EP1320616A2 - Sequences which code for the sige gene of corynebacterium glutamicum - 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 nucleotides of the polynucleotide sequence of a), b) or c), and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the sigE gene is present in enhanced form, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

Description

Sequences which code for the sigE Gene

Field of the Invention

The invention provides nucleotide sequences from coryneform bacteria which code for the sigE gene and a process for the fermentative preparation of amino acids using bacteria in which the sigE gene is enhanced.

Prior Art

L-Amino acids are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and especially in animal nutrition.

It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, -in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, 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 or are auxotrophic for metabolites of regulatory importance and produce amino acids 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 L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.

Object of the Invention

The inventors had the object of providing new measures for improved fermentative preparation of amino acids.

Summary of the Invention

Where L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L- asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L- isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L- histidine, L-lysine, L-tryptophan and L-arginine. Lysine is particularly preferred.

The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the sigE gene, 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 nucleotides of the polynucleotide sequence of a) , b) or c), the polypeptide preferably having the activity of sigma factor E.

The invention also provides the above-mentioned polynucleotide, this preferably being a DNA which is capable of replication, comprising:

(i) the nucleotide sequences shown in SEQ ID No. 1, SEQ ID NO. 3 or 4, 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, in particular DNA, which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No. 1;

a polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No. 2;

a vector containing the polynucleotide according to the invention, in particular a shuttle vector or plasmid vector, and

coryneform bacteria which contain the vector or in which the sigE gene is enhanced.

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 of a coryneform bacterium, which comprises the complete gene or parts thereof, with a probe which comprises the sequence of the polynucleotide according to the invention according 'to SEQ ID No.l or a fragment thereof, and isolation of the polynucleotide sequence mentioned.

Detailed Description of the Invention

Polynucleotides which comprise the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids or polynucleotides or genes which code for sigma factor E or to isolate those nucleic acids or polynucleotides or genes which have a high similarity of sequence with that of the sigE gene.

Polynucleotides which comprise the sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for sigma factor E can be prepared by the polymerase chain reaction (PCR) .

Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides which have a length of at least 40 or 50 nucleotides 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.

The polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom and also those which are at least 70%, preferably at least 80% and in particular at least 90% to 95% identical to the polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom.

"Polypeptides" are 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 sigma factor E, and also those which are at least 70%, preferably at least 80% and in particular at least 90% to 95% identical to the polypeptide according to SEQ ID No. 2 and have the activity mentioned.

The invention furthermore relates to a process for the fermentative preparation of amino acids chosen from the group consisting of L-asparagine, L-threonine, L-serine, L- glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan and L- arginine using coryneform bacteria which in particular already produce amino acids and in which the nucleotide sequences which code for the sigE gene are enhanced, in particular over-expressed.

The term "enhancement" 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 allele or of the genes or alleles, using a potent promoter or using a gene or allele which codes for a corresponding enzyme having a high activity, .and optionally combining these measures.

The microorganisms which the present invention provides can produce L-amino acids 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 experts for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum (C. glutamicum) , are in particular the known wild-type strains

Corynebacterium glutamicum ATCC13032

-Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoa inogenes FERM BP-1539 Corynebacterium melassecola ATCC17965. Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020

and L-amino acid-producing mutants or strains prepared therefrom.

The new sigE gene from C. glutamicum which codes for the enzyme sigma factor E has been isolated.

To isolate the sigE gene or also other genes of C. glutamicum, a gene library of this microorganism is first set up in Escherichia coli (E. coli) . The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Eine Einfϋhrung in die Gentechnologie (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 λ 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).

Bδrmann et al. (Molecular Microbiology 6(3), 317-326) (1992) ) 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 pϋC9 (Vieira 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 DH5αmcr, 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 in turn be subcloned in the usual vectors suitable for sequencing and then sequenced, 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 resulting DNA sequences can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217- 232(1986)), that of Marck (Nucleic Acids Research 16, 1829- 1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).

The new DNA sequence of C. glutamicum which codes for the sigE gene and which, as SEQ ID No. 1, is a constituent of the present invention has been found. 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 sigE 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-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. 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 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 nucleotides.

Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in CO ω K. ro -' μ» μ- π o Oi o cπ o Cπ

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Φ Φ 3 CO μ> ^ ^•< rt rt O CO φ H Ω Ω μ- μ- Φ Hi 3 CO CO SD O Q Ω 3

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1 Φ 3- o < > Φ μ- 3 o fD ω 3 3 Hi μ- ω fD fD Ω 3 O 13 Φ . tr

O I-¹ rt a M <£> -** D o <i 3 3 • Ω Φ o Φ h-¹ £D 13 3 μ- CD 3 _^-» .— Φ ιΩ co h-* Tl . — . •£- O Φ H Φ μ- i 3 X CO CO O φ 3 CO M μ-

