AU6135900A - New nucleotide sequences which code for the eno gene - Google Patents

Description

I S&FRef: 515637

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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Name and Address of Applicant: Actual Inventor(s): Address for Service: Degussa-Huls Aktiengesellschaft DE-60287 Frankfurt am Main Germany Bettina Mockel, Walter Pfefferle, Thomas Hermann, Alfred Puhler, Jorn Kalinowski and Brigitte Bathe Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 New Nucleotide Sequences which Code for the Eno Gene

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S Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 990152 BT 1 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.

10 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 S 15 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-(2aminoethyl)-cysteine, or are auxotrophic for amino acids of regulatory importance and produce L-lysine are obtained in this manner.

990152 BT 2 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, 115-14.2), 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)).

990152 BT 3 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 70% to the amino acid sequence of SEQ ID No. 2, 4 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 b) or c).

The invention also provides the polynucleotide this preferably being a DNA which is capable of replication, comprising: the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence which corresponds to sequence within the range of the degeneration of the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to sequence or and optionally (iv) sense mutations of neutral function in The invention also provides a polynucleotide comprising the nucleotide sequence as shown in SEQ ID 1 No. 1, a polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2, vectors containing the polypeptides of the invention in particular a shuttle vector or plasmid vector [I:\DayLib\LIBC]08639.doc:mef 990152 BT and coryneform bacteria serving as the host cell, which contain the vector.

The invention also provides polynucleotides 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 Spolynucleotide mentioned, according to SEQ ID no. 1, or a 10 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 e 15 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.

990152 BT 6 "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 10 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 80% 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 overexpressed.

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.

990152 BT 7 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 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.

S 10 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 15 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 o: Corynebacterium melassecola ATCC17965 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and 20 Brevibacterium divaricatum ATCC14020 and L-lysine-producing mutants or strains prepared therefrom, such as, for example Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM5715.

990152 BT 8 The inventors have succeeded in isolating the eno gene of C. glutamicum which codes for the enzyme enolase (EC 4.2.1.11).

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 und Klone, Eine Einfdhrung in die Gentechnologie [Genes and Clones, An Introduction to 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 15 up in X 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 20 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). B6rmann et al. (Molecular Microbiology 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- 990152 BT 9 defective. An example of these is the strain DH5amcr, 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).

10 The new DNA sequence of C. glutamicum which codes for the coo eno gene and which is a constituent of the present invention as SEQ ID NO 1-was obtained in this manner. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by 15 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 S" 20 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-Bassat 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 molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID No. 1 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. 2 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID No. 1 are 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, i 1993) and in Liebl et al. (International Journal of Systemic Bacteriology (1991)41: Is 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: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heideslbert, 20 Germany, 1994).

The inventors have found that coryneform bacterial produce amino acids, in particular L-lysine in an improved manner after over-expression of the eno gene.

°e 0 [I:\DayLib\LIBC]08639.doc:mef 990152 BT 11 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- RNA. Furthermore, the enzyme activity is also increased by

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e" 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 overexpression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.

0e Instructions in this context can be found by the expert, inter alia, in Martin et al. (Bio/Technology 5, 137-146

S.

20 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)) *0 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 Pihler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in Patent Application WO 96/15246, in Malumbres et al. (Gene 134, 15 24 (1993)), in Japanese Laid-Open Specification 229891, in Jensen and Hammer (Biotechnology and 990152 BT 12 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: 10 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. 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 pAG1 (US-A 15 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 13 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 Academic Press, London, UK, 1982).

The microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed 20 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 is described in the textbook by Chmiel (bioprozesstechnik 0.: 1.

Einf0hrung in die Bioverfahrenstechnik [Bioprocess Technology 1.

Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[I:\DayLib\L1C]08639.doc:mef 14 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 USA, 1981). 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.

1o These substances can be used individually or as a mixture. Organic nitrogencontaining compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, S°such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of is 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 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.

