AU6245100A - Novel nucleotide sequences coding for the irp gene - Google Patents

Description

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PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

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0 0 0 0600 Name and Address of Applicant: Actual Inventor(s): Address for Service: Degussa-Huls Aktiengesellschaft DE-60287Frankfurt am Main Germany Brigitte Bathe Jorn Kalinowski Alfred Piihler Bettina M6ckel Walter Pfefferle Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Invention Title: Novel Nucleotide Sequences Coding for the Irp Gene The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 990109 BT 1 Novel nucleotide sequences coding for the Irp gene The present invention provides nucleotide sequences coding for the Irp gene and a process for the fermentative production of amino acids, in particular lysine and isoleucine, using coryneform bacteria in which expression of the Irp gene is modified.

Prior art L-amino acids are used in animal nutrition, in the food industry, in human medicine and in the pharmaceuticals industry.

It is known that these amino acids are produced by fermentation of strains of coryneform bacteria in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve 15 the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the 20 product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.

The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant 25 selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-(2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant amino acids and produce L-amino acids. For some years, the methods of recombinant DNA technology have also been used for strain improvement of strains of Corynebacterium which produce Lamino acid.

990109 BT 2 LRP (leucine-responsive protein) is a global regulator, first described in Escherichia coli, which influences the transcription of a series of genes, the gene products of which are involved in the transport, biosynthesis and degradation of amino acids (Calvo and Matthews, Microbiological Reviews 58, 466-490, 1994).

In recent years, similar genes have also been identified in other organisms, such as Bradyrhizobium japonicum (King and O'Brian, Journal of Bacteriology, 179, 1828-1831, 1997), Klebsiella aerogenes (Janes and Bender, Journal of Bacteriology, 181, 1054-1058, 1999), Sulfolobus acidocaldarius (Charlier et al., Gene, 201, 63-68, 1997) and in the gram positive bacterium Bacillus subtilis (Belitsky et al., Journal of Bacteriology, 179, 5448-5457, 1997).

e In E. coli, the Irp protein regulates its own expression.

Lrp also has an either negative or positive influence on many genes in E. coli. In general, it is the expression of gene products which are active in biosynthetic pathways which is stimulated. Gene products having a catabolic action are generally correspondingly negatively controlled.

In some cases, the action of Irp is potentiated by the addition of L-leucine, but addition of L-leucine may also have a negative effect (Newman et al., In: Neidhardt et al.

25 Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. American Society for Microbiology, Washington 1513-1525, 1996). In E. coli, Irp regulates a large number of genes and operons which play a central role in amino acid biosynthesis and amino acid catabolism. The following operons in E. coli are, for example, negatively controlled: livJ, which codes for a binding protein in a highly sophisticated uptake system for branched-chain amino acids (Haney et al., Journal of Bacteriology, 174, 108-115, 1992) and lysU, which codes for lysine tRNA synthetase (Gazeau et al., FEBS Letters, 300, 254-258, 1994). Genes which have hitherto been known to be 990109 BT 3 positively influenced in E. coli include, inter alia, ilvIH, gltBDF and leuABCD (Lin et al., Journal of Bacteriology, 174, 1948-1955, 1992).

The last-stated operon is of fundamental interest in leucine biosynthesis and of particular interest for lysine biosynthesis in Corynebacterium glutamicum. It is suspected that there is an association between leucine auxotrophy and elevated lysine productivity values (Schrumpf et al., Applied Microbiology and Biotechnology, 37, 566-571, 1992) Patek et al. (Applied and Environmental Microbiology, 133-140, 1994) have demonstrated that inactivating leuA in some lysine producers of C. glutamicum results in increased lysine yields. In a mutant of Brevibacterium lactofermentum, Tosaka et al. (Agricultural Biological Chemistry, 43, 265-270, 1979) have been able to achieve a reduction in lysine formation with a simultaneous increase in threonine formation by addition of L-leucine.

Object of the Invention The inventors set themselves the object of providing novel measures for the improved fermentative production of L-amino acids, in particular L-lysine and Lisoleucine, with coryneform bacteria.

Description of the Invention The present invention provides an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical 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 containing at least 15 successive bases of the polynucleotide sequence of b) or c).

