SK10202000A3 - L-lysine producing coryneform bacteria and method for the production of l-lysine - Google Patents

Abstract

L-lysine producing coryneform bacterium (A) with an amplified pyc (pyruvate carboxylase) gene in which at least one of the additional genes dapA (dihydropicolinate synthase), lysC (aspartate kinase), lysE (lysine exporter-carrier) and/or dapB (dihydropicolinate reductase) is amplified, preferably overexpressed. Independent claims are also included for: (a) DNA (I), replicable in coryneform bacteria and preferably recombinant DNA of Corynebacterium origin, which includes a sequence (1; 795 bp), reproduced, that represents the 5'-region of the dapB gene; (b) production of L-lysine by fermenting (A); Escherichia coli K12 strains DH5 alpha /pECTlysE (DSM 12871) or DH5 alpha /pEC7dapBlysE (DSM 12875); C. glutamicum strains DSM5715/pJC23 (DSM 12869), DSM5715aecD::dapA(MA16) (DSM 12867), DSM5715aecD::dapA(MC20) (DSM12868) and DM678 (DSM 12866); and (c) replicable DNA sequences MC20 (79 bp) and MA16 (80 bp), representing mutations of the C. glutamicum dapA promoter.

Description

Technical field

The invention relates to L-lysine-producing strains of coryneform bacteria with an enhanced pyc gene (pyruvate carboxylase gene) in which they are amplified, in particular overexpressing additional genes selected from the group consisting of the dapA gene (dihydrodipicolinate synthase c), the lysE gene (export-lysine gene), the dapB gene (dihydrodipicolinate reductase gene), in particular the dapA gene, and a method for producing L-lysine.

BACKGROUND OF THE INVENTION

L-Lysine is a commercially important L-amino acid which is mainly used as a feed additive in animal nutrition. Its consumption has been steadily increasing in recent years.

L-Lysine is produced by fermentation using strains of L-lysine-producing coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance of this product, work is still ongoing to improve this process. Improvement of the way

They may relate to fermentation-technological measures such as mixing and oxygen supply, or nutrient composition such as sugar concentration during fermentation, or processing into product form, for example by ion exchange chromatography or the intrinsic performance of the microorganism itself.

• ······································································· ····················································

Mutagenesis, selection and mutant selection methods are used to improve the performance of these microorganisms. In this way strains are obtained which are resistant to antimetabolites, such as S- (2-aminoethyl) cysteine, or are auxotrophic to amino acids such as L-leucine, and produce L-lysine.

Recombinant DNA techniques have also been used for several years to strain-improve L-lysine-producing Corynebacterium strains by amplifying the individual biosynthesis genes and investigating the effect on L-lysine production.

EP-A-0 088 166 discloses an increase in performance after amplification of an aminoethylcysteine resistance mediating DNA fragment. EP-B-0 387 527 reports an increase in performance after amplification of the lysC allele encoding feed back resistant aspartate kinase. EP-B-0 197 335 reports an increase in performance after amplification of the dapA gene encoding dihydrodipicolinate synthase. EP-A-0 219 027 reports an increase in performance after amplification of the asd gene encoding aspartate semialdehyde dehydrogenase. Pisabarro et al. (Journal of Bacteriology, 175 (9), 2743-2749, (1993) describe the dapB gene encoding dihydrodipicolinate reductase.

The effect of amplification of primary Λ metabolism genes on L-lysine production was also investigated. EP-A-0 219 027 reports an increase in performance after amplification of the aspC gene encoding aspartate aminotransferase. EP-B-0 143 195 and EP-B-0 358 940 refer to an increase in performance after amplification of the ppc gene encoding phosphoenolpyruvate carboxylase. DE-A-198 31 609 refers to an increase in power after amplification of the pyc gene encoding pyruvate carboxylase. Anaplerotic catalysed reaction

• · « • · · • ·· ··. ·· «· · · • • • · • • • • • • · · · • · · • • • • • • · ·· • · · · · • · •

pyruvate carboxylase is of particular importance compared to phosphoenolpyruvate carboxylase catalyzed reaction. Wendisch et al. (FEMS Microbiology Letters 112, 269-274, (1993)) showed that elimination of the ppc gene did not impair lysine production of the MH2022B strain.

Finally, DE-A-195 48 222 discloses that the increased exportcarrier activity of L-lysine encoded by the lysE gene promotes lysine production.

In addition to these contributions to single gene amplification, the benefits of simultaneous amplification of two or more genes have been studied, thereby improving production

Of L-lysine in corynephormic bacteria. DEA-38 23 451 discloses an increase in performance following the simultaneous amplification of the asd gene and the dapA gene from Escherichia coli. DE-A-39 43 117 discloses an increase in performance following simultaneous amplification of the lysC allele encoding feed back resistant and dapA gene. In particular, EP-A-0 841 395 refers to an increase in performance while amplifying the lysC allele encoding feed back resistant and dapB gene; additional improvements could be achieved by additional amplification of the dapB, lysA and ddh genes.

EP-A-0 854 189 describes an increase in performance while amplifying the lysC allele encoding feed back resistant, dapA gene, dapB gene, lysA gene and aspC gene. In particular, EP-A-0 857 784 refers to an increase in performance while amplifying the ΛlysC allele encoding the feed back resistant and the lysA gene; by additional amplification of the ppc gene, further improvement could be achieved.

It is clear from the large number of methods described in the prior art that there is a need to develop new benefits and to improve existing methods of lysine production by coryneform bacteria.

• - · • · · • ·· • · · 9 9 999 · · • · • · · ·· ·· ·· ··· · · • ·

The inventors have set out to provide novel strains of L-lysine-producing coryneform bacteria and a method for producing L-lysine.

SUMMARY OF THE INVENTION

L-Lysine is a commercially important L-amino acid which is mainly used as a feed additive in animal nutrition.

When L-lysine or lysine is mentioned in the following, it is meant not only the base but also the corresponding salts, such as lysine hydrochloride or lysine sulfate.

The present invention provides L-lysine-enhanced pyc gene strains of coryneform bacteria (pyruvate carboxylase gene), characterized in that they are amplified, in particular overexpressing, additional genes selected from the group consisting of the dapA gene (dihydrysipicolinate synthase gene), aspartate kinase), the lysE gene (exportcarrier lysine gene), the dapB gene (dihydrodipicolinate reductase gene), in particular the dapA gene.

A

Furthermore, a new DNA sequence has been found which is upstream of the dapB gene (5'-region), which carries the -35-region of the dapB promoter and is advantageous for expression of the dapB gene. It is shown as SEQ-ID-No.1.

Therefore, the corresponding replicable DNA is also claimed.

with the nucleotide sequence shown in SEQ-ID-No. First

A further object of the invention are mutations of the MC20 and MA16 promoter of the dapA promoter shown in SEQ-ID-No. 5 and SEQ-ID-No. 6, stored in strains DSM 12868 and DSM 12867.