[!:\DayLib\LIBC08639.doc:mef 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 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 200C to 450C, and preferably 250C to 40 0 C. Culturing is continued until a 0o maximum of lysine has formed. This target is usually reached within 10 hours to 160 hours.

ae a [I:\DayLib\LIBC]08639.doc:mef 16 The analysis of L-lysine can be carried out by anion enhance 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 s preparation of amino acids, in particular L-lysine.

[I:\DayLib\LIBC08639.doc:mef 990152 BT 17 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 10 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 15 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 ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27- 990152 BT 18 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). For 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 MgSO, 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, 10 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the cells being plated out on LB agar (Lennox, S. 1955, Virology, 1:190) with 100 ig/ml ampicillin. After S/ incubation overnight at 37 0 C, recombinant individual clones were selected.

15 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 Pharmacia, 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 bp were isolated with the QiaExII Gel Extraction Kit (Product No.

20021, Qiagen, Hilden, Germany). The DNA of the sequencing 990152 BT 19 vector pZero-1, obtained from the company Invitrogen (Groningen, The Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) 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 0 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 DH5XMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 15 87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 pg/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 20 chain-stopping method of Sanger et al. (1977, Proceedings of the National Academy of Sciences 74:5463-5-467) 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 out in a "Rotiphoresis NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No.

A124.1, Roth, Karlsruhe, Germany) with the "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 sequence of the pZerol derivatives were assembled to a continuous contig. The computer-assisted coding region analyses were prepared with the s 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-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 0o the nucleotide sequence showed an open reading frame of 1275 base pairs, which was called the eno gene. The eno gene codes for a protein of 425 amino acids.

[I :\DayLib\LI3C]08639.doc mef 990152 BT 21 SEQUENCE PROTOCOL <110> Degussa-Hils AG <120> New nucleotide sequences which code for the eno gene <130> 990152 BT <140> <141> <160> 2 <170> Patentln Ver. 2.1 oo.. <210> <211> <212> <213> <220> <221> <222> 1 1578

DNA

Corynebacterium glutamicum

CDS

(151) (1425) <400> 1 ggctggggat atgggtagtt ttcgccacta atttcaactg attgcctcat cgaaacaaga ttcgtgcaac aattgggtgt agacgtgatt gaagacattt gatcacgtga ataattctag 120 ttagctccca agttggcata ggaggccaca gtg gct gaa atc atg cac gta ttc Val. Ala Glu Ile Met His Val Phe 174 0@ 0* .gct cgc 3 5 Ala Arg 10 gaa att ctc gac tcc cgc ggt aac cca Glu Ile Leu Asp Ser Arg Gly Asn Pro acc Thr gtc gag gca gag Val Giu Ala Glu 222 270 gtt Val 25 ttc ctg gat gac Phe Leu Asp Asp ggt Gly 30 tcc cac ggt gtc gca ggt gtt cca tcc Ser His Gly Val Ala Gly Vai Pro Ser gca tcc acc ggc gtc cac gag gct cat Ala Ser Thr Gly Val His Giu Ala His gag Glu ctg cgt gac ggt Leu Arg Asp Gly ggc gat Gly Asp 318 366 cgc tac ctg Arg Tyr Leu aag ggc gtt ttg Lys Gly Val Leu gca gtt gaa aac Ala Val Glu. Asn gtc aac gaa Val Asn Glu gaa atc ggc Glu Ile Gly gac gag ctc gct Asp Giu Leu Ala ggc Gly 80 cta gag gct gac gat cag cgc ctc Leu Glu Ala Asp Asp Gin Arg Leu 414 atc gac gaa gca atg atc Ile Asp Giu Ala Met Ile ctt gat ggc acc Leu Asp Gly Thr gcc Ala 100 aac aag tcc cgc Asn Lys Ser Arg 990152 BT ctg Leu 105 ggt gca aac goa Gly Ala Asn Ala atc Ile 110 ott ggt gtt too atg got gtt gca aag Leu Gly Val Ser Met Ala Val Ala Lys 115 gct Ala 120 510 got gct gat too Ala Ala Asp Ser goa Ala 125 ggc ctc ooa ctg Gly Leu Pro Leu ttc Phe 130 ogo tao ato ggt Arg Tyr Ile Gly gga ooa Gly Pro 135 558 aao goa oao Asn Ala His got oao got Ala His Ala 155 ott ooa gtt ooa Leu Pro Val Pro atg aac ato ato Met Asn Ile Ile aao ggt ggo Asn Gly Gly 150 ato got ooa Ile Ala Pro gao too ggt gtt Asp Ser Gly Val gao Asp 160 gtt oag gaa tto Val Gin Glu Phe