15 The present invention also provides a polynucleotide wherein it preferably Scomprises a replicable DNA containing: the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence which matches the sequence within the degeneration range of the genetic code, or 20 (iii) at least one sequence which hybridises with the sequence complementary to sequence or (ii) and optionally o o o oo# 00 [R:\LIBC]08334.doc:mcf (iv) functionally neutral sense mutations in The present invention also provides a polynucleotide containing the nucleotide sequence as shown in SEQ ID No. 1, a polynucleotide which codes for a polypeptide which contains the amino acid sequence as shown in SEQ ID No. 2.

The present invention also provides polynucleotides which substantially consist of one polynucleotide sequence and are obtainable by hybridisation screening of an appropriate gene library which contains the complete gene having the polynucleotide sequence corresponding to SEQ ID No. 1 with a probe which contains the sequence of the o0 stated polynucleotide according to SEQ ID No. 1 or a fragment thereof and isolation of the stated DNA sequence.

Polynucleotide sequences according to the invention are suitable as hybridisation probes for RNA, cDNA and DNA in order to isolate full length cDNA which code for the Irp protein and to isolate such cDNA or genes, the sequence of which exhibits a high level 15 of similarity with that of the Irp gene.

Polynucleotide sequences according to the invention are furthermore suitable as primers for the production of DNA of genes which code for Irp proteins by the polymerase chain reaction (PCR).

Such oligonucleotides acting as probes or primers contain at least 30, preferably at 20 least 20, very particularly preferably at least 15 successive bases. Oligonucleotides having a length of at least 40 or 50 base pairs are also suitable.

"Isolated" means separated from its natural surroundings.

a 9 a [R:\LIBC]08334.doc:mcf 990109 BT 6 "Polynucleotide" generally denotes polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be unmodified or modified.

"Polypeptides" is taken to mean peptides or proteins which contain two or more amino acids joined via peptide bonds.

The polypeptides according to the invention include a polypeptide according to SEQ ID no. 2, in particular those having the biological activity of the Irp protein and also those which are at least 70%, preferably at least identical to the polypeptide according to SEQ ID no. 2 and in particular which exhibit 90% to 95% identity to the polypeptide according to SEQ ID no. 2 and exhibit the stated activity.

The invention furthermore relates to a process for the 15 fermentative production of amino acids, in particular L- *lysine and L-isoleucine by means of coryneform bacteria, which in particular already produce amino acids, by amplifying or attenuating the novel Irp gene as a function of the target substance.

20 In this connection, the term "amplification" describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are coded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene which codes for a corresponding enzyme having elevated activity and optionally by combining these measures.

In this connection, the term "attenuation" means reducing or suppressing the intracellular activity of one or more enzymes (proteins) in a microorganism, which enzymes are coded by the corresponding DNA, for example by using a weak promoter or a gene or allele which codes for a corresponding enzyme which has a low activity or 990109 BT 7 inactivates the corresponding enzyme (protein) and optionally by combining these measures.

The microorganisms, provided by the present invention, may produce L-lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may comprise representatives of coryneform bacteria, in particular of the genus Corynebacterium.

Within the genus Corynebacterium, Corynebacterium glutamicum may in particular be mentioned, which is known in specialist circles 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.

oo 15 Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 20 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and amino acid producing mutants or strains produced therefrom, such as for example the L-lysine producing strains 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 or the L-isoleucine producing strains Corynebacterium glutamicum ATCC14309 Corynebacterium glutamicum ATCC14310 990109 BT 8 Corynebacterium glutamicum ATCC14311 Corynebacterium glutamicum ATCC15168 and Corynebacterium ammoniagenes ATCC 6871.

The inventors succeeded in isolating the novel Irp gene which codes for the Irp protein from C. glutamicum.

The Irp gene or also other genes are isolated from C.

glutamicum by initially constructing a gene library of this microorganism in E. coli. The construction of gene libraries is described in generally known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker, Gene und Klone, Eine Einfihrung in die Gentechnologie (Verlag Chemie, Weinheim, Deutschland, 1990) or the manual by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). One very well known gene library is that of E. coli K-12 strain W3110, which was constructed by Kohara et al.