M CU φ μ- — -J X fD Φ l-i α H3 φ o O μ- sQ CO rt rt CO Hi 13 ?o Hi rt μ^** H o SD -~- ^• 1 13 3 1 CO a CD Hi μ- Hi rt Φ φ hi Φ μ- o φ IT¹ o a μ- 00 μ> Φ tr Φ CO co μ- 3 rt Φ H O 3 3 ΪV 3 α-> CX⁾ j-*. H Φ •s φ rt O Φ rt ø) Hi 13 3 rt ^■τ) 3 rt cπ σi rt rt μ- ιQ a 3 φ 3 ø⁾ hi P. H hi

"_^ a ro •*« Φ Φ fD fD CO

-_* a rt 3 ft o 3 Φ φ φ a 3 tr Ω o a < 3 tr Φ 3 μ*- rt Hi Φ φ

SD 1 D*

1001-1007 (1993)), in WO 96/15246, in Malumbres et al. (Gene 134, 15 - 24 (1993)), in 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, for enhancement the sigE gene according to the invention was over-expressed with the aid of episomal 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-l (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.

Plasmid vectors which are furthermore suitable are also those with the aid of which the process of gene amplification by integration into the chromosome can be used, as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum. Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKlδmob or pK19mob (Schafer et al., Gene 145, 69-

73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), PCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5, 87, 993) , pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al.,1986, Gene 41: 337-342). The plasmid vector which contains the gene to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070

(1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross over" event, the resulting strain contains at least two copies of the gene in question.

In addition, it may be advantageous for the production of

L-amino acids to enhance, in particular over-express one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and optionally regulatory proteins, in addition to the sigE gene.

Thus, for example, for the preparation of L-amino acids, in addition to enhancement of the sigE gene, one or more genes chosen from the group consisting of

• the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0 197 335),

• the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) ,

• the tpi gene which codes for triose phosphate isomerase

(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

• the pgk gene which codes for 3-phosphoglycerate kinase

(Eikmanns (1992), Journal of Bacteriology 174:6076-6086), • the zwf gene which codes for glucose 6-phosphate dehydrogenase (JP-A-09224661) ,

• the pyc gene which codes for pyruvate carboxylase (DE-A- 198 31 609) ,

• the mqo gene which codes for malate-quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),

• the lysC gene which codes for a feed-back resistant aspartate kinase (Accession No.P265.12; EP-A-0699759) ,

• the lysE gene which codes for lysine export (DE-A-195 48 222),

• the horn gene which codes for homoserine dehydrogenase (EP-A 0131171) ,

• the ilvA gene which codes for threonine dehydratase (Mδckel et al., Journal of Bacteriology (1992) 8065-

8072) ) or the ilvA(Fbr) allele which codes for a "feed back resistant" threonine dehydratase (Mδckel et al., (1994) Molecular Microbiology 13: 833-842),

• the ilvBN gene which codes for acetohydroxy-acid synthase (EP-B 0356739) ,

• the ilvD gene which codes for dihydroxy-acid dehydratase (Sahm and Eggeling (1999) Applied and Environmental

Microbiology 65: 1973-1979),

• the zwal gene which codes for the Zwal protein (DE: 19959328.0, DSM 13115)

can be enhanced, in particular over-expressed.

It may furthermore be advantageous for the production of L- amino acids, in addition to the enhancement of the sigE gene, for one or more of the genes chosen from the group consisting of:

• the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1; DSM 13047),

• the pgi gene which codes for glucose 6-phosphate isomerase (US 09/396,478; DSM 12969),

• the poxB gene which codes for pyruvate oxidase (DE: 1995 1975.7; DSM 13114),

• the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2, DSM 13113)

to be attenuated, in particular for the expression thereof to be reduced.

In addition to over-expression of the sigE gene it may furthermore be advantageous for the production of amino acids 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 invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the 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. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfϋhrung in die

Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (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, 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. 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 sulfate, 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 above-mentioned 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. 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 of the culture. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as 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 the desired product has formed. This target is usually reached within 10 hours to 160 hours.

Methods for the determination of L-amino acids are known from the prior art. The analysis can thus be carried out, for example, as described by Spackman et al. (Analytical Chemistry, 30, (1958) , 1190) by ion exchange chromatography with subsequent ninhydrin derivation, or it can be carried out by reversed phase HPLC, for example as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167- 1174) .

The process according to the invention is used for fermentative preparation of amino acids.

The following microorganism was deposited as a pure culture on 11th April 2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty:

• Corynebacterium glutamicum DSM5715/pEC-T18mob2sigEexp as DSM 14229. The present invention is explained in more detail in the following with the aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques of restriction, Klenow and alkaline phosphatase treatment were carried out by the method of Sambrook et al. (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA) . Methods for transformation of Escherichia coli are also described in this handbook.

The composition of the usual nutrient media, such as LB or TY medium, can also be found in the handbook by Sambrook et al.

Example_ι 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 Diagnostics GmbH, 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 Stratagene (La Jolla, USA, Product Description SuperCosl Cosmid Vector Kit, Code no. 251301) was cleaved with the restriction enzyme Xbal (Amersham Pharmacia, Freiburg, Germany, Product Description Xbal, 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.27-0870-04) . The ligation mixture was then packed in phages with the aid of Gigapack II XL Packing Extract (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 MgS0 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 mg/1 aitipicillin. After incubation overnight at 37°C, recombinant individual clones were selected.

