SK362001A3 - Nucleotide sequences which code for the ptsh gene - Google Patents

Abstract

Isolated polynucleotide (I) from coryneform bacteria is a sequence at least 70% identical with a sequence that encodes an 89 amino acid (aa) polypeptide (S2) fully defined in the specification, a sequence that encodes a polypeptide at least 70% identical with (S2), its complement or fragment containing at least 15 consecutive nucleotides (nt) of (I). Independent claims are also included form the following: (1) vector (II) containing (I); (2) coryneform bacteria containing (II); and (3) production of L-aa by fermenting an appropriate coryneform in which the gene encoding component H of the phosphotransferase system is amplified, especially over expressed.

Description

Technical field

The present invention provides nucleotide sequences encoding ptsH and a method for fermentative production of L-amino acids, particularly L-lysine, using coryneform bacteria in which the ptsH gene is amplified.

BACKGROUND OF THE INVENTION

L-Amino acids, especially L-lysine, are used in human medicine and in the pharmaceutical industry, but especially in animal nutrition.

It is known that L-amino acids are produced by fermenting strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance, improvements are still being made to the production methods. Process improvements may even relate to fermentation technology measures such as mixing and supplying oxygen, or nutrient composition, such as sugar concentration during fermentation, or processing into a product form, for example by ion exchange chromatography, or the intrinsic utility of the microorganism itself.

Mutagenesis, selection and mutant selection methods are used to improve the utility properties of these microorganisms. In this way strains are obtained which are resistant to antimetabolites, such as the lysine analog of S- (2-aminoethyl) cysteine, or are auxotrophic with respect to regulatory important metabolites and produce L-lysine.

e b c * * * * * * * * * * * * * * * r

· 'C r

C r- r, r

For several years, recombinant DNA technology methods have also been used to improve L-amino acid-producing Corynebacterium strains by amplifying individual L-amino acid biosynthesis genes and investigating the effect on L-amino acid production. A review of this can be found, inter alia, in Kinoshita (Glutamic Acid Bacteria, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, United Kingdom, 1985, 115-142), Hilliger (BioTec 2 40-44 (1991), Eggeling (Amino Acids 6: 261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15: 73-103 (1995)) and Sahm et al. (Annuals of New York) Academy of Science 782, 2539 (1996)).

The inventors have set out to provide new measures for improved fermentative production of L-amino acids, in particular L-lysine.

L-Amino acids, especially L-lysine, are used in human medicine, in the pharmaceutical industry, and in particular in animal nutrition. There is therefore a general interest in providing new, improved methods for producing L-amino acids, especially L-lysine.

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

SUMMARY OF THE INVENTION

The present invention provides an isolated polynucleotide from coryneform bacteria comprising a polynucleotide sequence selected from the group consisting of:

a) a polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO. 2

b) a polynucleotide that encodes a polypeptide that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO. 2

(c) a polynucleotide that is complementary to polynucleotides (a) or (b); and

d) a polynucleotide comprising at least 15 contiguous nucleotides of the polynucleotide sequence a), b) or c).

The present invention also provides a polynucleotide, which is preferably recombinant DNA, replicable in coryneform bacteria.

The invention also provides a polynucleotide which is RNA.

The present invention also provides a polynucleotide, preferably a replicable DNA comprising:

(i) the nucleotide sequence shown in SEQ ID NO. 1 or i

(ii) at least one sequence that corresponds to sequence (i) in the region of degeneracy of the genetic code, or (iii) at least one sequence that hybridizes to a secretion.

· C f - rcro 'eecr Γ> C .. · r · RPS, _r sequences set complementary to sequence (i) or (ii), and optionally (iv) functionally neutral sense mutations in (i).

Further objects are a vector comprising one of the said nucleotides and coryneform bacteria serving as host cells containing the vector.

The present invention also provides polynucleotides consisting essentially of a polynucleotide sequence obtainable by screening by hybridization of an appropriate gene bank containing the complete gene with a polynucleotide sequence corresponding to SEQ ID NO. 1, using a probe comprising the sequence of said polynucleotide of SEQ ID NO. 1 or a fragment thereof, and isolating said DNA sequence.