S:o. (Cell 50, 495-508 (1987)) in X-vectors. Bathe et al.

(Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was constructed using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bathe et al. (Molecular and General Genetics, 6:3-317,. 326) also 25 describe a gene library of C. glutamicum ATCC13032, using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).

Plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807- 818 (1979)) or pUC9 (Viera et al., 1982, Gene, 19:259-268) may also be used to produce a gene library of C. glutamicum in E. coli. Particularly suitable hosts are those strains of E. coli which exhibit defects with regard to restriction and recombination. One example of such a strain 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 assistance of cosmids may then in turn be sub-cloned in 990109 BT 9 vectors suitable for sequencing and then be sequenced, as described, for example, in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).

The novel DNA sequence from C. glutamicum which codes for the Irp gene and, as SEQ ID no. 1, is provided by the present invention, was obtained in this manner. The amino acid sequence of the corresponding protein was furthermore deduced from the above DNA sequence using the methods described above. The resultant amino acid sequence of the Irp gene product is shown in SEQ ID no. 2.

Coding DNA sequences arising from the degeneracy of the genetic code are also provided by the present invention.

DNA sequences which hybridise with SEQ ID no. 1 or parts of SEQ ID no. 1 are similarly also provided by the invention.

*o *Conservative substitutions of amino acids in proteins, for example the substitution of glycine for alanine or of aspartic acid for glutamic acid, are known in specialist circles as "sense mutations", which result in no fundamental change in activity of the protein, i.e. they are functionally neutral. It is furthermore known that changes to the N and/or C terminus of a protein do not substantially impair or may even stabilise the function thereof. The person skilled in the art will find 25 information in this connection 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 arising in a corresponding manner from SEQ ID no. 2 are also provided by the present invention.

DNA sequences which hybridise with SEQ ID no. 1 or parts of SEQ ID no. 1 are also provided by the invention. Finally, DNA sequences produced by the polymerase chain reaction 990109 BT (PCR) using primers obtained from SEQ ID no. 1 are also provided by the present invention. Such oligonucleotides typically have a length of at least 15 base pairs.

The person skilled in the art will find instructions for identifying DNA sequences by means of hybridisation inter alia in the manual "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). The person skilled in the art will find instructions for amplifying DNA sequences by means of the polymerase chain reaction (PCR) inter alia in the textbook by Gait, Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham, PCR (Spektrum Akademischer 15 Verlag, Heidelberg, Deutschland, 1994).

The inventors discovered that the Irp gene is important for the production of amino acids, in particular L-lysine and L-isoleucine.

The inventors furthermore discovered that the additional 20 overexpression of one or more enzymes of the particular biosynthetic pathway, of glycolysis, of the citric acid cycle, of anaplerotic metabolism or of amino acid export, individually or in combination, results in further advantages for the production of amino acids, in particular 25 L-lysine and L-isoleucine.

Thus, for example, for the production of L-lysine the dapA gene (EP-B 0 197 335) which codes for dihydropicolinate synthase may simultaneously be overexpressed, or the gap gene (Schwinde et al., Journal of Bacteriology 175: 3905-3908 (1993)) which codes for glyceraldehyde 3phosphate dehydrogenase may simultaneously be overexpressed, or 990109 BT 11 the mqo gene (Molenaar et al., European Journal of Biochemistry 254, 395 403 (1998)) which codes for malate:quinone oxidoreductase may simultaneously be overexpressed, or the pyc gene (DE-A-19 831 609) which codes for pyruvate carboxylase may simultaneously be overexpressed, or the lysE gene (DE-A-195 48 222) which codes for lysine export may simultaneously be overexpressed.

10 Thus, for example, for the production of L-isoleucine the ilvA gene (M6ckel et al., Journal of Bacteriology (1992) 8065-8072)) which codes for threonine dehydratase or the "feed back resistant" ilvA(Fbr) allele (M6ckel et al., (1994)Molecular Microbiology 13: 833-842) may 15 simultaneously be overexpressed, or the gap gene (Schwinde et al., Journal of Bacteriology 175: 3905-3908 (1993)) which codes for glyceraldehyde 3phosphate dehydrogenase may simultaneously be overexpressed, or the mqo gene (Molenaar et al., European Journal of Biochemistry 254, 395 403 (1998)) which codes for malate:quinone oxidoreductase may simultaneously be overexpressed, or the pyc gene (DE-A-19 831 609) which codes for pyruvate carboxylase may simultaneously be overexpressed.