Example 2

Isolation and sequencing of the sigE 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 Diagnostics GmbH, 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 vector pZero-1, obtained from Invitrogen (Groningen, Holland, 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 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 DH5αMCR (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 mg/1 zeocin.

The plasmid preparation of the recombinant clones was carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) . The sequencing was carried out by the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467) with modifications according to Zimmer ann 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 sequences of the pZerol derivatives were assembled to -a continuous contig. The computer-assisted coding region a'nalysis was prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231).

The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed an open reading frame of 651 base pairs, which was called the sigE gene. The sigE gene codes for a protein of 216 amino acids (SEQ ID NO. 2) .

The DNA sections lying upstream and downstream of SEQ ID NO. 1, which are shown in SEQ ID NO. 3 and SEQ ID NO. 4, were identified in the same manner. The sigE gene region extended by SEQ ID NO. 3 and SEQ ID NO. 4 is shown in SEQ ID NO. 5.

Example 3

Preparation of a shuttle vector pEC-T18mob2sigEexp for enhancement of the sigE gene in C. glutamicum

3.1. Cloning of the sigE gene

From the strain ATCC 13032, chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817 -1828 (1994)). On the basis of the sequence of the sigE gene known for C. glutamicum from example ^'2, the following oligonucleotides were chosen for the polymerase chain reaction (see SEQ ID No. 7 and SEQ ID No. 8) .

sigEl: 5 "TAG TCA CCA CGG TTA AGC CT 3^N sigE2: 5 GCC TTG GTT CTT ACG AAC TG 3^s

The primers shown were synthesized by ARK Scientific GmbH Biosystems (Darmstadt, Germany) and the PCR reaction was carried out by the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) with Taq-Polymerase from Qiagen (Hilden, Germany) . With the aid of the polymerase chain reaction, the primers allow amplification of a DNA fragment approx. 2.03 kb in size, which carries the sigE gene.

The amplified DNA fragment of approx. 2.03 kb in size which carries the sigE gene was ligated with the TOPO TA Cloning® Kit from Invitrogen Corporation (Carlsbad, CA, USA) in the vector pCR®2.1TOPO (Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)). The E. coli strain ToplO (Grant et al., Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) was then transformed with the ligation batch in accordance with the instructions of the manufacturer of the kit (Invitrogen Corporation, Carlsbad, CA, USA) . Selection of plasmid-carrying cells was carried out by plating out the transformation batch on LB Agar (Sambrook et al., Molecular cloning: a laboratory manual. 2^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 50 mg/1 kanamycin. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany) and checked by treatment with the restriction enzyme Sphl and EcoRI with subsequent agarose gel electrophoresis (0.8 %) . The DNA sequence of the amplified DNA fragment was checked by sequencing. The plasmid was called pCR2. IsigEexp. The strain was called E. coli ToplO / pCR2. IsigEexp.

3.2. Preparation of the E. coli - C. glutamicum shuttle vector pEC-T18mob2

The E. coli - C. glutamicum shuttle vector was constructed according to the prior art. The vector contains the

replication region reg of the plasmid pGAl including the replication effector per (US-A- 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline resistance-imparting tetA(Z) gene of the plasmid pAGl (US-A- 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with the accession number AF121000), the replication region oriV of the plasmid pMBl (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43,

I

77-90 (1979) ) , the lacZα gene fragment including the lac promoter and a multiple cloning site (mcs) (Norrander, J.M. et al. Gene 26, 101-106 (1983)) and the mob region of the plasmid RP4 (Simon et al.,(1983) Bio/Technology 1:784-791). The vector constructed was transformed in the E. coli strain DH5α (Hanahan, In: DNA cloning. A Practical Approach. Vol. I. IRL-Press, Oxford, Washington DC, USA).

Selection for plasmid-carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al., Molecular cloning: A Laboratory Manual. 2^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which had been supplemented with 5 mg/1 tetracycline. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzymes EcoRI and Hindlll and subsequent agarose gel electrophoresis (0.8 %) . The plasmid was called pEC-T18mob2 and is shown in figure 1.

3.3. Cloning of sigE in the E. coli-C. glutamicum shuttle vector pEC-T18mob2

The E. coli - C. glutamicum shuttle vector pEC-T18mob2 described in example 3.2 was used as the vector. DNA of this plasmid was cleaved completely with the restriction enzymes BamHI and Sail and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .

The sigE gene was isolated from the plasmid pCR2. IsigEexp described in example 3.1. by complete cleavage with the enzymes BamHI and Sail. The sigE fragment 1930 bp in size was isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .

The sigE fragment obtained in this manner was mixed with the prepared vector pEC-T18mob2 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 batch was transformed in the E. coli strain DH5αMCR (Hanahan, In: DNA cloning. A Practical Approach. Vol. I. IRL-Press, Oxford, Washington DC, USA).

Selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 5 mg/1 tetracycline. After incubation overnight at 37°C, recombinant individual clones were selected. Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and cleaved with the restriction enzymes BamHI and Sail to check the plasmid by subsequent agarose gel electrophoresis. The plasmid obtained was called pEC- T18mob2sigEexp. It is shown in figure 2.

Example 4

Transformation of the strain DSM5715 with the plasmid pEC- Tl8mob2sigEexp

The strain DSM5715 was transformed with the plasmid pEC- T18mob2sigEexp using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989) ) . Selection of the transformants took place on LBHIS agar comprising 18.5 g/1 brain-heart infusion broth, 0.5 M sorbitol, 5 g/1 Bacto-tryptone, 2.5 g/1 Bacto-yeast

extract, 5 g/1 NaCl and 18 g/1 Bacto-agar, which had been supplemented with 5 mg/1 tetracycline. Incubation was carried out for 2 days at 33°C. Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al . , 1998, Microbiology, 144, 915 -927), cleaved with the restriction endonucleases BamHI and Sail, and the plasmid was checked by subsequent agarose gel electrophoresis. The strain obtained was called DSM5715/pEC-Tl8mob2sigEexp.

Example 5

Preparation of lysine

The C. glutamicum strain DSM5715/pEC-T18mob2sigEexp obtained in example 4 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined.

For this, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with tetracycline (5 mg/1)) for 24 hours at 33°C. Starting from this agar plate culture, a pre-culture was seeded (10 ml medium in a 100 ml conical flask) . The complete medium Cglll was used as the medium for the pre-culture.

Medium Cg III

NaCl 2.5 g/1

Bacto-Peptone 10 g/1

Bacto-Yeast extract 10 g/1

Glucose (autoclaved separately) 2 % (w/v)

The pH was brought to pH 7.4

Tetracycline (5 mg/1) was added to this. The pre-culture was incubated for 16 hours at 33°C at 240 rpm on a shaking machine. A main culture was seeded from this pre-culture such that the initial OD (660 nm) of the main culture was 0.05. Medium MM was used for the main culture.

Medium MM

CSL (corn steep liquor) 5 g/1

MOPS (morpholinopropanesulfonic acid) 20 g/1

Glucose (autoclaved separately) 50 g/1

(NH₄)₂S0₄ 25 g/1

KH₂P0 0.1 g/1

MgS0₄ * 7 H₂0 1.0 g/1

CaCl₂ * 2 H₂0 10 mg/1

FeS0 * 7 H₂0 10 mg/1

MnS0 * H₂0 5.0mg/l

Biotin (sterile-filtered) 0.3 mg/1

Thiamine * HC1 (sterile-filtered) 0.2 mg/1

L-Leucine (sterile-filtered) 0.1 g/1

CaC0₃ 25 g/1

The CSL, MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions were then added, as well as the CaC0₃ autoclaved in the dry state.

Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Tetracycline (5 mg/1) was added. Culturing was carried out at 33°C and 80 % atmospheric humidity.

After 48 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich) . The amount of lysine formed was determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection.

The result of the experiment is shown in Table 1.

Table 1

Brief Description of the Figures

Figure 1: Map of the plasmid pEC-T18mob2

Figure 2: Map of the plasmid pEC-T18mob2sigEexp

The abbreviations and designations used have the following meaning.

per: Gene for controlling the number of copies from PGAl

oriV: ColEl-similar origin from pMBl

rep: Plasmid-coded replication region from C glutamicum plasmid pGAl

RP4mob: RP4 mobilization site lacZ-alpha: lacZ gene fragment from E. coli

Tet: Resistance gene for tetracycline sigE: sigE gene of C. glutamicum BamHI : Cleavage site of the restriction enzyme BamHI Sail: Cleavage site of the restriction enzyme Sail

sigE: sigE gene of C. glutamicum

BamHI: Cleavage site of the restriction enzyme BamHI

Sail: Cleavage site of the restriction enzyme Sail

SEQUENCE PROTOCOL

<110> Degussa AG <120> Nucleotide sequences which code for the sigE gene

<130> 000445 BT

<140> <141>

<160> 8

<170> Patentln Ver. 2.1

<210> 1

<211> 1330

<212> DNA

<213> Corynebacterium glutamicum

<220>

<221> CDS

<222> (302) .. (949)