• ·· • · I • ·· • · • · • · • • ea • · · · ·· · • · ·· • · · • · ·· · · · · · • · · ·

Another object of the invention are L-lysine-producing strains of coryneform bacteria with an enhanced pyc gene, characterized in that they additionally simultaneously amplify, in particular overexpress the dapA and dapB gene.

The invention also relates to L-lysine-producing strains of coryneform bacteria with an enhanced pyc gene, characterized in that they additionally simultaneously amplify, in particular overexpress, the dapA, dapB and lysE gene.

The term "enhancement" in this context describes the enhancement of the intracellular activity of one or more enzymes in a microorganism that are encoded by the DNA of interest by increasing the copy number of the gene (s), using a strong promoter, or using a gene encoding a particular high-enzyme enzyme. activity and these measures are combined where appropriate.

A method for producing L-lysine using the bacteria described above is also an object of the invention.

The microorganisms of the present invention can form L-lysine from glucose, sucrose, lactose, lactose, glucose

fructose, maltose, molasses, starch, cellulose or glycerol and ethanol. They are representatives of coryneform bacteria, especially of the genus Corynebacterium. In the genus Corynebacterium, mention should be made in particular of the species Corynebacterium glutamicum, which is known in the art for its ability to produce amino acids.

This species includes wild-type strains such as Corynebacterium glutamicum ATCC13032, Brevibacterium flavum ATCC14067, Corynebacterium melassecola ATCC17965 and strains or mutants derived therefrom. Examples of mutants of L-lysine-producing coryneform bacteria are, for example,. ···· · · · · ···. ·· ·· ··· ·· ··· ·· ·· ···

Corynebacterium glutamicum FERM-P 1709

Brevibacterium flavum FERM-P 1708

Brevibacterium lactofermentum FERM-P 1712

Brevibacterium flavum FERM-P 6463

Brevibacterium flavum FERM-P 6464

Corynebacterium glutamicum DSM 5714 a

Corynebacterium glutamicum DSM 12866.

The inventors have now found that enhanced expression of the lysE gene additional to the pyc gene or additional enhanced expression of the lysC allele encoding feed back resistant aspartate kinase or additional enhanced expression of the dapB gene, and in particular additionally enhanced expression of the dapA gene, individually or co-production.

It has also been found that, given a given overexpression of the pyc gene, the concomitant, additionally enhanced expression of the dapA and dapB gene provides additional advantages for L-lysine production.

Furthermore, the present inventors have found that, given a given overexpression of the pyc gene, the simultaneous, additionally enhanced expression of the dapA, dapB and lysE gene is particularly advantageous for L-lysine production.

To achieve amplification (overexpression), for example, the copy number of the respective genes is increased or the promoter and regulatory region or ribosome binding site is mutated upstream of the structural gene. Expression cassettes which are inserted upstream of the structural gene act in the same way. Inducible promoters can additionally increase expression during fermentative L-lysine production. Expression measures also improve m-RNA durability measures. Next,

• ·· • · · · • ·· · · • · • I ···· • · · · • · • · · • · • · · • · • • · ·· · ·· · · · · · ·· ·

the enzyme activity also enhances by preventing the degradation of the enzyme protein. The genes or gene constructs are either in plasmids (shuttle vectors) with different copy numbers, or they are integrated and amplified in the chromosome. Further, overexpression of the respective genes may alternatively be achieved by changing the composition of the media and changing the culture.

Instructions for this can be found by one skilled in the art in, inter alia, Martin et al. (Bio / Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European Patent Specification EPS 0 472 869, in US Patent 4,601,893, in Schwarzer and Puhler (Bio / Technology 9, 84-87 (1991)), in Reinschéid et al. . (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), Japanese JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) or the Manual of Methods for General. Bacteriology of the American Society for Bacteriology (Washington DC, USA, 1981) and in well-known textbooks of genetics and molecular biology.

The Corynebacterium glutamicum genes used according to the invention are described and can be isolated, optionally generated or synthesized by known methods.

Methods for site-specific mutagenesis are described, inter alia, in Higuchi et al. (Nucleic Acids Research 16: 7351-7367 (1988)) or in Silver et al. in Innis, Glefand and Sninský (eds.): PCR Strategies (Academic Press, London, United Kingdom)

Britain, 1995).

· ··. ················································

In order to isolate the C. glutamicum gene of interest, a gene bank of this microorganism is first established in, for example, E. coli or possibly in C. glutamicum. The establishment of gene banks is described in commonly known textbooks and manuals. Examples include Winnacker's textbook: Gene und Klone, Eine Einfíihrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual from Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) . Bathe et al. (Molecular and General Genetics,

252: 255-265, 1996) describe a C. glutamicum gene bank ATCC13032 which was established 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 NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Bormann et al. (Molecular Microbiology 6 (3), 317-326)) in turn describe a C. glutamicum gene bank ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). Plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979) or pUC19 (Norrander et al., 1983, Gene, 26: 101-) may also be used to generate the C. glutamicum gene bank in E. coli. Shuttle vectors such as pJCl (Cremer et al., Molecular and General Genetics 220, 478-480 (1990)) or pEC5 (Eikmanns et al., 1991, Gene 102, 93-98) may also be used, E. coli strains which are defective in terms of restriction and expression are particularly suitable as hosts, such as the DH5amcr strain described by Grant et al. (Proceedings of the National). Acadeins of Sciences USA, 87 (1990) 4645-4649) Another example of this is the restriction defective strains of C. glutamicum RM3 and RM4, as described in Schafer et al., (Applied and Environmental Microbiology 60 (2), 756-759). , (1994)).

• ·· ·· · · • ·· • · · • · ···· • · · · • · · · • · · • · • · • • • · • • • ·· · ·· · · · · · ·· ·

The gene bank is then transferred to the indicator strain by transformation (Hanahan, Journal of Molecular Biology 166, 557580, 1983) or by electroporation (Tauch et al. 1994, FEMS Microbiological Letters, 123: 343-347). The indicator strain is characterized in that it has a mutation in the gene of interest that induces a detectable phenotype, for example auxotrophy. Indicator strains or mutants are available from published sources or strain collections, such as the Genetic Stock Center of Yale University (New Haven, Connecticut, USA) or are self-generated. An example of such an indicator strain is the E. coli strain RDA8 requiring mesodiaminopimelic acid (Richaud et al. CR Acad. Sci. Paris Ser. III 293: 507-512 (1981)) which carries a mutation (dapA :: Mu) in the gene dapA.

Upon transformation of the indicator strain with the recombinant plasmid carrying the gene of interest and expression of the gene of interest, the indicator strain becomes prototrophic with respect to the property in question. If the cloned DNA fragment mediates resistance to, for example, antimetabolites such as S- (2-aminoethyl) cysteine, the identification of the indicator strain carrying the recombinant plasmid may be performed by selection for a suitable

Ffilled media.