990109 BT 12 In may furthermore be advantageous for the production of amino acids, in particular L-lysine and L-isoleucine, to suppress unwanted secondary reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek Academic Press, London, UK, 1982).

For the purposes of amino acid production, in particular of L-lysine and L-isoleucine, the microorganisms produced according to the invention may be cultured continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process. A summary of known culture methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfahrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 15 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must adequately satisfy the requirements of the particular strains. Culture media for various microorganisms are described in "Manual of Methods for General Bacteriology" from American Society for Bacteriology (Washington USA, 1981). Carbon sources which may be used include sugars and carbohydrates, such as for example glucose, sucrose, lactose, fructose, maltose, 25 molasses, starch and cellulose, oils and fats, such as for example soya oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as for example palmitic acid, stearic acid and linoleic acid, alcohols, such as for example glycerol and ethanol, and organic acids, such as for example acetic acid. These substances may be used individually or as a mixture. Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, 990109 BT 13 ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used comprise phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The culture medium must furthermore contain salts of metals, such as for example magnesium sulfate or iron sulfate, which are required for growth.

Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. In addition, suitable precursors may be added to the culture medium. The stated materials may be added to the culture in the form of a single batch or may be supplied in a suitable manner during culturing.

The pH of the culture is controlled by using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia solution or acidic compounds such as phosphoric acid or sulfuric acid in a suitable manner.

Antifoaming agents, such as for example fatty acid polyglycol esters, may be used to control foaming.

Suitable, selectively acting substances, for example antibiotics, may be added to the medium in order to •maintain plasmid stability. Oxygen or gas mixtures containing oxygen, such as for example air, are introduced into the culture in order to maintain aerobic conditions.

-The temperature-of the culture is normally from 200C to 450C and preferably from 250C to 400C. The culture is continued until the maximum quantity of the desired amino acid has formed. This objective is normally achieved within hours to 160 hours.

The amino acids may be analysed by anion exchange chromatography with subsequent ninhydrin derivatisation, as described in Spackman et al. (Analytical Chemistry, (1958), 1190) or by reversed phase HPLC, as described in 990109 BT 14 Lindroth et al. (Analytical Chemistry (1979) 51: 1167- 1174).

The purpose of the process according to the invention is the fermentative production of amino acids, in particular L-lysine and L-isoleucine.

990109 BT Examples The present invention is illustrated in greater detail by the following practical examples.

Example 1 Production of a genomic cosmid gene library from Corynebacterium glutamicum ATCC13032 Chromosomal DNA from Corynebacterium glutamicum ATCC13032 was isolated as described in Tauch et al., (1995, Plasmid 33:168-179) and partially cleaved with the restriction i 10 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, production description SAP, code no. 1758250). The 15 DNA of cosmid vector SuperCosl (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164), purchased from Stratagene (La Jolla, USA, production description SuperCosl Cosmid Vector Kit, code Sno. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,-product description XbaI, Code no. 27-0948-02) and also 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). Cosmid DNA treated in this manner was mixed with the treated ATCC 13032 DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA Ligase, code no. 27- 0870-04). The ligation mixture was then packed in phages using Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, product description Gigapack II XL Packing Extract, code no. 200217). E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Res. 16:1563-1575) was infected by suspending the cells in 10 mM MgSO, and mixing them with an aliquot of the phage suspension. The cosmid library was 990109 BT 16 infected and titred as described in Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), wherein the cells were plated out on LB agar (Lennox, 1955, Virology, 1:190) 100g/ml of ampicillin.

After overnight incubation at 37 0 C, individual recombinant clones were selected.

Example 2 Isolation and sequencing of the Irp gene.

Cosmid DNA from an individual colony was isolated in accordance with the manufacturer's instructions using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, code no. 27-0913-02). The DNA 15 fragments were dephosphorylated with shrimp alkaline "phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, production description SAP, code no. 1758250).