<223> sigE gene

<400> 1 accagtggag ccgttgccat tggtggtggc agccaaagtg gttagcagct ggccagtcat 60 ttcatccggg gcggggagac cgaactcggc ggcgtcttca cgagcgcgcg ctacagcagc 120 gtcggtttca gtagtggact cgacataagt gcgaagatac tcgaaggcgt tactcacgcg 180 ttatagtcta gagcgagcag gcgagatgtg aagtacctac acgcattaag tgcaaatgaa 240 ttcacaattg ccagaagatg cacaggatgt aatctagatt tcccaagttc agtggggcaa 300 a atg act tat atg aaa aag aag tec cga gat gac gca ccc gtc gta ate 349 Met Thr Tyr Met Lys Lys Lys Ser Arg Asp Asp Ala Pro Val Val Ile 1 5 10 15 gaa ace gtt caa gca gaa cat get gaa gaa etc acg ggc act gca gca 397 Glu Thr Val Gin Ala Glu His Ala Glu Glu Leu Thr Gly Thr Ala Ala 20 25 30 ttc gat get gga cag gca gac atg cca aca tgg ggc gag eta gtc gca 445 Phe Asp Ala Gly Gin Ala Asp Met Pro Thr Trp Gly Glu Leu Val Ala 35 40 45 gaa cat gca gat age gtt tac cgc etc gcg tac cgt ctt tec ggc aac 493 Glu His Ala Asp Ser Val Tyr Arg Leu Ala Tyr Arg Leu Ser Gly Asn 50 55 60 cag cac gat get gaa gac ctg ace caa gaa aca ttc atg cgt gtc ttc 541 Gin His Asp Ala Glu Asp Leu Thr Gin Glu Thr Phe Met Arg Val Phe 65 70 75 80 cgc teg ttg aag age tac cag cca ggc ace ttt gag ggc tgg ctg cac 589 Arg Ser Leu Lys Ser Tyr Gin Pro Gly Thr Phe Glu Gly Trp Leu His 85 90 95 cgc ate ace ace aac ttg ttc ctt gat atg gtt cgc cac cgc ggc aag 637

Arg Ile Thr Thr Asn Leu Phe Leu Asp Met Val Arg His Arg Gly Lys

100 105 110 ate cgc atg gag gcg ctg cct gaa gat tat gag cgc gtt ccg ggc aat 685

Ile Arg Met Glu Ala Leu Pro Glu Asp Tyr Glu Arg Val Pro Gly Asn

115 120 125 gac ate ace cca gag cag gca tac ace gaa get aac ctt gac cca get 733 Asp Ile Thr Pro Glu Gin Ala Tyr Thr Glu Ala Asn Leu Asp Pro Ala 130 135 140 ctg cag gca gcc etc gat gag ttg age cca gac ttc cgc gtg gca gtg 781 Leu Gin Ala Ala Leu Asp Glu Leu Ser Pro Asp Phe Arg Val Ala Val 145 150 155 160 ate etc tgt gat gtt gtt ggt atg age tat gac gaa ate gca gag ace 829 Ile Leu Cys Asp Val Val Gly Met Ser Tyr Asp Glu Ile Ala Glu Thr 165 170 175 etc gga gtg aaa atg ggt ace gtg cgt tec cgt att cac cgt gga cgc 877

Leu Gly Val Lys Met Gly Thr Val Arg Ser Arg Ile His Arg Gly Arg 180 185 190 age cag ctt cgt gca agt ttg gaa get gca gca atg ace age gag gaa 925

Ser Gin Leu Arg Ala Ser Leu Glu Ala Ala Ala Met Thr Ser Glu Glu 195 200 205 gtt tct ttg ttg gtt cca ace cac taaagttggt gtgttttctg acacgacaaa 979 Val Ser Leu Leu Val Pro Thr His 210 215 cgcaaatgtc gtgteatttt tgcagctcag tgcattattt tggggttegt ggtgcggaea 1039 gggaacttat cacaggcgac atccgttttg agtagtaggt atcttggata agaagttacc 1099 cacatccttg aaagtcgaga cacaggaggt catcggaaga tatgttcaat tccgacacca 1159 ecgcgaatct ecaagctaaa agtegagatc gtgcaggatc taaagcaaag cgcagcagge 1219 caagttttga ttcagtageg cgggatgttt tggatgttcg aaeaaaaaca gcaeaagtta 1279 aaaacaaggc taaagagttt tcctctgttg atcacctttc agcagacgcc g 1330

<210> 2 <211> 216 <212> PRT <213> Corynebacterium glutamicum

<400> 2

Met Thr Tyr Met Lys Lys Lys Ser Arg Asp Asp Ala Pro Val Val lie 1 5 10 15

Glu Thr Val Gin Ala Glu His Ala Glu Glu Leu Thr Gly Thr Ala Ala 20 25 30 Phe Asp Ala Gly Gin Ala Asp Met Pro Thr Trp Gly Glu Leu Val Ala 35 40 45

Glu His Ala Asp Ser Val Tyr Arg Leu Ala Tyr Arg Leu Ser Gly Asn 50 55 60

Gin His Asp Ala Glu Asp Leu Thr Gin Glu Thr Phe Met Arg Val Phe 65 70 75 80 Arg Ser Leu Lys Ser Tyr Gin Pro Gly Thr Phe Glu Gly Trp Leu His