The polynucleotide sequences of the invention are useful as hybridization probes for RNA, cDNA and DNA to isolate full-length cDNAs that encode the H component of the phosphotransferase system (ptsH), and to isolate such cDNAs or genes that exhibit high sequence similarity to the sequence gene for component H of the phosphotransferase system.

The polynucleotide sequences of the invention are further suitable as primers for the preparation of DNA genes that code for the H component of the phosphotransferase system by polymerase chain reaction (PCR).

Such oligonucleotides serving as probes or primers comprise at least 30, preferably at least 20, very particularly preferably

O r CC ·

Preferably, at least 15 consecutive nucleotides. Oligonucleotides of at least 40 or 50 nucleotides in length are also suitable.

“Isolated means separated from its natural environment.

"A polynucleotide generally refers to polyribonucleotides and polydeoxyribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.

The term "polypeptides" refers to peptides or proteins that contain two or more amino acids linked by peptide bonds.

Polypeptides of the invention include the polypeptide of SEQ ID NO. 2, further having the biological activity of the H component of the phosphotransferase system and also those which are at least 70% identical to the polypeptide of SEQ ID NO. 2, preferably at least at. 80%, and particularly those that exhibit at least 90% to 95% identity to the polypeptide of SEQ ID NO. 2 and said activity.

The invention further relates to a method for the fermentative production of L-amino acids, in particular L-lysine, using coryneform bacteria which in particular already produce L-amino acids and in which they amplify, in particular overexpress, the nucleotide sequences encoding the ptsH gene.

The term "enhancement" in this context describes the enhancement of the intracellular activity of one or more enzymes in a microorganism which are encoded by the DNA of interest, for example by increasing the copy number of the gene or genes, ο ι>

r. c r r t f e r o e * Γ.

c '· o c r c r' c r <r. c c r λ r a strong promoter or a gene that encodes a particular high-activity enzyme is used and these measures are combined, if necessary.

The microorganisms of the present invention can produce L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may be representatives of coryneform bacteria, in particular 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 L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are for example known wild-type strains.

Corynebacterium Corynebacterium Corynebacterium Corynebacterium, Brevibacterium Corynebacterium glutamicum ATCC13032 Brevibacterium Brevibacterium, acetoglutamicum ATCC15806, ATCC13870 acetoacidophilum, thermoaminogenes FERM BP-1539 melassecola ATCC17965, ATCC14067 flavum, lactofermentum ATCC13869 and ATCC14020 divaricatum and are produced mutants or strains producing L-lysine, such as e.g.

Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708

Brevi bacleri lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464, and

Β β r c e c c- <* r. λ r to r e f f>

c t 'f * CC f r e r. Γ o i '. r r c

Corynebacterium glutamicum DSM5715.

The inventors have succeeded in isolating a novel ptsH gene from C. glutamicum, coding for the H component of the phosphotransferase system.

To isolate the ptsH gene or other genes from C. glutamicum, a gene bank of this microorganism is first

E. coli. The establishment of gene banks is described in commonly known textbooks and manuals. Examples include Winnacker's textbook: Gene und Klone, Eine Einfuhrung 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). A well known gene bank is the gene bank of strain W3110 of E. coli K-12, founded by Kohara et al. (Cell 50, 495-508 (1987)) in λ-vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) disclose 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 the NM554 strain of E. coli K-12 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Bormann et al. (Molecular Microbiology 6 (3), 317-326 (1992)), 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 pUC9 (Viera ¹ et al., 1982, Gene, 19: 259) may also be used to generate a C. glutamicum gene bank in E. coli. Particularly suitable as hosts are those E. coli strains that are restriction and recombinantly defective, as exemplified by the DH5aMCR strain described by Grant et al (Proceedings of the National Academy of Sciences, USA, 87 (1990) 4645-). The long DNA fragments cloned with cosmids can then be rr subcloned into conventional sequencing vectors and then sequenced as described, for example, in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America 74: 5463-5467 (1977)).

