl Cosmid Vektor Kit, Code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, , Code no. 27-0948-02) and likewise dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). The cosmid DNA treated in this manner was mixed with the treated ATCC13032 DNA and the batch was treated with T4 DNA lipase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Lipase, Code no.27-0870-04). The ligation mixture was then packed in phages with the aid of Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, Product Description Gigapack II
XL Packing Extract, Code no. 200217). F'or infection of the E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575) the cells were taken up in 10 mM
MgS04 and mixed with an aliquot of the phage suspension.
The infection and titering of the cosmid library were carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the cells being plated out on LB agar (Lennox, 1955, Virology, 1:190) with 100 ~g/ml ampicillin. After incubation overnight at 37°C, recombinant individual clones were selected.
Example 2 Isolation and sequencing of the eno gene The cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacies, Freiburg, Germany, Product .
Description Sau3AI, Product No. 27-0913-02). The DNA
fragments were dephosphorylated with shrimp alkaline phosphatase -(Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250).
After separation by gel electrophoresis, the cosmid fragments in the size range of 1500 to 2000 by were isolated with the QiaExII Gel Extraction Kit (Product No.
20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1, obtained from the company Invitrogen (Groningen, The Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-O1) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No.
27-0868-04). The ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli strain DHSaMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 ~g/ml zeocin. The plasmid preparation of the recombinant clones was carried out with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out by the dideoxy chain-stopping method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-567) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067). The "RR dRhodamin Terminator Cycle Sequencing Kit" from PE Applied Biosystems(Product No. 403044, Weiterstadt, Germany) was used. The separation by gel electrophoresis and analysis of the sequencing reaction were carried outs in a "Rotiphoresis NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No.
A124.1, Roth, Karlsruhe, Germany) with t:he "ABI Prism 377"
sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0. The individual sequences of the pZerol derivatives were assembled to a continuous contig.
5 The computer-assisted coding region analysis [sic] were prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyses were carried out with the "BLAST search program" (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402), against the non-10 redundant databank of the "National Center for Biotechnology Information" (NCBI, Bethesda, MD, USA).
The nucleotide sequence obtained is shown in SEQ ID NO 1.
Analysis of the nucleotide sequence showed an open reading frame of 1275 base pairs, which was called the eno gene.
15 The eno gene codes for a protein of 425 amino acids.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Degussa-Hiils Aktiengesellschaft (B) CITY: Frankfurt: am Main (C) COUNTRY: Germany (D) POSTAL CODE (ZI_f): DE-60287 (ii) TITLE OF INVENTION: NEW NUCLEOTIDE SEQUENCES WHICH
CODE FOR THE ENO GENE
(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) NAME: Marks & Clerk (B) STREET: 280 Slat:er Street, Suite 1800 (C) CITY: Ottawa (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (Z:CP): K1P 1C2 (v) COMPUTER-READABLE FORM:
(A) MEDIUM TYPE: D_L:~kette (B) COMPUTER: IBM I?C
(C) OPERATING SYSTEM: MS DOS
(D) SOFTWARE: Patent:In Ver. 2.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,319,716 (B) FILING DATE: 2000-10-04 (C) CLASSIFICATION: Unknown (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 199 47 791.4 (B) FILING DATE: 1999-10-05 (C) CLASSIFICATION: Unknown (viii) PATENT AGENT INFORhIATION:
(A) NAME: Richard .J. Mitchell (B) REGISTRATION NI7MBER:
(C) REFERENCE/DOCKET NUMBER: 99472-3 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 236-9561 (B) TELEFAX: (613) 230-8821 (2) INFORMATION FOR SEQ ID NO.: 7.:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1578 (B) TYPE: nucleic.~ acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium glutamicum (ix) FEATURE:

(A) CDS
NAME/KEY:

(B) (151)..(1425) LOCATION:

(xi ) SEQUENCE SEQID O.: l:
DESCRIPTION: N

ATGGGTAGTT C
TTCGCCACTA
A'CT

AATTGGGTGT A
AGACGTGATT

AGTTGGCATA ATC TTC
GGAGGCCACA ATG
G'PG

Va31AlaGlu IleMetHis ValPhe GCTCGC GAA CTC GACTCCCGC GG'L'AACCCA ACCGTCGAG GCAGAG 222 ATT

AlaArg Glu Leu AspSerArg GlyAsnPro ThrValGlu AlaGlu Ile GTTTTC CTG GAC GGTTCCCAC GG'PGTCGCA GGTGTTCCA TCCGGC 270 GAT

ValPhe Leu Asp GlySerHis GlyValAla GlyValPro SerGly Asp GCATCC ACC GTC CACGAGGCT CA'PGAGCTG CGTGACGGT GGCGAT 318 GGC

AlaSer Thr Val HisGluAla HisGluLeu ArgAspGly GlyAsp Gly CGCTAC CTG AAG GGCGT'PTTG AAGGCAGTT GAAAACGTC AACGAA 366 GGC