For the nucleotide sequence of the gene region of interest known or available in the database, the chromosomal DNA can be isolated by known methods, as described, for example, in Eikmanns et al. (Microbiology 140, 1817-1828, (1994)), and said gene can be synthesized by polymerase chain reaction (PCR) using suitable primers and cloned into a suitable plasmid vector such as pCRIITOPO from Invitrogen (Groningen, The Netherlands). Summary of PCR methodology

• ·· • · · • ·· · · • · • · ···· • · · · · · • · • · • • · · · · · · · · • ·

can be taken from a book by Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

Publicly available databases for nucleotide sequences are, for example, from the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany) or the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).

The isolation and cloning of the C. glutamicum ATCC13032 pyc gene is described in DE-A-198 31 609 and in Koffas et al. (Applied Microbiology and Biotechnology 50, 346-352 (1998). The pyc gene nucleotide sequence is available under accession number AF038548 or Y09548.

The isolation and cloning of the C. glutamicum lysE gene ATCC13032 is described in DE-A-195 48 222. The nucleotide sequence of the lysE gene is available under accession number X96471.

The isolation, cloning and sequencing of the dapA gene of various strains of C. glutamicum is described in Cremer et al. (Molecular and General Genetics 220, 478-480 (1990)), in Pisabarro et al. (Joúrnal of Bacteriology, 175: 2743-2749, (1993)) and in Bonnassie et al. (Nucleic Acids Research 18: 6421, 1990). DE-A-39 43 117 describes the enhancement of the dapA gene by means of plasmid pJC23. The nucleotide sequence of the dapA gene is available under accession number X53993. , υ

The isolation, cloning and sequencing of the dapB gene of Brevibacterium lactofermentum is described in Pisabarro et al. (Journal of Bacteriology, 175: 2743-2749, (1993)). The nucleotide sequence of the dapB gene is available under accession number X67737.

* 9 ‘· ·· yy * <« <

• 9 9 • · ··· · · · ··· ··· ·· ·

Several authors describe the isolation, cloning, and sequencing of the lysC gene and the lysC alleles that encode a feed back resistant aspartate enzyme. Kalinowski et al. (Molecular and General Genetics 224: 317-324 (1990)) reports on the lysC allele of C. glutamicum DM53-1. DE-A-39 43 117 reports the cloning of the lysC allele of C. glutamicum MH20. Follettie et al. (Journal of Bacteriology, 175: 4096-4103 (1993)) refer to the lysC allele of C. flavum N13, which is referred to there as ask.

The nucleotide sequences of the lysC gene and the various lysC alleles are available under accession numbers X57226 and E06826.

The genes thus obtained can then be inserted, individually or in suitable combinations, inter alia into plasmid vectors such as pJCl (Cremer et al., Molecular and General Genetics 220, 478-480 (1990)) or pEC5 (Eikmanns et al., 1991). , Gene 102, 93-98) and introduced into desired strains of coryneform bacteria, for example strain MH20-22B (Schrumpf et al., Applied Microbiology and Biotechnology 37: 566-571 (1992)) by transformation, such as in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)) or by electroporation such as Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and expressed. Alternatively, the selected strain can be transformed with two plasmid vectors, which contain a particular gene or genes, thereby providing advantageous, additionally enhanced expression of two or more genes in addition to the known enhancement of the pyc gene.

, I.

Examples of such strains are the MH20-22B / pJC23 / pEC7pyc strain, in which the expressed pyc and dapA genes are simultaneously amplified, or Strain MH20-22B / pJC33 / pEC7pyc in which they are simultaneously amplified, in particular overexpressed the pyc gene and the lysC allele (FBR), or strain MH20-22B / pJC23 / pEC7dapBpyc in which they are simultaneously amplified in particular, overexpressed pyc, dapA and dapB genes, or strain MH20-22B / pJC23 / pEC71ysEdapBpyc in which they are co-amplified, particularly overexpressed pyc, dapA, dapB and lysE genes.

The microorganisms produced according to the invention can be cultivated continuously or discontinuously for the production of L-lysine by the batch method or the fed batch method or the repeated fed batch method. A summary of known cultivation methods is described in a textbook by Hop (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in a textbook from Storhas (Bioreactor and Periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium used must suitably meet the requirements of the microorganisms. Descriptions of the culture media of various microorganisms can be found in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington, D.C., USA, 1981). As carbon sources, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose can be used; oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid; alcohols such as glycerol and ethanol; and organic

• ·· · · ···· · · • · · • • • • • • ·· • • · · · • • • · · • • • • • ·· ·· · · · · · · ·

acids such as acetic acid. These substances can be used as individual components or as a mixture. Nitrogen-containing organic compounds such as peptones, yeast extract, meat extract, malt extract, corn extract, soy flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate can be used as nitrogen sources. and ammonium nitrate. The nitrogen sources may be used singly or as a mixture. As the phosphorus source, potassium dihydrogen phosphate or potassium hydrogen phosphate or the corresponding sodium salts may be used. The culture medium must further comprise metal salts such as magnesium sulfate or iron sulfate, which are needed for growth. Finally, essential growth agents such as amino acids and vitamins may be used in addition to the above-mentioned substances. The raw materials used can be added to the culture as a single batch or added in a suitable manner during the cultivation.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acidic compounds such as phosphoric acid or sulfuric acid are suitably used to control the pH of the culture. Antifoams, such as polyglycol esters of fatty acids, can be used to control foaming.

Suitable selective agents, for example antibiotics, may be added to the medium to maintain the stability of the plasmids. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, for example air, are introduced into the culture. The temperature of the culture is usually about 20 ° C to 45 ° C, and especially about 25 ° C to 40 ° C. The culture is maintained until the maximum of L-lysine is formed. This target is usually achieved in 10 hours to 160 hours.

• · · • ·· • · · - -. ··. ···· · · • · • · • · • • • • · • · • »·· • • · • · ·· · · · · · · ·

The concentration of L-lysine formed can be determined using amino acid analyzers by ion exchange chromatography and post-reaction on a ninhydrin detection column as described by Spackman et al. (Analytical Chemistry, 30,

1190 (1958)).

The following microorganisms were deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig,

Germany) under the Budapest Treaty:

• Escherichia coli K-12 DH5α / pEC7pyc strain as DSM 12870 • Escherichia coli K-12 DH5α / pEC7dapBpyc strain as DSM 12873 • Escherichia coli K-12 DH5α / pEC71ysEdapBpyc strain as DSM

12874, a strain of Corynebacterium glutamicum DSM5715 / pJC23 as

DSM 12869, • Corynebacterium glutamicum strain DSM5715aecD :: dap (MA16) »as DSM 12867, • Corynebacterium glutamicum strain DSM5715aecD :: dapA (MC20) as DSM 12868,

Strain Corynebacterium glutamicum DM 678 as

DSM 12866, strain Escherichia coli K-12 DH5α / pEC71ysEpyc as DSM 12872, 9 · 9 Escherichia coli strain K-12 DH5α / pEC7dapBlysE deposited as DSM 12875, Escherichia coli strain K-12 DH5α / pEC71ysE deposited as DSM 12871.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in more detail below with reference to exemplary embodiments.