Once separated by gel electrophoresis, the cosmid fragments of a size of approx. 1500 to 2000 bp were isolated using the QiaExII Gel Extraction Kit (product no. 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1 purchased from Invitrogen (Groningen, Netherlands, product description Zero Background Cloning Kit, product no. K2500- 01) was cleaved with the restriction enzyme BamHI (Amersham 9 25 Pharmacia, Freiburg, Deutschland, product description BamHI, Product No. 27-0868-04). Ligation of the cosmid fragments into the sequencing vector pZero-1 was performed as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), wherein the DNA mixture was incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was then electroporated into the E. coli strain DH5aMCR (Grant, 1990, Proceedings of the National Academy of Sciences 87:4645-4649) (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) and plated out onto LB agar (Lennox, 990109 BT 3'- 17 1955, Virology, 1:190) 50 [tg/ml of Zeocin. Plasmids of the recombinant clones were prepared using the Biorobot 9600 (product no. 900200, Qiagen, Hilden, Germany).

Sequencing was performed using the dideoxy chain termination method according to Sanger et al. (1977, Proceedings of the National Academies of Sciences U.S.A., 74:5463-5467) as modified by 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.

Separation by gel electrophoresis and analysis of the sequencing reaction was performed in a "Rotiphorese NF" acrylamide/bisacrylamide gel (29:1) (product no. A124.1, Roth, Karlsruhe, Germany) using the "ABI Prism 377" sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The resultant raw sequence data were then processed using the Staden software package (1986, Nucleic Acids Research, 14:217-231), version 97-0. The individual sequences of the 20 pZero 1 derivatives were assembled into a coherent contig.

Computer-aided coding region analysis was performed using XNIP software (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analysis was performed using the o "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402), against the non-redundant *NCBI database of the "National Library of Medicine" (USA).

The resultant nucleotide sequence is stated in SEQ ID no.

1. Analysis of the nucleotide sequence revealed an open reading frame of 462 base pairs, which was designated the Irp gene. The Irp gene codes for a polypeptide of 154 amino acids.

990109 BT SEQUENCE LISTING <110> Degussa-Hais AG <120>Novel nucleotide sequences coding for the lrp, gene <130> 990126 BT <140> <141> <160> 2 <170> Patentln Ver. 2.1 <210> <211> <212> <213> 9* <220> <221> <222> <220> <221> <222> <220> 30 <221> <222> 1 715

DNA

Corynebacterium glutamicum (62) .(67) (88) .(93)