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*5 5O S *5 S. 0*

S.

ato ggt Ile Gly 170 goa gag aoo tto Ala Giu Thr Phe tot Ser 175 gag gct oto ogo Glu Ala Leu Arg aao Asn 180 ggo gog gag gto Gly Ala Glu Val oao goa otg aag His Ala Leu Lys too Ser 190 gto ato aag gaa Val Ire Lys Giu ggo otg too aoo Gly Leu Ser Thr gga Gly 200 ott ggo gat gag Leu Gly Asp Giu ggo Gly 205 ggo tto got oot Gly Phe Ala Pro too Ser 210 gto ggo too aoo Vai Gly Ser Thr ogt gag Arg Giu 215 30 got ott gao Ala Leu Asp ggo aag gao 35 Gly Lys Asp 235 ott Leu 220 ato gtt gag goa Ile Val Glu Ala ato Ile 225 gag aag got ggo Giu Lys Ala Gly tto aoo ooa Phe Thr Pro 230 gag tto tto Glu Phe Phe ato got ott got Ile Ala Leu Ala otg Leu 240 gao gtt got too Asp Vai Ala Ser tot Ser 245 846 894 942 aag gao Lys Asp 250 ggo aoo tao oao Gly Thr Tyr His tto Phe 255 gaa ggt ggo oag Glu Gly Giy Gin toc goa got gag Ser Ala Ala Giu atg Met 265 goa aao gtt tao Ala Asn Val Tyr got Al a 270 gag oto gtt gao Glu Leu Val Asp gog Al a 275 tao ooa ato gto Tyr Pro Ile Val 990 1038 ato gag gao ooa Ile Glu Asp Pro o tg Leu 285 oag gaa gat gao Gin Glu Asp Asp tgg gag Trp Giu 290 ggt tao aoo Gly Tyr Thr aao oto Asn Leu 295 aoo goa aoo Thr Ala Thr ggo gao aag gtt oag ato gtt. ggo gao Gly Asp Lys Val Gin Ile Val Gly Asp 305 gao tto tto Asp Phe Phe 310 aag got goo Lys Aia Ala 1086 gtc acc aac Val Thr Asn 315 cot gag ogo otg Pro Glu Arg Leu gag ggo ato got Giu Gly Ilie Ala 1134 aac tce ate etg gtt aag gtg aac cag ate ggt ace etc ace gag acc 1182 Asn Ser Ile Leu Vai Lys Val Asn Gin Ile Gly Thr Leu Thr Giu Thr 330 335 340 ttc gac gct gtc gac atg get cac cgc gca ggc tac acc tee atg atg 1230 Phe Asp Ala Val Asp Met Ala His Arg Ala Gly Tyr Thr Ser Met Met 345 350 355 tee eac cgt tee ggt gag ace gag gac ace acc att get gae etc gca 1278 Ser His Arg Ser Gly Giu Thr Glu Asp Thr Thr Ile Ala Asp Leu Ala 370 375 gtt gca etc aae tgt ggc cag ate aag act ggt get eca gca cgt tee 1326 Val Ala Leu Asn Cys Gly Gin Ile Lys Thr Gly Ala Pro Ala Arg Ser 380 385 390 gac egt gte gca aag tac aac cag ett etc egc ate gag cag etg ctt 1374 *Asp Arg Vai Ala Lys Tyr Asn Gin Leu Leu Arg Ile Giu Gin Leu Leu 20 395 400 405 gge gac gee ggc gtc tac gca ggt cgc age gca ttc eea ege ttt cag 1422 **Gly Asp Ala Gly Val Tyr Ala Gly Arg Ser Ala Phe Pro Arg Phe Gin *410 415 420 ggc taaataaaag cgcttttcga cgeccggtaa ccteaaggtt geegggegte 1475 ****Gly 425 gttgcettae taetgttact ggtgt gacta. tgatcgagga ttatggcaaa geagaagaaa 1535 *actcataaag geettgttcc tgtctcaagc agggaaegtg ctt 1578 <210> 2 425 <212> PRT <213> Corynebacterium giutamicum <400> 2 Val Ala Giu Ile Met His Val Phe Ala Arg Giu Ile Leu Asp Ser Arg 1 5 10 Gly Asn Pro Thr Vai Giu Ala Giu Val Phe Leu Asp Asp Gly Ser His 20 25 Giy Val Ala Gly Val Pro Ser Gly Ala Ser Thr Gly Val His Glu Ala 40 His Giu Leu Arg Asp Gly Gly Asp Arg Tyr Leu Gly Lys Gly Val Leu 55 Lys Ala Val Giu Asn Val Asn Giu Giu Ile Gly Asp Giu Leu Ala Gly 70 75 Leu Glu Ai~a Asp Asp Gin Arg Leu Ile Asp Glu Ala Met Ile Lys Leu 90 24 Asp Gly Thr Ala Asn Lys Ser Arg Leu Gly Ala Asn Ala Ile Leu Gly 100 110