ArgTyr Leu Lys GlyVa.1Leu LysAlaVal GluAsnVal AsnGlu Gly 60 6.5 70 GAAATC GGC GAG CTCGC'rGGC CTAGAGGCT GACGATCAG CGCCTC 414 GAC

GluIle Gly Glu LeuAlaGly LeuGluAla AspAspGln ArgLeu Asp ATCGAC GAA ATG ATCAAGCTT GA'rGGCACC GCCAACAAG TCCCGC 962 GCA

IleAsp Glu Met IleLysLeu AspGlyThr AlaAsnLys SerArg Ala CTGGGT GCA GCA ATCCTTGGT GT'rTCCATG GCTGTTGCA AAGGCT 510 AAC

LeuGly Ala Ala IleLeuGly ValSerMet AlaValAla LysAla Asn TCC

AlaAla Asp Ala GlyLeuPro LeuPheArg TyrIleGly GlyPro Ser GTT

AsnAla His Leu ProVa1Pro MetMetAsn I:leIleAsn GlyGly Val GAC

AlaHis Ala Ser GlyVa.LA.spValGlnGlu PheMetIle AlaPro Asp GAG

IleGly Ala Thr PheSerGlu AlaLeuArg AsnGlyAla GluVal Glu AAG

TyrHisAla LeuLysSerVal :I:LeLysoGlu LysGlyLeu SerThrGly LeuGlyAsp GluGlyGlyPheeAlaProSer ValGlySer ThrArgGlu GCTCTTGAC CTTATCGTTGAG (~CAATCGAG AAGGCTGGC TTCACCCCA 846 AlaLeuAsp LeuIleValGlu RlaIleGlu LysAlaGly PheThrPro GGCAAGGACATC GCTCTTGCT (.'CGGAC:GTT GCTTCCTCT GAGTTCTTC 894 GlyLysAspIle AlaLeuAla LeuAspVal AlaSerSer GluPhePhe LysAspGlyThr TyrHisPhe GluGlyGly GlnHisSer AlaAlaGlu ATGGCAAACGTT TACGCTGAG (~TCGTTGAC GCGTACCCA ATCGTCTCC 990 MetAlaAsnVal TyrAlaGlu LeuVal.Rsp AlaTyrPro IleValSer IleGluAspPro LeuGlnGlu AspAspTrp GluGlyTyr ThrAsnLeu ACCGCAACCATC GGCGACAAG (iTTCAGATC GTTGGCGAC GACTTCTTC 1086 ThrAlaThrIle GlyAspLys ValGlr~Ile ValGlyAsp AspPhePhe Val Thr Asn Pro Glu Arg Leu Lys Glu Gly Ile Ala Lys Lys Ala Ala Asn Ser Ile Leu Val Lys Val. Asn Gln Ile Gly Thr Leu Thr Glu Thr PheAspAlaVal AspMetAla l3isArgAla GlyTyrThr SerMetMet TCCCACCGTTCC GGTGAGACC GAGGAC:ACC ACCATTGCT GACCTCGCA 1278 SerHisArgSer GlyGluThr GluAs~>Thr ThrIleAla AspLeuAla ValAlaLeuAsn CysGlyGln :LleLysThr GlyAlaPro AlaArgSer GACCGTGTCGCA AAGTACAAC:CRGCTTCTC CGCATCGAG CAGCTGCTT 1374 AspArgValAla LysTyrAsn (~lnLeuLeu ArgIleGlu GlnLeuLeu GGCGACGCCGGC GTCTACGCA GGTCGC:AGC GCATTCCCA CGCTTTCAG 1922 GlyAspAlaGly ValTyrAla (~lyArgSer AlaPhePro ArgPheGln Gly (2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 425 (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: Corynebacterium glutamicum (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
Val Ala Glu Ile Met His Val Phe Ala Arg Glu Ile Leu Asp Ser Arg Gly Asn Pro Thr Val Glu Ala Glu Val Phe Leu Asp Asp Gly Ser His Gly Val Ala Gly Val Pro Ser Gly Ala Ser Thr Gly Val His Glu Ala His Glu Leu Arg Asp Gly Gly Asp Arg Tyr Leu Gly Lys Gly Val Leu Lys Ala Val Glu Asn Val Asn Glu Glu Ile Gly Asp Glu Leu Ala Gly Leu Glu Ala Asp Asp Gln Arg heu Ile Asp Glu Ala Met Ile Lys Leu Asp Gly Thr Ala Asn Lys Ser Arg Leu Gly Ala Asn Ala Ile Leu Gly Val Ser Met Ala Val Ala Lys Ala Ala Ala Asp Ser Ala Gly Leu Pro 115 1.20 125 Leu Phe Arg Tyr Ile Gly Gly Pro Asn Ala His Val Leu Pro Val Pro Met Met Asn Ile Ile Asn Gly Gly Ala His Ala Asp Ser Gly Val Asp Val Gln Glu Phe Met Ile Al.a f'ro Ile Gly Ala Glu Thr Phe Ser Glu Ala Leu Arg Asn Gly Ala Glu Val Tyr His Ala Leu Lys Ser Val Ile Lys Glu Lys Gly Leu Ser Thr Gl.y Leu Gly Asp Glu Gly Gly Phe Ala Pro Ser Val Gly Ser Thr Arg Glu Ala Leu Asp Leu Ile Val Glu Ala Ile Glu Lys Ala Gly Phe Thr E'ro Gly Lys Asp Ile Ala Leu Ala Leu Asp Val Ala Ser Ser Glu Phe E'he Lys Asp Gly Thr Tyr His Phe Glu Gly Gly Gln His Ser Ala Ala Gl.u Met Ala Asn Val Tyr Ala Glu Leu Val Asp Ala Tyr Pro Ile Val :>er Ile Glu Asp Pro Leu Gln Glu Asp Asp Trp Glu Gly Tyr Thr Asn Leu Thr Ala Thr Ile Gly Asp Lys Val Gln Ile Val Gly Asp Asp Phe Phe Val Thr Asn Pro Glu Arg Leu Lys Glu Gly Ile Ala Lys Lys Ala F~l.a Asn Ser Ile Leu Val Lys Val Asn Gln Ile Gly Thr Leu Thr Glu Thr Phe Asp Ala Val Asp Met Ala His Arg Ala Gly Tyr Thr Ser Met Met Ser His Arg Ser Gly Glu Thr Glu 355 ..60 365 Asp Thr Thr Ile Ala Asp Leu Al.a Val Ala Leu Asn Cys Gly Gln Ile Lys Thr Gly Ala Pro Ala Arg ~le~r Asp Arg Val Ala Lys Tyr Asn Gln Leu Leu Arg Ile Glu Gln Leu Leu Gly Asp Ala Gly Val Tyr Ala Gly Arg Ser Ala Phe Pro Arg Phe Gl.n Gly