Example 1

Obtaining DNA encoding lysE

Chromosomal DNA was isolated from ATCC 13032 strain by conventional methods (Eikmanns et al., Microbiology 140: 1817-1828, (1994)). A DNA fragment carrying the lysE gene was amplified by polymerase chain reaction (PCR). Based on the sequence of the lysE gene known for C. glutamicum (Vrljic et al., Molecular Microbiology 22 (5), 815-826 (1996)) (Accession No. X96471), the following primer oligonucleotides were selected for PCR:

LysBaml:

5 'CTC GAG AGC (GGA TCC)

LysBam2:

5 'GGA GAG TAC GGC (GGA TCC) ACC GTG. ACC 3 '

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany) and according to the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990,

Academic Press) a PCR reaction was performed. Primers allow • 4

• · 0000 00 · • · • 0 0 00 • 00 • 0 · · · 0 0 0 00 ·· 00 000 ·· ··

amplifying a DNA fragment of approximately 1.1 kb that carries the lysE gene. In addition, the primers contain a BamHI restriction endonuclease cleavage sequence, which is indicated by parentheses in the nucleotide sequence shown above.

The amplified approximately 1.1 kb DNA fragment carrying the lysE gene was identified by 0.8% agarose gel electrophoresis, isolated from the gel, and purified by QIAquick Gel Extraction Kit (Cat. No. 28704) from Qiagen (Hilden) , Germany).

The fragment was then ligated with T4 DNA ligase from Boehringer Mannheim (Mannheim, Germany) into the pUC18 vector (Norrander et al., Gene (26) 101-106 (1983)).

For this purpose, the pUC18 vector was completely digested with restriction endonuclease SmaI and treated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim (Mannheim, Germany). The ligation batch was transformed into E. coli strain DH5α (Hanahan, In: DNA cloning. A practical approach). I. IRLPress, Oxford, Washington, DC) Selection of plasmid-bearing cells was performed by spreading 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, NY), which was supplemented with 50 mg / L ampicillin. Plasmid DNA was isolated from the transformant by treatment with a restriction enzyme BamHI followed by agarose gel electrophoresis. The plasmid was named pUC181ysE.

Example 2

Obtaining dapB ······················· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Chromosomal DNA was isolated from Corynebacterium glutamicum ATCC 13032 as described in Example 1.

The sequence of the dapB gene as such from Corynebacterium glutamicum is known (accession number X67737). However, the disclosed sequence contains only 56 bp before translation starts. Therefore, the 5'-region is additionally sequenced prior to the start of translation.

To do this, sequencing was performed using plasmid pJC25 (EP-B 0 435 132) and using a primer oligonucleotide that binds in the region of the known dapB sequence (accession number X67737). Sequence of sequence primer used:

5 'GAA CGC CAA CCT TGA TTC C 3'

Sequencing was performed by the chain disruption method described in Sanger et al., Proc. Natl. Acad. Sci. USA, (74) 5463-5467 (1977). Sequencing was performed using the AutoRead Sequencing Kit (Pharmacia, Freiburg). Electrophoretic analysis and detection of sequence products was performed using an A.L.F. DNA sequencing apparatus. from Pharmacia (Freiburg, Germany).

The obtained DNA sequence was used to select a second primer to obtain additional sequence data before the start of transcription. The following primer was selected for this purpose:

5 'CTT TGC CGC

Sequencing was performed as described above. The new upstream sequence of the dapB gene is shown as SEQ ID NO 1. The sequence including the nucleotide sequence of the dapB gene is shown as SEQ ID NO 2.

The dapB gene was amplified by the polymerase chain reaction. For this, two oligonucleotides were synthesized by MWG Biotech and were selected on the basis of the known DNA sequence of the dapB gene: &lt; tb &gt; ______________________________________ &lt; tb &gt;

P-dap:

5 '(AAG CTT) AGG TTG TAG

5 * TTA ACT TGT TCG GCC ACA GC 3 '

Primer 5 '(primer P-dap) contains a HindIII cleavage site, indicated in parentheses in the sequence shown. The PCR was performed according to Example 1. In this way, an approximately 1.1 kb DNA fragment carrying the dapB gene and containing a HindIII restriction endonuclease cleavage site was amplified. The obtained PCR fragment was purified from a 88% agarose gel (QIAquick Gel Extraction Kit from Qiagen, Hilden, Germany) and cloned using the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. No K4550-01) into the pCR2 cloning vector. .TOPO (Invitrogen, Leek, The Netherlands). The ligation batch was transformed into E. coli TOPIOF 'strain from Invitrogen, the transformation batch was plated on LB agar with IPTG (0.16 mM) and X-Gal (64 mg / l) containing kanamycin (50 mg / l) and isolated is a white colored kanamycin resistant colony. Plasmid DNA was isolated from a single transformant using QIAprep Spin Miniprep Kit from Qiagen and examined by restriction digestion with HindIII and subsequent agarose gel electrophoresis. The DNA sequence of the amplified DNA fragment was examined by sequencing. The sequence of the PCR product was identical to that shown in SEQ ID NO 1. The plasmid obtained was named pCR2. lTOPOdapB.

Example 3

Obtaining the DNA encoding pyc · · · · yc yc yc yc yc yc yc yc yc yc yc yc yc

The Corynebacterium glutamicum strain ATCC 13032 was used as a donor for chromosomal DNA. Chromosomal DNA was isolated from ATCC 13032 as described in Example 1. A DNA fragment carrying the pyc gene was amplified by polymerase chain reaction (PCR). Based on the sequence of the pyc gene known for C. glutamicum (Peters-Wendisch et al., 1998. Microbiology, 144, 915-927) (Accession No. Y09548), the following primer oligonucleotides were selected for PCR:

5-PYC-IN:

5 'GC (T CTA GA) & GTG TCG CAA CCG TGC TTG A 3'

3-PYC-IN:

5 'GC (T-CTA GA) T TGA GCC TTG GTC TCC ATC T 3'

The primers shown were synthesized by MWG Biotech and the PCR reaction was carried out according to the standard PCR method of Innis et al., (PCR protocols. A guide to methods and applications, 1990, Academic Press). The primers allow amplification of a DNA fragment of approximately 3.8 kb that carries the pyc gene. In addition, the primers contain a sequence for the restriction endonuclease XbaI cleavage site, which is indicated by parentheses in the nucleotide sequence shown above.

An amplified approximately 3.8 kb DNA fragment carrying the pyc gene was identified by gel electrophoresis in a 0.8% agarose gel, isolated from the gel, and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).

The fragment was then ligated with Dual

Promotor Topo TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat.

Number K4600-01) into pCRII-TOPO vector. The ligation batch was transformed into E. coli strain TOP10 (Invitrogen, Leek, -1)

- <··· ·· · · · · ··· a · a · a a · · · · ·······

Netherlands). Selection of plasmid-bearing cells was performed by spreading the transformation batch on LB agar with X-Gal (64 mg / L) containing kanamycin (50 mg / L).