In this way, a new C. glutamicum DNA sequence encoding ptsH was obtained, which is as SEQ ID NO. 1 of the present invention. Further, the amino acid sequence of the protein of interest was derived from the present DNA sequence using the methods described above. In SEQ ID NO. 2 shows the resulting amino acid sequence of the ptsH gene product.

The DNA coding sequences resulting from SEQ ID NO. 1 due to the degeneracy of the genetic code, they are also part of the invention. Furthermore, in the art world, conservative amino acid exchanges, such as glycine to alanine or aspartic acid to glutamic acid, in proteins are known as "sense mutations" that do not lead to any major change in protein activity, i. no. are functionally neutral. Furthermore, it is known that changes at the N-terminus and / or C-terminus of a protein cannot significantly limit its function or even stabilize it. Data to this can be found by one skilled in the art, among others, 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: 13211325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences that result, respectively, from SEQ ID NO. 2, and the DNA sequences encoding these amino acid sequences are also part of the invention.

r r, c

DNA sequences that hybridize to SEQ ID NO. 1 or parts of SEQ ID NO. Finally, DNA sequences which are prepared by polymerase chain reaction (PCR) using primers resulting from SEQ ID No. 1 are part of the invention. Such oligonucleotides are typically at least 15 nucleotides in length.

Guidelines for identifying DNA sequences by hybridization can be found by one skilled in the art, inter alia, in &quot; The DIG System Users Guide for Filter Hybridization by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). Instructions for amplifying DNA sequences by polymerase chain reaction (PCR) can be found by one skilled in the art, among others, in the Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

The inventors have found that coryneform bacteria produce L-amino acids, especially L-lysine, in an improved manner after overexpression of the ptsH gene.

To achieve overexpression, the copy number of the respective genes may be increased or the promoter and regulatory region or ribosome binding site that is upstream of the structural gene may be mutated. Same. in this way, expression cassettes that are inserted upstream of the structural gene act. By means of inducible promoters, it is additionally possible to increase expression during the fermentative production of L-amino acids. Expression measures also improve m-RNA durability measures. Further, the enzyme activity is also enhanced by preventing the degradation of the enzyme protein. The genes or gene constructs can either orrrrrc to Cr _y ^y - I t rr rr r r L. rcrrr exist in plasmids with different copy numbers or can be 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, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European Patent Specification EPS 0 472 869, in US Patent 4, 601,893, in Schwarzer and Pihler (Bio / Technology 9, 84-87 (1991)), in Reinscheid et al. . (Applied and Environmental Microbiology 60, 126-132 (1994)), in 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)), in Makrides (Microbiological Reviews 60: 512 -538 (1996)) and in well-known textbooks of genetics and molecular biology.

For example, the ptsH gene of the invention was overexpressed with plasmids.

Suitable plasmids are those which replicate in coryneformic bacteria. Numerous known plasmid vectors such as pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 ( Sonnen et al., Gene 107: 69-74 (1991)) are based on cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors may also be used, such as those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al., FEMS et al.).

P Γ r! c.

O © C Γ r <? p f c r y r. c C ..

Cl

Q

Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891).

Further suitable are plasmid vectors by which a method of gene amplification by integration into a chromosome can be used, as described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the homthrB operon. In this method, the entire gene is cloned into a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum. Suitable vectors are, for example, pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob 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 Patent 5,487,993), pCR ® Blunt (Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516). The plasmid vector containing the gene to be amplified is then transferred by conjugation or transformation into the desired C. glutamicum strain. The method of conjugation is described, for example, in Schäfer et al., (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods are described, for example, in 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 said gene.

A further object of the invention is therefore a process for the fermentative production of L-amino acids, in particular L-lysine, in which r r r r r r is used.

r. y r y r <- n <p * Γ C r c

C 'r. a strain transformed with a plasmid vector, and a plasmid vector that carries the nucleotide sequence of the gene encoding the H component of the phosphotransferase system.