Claims (17)

1. An isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of a) a polynucleotide which is identical to the extent of at least 70 % to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) a polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70 % to the amino acid sequence of SEQ ID No. 2, c) a polynucleotide which is complementary to the polynucleotides of a) or b) and e [sic]) a polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of a), b) or c).

2. The polynucleotide as claimed in claim 1, wherein the polynucleotide is a preferably recombinant DNA which is capable of replication in coryneform bacteria.

3 The polynucleotide as claimed in claim 1, wherein the polynucleotide is an RNA.

4. The polynucleotide as claimed in claim 2, comprising the nucleic acid sequence as shown in SEQ
ID No. 1.

5. DNA as claimed in claim 2 which is capable of replication, comprising (i) the nucleotide sequence shown in SEQ ID No.1, or (ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or (iii)at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and optionally (iv) sense mutations of neutral function in (i).

6. The polynucleotide sequence as claimed in claim 2, which codes for a polypeptide which comprises the amino acid sequence in SEQ ID No. 2.

7. A process for the fermentative preparation of L-amino acids, in particular L-lysine, wherein the following steps are carried out:
a) fermentation of the L-lysine-producing coryneform bacteria in which at least the eno gene or nucleotide sequences which code for it are amplified, in particular over-expressed. (sic) b) concentration of L-amino acid in the medium or in the cells of the bacteria and c) isolation of the L-amino acid.

8. The process as claimed in claim 7, wherein bacteria in which further genes of the biosynthesis route of the desired L-amino acid are additionally amplified are employed.

9. The process as claimed in claim 7, wherein bacteria in which the metabolic routes which reduce the formation of L-lysine are at least partly eliminated are employed.

10. The process as claimed in claim 7, wherein a strain transformed with a plasmid vector is employed, and the plasmid vector carries the nucleotide sequence which codes for the eno gene.

11. The process as claimed in one or more of claims 7 to 10, wherein coryneform bacteria which produce L-lysine are used.

12. The process as claimed in claim 8, wherein at the same time the dapA gene which codes for dihydrodipicolinate synthase is over-expressed.

13. The process as claimed in claim 8, wherein at the same time a DNA fragment which imparts S-(2-aminoethyl)-cysteine resistance is amplified.

14. The process as claimed in claim 8, wherein at the same time the gap gene which codes for gyceraldehyde [sic] 3-phosphate is over-expressed.

15. The process as claimed in claim 8, wherein at the same time the tpi gene which codes for triose phosphate isomerase is over-expressed.

16. The process as claimed in claim 8, wherein at the same time the pgk gene which codes for 3-phosphate glycerate kinase is over-expressed.

17. The process as claimed in claim 8, wherein at the same time the pyc gene which codes for pyruvate carboxylase is over-expressed.