The plasmid obtained after restriction digestion of the DNA was examined and identified in an agarose gel. The plasmid was named pCRII-TOPOpyc and the DNA sequence of the cloned insert was sequenced for control. Since the searched sequence of the pyc insert in pCRII-TOPOpyc coincided with the gene bank write sequence, this plasmid was used below.

Example 4

Cloning of dapB in pEC7 vector

An approximately 1.1 kb fragment carrying the dapB gene was isolated from plasmid pCR2.1TOPOdapB (Example 2). For this purpose, plasmid pCR2.lTOPOdapB was completely digested with the restriction enzyme HindIII and an approximately 1.1 kb DNA fragment with the dapB gene was isolated.

The dapB fragment was inserted into the vector pEC7. The pEC7 vector is based on the E. coli-C. glutamicum pEC5 shuttle vector (Eikmanns et al., 1991, 102: 93-98). The BamHI cleavage site not located in the polylinker was removed from plasmid pEC5 as follows: Plasmid pEC5 was partially digested with the restriction enzyme BamHI. A DNA fragment of approximately 7.2 kb was isolated from an agarose gel and the overlapping ends were treated with Klenow polymerase (Boehringer Mannheim). The DNA fragment thus obtained was ligated (T4-ligase, Boehringer Mannheim). The ligation batch was transformed into E. coli strain DH5α and kanamycin-resistant colonies were isolated on kanamycin LB agar (50 mg / L). Plasmid DNA was isolated from a transformant (QIAprep Spin Miniprep Kit from Qiagen). And examined by restriction digestion with BamHI and PstI. The plasmid thus obtained was called pEC6.

Plasmid pEC6 was completely digested with the restriction enzyme XhoI. The DNA fragment carrying the trp terminator was ligated with the vector DNA fragment (T4-ligase, Boehringer Mannheim). The ligation batch was transformed into E. coli strain DH5α and kanamycin-resistant colonies were isolated on kanamycin LB agar (50 mg / L). Plasmid DNA was isolated from one transformant (QIAprep Spin Miniprep Kit from Qiagen) and examined by restriction digestion with BamHI and XhoI restriction enzymes. The plasmid thus obtained was called pEC7.

The obtained dapB-bearing DNA fragment was ligated with the pEC7 vector (T4-ligase, Boehringer Mannheim), which was also completely digested with the restriction enzyme HindIII and treated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim). The ligation batch was transformed into E. coli strain DH5α and kanamycin-resistant colonies were isolated on kanamycin LB agar (50 mg / L). Plasmid DNA was isolated from a single transformant (QIAprep Spin Miniprep Kit from Qiagen) and examined by restriction digestion with the restriction enzyme HindIII. The plasmid thus obtained was called pEC7dapB (Figure 1). The obtained Escherichia coli strain was named DH5α / pEC7dapB.

Example 5

Cloning of lysE in pEC7 vector

The plasmid pUC181ysEneu described in Example 1 was completely digested with the restriction enzyme BamHI and, as in Example 1, a 1.1 kb BamHI DNA fragment with the lysE gene was isolated. The vector pEC7 was also completely digested with the restriction enzyme BamHI and treated with alkaline phosphatase. BamHI vector fragment and fragment e · H I ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ·· ··

BamHI lysE was ligated (Rapid DNA Ligation Kit, Boehringer Mannheim) and transformed into E. coli strain DH5α. Plasmid-bearing transformants were selected on LB agar containing chloramphenicol (10 mg / L). Plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen) and examined by restriction digestion with BamHI. The plasmid thus obtained was called pEC71ysE (Figure 2). The strain obtained by transforming plasmid pEC71ysE into E. coli strain DH5α was named DH5α / pEC71ysE.

Example 6

Cloning of pyc in pEC7 vector

From the plasmid pCRII-TOPOpyc (from Example 3), a 3.8 kb DNA fragment carrying the C. glutamicum ATCC pyc gene ATCC 13032 was obtained by digestion with XbaI. The DNA fragment of 3.8 kb was identified by gel electrophoresis, isolated. from the gel and purified by conventional methods and the Klenow polymerase were treated with overlapping ends. The pEC7 vector was also completely digested with the restriction enzyme SmaI and treated with alkaline phosphatase. The Smal and XbaI vector fragment and the Klenow phosphatase treated pyc fragment were ligated (T4 ligase, Boehringer Mannheim) and transformed into E. coli strain DH5α. Plasmid-bearing transformants were selected on LB agar containing chloramphenicol (10 mg / L). Plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and examined by restriction digestion with SalI restriction enzyme. The plasmid thus obtained was called pEC7pyc (Figure 3). The E. coli strain obtained by transforming the plasmid pEC7pyc into an E. coli strain DH5α was named DH5α / pEC7pyc.

Example 7

Generation of a plasmid containing lysE and dapB · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The dapB gene was isolated as a HindIII fragment from plasmid pCR2.1TOPOdapB, which contains the dapB gene from C. glutamicum ATCC 13032. For this purpose, the plasmid was completely digested with the restriction enzyme HindIII and the DNA fragment carrying dapB was isolated from a 0.8% agar gel (QIAquick Gel Extraction Kit, Qiagen).

In addition, the vector pEC71yC was completely digested with the restriction enzyme HinII and treated with alkaline phosphatase. The 1.1 kb fragment containing dapB (T4-ligase, Boehringer Mannheim) was ligated with the linear vector fragment thus obtained, and the ligation batch was transformed into E. coli DH5α strain. Plasmid-bearing transformants were selected on LB agar containing chloramphenicol (10 mg / L). Plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen, Hilden) and examined by restriction digestion with the restriction enzyme HindIII. The plasmid thus obtained was called pEC71ysEdapB. This plasmid is autonomously replicable in Escherichia coli and Corynebacterium glutamicum and confers resistance to the antibiotic chloramphenicol.

Plasmid pEC71ysEdapB simultaneously contains the dapB gene, which encodes dihydrodipicolinate reductase, and the lysE gene, which encodes a lysine exporter.

The strain obtained by transforming E. coli DH5α with pEC71ysEdapB was deposited as DH5α / pEC71ysEdapB. p

Example 7

Generation of a plasmid containing both dapB and pyc

The plasmid carrying the pyc gene, which encodes pyruvate carboxylase from C. glutamicum ATCC 13032, was completely digested with the restriction enzyme XbaI and the overhanging ends were digested with Klenow's. Thus, the 3.8 kb DNA fragment containing the pyruvate carboxylase gene could be isolated.