In addition to the production of L-amino acids, especially L-lysine, it may be advantageous to enhance other genes for the biosynthetic pathway of the desired L-amino acid in addition to the ptsH gene to overexpress one or more enzymes of the given biosynthetic pathway, glycolysis, anaplerotics or L-amino acid transfer.

Thus, for example, one or more genes selected from the group consisting of the dapA gene encoding dihydrodipicolinate synthase (EP-B 0 197 335), the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal) can be overexpressed simultaneously for L-lysine production. of Bacteriology 174

6076-6086), the tpi gene encoding triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), the pgk gene encoding 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086). ptsM gene encoding the M component of the phosphoenolpyruvate-sugarphosphotransferase system (ptsM) (Lee et al. (1994), FEMS Microbiology Letters 1-2, 137-145), • the pyc gene encoding pyruvate carboxylase (DE-A-198 31 609) and III C Λ.

r i r r c o r t r.

The lysE gene encoding lysine transfer (DE-A-195 48 222).

Furthermore, it may be advantageous for the production of L-amino acids, especially L-lysine, in addition to the ptsH gene, to simultaneously attenuate the pck gene encoding phosphoenolpyruvate carboxykinase (DE).

199 50 409.1, DSM 13047) and / or the pgi gene encoding glucose-6-phosphate isomerase (US 09 / 396,478, DSM 12969) the poxB gene encoding pyruvate oxidase (DE 19846499.1, DSM 13114).

Furthermore, for the production of L-amino acids, especially L-lysine, in addition to overexpression of the ptsH gene, it may be advantageous to avoid undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek. eds.), Academic Press, London, UK, 1982).

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

rrrreerrr © c _r cerfncpr <<'rrc' ~ t '- r • --r Γ f? The culture medium used must suitably suit the requirements of the strains. 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 DC, 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 and ammonium nitrate. The nitrogen sources may be used singly or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or potassium hydrogen phosphate or the corresponding sodium salts can be used as phosphorus sources. 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 Laminic acids and vitamins may be used in addition to the above-mentioned substances. In addition, suitable precursors may be added to the culture medium. The raw materials used can be added to the culture as a single batch or added in a suitable manner during the cultivation.

Basically, basic compounds such as sodium hydroxide, potassium hydroxide, ammonium and potassium hydroxide are suitably used to control the pH of the culture.

O C c r c c c c c c r c C c.

and optionally acidic compounds such as phosphoric acid or sulfuric acid. 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.

A further object of the invention is therefore a process for the fermentative production of L-amino acids, in particular L-lysine, in which the following steps are carried out:

(a) fermentation of coryneform producing bacteria

The L-amino acid in which it is amplified, in particular overexpresses at least the ptsH gene encoding the H component of the phosphotransferase system,

b) concentrating the L-amino acid in the medium or in the cells of the bacteria; and

c) isolating the L-amino acid.

L-lysine analysis can be performed by anion exchange chromatography followed by ninhydrin derivatization as described by Spackman et al. (Analytical Chemistry, 30, (1958) 1190).

The process according to the invention serves for the fermentative production of L-amino acids, in particular L-lysine.

Examples: (. C. <* ·

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

Example 1

Generation of genomic cosmid gene bank from Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described in Tauch et al., (1995, Plasmid 33: 168-179), and partially digested with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, Code No. 27-0913-02). DNA fragments were dephosphorylated using shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, SAP product description, Code no. 1758250). SuperCosl cosmid vector DNA (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164), obtained from Stratagene (La Jolla, USA, description of SuperCosl Cosmid Vector Kit, Code no. 251301), was digested with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, Code no. 27-0948-02) and also dephosphorylated with shrimp alkaline phosphatase. Subsequently, the cosmid DNA was digested with BamHI restriction enzyme (Amersham Pharmacia, Freiburg, Germany, BamHI product description, Code no. 27-0868-04). The cosmid DNA treated in this way was mixed with the treated ATCC13032 DNA and treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, Code no. 27-0870-04). The ligation mixture was then packaged into phage using Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). For infection of E. coli c. F

e c r tO

NM554 (Raleigh et al. 1988, Nucleic Acid Research 16: 15631575) cells were captured in 10mM MgSO ₄ and mixed with an aliquot of phage suspension. Infection and titration of the cosmid bank were performed as described in Sambrook et al. (1989,