85 90 95

Arg Ile Thr Thr Asn Leu Phe Leu Asp Met Val Arg His Arg Gly Lys 100 105 110

Ile Arg Met Glu Ala Leu Pro Glu Asp Tyr Glu Arg Val Pro Gly Asn 115 120 125

Asp Ile Thr Pro Glu Gin Ala Tyr Thr Glu Ala Asn Leu Asp Pro Ala 130 135 140

Leu Gin Ala Ala Leu Asp Glu Leu Ser Pro Asp Phe Arg Val Ala Val

145 150 155 160 Ile Leu Cys Asp Val Val Gly Met Ser Tyr Asp Glu Ile Ala Glu Thr

165 170 175

Leu Gly Val Lys Met Gly Thr Val Arg Ser Arg Ile His Arg Gly Arg 180 185 190

Ser Gin Leu Arg Ala Ser Leu Glu Ala Ala Ala Met Thr Ser Glu Glu 195 200 205

Val Ser Leu Leu Val Pro Thr His 210 215

<210> 3 <211> 457 <212> DNA <213> Corynebacterium glutamicum

<220> <223> upstream region

<400> 3 tagtcaccac ggttaagcct gcaccaaggg gcaggcgagc aacgtgtgcg ccttcaatgg 60 aacgaatata ttcatcggcg tcaegtgctg cttgggtgtc acgatccttg cgggtttgat 120 ccgcaatggt gccgtcaagg agcgcategg cgagcaceag egcaecgcet cgtegaagaa 180 gcggccaggc ggcgtcgaca agegccttta aatccatggg ggagacttgg ecgaagacaa 240 gctgatagct gtcgttggca aggcgactca tcacgtcgag cgggcgcgag agcaagaagc 300 gtacgcggct gggggaatag ccggcctcgc ggaagagtgc tttggcctgg cgctgatgct 360 ctgattcagg atcaatgcag gtcagtgtgg tgttatcggc cagtccgttc aggatataca 420 gacccaccaa cccggcagcc ggggtaatcg cgatggc 457

<210> 4 <211> 299 <212> DNA

<213> Corynebacterium glutamicum

<220> <223> downstream region

<400> 4 cagccatgtt tgtagacaat gaactgtccc gtggcgccat gcatcgcgcc aggctgcaca 60 ttgtgcactg cgctgaatgt agggaagaga ttaaecgtca gcgggaaacc gttgattatc 120 tccgcteaga gtgcaaaaac gaagaagtgt ccgccccaat ggacctcaaa gcacggcttg 180 ccagcctcgc cactgagtgc atgcctggcc ctggcgcaga gaatttagca atgcagcgcc 240 cagagtcttt tgtggctaaa gttgagtccg tagtgcgcgc agttcgtaag aaecaagge 299

<210> 5

<211> 2086

<212> DNA

<213> Corynebacterium glutamicum <220>

<221> CDS

<222> (759) .. (1406)

<223> sigE <400> 5 tagtcaccac ggttaagcct gcaccaaggg gcaggcgagc aacgtgtgcg ccttcaatgg 60 aacgaatata ttcatcggcg tcaegtgctg cttgggtgtc acgatccttg cgggtttgat 120 ccgcaatggt gccgtcaagg agcgcategg cgagcaceag egcaecgcet cgtegaagaa 180 gcggccaggc ggcgtcgaca agegccttta aatccatggg ggagacttgg ecgaagacaa 240 gctgatagct gtcgttggca aggcgactca tcacgtcgag cgggcgcgag agcaagaagc 300 gtacgcggct gggggaatag ccggcctcgc ggaagagtgc tttggcctgg cgctgatgct 360 ctgattcagg atcaatgcag gtcagtgtgg tgttatcggc cagtccgttc aggatataca 420 gacccaccaa cccggcagcc ggggtaatcg cgatggcacc agtggagccg ttgccattgg 480 tggtggcagc caaagtggtt agcagctggc cagtcatttc atccggggcg gggagaccga 540 actcggcggc gtcttcacga gcgcgcgcta cagcagcgtc ggtttcagta gtggactcga 600 cataagtgcg aagatactcg aaggcgttac tcacgcgtta tagtctagag cgagcaggcg 660 agatgtgaag tacctacacg cattaagtgc aaatgaattc acaattgcca gaagatgcac 720 aggatgtaat ctagatttcc caagttcagt ggggcaaa atg act tat atg aaa aag 776

Met Thr Tyr Met Lys Lys 1 5 aag tec cga gat gac gca ccc gtc gta ate gaa ace gtt caa gca gaa 824 Lys Ser Arg Asp Asp Ala Pro Val Val Ile Glu Thr Val Gin Ala Glu

10 15 20 cat get gaa gaa etc acg ggc act gca gca ttc gat get gga cag gca 872 His Ala Glu Glu Leu Thr Gly Thr Ala Ala Phe Asp Ala Gly Gin Ala 25 30 35 gac atg cca aca tgg ggc gag eta gtc gca gaa cat gca gat age gtt 920 Asp Met Pro Thr Trp Gly Glu Leu Val Ala Glu His Ala Asp Ser Val 40 45 50 tac cgc etc gcg tac cgt ctt tec ggc aac cag cac gat get gaa gac 968 Tyr Arg Leu Ala Tyr Arg Leu Ser Gly Asn Gin His Asp Ala Glu Asp 55 60 65 70 ctg ace caa gaa aca ttc atg cgt gtc ttc cgc teg ttg aag age tac 1016 Leu Thr Gin Glu Thr Phe Met Arg Val Phe Arg Ser Leu Lys Ser Tyr 75 80 85 cag cca ggc ace ttt gag ggc tgg ctg cac cgc ate ace ace aac ttg 1064