Plasmid pEC7dapB (from Example 4) was also completely digested with the restriction enzyme SmaI and the ends treated with alkaline phosphatase. The linear vector fragment thus obtained was ligated with a 3.8 kb DNA fragment containing the pyc gene using T4 DNA ligase (Boehringer Mannheim, Mannheim, Germany). This resulted in a plasmid containing both the dapB gene and the pyc gene from corynebacteria. Plasmid-bearing transformants were selected on LB agar containing chloramphenicol (10 mg / L). Plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and examined by restriction digestion with the restriction enzyme SalI. The plasmid is shown in Figure 4 and was called pEC7dapBpyc. The E. coli strain obtained by transforming the plasmid pEC7dapBpyc into an E. coli strain DH5α was named DH5α / pEC7dapBpyc.

Example 8

Generation of a plasmid containing sequences encoding simultaneously lysE, dapB and pyc

Plasmid pCRII-TOPOpyc (from Example 3), which carries the pyc gene, which encodes pyruvate carboxylase from C. glutamicum ATCC 13032, was completely digested with the restriction enzyme XbaI and the overhanging ends were treated with Klenow polymerase as described in Example 6. Thus, it could be isolated a 3.8 kb DNA fragment containing the pyruvate carboxylase gene.

Plasmid pEC7dapBlysE was also completely digested with the restriction enzyme SmaI and the ends treated with alkaline phosphatase. The linear vector fragment thus obtained was ligated with a 3.8 kb DNA fragment containing the pyc gene using T4-DNA. .. ..... - * e - · · · é a é a a a r r r r r r r r r r r r r r r · ·

4 • · ·

4 ·· · ligases (Boehringer Mannheim). This produces a plasmid containing the lysE gene and the dapB gene as well as the pyc gene from Corynebacterium glutamicum. Plasmid-bearing transformants were selected on LB agar containing chloramphenicol (10 mg / L). Plasmid DNA was isolated (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and examined by restriction digestion with the restriction enzyme SmaI. The plasmid is shown in Figure 5 and was called pEC7dapBlysEpyc. The E. coli strain obtained by transformation of plasmid pEC7dapBlysEpyc into E. coli strain DH5a was named

DH5a / pEC7dapBlysEpyc.

Example 9

Transformation of strain MH20-22B with plasmids pJC23 and pJC33

Plasmid pJCl is a plasmid replicable in Escherichia coli and Corynebacterium glutamicum (Cremer et al., 1990, Molecular and General Genetics 220: 478-480). Derived from it is the plasmid pJC33 (Cremer et al., 1991. Applied and Environmental Microbiology 57 (6) 1746-1752), which carries the lysC (Fbr) gene from C. glutamicum MH20-22B.

Plasmid pJC23 is also based on the pJCl vector and carries the dapA gene from C. glutamicum ATCC 13032 (Cremer et al., 1990, Molecular and General Genetics 220: 478-480) (EP-B 0 435 132). The plasmids pJCl, pJC33 and pJC23 were introduced into strain MH-20-22B by electroporation method (Haynes und Britz, FEMS Microbiology Letters f-61) 329-334 (1989)). The strain C. glutamicum MH20-22B is an AEC resistant lysine producer, which is deposited under the number DSM5715.

Transformants obtained by electroporation were isolated on selection agar (LBHIS agar (18.5 g / L brain-heart infusion broth, 0.5 M sorbitol, 5 g / L Bacto-tryptone, 2.5 ^). • Bac to / to to to to to to to to to to to to to to to to to to to to to to to to to yeast extract, 5 g / l NaCl, 18 g / l Bactoagar with 15 mg / l kanamycin The plasmid DNA was isolated by conventional methods (Peters-Wendisch et al., 1998. Microbiology, 144, 915-927), digested with suitable The strains obtained were named MH20-22B / pJC1, MH2022B / pJC33 and MH20-22B / pJC23.

Example 10

Transformation with plasmids pEC7pyc, pEC7dapBpyc and pEC7dapBlysEpyc

In addition, the strains produced in Example 9 were used to provide a second plasmid.

The plasmids pEC7pyc (see Example 6) pEC7dapBpyc (see Example 7) pEC7dapBlysEpyc (see Example 8) were introduced into the strains MH20-22B / pJC1, MH20-22B / pJC33 and MH2022B / pJC23 described above.

Transformed bacteria are selected based on the resistance of the contained plasmids to antibiotics. Transformants obtained by electroporation were isolated on selection agar (LBHIS agar with 15 mg / l kanamycin and 7.5 mg / l chloramphenicol). Plasmid DNA was isolated, digested with appropriate restriction endonucleases, and screened.

The strains obtained are as follows:

DSM5715 / pJCl / pEC7pyc

DSM5715 / pJC33 / pEC7pyc

DSM5715 / pJC23 / pEC7pyc ·· ···

DSM5715 / pJC2 3 / pEC7dapBpyc

DSM5715 / pJC23 / pEC71ysEdapBpyc

Example 11

L-lysine production

The various C. glutamicum strains obtained in Examples 9 and 10 were cultured in a nutrient medium suitable for lysine production, and the lysine content was determined in the culture supernatant.

To this end, different strains were first incubated for 24 hours at 33 ° C on agar plates with appropriate antibiotics (brain-heart agar with kanamycin (25 mg / l), chloramphenicol (10 mg / l)). Starting from these cultures from agar plates, a preculture (10 ml of medium in a 100 ml Erlemeyer flask) was seeded. Complete CglII medium was used as the preculture medium. Kanamycin (25 mg / L) and chloramphenicol (10 mg / L) were added. The preculture was incubated for 24 hours at 33 ° C at 240 rpm on a shaker. The main culture was inoculated from this preculture so that the initial optical density (660 nm) of the main culture was 0.2. MM medium was used for the main culture.

MM Medium: CSL 5 g / l t. MO PS 20 g / l glucose 50 g / l (separately autoclaved) Soli: (NH ₄ ) ₂ SO ₄ 25 g / l kh ₂ Mon ₄ 0.1 g / l MgSO ₄ .7H ₂ O 1.0 g / l CaCl ₂ 2H ₂ O 10 mg / l FeSO ₄ .7H ₂ O 10 mg / l

* ·· '··· ⁴ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · ·· ·· ···

MnSO ₄ .H ₂ O 5, 0 mg / l biotin 0.3 mg / l (sterile filtered) Tiamín.HCI 0.2 mg / l (sterile filtered)

CaCO ₃ 25 g / L

abbreviations:

CSL: Corn Steep Liquor

MOPS: morpholinopropanesulfonic acid

CSL, MOPS and salt solution were adjusted to pH 7 with ammonia water and autoclaved. Sterile substrate and sterile vitamin solutions were then added, as well as dry autoclaved CaCO ₃ .

Cultivation is carried out in a 10 ml volume in a 100 ml Erlenmeyer flask with baffles. Kanamycin (25 mg / L) and chloramphenicol (10 mg / L) were added. The cultivation was carried out at 33 ° C and 80% humidity.

After 72 hours, the optical density (OD) was determined at a measuring wavelength of 660 nm. The amount of lysine produced was determined using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange choromatography and additional derivatization on a ninhydrin detection column. The glucose content was determined using a sugar analyzer from Skalar Analytik GmbH (Erkelenz, Germany).