CDS

(151) (612) <400> 1 gccgataacc tttatcatct ggttccaggg ^ctgccttgga tggcgacacc tccaggcttg aatgaatctc ttgcgttttt tgcacactac aatcatcaca caattgccgg gtagttttgt 120 tgccagtttg cgcacctcaa ctaggctatt gtg caa tat atg aag cta gat tcc Val Gin Tyr Met Lys Leu Asp Ser att gat cgc gca att att gcg gag ctt agc gcg aat gcg cgc atc tca Ile Asp Arg Ala Ile Ile Ala Giu Leu Ser Ala Asn Ala Arg Ile Ser 15 aat ctc gca ctg gct gac aag gtg cat ctc act ccg gga cct tgc ttg Asn Leu Ala Leu Ala Asp Lys Val His Leu Thr Pro Gly Pro Cys Leu 30 35 agg agg gtg cag cgt ttg gaa gcc gaa gga atc att ttg ggc tac agc Arg Arg Val Gin Arg Leu Giu Ala Giu Gly Ile Ile Leu Gly Tyr Ser 50 gcg gac att cac cct gcg gtg atg aat cgt gga ttt gag gtg acc gtg Ala Asp Ile His Pro Ala Val Met Asn Arg Gly Phe Giu Val Thr Val 65 990109 BT 19 gat gto act cto agc aac tto gac cgc tcc act gta gac aat ttt gaa 414 Asp Val Thr Leu Ser Asn Phe Asp Arg Ser Thr Val Asp Asn Phe Giu 80 agc toc gtt gog cag cat gat gaa gta Ctg gag ttg cac agg ctt ttt 462 Ser Ser Val Ala Gin His Asp Giu Val Leu'Giu Leu His Arg Leu Phe 95 100 ggt tog cca gat tat ttt gtc cgc atc ggo gtt gct gat ttg gag gcg 510 Gly Ser Pro Asp Tyr Phe Val Arg Ile Gly Val Ala Asp Leu Giu Ala 105 110 115 120 tat gag caa ttt tta tcc agt cac att caa acc gtg oca gga att gca 558 Tyr Giu Gin Phe Leu Ser Ser His Ile Gin Thr Val Pro Gly Ile Ala 125 130 135 aag ato tca tca cgt ttt got atg aaa gtg gtg aaa oca got ogo ccc 606 Lys Ile Ser Ser Arg Phe Ala Met Lys Val Vai Lys Pro Ala Arg Pro 140 145 150 *cag gtg tgaagcatgc attttgaagc atgaatottt ttcatctagt gaaggactga 662 :Gin Val toooatgcgt atgaaatoaa tcgcagcaat tgcaatogct acogoogco tgg 715 <210> 2 <211> 154 <212> PRT <213> Corynebacterium giutamicum <400> 2 Vai Gin Tyr Met Lys Leu Asp Ser le Asp Arg Ala Ile Ile Ala Giu 1 5 10 **Leu Ser Ala Asn Ala Arg Ile Ser Asn Leu Ala Leu Ala Asp Lys Val 25 40 His Leu Thr Pro Gly Pro Cys Leu Arg Arg Vai Gin Arg Leu Glu Ala *35 40 Glu Gly Ile Ile Leu Gly Tyr Ser Ala Asp Ile His Pro Ala Val Met 55 Asn Arg Gly Phe Glu Vai Thr Vai Asp Val Thr Leu Ser Asn Phe Asp 70 75 Arg Ser Thr Val Asp Asn Phe Glu Ser Ser Val Ala Gin His Asp Giu 85 90 Val Leu Giu Leu His Arg Leu Phe Gly Ser Pro Asp Tyr Phe Val Arg 100 105 110 Ile Gly Val Ala Asp Leu Giu Ala Tyr Gliu Gin Phe Leu Ser Ser His 115 120 125 990109 BT Ile Gin Thr Val Pro Gly Ile Ala Lys Ile Ser Ser Arg Phe Ala Met 130 135 140 Lys Val Val Lys Pro Ala Arg Pro Gin Val 145 150 too.

too

Claims (16)

1. An isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical 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 containing 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 .9 DNA replicable in coryneform bacteria.

3. The polynucleotide as claimed in claim 2, wherein the polynucleotide is recombinant.

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

5. The polynucleotide as claimed in claim 2 or claim 2, containing the nucleic acid sequence as shown in SEQ ID No. 1.

6. The replicable DNA as claimed in claim 2 or claim 3, containing the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence which matches the sequence within the "degeneration range of the genetic code, or (iii) at least one sequence which hybridises with the sequence complementary to sequence or (ii) and optionally (iv) functionally neutral sense mutations in

7. The polynucleotide sequence as claimed in claim 2 or claim 3 which codes for a polypeptide which contains the amino acid sequence as shown 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 production of L-amino acids, wherein the following steps are performed: [R\LIBC]08334.doc:mef 22 a) fermentation of the bacteria producing the desired L-amino acid bacteria, in which at least the Irp gene is attenuated or amplified. b) accumulation of the desired product 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 bacteria are used in which further genes of the biosynthetic pathway of the desired L-amino acid are additionally amplified.

11. The process as claimed in claim 9, wherein bacteria are used in which the metabolic pathways which reduce the formation of the desired amino acid are at least partially suppressed.

12. The process as claimed in claim 9, wherein a strain transformed with a plasmid vector is used and the plasmid vector bears the nucleotide sequences which code for the Irp gene.

13. The process as claimed in claim 9, wherein a deletion or insertion is made in is the Irp gene of the bacterium for the purpose of attenuation.

14. The process as claimed in any one of claims 10 to 13, wherein coryneform bacteria which produce L-amino acids are used.

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

16. L-amino acids produced by the process of any one of claims 9 to Dated 28 September, 2000 SDegussa-Huls Aktiengesellschaft Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\LIBC]08334.doc:mef