Molecular Cloning: and Laboratory Manual, Cold Spring Harbor), where cells were plated on LB agar (Lennox, 1955, Virology, 1: 190) with 100 µg / ml ampicillin. After incubation overnight at 37 ° C, individual recombinant clones were selected.

Example 2

Isolation and sequencing of the ptsH gene

Single colony cosmid DNA was isolated using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and partially digested with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913). -02). DNA fragments were dephosphorylated using shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, SAP Product Description, Product No. 1758250). After separation by gel electrophoresis, cosmid plasmids ranging in size from 1,500 to 2,000 bp were isolated using the QiaExII gel Extraction Kit (Product No. 20021,

Qiagen, Hilden, Germany). The DNA sequence vector pZero-1, obtained from Invitrogen (Groningen, The Netherlands, product description Zero Background Cloning Kit, product # K2500-01), was digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, product no. 27-086804). Ligation of cosmid fragments into the pZero-1 sequence vector was performed as described in Sambrook et al. (Molecular Cloning: A laboratory manual, Cold Spring Harbor), wherein a mixture of DNA with T4 ligase (Pharmacia Biotech,

Λ (_ t; úr r (· e r f I * <. 'R é β t

O f r o r r · n / ·. r t or>

r. C

C r GC

Freiburg, Germany) was incubated overnight. This ligation mixture was subsequently electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123: 343-7) into E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649) and plated on LB agar (Lennox, 1955, Virology, 1: 190) with 50 pg / ml zeocin. Plasmid preparation of recombinant clones was performed with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Sequencing was performed by the dideoxy chain termination method of Sanger et al. (1997, Proceedings of the National of Sciences)

U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). The RR dRhodamine Terminator Cycle Sequencing Kit from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) was used. Gel electrophoresis and sequencing reaction analysis was performed in a Rotiphorese NF Acrylamide / Bisacrylamide (29: 1) gel (Product No. A124.1, Roth, Karslruhe, Germany) with a sequencing apparatus "ABI prism 377 from PE Applied Biosystems (Weiterstadt, Germany).

The obtained raw sequence data was then processed by the processor using the Staden (1986, Nucleic Acids Research, 14: 217-231) Version 97-0 software package. The individual sequences of the pZerol derivatives were assembled into one contiguous whole. Computer analysis of coding regions was performed using the XNIP program (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analyzes were performed using BLAST search programs (Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402) against a non-redundant database from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).

• t

r. r t e e r r e r b * i ·

C ft l f ’

The nucleotide sequence obtained is shown in SEQ ID NO 1. Nucleotide sequence analysis yielded an open reading frame of 267 base pairs, which was designated as the ptsH gene. The ptsH gene encodes a protein of 89 amino acids.

Example 3

Preparation of shuttle vector pEC-K18mob2ptsHexp for amplification of ptsH gene in C.glutamicum

3.1 Cloning of the ptsH gene into the pCR®Blunt II vector

Chromosomal DNA was isolated from ATCC 13032 by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). Based on the sequence of the ptsH gene known from Example 2 for C. glutamicum, the following oligonucleotides for the polymerase chain reaction were selected:

PtsHexpl:

5 'ACC GGT GCA ATC TCC AT 3' ptsHexp2:

5 'TTT CAG CGT CAA GGT CC 3'

The shown primers were synthesized by ARK Scientific GmbH Biosystems (Darmstadt, Germany) and the PCR reaction was performed according to the standard PCR method of Innis et al. (PCR protocols. A guide to Methods and Applications, 1990, Academic Press) with Pwo polymerase from Roche Diagnostics GmbH (Mannheim, Germany). Using this polymerase reaction, the primers allowed the amplification of the 686 bp DNA fragment carrying the ptsH gene with a potential promoter region. The DNA sequence of the amplified DNA fragment was analyzed by sequencing.