Gin Pro Gly Thr Phe Glu Gly Trp Leu His Arg Ile Thr Thr Asn Leu

90 95 100 ttc ctt gat atg gtt cgc cac cgc ggc aag ate cgc atg gag gcg ctg 1112

Phe Leu Asp Met Val Arg His Arg Gly Lys Ile Arg Met Glu Ala Leu

105 110 115 cct gaa gat tat gag cgc gtt ccg ggc aat gac ate ace cca gag cag 1160 Pro Glu Asp Tyr Glu Arg Val Pro Gly Asn Asp Ile Thr Pro Glu Gin 120 125 130 gca tac ace gaa get aac ctt gac cca get ctg cag gca gee etc gat 1208 Ala Tyr Thr Glu Ala Asn Leu Asp Pro Ala Leu Gin Ala Ala Leu Asp 135 140 145 150 gag ttg age cca gac ttc cgc gtg gca gtg ate etc tgt gat gtt gtt 1256 Glu Leu Ser Pro Asp Phe Arg Val Ala Val Ile Leu Cys Asp Val Val 155 160 165 ggt atg age tat gac gaa ate gca gag ace etc gga gtg aaa atg ggt 1304

Gly Met Ser Tyr Asp Glu Ile Ala Glu Thr Leu Gly Val Lys Met Gly 170 175 180 ace gtg cgt tec cgt att cac cgt gga cgc age cag ctt cgt gca agt 1352

Thr Val Arg Ser Arg Ile His Arg Gly Arg Ser Gin Leu Arg Ala Ser 185 190 195 ttg gaa get gca gca atg ace age gag gaa gtt tct ttg ttg gtt cca 1400 Leu Glu Ala Ala Ala Met Thr Ser Glu Glu Val Ser Leu Leu Val Pro 200 205 210 ace cac taaagttggt gtgttttctg acacgacaaa cgcaaatgtc gtgteatttt 1456 Thr His 215 tgcagctcag tgcattattt tggggttegt ggtgcggaea gggaaettat cacaggegae 1516 atccgttttg agtagtaggt atcttggata agaagttacc cacatecttg aaagtcgaga 1576 cacaggaggt catcggaaga tatgttcaat tccgacacca ecgcgaatct ecaagctaaa 1636 agtegagatc gtgcaggatc taaagcaaag cgcagcagge caagttttga ttcagtageg 1696 cgggatgttt tggatgttcg aaeaaaaaca gcaeaagtta aaaacaaggc taaagagttt 1756 tectctgttg ateacctttc agcagaegcc gcagccatgt ttgtagacaa tgaactgtcc 1816 cgtggcgeca tgeatcgcgc caggctgcac attgtgcact gcgctgaatg tagggaagag 1876 attaaccgtc agcgggaaac cgttgattat ctccgctcag agtgcaaaaa cgaagaagtg 1936 tecgccecaa tggacctcaa agcacggett gccagcctcg ccactgagtg catgcetggc 1996 cctggcgcag agaatttagc aatgcagcgc ccagagtctt ttgtggctaa agttgagtcc 2056 gtagtgcgcg cagttcgtaa gaaccaaggc 2086

<210> 6

<211> 216

<212> PRT <213> Corynebacterium glutamicum

<400> 6

Met Thr Tyr Met Lys Lys Lys Ser Arg Asp Asp Ala Pro Val Val Ile 1 5 10 15

Glu Thr Val Gin Ala Glu His Ala Glu Glu Leu Thr Gly Thr Ala Ala 20 25 30

Phe Asp Ala Gly Gin Ala Asp Met Pro Thr Trp Gly Glu Leu Val Ala 35 40 45

Glu His Ala Asp Ser Val Tyr Arg Leu Ala Tyr Arg Leu Ser Gly Asn

50 55 60 Gin His Asp Ala Glu Asp Leu Thr Gin Glu Thr Phe Met Arg Val Phe

65 70 75 80

Arg Ser Leu Lys Ser Tyr Gin Pro Gly Thr Phe Glu Gly Trp Leu His 85 90 95

Arg Ile Thr Thr Asn Leu Phe Leu Asp Met Val Arg His Arg Gly Lys 100 105 110

Ile Arg Met Glu Ala Leu Pro Glu Asp Tyr Glu Arg Val Pro Gly Asn 115 120 ^' 125

Asp Ile Thr Pro Glu Gin Ala Tyr Thr Glu Ala Asn Leu Asp Pro Ala 130 135 140 Leu Gin Ala Ala Leu Asp Glu Leu Ser Pro Asp Phe Arg Val Ala Val 145 150 155 160