S

*5*S S S S. S. S Val Leu Met 145 Val1 Ala 20 Lys Pro Ie 225 Asp 30 Gly 35 Val Asp Gin 305 Glu Gin Arg Asp Ser Phe 130 Met Gin Leu Glu Ser 210 Glu Vai Giy Asp Trp 290 Ile Gly Ile Ala Thr 370 Met 115 Arg Asn Glu- Arg Lys 195 Val Lys Ala Gin Ala 275 Glu Val Ile Gly Gly 355 Thr Al a Tyr Ile Phe Asn 180 Gly Gly Ala Ser His 260 Tyr Gly Gly Aa rhr 340 Tyr Ilie *Val Ile Ile Met 165 Gly Leu Ser Gly Ser 245 Ser Pro Tyr Asp Lys 325 Leu Thr Ala Ala Gly Asn 150 Ile Ala Ser Thr Phe 230 Glu Ala Ile Thr Asp 310 Lys Thr Ser Asp *Lys Gly 135 Gly Ala Glu Thr Arg 215 Thr Phe Ala Val Asn 295 Phe Ala Giu Met Leu 375 Al a 120 Pro Gly Pro Val Gly 200 Gi7u Pro Phe Giu Ser 280 Leu Phe Ala Thr Met 360 Ala Ala Ala Asp Sex *Asn Ala Ile Tyr 185 Leu Ala Gly Lys Met 265 Ile Thr Val Asn Phe 345 Ser Val1 Ala His Gly 170 His Gly Leu Lys Asp 250 Ala Giu Ala Thr Ser 330 Asp His Ala His Ala 155 Al a Ala Asp Asp Asp 235 Gly Asn Asp Thr Asn 315 Ile Ala Arg Leu Vai 140 Asp Giu Leu Giu Leu 220 Ile Thr Vai Pro Ile 300 Pro Leu Val Ser Asn 380 Ala 125 Leu Ser Thr Lys Gly 205 Ile Ala Tyr Tyr Leu 285 Giy Giu Val Asp Gly 365 Cys *Gly Leu Pro Val Gly Val Phe Ser 175 Ser Val 190 Gly Phe Val Giu Leu Ala His Phe 255 Ala Giu 270 Gin Glu Asp Lys Arg Leu Lys Val 335 Met Ala 350 Giu Thr Gly Gin Pro Pro Asp 160 Giu Ile Ala Ala Leu 240 Giu Leu Asp Val Lys 320 Asn His Glu Ile Lys Thr Gly Ala Pro Ala Arg Ser Asp Arq Val Ala Lys Tyr Asn Gin 385 390 395 4-09 Leu Leu Arg Ile Glu Gin Leu Leu Gly Asp Ala Gly Val Tyr Ala Gly 405 410 415 Arg Ser Ala Phe Pro Arg Phe Gin Gly 420 425 0