C r ·

C r r r r r r r r r r r, (. · O

The amplified DNA fragment was ligated using the Zero Blunt ™ Kit from Invitrogen Corporation (Carlsbad, CA, USA; catalog number K2700-20) to the pCR ® Blunt II vector (Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993) )).

The E. coli TOPIO strain was then electroporated with the ligation mixture (Hanahan, in: DNA Cloning. A Practical Approach, Vol. I, IRL-Press, Oxford, Washington DC, USA, 1985). The plasmid-bearing cells were selected by plating the transformation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) supplemented with 25 mg / L. kanamycin. Plasmid DNA was isolated from the transformant using the QIAprep Spin Miniprep Kit from Qiagen and analyzed by restriction enzyme EcoRI followed by agarose gel electrophoresis (0.8%). The plasmid was called pCRB1ptsHexp and is shown in Figure 1.

3.2 Preparation of the shuttle vector pEC-K18mob2 from E. coli - C. glutamicum

The E. coli-C. glutamicum shuttle vector was constructed according to the prior art. The vector comprises the rep plasmid region of the rep plasmid pGA1 comprising a replication effector per (US-A-5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the aph (3 ') - IIa gene of transposon Tn5 conferring kanamycin resistance. (Beck et al., Gene 19, 327-336 (1982)), the oriV replication region of plasmid pMB1 (Sutcliffe, Cold Spring Harbor Symposium on Quntitative Biology 43, 77-90 (1979)), the lacZa gene fragment comprising the lac promoter and multiple cloning site (mcs) (Norrander, JM et al., Gene 26, 101-106 (1983)) and

mob region of plasmid RP4 (Simon et al. _of Biol / Technology 1: 784-791 (1983)). The constructed vector was transformed into an E. coli strain DH5ctmcr (Hanahan, in: DNA Cloning. A Practical Approach, Vol. I, IRL-Press, Oxford, Washington DC,

USA). The plasmid-bearing cells were selected by plating the transformation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) supplemented with 25 mg / L kanamycin. Plasmid DNA was isolated from the transformant using the QIAprep Spin Miniprep Kit from Qiagen and analyzed by restriction enzyme EcoRI and HindIII and then agarose gel electrophoresis (0.8%). The plasmid was called pEC-K18mob2 and is shown in Figure 2.

The following microorganism was deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty:

In strain C. glutamicum DSM 5715 / pEC-K18mob2 as DSM 13245.

3.3 Cloning of ptsH into shuttle vector pEC-K18mob2 from E. coli - C. glutamicum

To clone the ptsH gene into the shuttle vector pECK18mob2 from E. coli - C. glutamicum described in Example 3.2, plasmid DNA from pEC-K18mob2 was completely digested with restriction endonucleases KpnI and XbaI and treated with alkaline phosphatase (Alkaline phosphatase, Mannheim, Roche Diagnostics GmbH). .

The vector pCRB1-ptsHexp was isolated from Escherichia coli

Top10 and completely digested with restriction endonucleases KpnI and

XbaI and a 0.8% agarose gel (QIAquick Gel Extraction Kit from Qiagen, Hilden, Germany) was purified with a 788 bp fragment with the e r o r e o f i c e r i o e e.

The ptsH gene is the yr yr yr yr yr yr yr yr yr. The ptsH gene fragment was then ligated with the pEC-K18mob2 vector (T4-ligase, Roche Diagnostics GmbH, Mannheim; Germany). The ligation mixture was transformed into an E. coli strain DH5? TMcr (Hanahan, in: DNA Cloning. A Practical Approach, Vol. I, IRL-Press, Oxford, Washington DC, USA). The plasmid-bearing cells were selected by plating the transformation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) supplemented with 25 mg / L kanamycin. Plasmid DNA was isolated from the transformant using QIAprep Spin Miniprep Kit from Qiagen and analyzed by restriction enzyme EcoRI treatment and then agarose gel electrophoresis. The plasmid was called pEC-K18mob2ptsHexp and is shown in Figure 3.