Ile Leu Cys Asp Val Val Gly Met Ser Tyr Asp Glu Ile Ala Glu Thr 165 170 175

Leu Gly Val Lys Met Gly Thr Val Arg Ser Arg He His Arg Gly Arg 180 185 190 Ser Gin Leu Arg Ala Ser Leu Glu Ala Ala Ala Met Thr Ser Glu Glu 195 200 205

Val Ser Leu Leu Val Pro Thr His 210 215

<210> 7 <211> 20 <212> DNA <213> Corynebacterium glutamicum

<220> <223> Primer sigEl

<400> 7 tagtcaccac ggttaagcct 20

<210> 8

<211> 20

<212> DNA

<213> Corynebacterium glutamicum

<220>

<223> Primer sigE2

<400> 8 gccttggttc ttacgaactg 20

Claims

What is claimed is :

1. An isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the sigE gene, 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 nucleotides of the polynucleotide sequence of a) , b) or c) ,

the polypeptide preferably having the activity of sigma factor E.

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

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

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

5. A 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. A polynucleotide sequence as claimed in claim 2, which codes for a polypeptide which comprises the amino acid sequences shown in SEQ ID No. 2.

7. A coryneform bacterium in which the sigE gene is enhanced, in particular over-expressed.

8. The shuttle vector pEC-T18mob2sigEexp, which

8.1. comprises a DNA fragment 1930 bp in size which carries the sigE gene,

8.2 the restriction map of which is reproduced in figure 2, and

8.3 is deposited in strain DSM5715/pEC-Tl8mob2sigEexp under no. DSM 14229 at the Deutsche Sammlung fϋr Mikroorganismen und Zellenkulturen [German Collection of Microorganisms and Cell Cultures] .

9. A process for the fermentative preparation of L-amino acids, in particular lysine, which comprises carrying out the following steps:

a) fermentation of the coryneform bacteria which produce the desired L-amino acid and in which at least the sigE gene or nucleotide sequences which code for it are enhanced, in particular over- expressed;

b) concentration of the L-amino acid in the medium or in the cells of the bacteria, and

c) isolation of the L-amino acid.

10. A process as claimed in claim 9, wherein bacteria in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally enhanced are employed.

11. A process as claimed in claim 9, wherein bacteria in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated are employed.

12. A process as claimed in claim 9, wherein a strain transformed with a plasmid vector is employed, and the plasmid vector carries the nucleotide sequence which codes for the sigE gene.

13. A process as claimed in claim 9, wherein the expression of the polynucleotide (s) which code(s) for the sigE gene is enhanced, in particular over-expressed.

14. A process as claimed in claim 9, wherein the regulatory properties of the polypeptide (enzyme protein) for which the polynucleotide sigE codes are increased.

15. A process as claimed in claim 9, wherein for the preparation of L-amino acids, coryneform microorganisms in which at the same time one or more of the genes chosen from the group consisting of

15.1 the dapA gene which codes for dihydrodipicolinate synthase,

15.2 the gap gene which codes for glyceraldehyde 3- phosphate dehydrogenase,

15.3 the tpi gene which codes for triose phosphate isomerase,

15.4 the pgk gene which codes for 3-phosphoglycerate kinase,

15.5 the zwf gene which codes for glucose 6- phosphate dehydrogenase,

15.6 the pyc gene which codes for pyruvate carboxylase, '

15.7 the mqo gene which codes for malate-quinone oxidoreductase,

15.8 the lysC gene which codes for a feed-back resistant aspartate kinase,

15.9 the lysE gene which codes for lysine export,

15.10 the horn gene which codes for homoserine dehydrogenase

15.11 the ilvA gene which codes for threonine dehydratase or the ilvA(Fbr) allele which codes for a feed back resistant threonine dehydratase,

15.12 the ilvBN gene which codes for acetohydroxy- acid synthase,

15.13 the ilvD gene which codes for dihydroxy-acid dehydratase,

15.14 the zwal gene which codes for the Zwal protein

is or are enhanced or over-expressed are fermented.

6. A process as claimed in claim 9, wherein for the preparation of L-amino acids, coryneform microorganisms in which at the same time one or more of the genes chosen from the group consisting of

16.1 the pck gene which codes for phosphoenol pyruvate carboxykinase,

16.2 the pgi gene which codes for glucose 6- phosphate isomerase,

16.3 the poxB gene which codes for pyruvate oxidase,

16.4 the zwa2 gene which codes for the Zwa2 protein

is or are attenuated are fermented.

17. A coryneform bacterium which contains a vector which carries a polynucleotide as claimed in claim 1.

18. A process as claimed in one or more of the preceding claims, wherein microorganisms of the genus

Corynebacterium are employed.

19. A process for discovering RNA, cDNA and DNA in order to isolate nucleic acids, or polynucleotides or genes which code for sigma factor E or have a high similarity with the sequence of the sigE gene, wherein the polynucleotide comprising the polynucleotide sequences as claimed in claim 1, 2, 3 or 4 is employed as hybridization probes.

20. A process as claimed in claim 19, wherein the hybridization is carried out under a stringency corresponding to at most 2x SSC.