Table 1 shows the result of the experiment.

Table 1

tribe gene OD (660) Lysine-HCl. g / l DSM5715 / pJCl / pEC7pyc pyc 9/3 11.3 DSM5715 / pJC33 / pEC7pyc pyc, lys (Fbr) 9/2 11/9 DSM5715 / pJC23 pEC7pyc pyc, dapA 9/3 14.4 DSM5715 / pJC23 pEC7dapBpyc pyc, dapA, dapB 9/3 16.9 DSM5715 / pJC23 / pEC71ysEdapBpyc pyc, dapA, dapB, lysE 9.1 17.6

.- 9 ·· · - ·· --f ·· 9

9 9 9

9 9

• · ·

Example 12

Cloning of the aecD gene into pUC18 vector

Plasmid pSIR21 (Rossol, Dissertation, University of Bielfeld 1992) was completely digested with BglII and EcoRV and a 1.4 kb DNA fragment containing the aecD gene (accession number M89931) was isolated (Rossol and Puhler, Journal of Bacteriology 174 (9)). 1, 2968-2977 (1992)) from C. glutamicum ATCC 13032. The isolated DNA fragment was ligated with plasmid pUC18, which had been completely digested with BamHI and SmaI, with T4 DNA ligase as described in Sambrook et al. (Molecular Cloning: a Laboratory Manual (1989) Cold Spring Harbor Laboratory Press). The ligation batch was transformed into E. coli strain DH5α.

Transformant selection was performed on brain-heart plates with ampicillin (100 mg / L). A plasmid DNA was isolated from one colony. The plasmid obtained was named pUC18 :: aecD.

Example 13

Cloning of the dapB gene in plasmid pSP72

The dapA gene fragment was isolated from plasmid pJC20 (Cremer, J. Dissertation 1989, University of Dusseldorf) as a Sph1BamHI fragment. The vector pSP72 (Promega Corporation, USA) was completely digested with SphI and BamHI and treated with alkaline phosphatase. A fragment carrying dapA was ligated into this vector with T4 DNA ligase. The DNA was then transformed _; gio strain E. coli XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). Transformant selection was performed on LB medium with ampicillin (100 mg / L). Plasmid DNA was isolated from one transformant and designated pSP72 :: dapA.

Example 14

DapA promoter mutagenesis and generation of plasmids pSP72 :: dapA (MC20) and pSP72 :: dapA (MA16)

A Quickchange site directed mutagenesis kit from Stratagene was used for mutagenesis of the promoter region. Based on the existing dapA sequence, the following primers were determined and used for mutagenesis:

To create pSP72 :: dapA (MC20)

Primer dapl for MC20

CCA AAT GAG AGA TGG TAA CCT TGA ACT

Primer dap2 for MC20

GTG CTC ATA GAG TTC AAG

To create pSP72 :: dapA (MA16)

Primer dap3 for MA16

CCA AAT GAG GGA AGA AGG TAT AAT TGA ACT

Primer dap4 for MA16

GTG CTC ATA GAG TTC AAT TAT ACC TTC TTC

CTA TGA GCA

CCT CAT TTG G

The PCR reaction was performed as described by the manufacturer (Stratagene) of the Quickchange site directed mutagenesis kit and using the plasmid pSP72 :: dapA (from Example 13) as a template.

I ·

The mutagenesis batches were transformed into E. coli XL1-Blue strain. Transformant selection was performed on LB medium with carbenicillin (100 mg / L). A plasmid DNA was isolated from one transformant, and BstEII digestion was checked for the BstEII cleavage site control. Plasmids that no longer carry any BstEII cleavage site showed the desired mutation.

·· ···· · · · · ···

The obtained plasmids were transformed into a dapA-defective E. coli RDA8 mutant. Transformation batches were plated on LB with carbenicillin (100 mg / L) to test the complementation of the dapA mutation. DNA was isolated from each transformant and the plasmids obtained were named pSP72 :: dapA (MC20) and pSP72 :: dapA (MA16). Plasmid sequencing was performed using reverse and universal sequencing primers according to the chain cleavage method described in Sanger et al., Proceedings of the National Academy of Sciences (74 5463-5467 (1977)). AutoRead Sequencing Kit (Pharmacia / Freiburg) Electrophoretic analysis and detection of sequence products was performed using an A? LF DNA sequencing apparatus (Pharmacia, Freiburg, Germany).

Example 15

Generation of plasmids pK19mobsacBaecD :: dapA (MC20) and pK19mobsacBaecD :: dapA (MA16) (recloning of mutagenized fragments)

Plasmids pSP72 :: dapA (MC20) and pSP72 :: dapA (MA16) (from Example 14) were completely digested with the restriction enzymes PvuII and SmaI.

I »

1450 bp PvuII-SmaI fragments carrying the mutated MC20 promoter or MA16 dapA gene were ligated with StuI cleaved pUC18 :: aecD vector (from Example 12) by T4 DNA ligase. The ligation batch was transformed into an E. coli strain

DH5a. Transformant selection was performed on LB medium with ampicillin (100 mg / L). One plasmid of DNA was isolated from transformants. In this way, plasmids pUC18aecD :: dapA (MC20) and pUC18aecD :: dapA (MA16) were obtained.

The plasmids pUC18aecD :: dapA (MC20) and pUC18aecD :: dapA (MA16) were partially digested with the restriction enzyme EcoRI and completely with the enzyme.

.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Sal to obtain a 3.0 kb aecD :: dapA (MA16) or aecD :: dapA (MC20) fragment, respectively. The fragment was ligated with T4DNA ligase into the cleaved and alkaline phosphatase-treated vector pK19mobsacB (Schafer et al., Gene (145) 6973 (1994)). The ligation batch was transformed into E. coli DH5 strain (Hanahan (1985), In: DNA cloning. A practical approach., To form I. IRL-Press, Oxford, Washington DC, USA). Transformant selection was performed on LB medium with kanamycin (50 mg / L). One plasmid of DNA was isolated from transformants. In this way, plasmids pK19mobsacBaecD :: dapA (MC20) and pK19mobsacBaecD :: dapA (MA16) were obtained.

The plasmid DNA was transformed into E. coli strain S17-1 (Simon, Priefer and Puhler / Bio / Tehology, (1), 784-791 (1983). Transformant selection was performed on LB medium with kanamycin (50 mg / ml). One DMA plasmid was isolated from the transformants and examined and the strains obtained were named S17-1 / pK19mobsacBaecD :: dapA (MC20) and

S17-l / pKl9mobsacBaecD :: dapA (MA16).