The strain was named E. coli DH5amcr / pEC-K18mob2ptsHexp and deposited as pure culture on November 28, 2000 at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty as DSM 13878.

Example 4

Transformation of strain DSM5715 with plasmid pEC-K18mob2ptsHexp

The DSM5715 strain was transformed with the plasmid pEC-K18mob2ptsHexp using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53: 299-303 (1989)). Transformants were selected on LBHIS agar composed of 18.5 g / l brain-heart infusion broth, 0.5 M sorbitol, 5 g / l Bacto-tryptone, 2.5 g / l Bacto-yeast extract, 5 g / l. 1 NaCl and 18 g / l Bacto-agar supplemented • *

t e e c c e e r c

mg / l kanamycin. Incubation was performed for 2 days at 33 ° C.

Plasmid DNA was isolated from the transformant by a conventional method (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), digested with restriction endonuclease EcoRI, and the plasmid was then analyzed by agarose gel electrophoresis. The strain obtained was named DSM5715 / pEC-K18mob2ptsHexp.

Example 5

Preparation of lysine

The C. glutamicum DSM5715 / pEC-K18mob2ptsHexp strain obtained in Example 4 was cultured in a nutrient medium suitable for lysine production and the lysine content was determined in the culture supernatant.

For this purpose, the strain was initially incubated for 24 hours at 33 ° C on an agar plate with a suitable antibiotic (brain-heart agar with kanamycin (25 mg / L)). Starting from this agar plate, a preculture (10 ml of medium in a 100 ml Erlenmeyer flask) was seeded. The medium used for the preculture was solid medium Cg III.

Medium Cg III 2.5 g / l 10 g / l 10 g / 1% (w / v)

NaCl

Bacto-peptone Bacto-yeast extract

Glucose (separately autoclaved) The pH was adjusted to 7.4.

To this was added kanamycin (25 mg / L). The preculture was incubated for 16 hours at 33 ° C at 240 rpm in a shaker. The main culture was inoculated with the preculture so that the initial optical density (660 nm) of the main culture was 0.05. MM medium was used for the main culture.

MM Medium:

CSL (Corn Steep Liquor) 5 g / l MOPS (morpholinopropane- 20 g / l sulfonic acid) Glucose (separately autoclaved) 100 g / l (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 MnSO ₄ .H ₂ O 5.0 mg / L Biotin (filter sterilized) 0.3 mg / l Thiamine.HCl (filter sterilized) 0.2 mg / l L-Leucine (filter sterilized) θ · 1 g / l CaCO ₃ 25 g / l

p e r> rc c t t: (c c r nc c © cc Λ r

C rr y r C r y r -i

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

Cultivation was performed in 10 ml volumes in a 100 ml baffled Erlenmeyer flask. Kanamycin (25 mg / L) was added. The cultivation was carried out at 33 ° C and 80% humidity.

. to 72 hours

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

The result of the experiment is shown in Table 1.

Table 1

tribe Optical density (660 nm) Lysine-HCl g / l DSM5715 / pEC-K18mob2 (48 hours) ^11/4 14.14 DSM5715 / pEC-Kl8mob2ptsHexp (48 hours) 10.7 15.98 DSM5715 / pEC-K18mob2mob2 (72 hours) 10.1 15.24 DSM5715 / pEC-K18mob2ptsHexp (72 hours) 10, 0 17.13

e c e e • e σ c c · o r r c r

BRIEF DESCRIPTION OF THE DRAWINGS

The following images are attached:

Figure 1: Map of plasmid pCRB1-ptsHexp

Figure 2: Map of plasmid pEC-K18mob2

Figure 3: Map of plasmid pEC-K18mob2ptsHexp

Abbreviations and names used have the following meanings:

Kan:

Zeocin: pts

ColEl: laZ-alpha: laZ-alpha 'per: oriV: rep:

EcoRI: HindIII: KpnI:

XbaI:

kanamycin resistance gene zeocin resistance gene ptsH gene from C. glutamicum plasmid origin of replication CelEl lacZ gene fragment from E. coli: lacZ gene fragment fragment from E. coli copy number gene from pGAl origin from pMB1 identical to ColE1 plasmid encoded replication region from plasmid C. glutamicum restriction enzyme cleavage site EcoRI restriction enzyme cleavage site Hindlll restriction enzyme cleavage site KpnI restriction enzyme cleavage site XbaI

Claims (17)

PATENT CLAIMS An isolated polynucleotide from coryneform bacteria comprising a polynucleotide sequence selected from the group consisting of: a) a polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO. 2 b) a polynucleotide that encodes a polypeptide that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO. 2 c) a polynucleotide that is complementary to the polynucleotides (a) or (b), and d) a polynucleotide comprising at least 15 consecutive nucleotides into the polynucleotide sequence a), b) or c). The polynucleotide of claim 1, wherein the polynucleotide is preferably recombinant DNA replicable in coryneform bacteria. The polynucleotide of claim 1, wherein the polynucleotide is RNA. 4. The replicable DNA of claim 2 comprising: (i) the nucleotide sequence shown in SEQ ID NO. Or (ii) at least one sequence that corresponds to sequence (i) in the region of degeneracy of the genetic code, or (iii) at least one sequence that hybridizes to sec30 e · λ r P · r V * f Γ C C r C c r e r e r p r r r r r r r c r r c c r by a complement complementary to sequence (i) or (ii), and optionally (iv) functionally neutral mutations in sense in (i). The polynucleotide sequence of claim 2, which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO. Second A vector comprising the polynucleotide sequence of claim 1. A coryneform bacterium comprising the vector of claim 1 6th 9. A method for the fermentative production of L-amino acids, characterized in that the following steps are performed: (a) fermentation of coryneform producing bacteria The L-amino acid in which it is amplified, in particular overexpresses at least the ptsH gene encoding the H component of the phosphotransferase system, b) concentrating the L-amino acid in the medium or in the cells of the bacteria; and c) isolating the L-amino acid. The method according to claim 9, characterized in that bacteria are used in which additional genes for the biosynthetic pathway of the desired L-amino acid are additionally amplified. .CC C ------ Γ -ľ · Method according to claim 9, characterized in that bacteria are used in which at least partially the metabolic pathways which reduce the formation of L-amino acids are eliminated. Method according to claim 9, characterized in that a strain transformed with a plasmid vector is used and the plasmid vector carries the nucleotide sequence of the gene encoding the H component of the phosphotransferase system. Method according to one or more of claims 9 to 12, characterized in that coryneform bacteria which produce L-lysine are used. Method according to claim 10, characterized in that one or more genes selected from the group consisting of the dapA gene encoding dihydrodipicolinate synthase, the pyc gene encoding pyruvate carboxylase, the tpi gene encoding triosaphosphate isomerase, the gap-3-encoding glyde gene, -phosphate dehydrogenase, the ptsM gene encoding component M of the phosphoenolpyruvate-sugar phosphor transferase system (ptsM), the pgk gene encoding 3-phosphoglycerate kinase, and the lysE gene encoding lysine transfer. CO r r f 'β r o C C C c r r o r 15. The method of claim 11 wherein bacteria are fermented to produce L-lysine, wherein one or more genes selected from the group consisting of the pck gene coding for phosphoenolpyruvate carboxykinase, the pgi gene coding for glucose-6-phosphate isomerase, the poxB gene are fermented. encoding pyruvate oxidase. Method according to one or more of the preceding claims, characterized in that microorganisms of the genus Corynebacterium glutamicum are used. Use of the polynucleotide sequences according to claim 1 as primers for the preparation of DNA genes which encode the ptsH gene product by a polymerase chain reaction. Use of the polynucleotide sequences of claim 1 as hybridization probes.