Claims (21)

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 d) a polynucleotide comprising at least 15 successive bases of the polynucleotide sequence of b) or c).

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

3. The polynucleotide as claimed in claim 2 which is recombinant.

4. The polynucleotide as claimed in claim 1, wherein the polynucleotide is an RNA. The polynucleotide as claimed in claim 2 or claim 3, comprising the nucleic acid sequence as shown in SEQ ID No. 1.

6. DNA as claimed in claim 2 or claim 3 which is capable of replication, comprising: the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence which corresponds to sequence within the range of the degeneration of the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to sequence or and optionally (iv) sense mutations of neutral function in

7. The polynucleotide sequence as claimed in claim 2 or claim 3 which codes for a polypeptide which comprises the amino acid sequence in SEQ ID No. 2.

8. An isolated polynucleotide from coryneform bacteria, said polynucleotide being substantially as hereinbefore described with reference to any one of the examples.

9. A process for the fermentative preparation of L-amino acids, wherein (I:\DayLib\LIBC]08639.doc:mef the following steps are carried out; a) fermentation of the L-amino acid producing coryneform bacteria in which at least the eno gene or nucleotide sequences which code for it are amplified, b) concentration of L-amino acid in the medium or in the cells of the bacteria and c) isolation of the L-amino acid. The process as claimed in claim 9 wherein the L-amino acid is L-lysine.

11. The process as claimed in claim 9 or claim 10, wherein the eno gene or nucleotide sequences which code for it are over expressed.

12. The process as claimed in any one of claims 9 to 11 wherein bacteria in which further genes of the biosynthesis route of the desired L-amino acid are additionally amplified are employed.

13. The process as claimed in any one of claims 9 to 11 wherein bacteria in which the metabolic routes which reduce the formation of L-lysine are at least partly eliminated are employed.

14. The process as claimed in any one of claims 9 to 11 wherein a strain transformed with a plasmid vector is employed, and the plasmid vector carries the nucleotide sequence which codes for the eno gene.

15. The process as claimed in any one of claims 7 to 14, wherein coryneform bacteria which produce L-lysine are used. a.

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

17. The process as claimed in claim 12, wherein at the same time a DNA fragment which imparts S-(2-aminoethyl)-cystein resistance is amplified.

18. The process as claimed in claim 12, wherein at the same time the gab gene which codes for glyceraldehyde 3-phosphate is over-expressed.

19. The process as claimed in claim 12, wherein at the same time the tpi gene which codes for triose phosphate isomerase is over-expressed.

20. The process as claimed in claim 12, wherein at the same time the pgk gene which codes for 3-phosphate glycerate kinase is over-expressed.

21. The process as claimed in claim 12, wherein at the same time the pyc gene which codes for pyruvate carboxylate is over-expressed. [I:\DayLib\LIBC ]08639.doc:mef 28

22. A process for the fermentative preparation of L-amino acids, said process being substantially as hereinbefore described with reference to any one of the examples.

23. L-amino acids prepared by the process of any one of claims 9 to 22 Dated 1 September, 2000 Degussa-Huls Akteingessellschaft Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON '00:0 fI:DayLib\LIBC]08639.doc:me f