Example 16

Generation of C. glutamicum strains DSM5715aecD :: dapA (MC20) and DSM5715aecD :: dapA (MA16)

Plasmids pK19mobsacBaecD :: dapA (MC20) and pK19mobsacBaecD :: dapA (MA16) with conjugation method (Schäfer et al., Journal of Bacteriology, (172) 1663-1666 (1990)) S17-1 / pK19mobsacBaecD :: dapA (MC20) and

S17-1 / pK19mobsacBaecD :: dapA (MA16) (from Example 15) was transferred to C. glutamicum DSM5715 strain. For selection of transconjugates, conjugation batches were plated on brain-heart medium with nalidixinic acid and kanamycin. The obtained transconjugates were incubated in 10 ml of brain-heart medium overnight. Then, aliquots were applied to the aliquots. • • • • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · on plates containing sucrose (cerebrospinal medium with 10% sucrose) to select for loss of sucrose sensitivity. Sucrose resistant clones were isolated and re-examined on agar plates containing chloramphenicol and kanamycin (brain-heart medium with 15 mg / l kanamycin and brain-heart medium with chloramphenicol (10 mg / l)).

Colonies were isolated which exhibited the following phenotype: sucrose resistant, kanamycin sensitive, chloramphenicol sensitive.

The insertion of the dapA gene fragment into the aecD gene was examined by the Southern-Blot method (Sambrook et al., Molecular Cloning: and Laboratory Manual (1989) Cold Spring Harbor Laboratory Press).

Example 17

Generation of C. glutamicum strains

DSM5715aecD :: dapA (MC20) / pEC7pyc.

:: DSM5715aecD dapA (MA16) / pEC7pyc.

DSM5715aecD :: dapA (MC20) / pEC7, DSM5715aecD :: dapA (MC16) / pEC7 and DSM5715 / pEC7

The pyc gene is as in Example 6 in the pEC7 vector. This plasmid pEC7pyc, as well as plasmid pEC7 with electroporation (Haynes 1989, FEMS Microbiology Letters 61: 329-334), were incorporated into strains DSM5715aecD:: dapA (MC20), DSM5715aecD :: dapA (MA16), and DSM5715 (from Example 16) to C. glutamicum DSM5715aecD :: dapA (MC20) / pEC7pyc was obtained,

DSM5715aecD :: dapA (MA16) / pEC7pyc.

DSM5715aecD: dapA (MC20) / pEC7, DSM5715aecD: dapA (MA16) / pEC7 and DSM5715 / pEC7. Transformant selection was performed on

• ·· • · ···· ·· · • · · • • · • · ·· • ·· • • ··· • · · ·· ·· • · · · · ·· ··

kanamycin brain-heart agar (25 mg / L). A plasmid DNA was isolated from each transformant and screened.

In this way, strains DSM5715aecD :: dapA (MC20) / pEC7pyc were obtained,

DSM5715aecD :: dapA (MA16) / pEC7pyc.

DSM5715aecD :: dapA (MC20) / pEC7.

DSM5715aecD :: dapA (MA16) / pEC7 and DSM5715 / pEC7.

Example 18

Lysine production using the strains generated in Example 17

Strains DSM5715aecD :: dapA (MC20) / pEC7pyc

DSM5715aecD: dapA (MA16) / pEC7pyc

DSM5715aecD :: dapA (MC20) / pEC7, DSM5715aecD :: dapA (MA16) / pEC7 and DSM5715 / pEC7 were cultured as described in Example 11 after pre-culture in CglII medium (Kase & Nakayama, Agricultural and Biological Chemistry 36 ( 9) 1611-1621 (1972)) in MM production medium. After 48 hours of incubation, the optical density at 660 nm and the concentration of L-lysine formed were determined.

Table 2 shows the result of the experiment.

Claims (22)

PATENT CLAIMS Coryneform L-lysine-producing bacteria with an enhanced pyc gene, in which additionally, individually or together, are amplified, in particular overexpressing genes selected from the group consisting of the dapA gene, the lysC gene, the lysE gene, and the dapB gene. Coryneform bacteria according to claim 1, in which the dapA gene and optionally the dapB gene are amplified, in particular overexpressed. Coryneform bacteria according to claim 1, in which the dapA gene, the dapB gene and the lysE gene are amplified, in particular overexpressed. ; The coryneform bacteria according to claim 1, which exhibit mutations of the MC20 or MA16 dapA promoter shown in SEQ-ID-No. 5 and SEQ-ID-No. 6th Coryneform bacteria according to claim 1, in which the dapB gene is additionally amplified, in particular overexpressing, which additionally comprises a 5'-region before the translation of this gene, shown in SEQ-ID-No. First 6. DNA which is replicable in coryneform bacteria, preferably recombinant, derived from Corynebacterium, which contains at least the nucleotide sequence which additionally comprises a 5 ' region before the translation of the dapB gene shown in SEQ-ID-No. First 7. The replicable DNA of claim 5 having the nucleotide sequence depicted in SEQ-ID-No. First · · 8. A process for the production of L-lysine by fermentation of coryneform bacteria with an enhanced pyc gene, characterized in that bacteria are used in which they are individually or co-amplified, in particular overexpressing the nucleotide sequences coding for genes selected from dapA, lysC, lysE and dapB. Method according to claim 8, characterized in that bacteria are used in which the dapA gene is amplified, in particular overexpresses, and optionally simultaneously the lysC gene. Method according to claim 8, characterized in that bacteria are used in which the dapA gene, the dapB gene and the lysE gene are simultaneously amplified, in particular overexpressing. Method according to claim 8, characterized in that a strain transformed with one or more plasmid vectors is used, said plasmid vector (s) carrying nucleotide sequences for one or more genes to be amplified. Method according to claim 8, characterized in that a strain transformed with one or more plasmid vectors is used and the plasmid vector carries nucleotide sequences which encode one or more genes selected from the c set comprising pyc, dapA, dapB and / or lysE genes. Method according to one or more of the preceding claims, characterized in that bacteria of the genus Corynebacterium glutamicum are used. • ·· • · · • ·· • · · · · • · • · • · · ···· • · · · • · · · • · · • · • · • · ·· · · · · · ·· · Process for the fermentative production of L-lysine according to one or more of the preceding claims, characterized in that the following steps are carried out: (a) fermentation of producing bacteria L-lysine, in which the described genes are amplified, b) concentrating L-lysine in the medium or in the cells of the bacteria; and c) isolating L-lysine. 15. Escherichia coli strain K-12 DH5α / pEC71ysE deposited as DSM 12871. 16. Escherichia coli strain K-12 DH5α / pEC7dapBlysE deposited as DSM 12875. Corynebacterium glutamicum strain DSM5715 / pJC23 deposited as DSM 12869. 18. Corynebacterium glutamicum strain DSM5715aecD :: dap (MA16) deposited as _DSM 12867. Corynebacterium glutamicum strain DSM5715aecD :: dapA (MC20) deposited as DSM 12868. Λ 20. Corynebacterium glutamicum 678 strain deposited as DSM 12866. 21. Replicable DNA with the nucleotide sequence of MC20 shown in SEQ-ID-No. 5th • ·· • · · · · • • ···· • · · • · · • ·· • • · · · • • • · · • · • • • · • ·· ·· · · · · · · · • 22. Replicable DNA with the nucleotide sequence of MA16 shown in SEQ-ID-No. 6th