NUCLEOTIDE SEQUENCES WHICH CODE FOR THE MENE GENE

Abstract: The invention relates to an isolated polynucleotide comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70 % to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70 % to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b), and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c), and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the menE gene coding for an O-succinylbenzoic acid C0A ligase is present in attenuated form, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

Description: Nucleotide sequences which code for the menΞ gene Field of the Invention The invention provides nucleotide sequences from coryneform bacteria which code for the menE gene and a process for the fermentative preparation of amino acids using bacteria in which the menE gene is attenuated. Prior Art L-Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition. It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself. Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce amino acids are obtained in this manner. Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production. Object of the Invention The inventors had the object of providing new measures for improved fermentative preparation of amino acids. Summary of the Invention Where L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L- asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L- isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L- histidine, L-lysine, L-tryptophan and L-arginine. L-Lysine is particularly preferred. When L-lysine or lysine are mentioned in the following, not only the bases but also the salts, such as e.g. lysine monohydrochloride or lysine sulfate, are meant by this. The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the menE gene, chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b) , and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c), the polypeptide preferably having the activity of 0- succinylbenzoic acid CoA ligase. The invention also provides the above-mentioned polynucleotide, this preferably being a DNA which is capable of replication, comprising: (i) the nucleotide sequence, shown in SEQ ID No.l, or (ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or (iii) at least one sequence which hybridizes with the sequences complementary to sequences (i) or (ii) , and optionally (iv) sense mutations of neutral function in (i) . The invention also provides: a polynucleotide, in particular DNA, which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No.l; a polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No . 2 ; a vector containing parts of the polynucleotide according to the invention, but at least 15 successive nucleotides of the sequence claimed, and coryneform bacteria in which the menE gene is attenuated, in particular by an insertion or deletion. The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library of a coryneform bacterium, which comprises the complete gene or parts thereof, with a probe which comprises the sequence of the polynucleotide according to the invention according to SEQ ID No.l or a fragment thereof, and isolation of the polynucleotide sequence mentioned. Detailed Description of the Invention Polynucleotides which comprise the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids or polynucleotides or genes which code for 0- succinylbenzoic acid CoA ligase or to isolate those nucleic acids or polynucleotides or genes which have a high similarity with the sequence the menE gene. They are also suitable for incorporation into so-called "arrays", "micro arrays" or "DNA chips" in order to detect and determine the corresponding polynucleotides. Polynucleotides which comprise the sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for O-succinylbenzoic acid CoA ligase can be prepared by the polymerase chain reaction (PCR) . Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides which have a length of at least 40 or 50 nucleotides are also suitable. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are optionally also suitable. "Isolated" means separated out of its natural environment. "Polynucleotide" in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA. The polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom. "Polypeptides" are understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds. The polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those with the biological activity of O-succinylbenzoic acid CoA ligase, and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polypeptide according to SEQ ID No. 2 and have the activity mentioned. The invention furthermore relates to a process for the fermentative preparation of amino acids chosen from the group consisting of L-asparagine, L-threonine, L-serine, L- glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan and L- arginine using coryneform bacteria which in particular already produce amino acids and in which the nucleotide sequences which code for the menE gene are attenuated, in particular eliminated or expressed at a low level. ω cv> t\) I- 1 o cn O n O cπ ιQ TJ CΛ TJ ιQ 3 h- 1 OJ d hi l- > φ d hi H- O d 3 rt rt ft On rt ft OJ H- OJ c & ) H- C ω trJ O O O O O 3 Ω tr Ω 3 O H hi hi 0 o 0 0 o H- d r- 1 Φ H- 3 Φ CD Φ hi hi hi H hi Ω H Φ Ω Φ <! <! <! •< ■ < ^ •< ^ d OJ t- 1 d On H- P- H- 3 3 3 3 3 hi ω 1 tr tr tr 3 CD CD φ φ Φ rt 0 ) *« H- ft> cu CD tr tr tr tr tr -< O hi 3 3 Ω o Ω OJ OJ OJ OJ J Hi J H- rt et rt =s Ω Ω Ω Ω Ω OJ H- 3 T CD Φ CD r+ f+ r+ r+ rt σ hi rt 3 O H- 0 ) l-i H hi CD CD Φ Φ Φ Φ tr O Ω hi H- H- H- hi hi H H H φ 0 ) t rt d d G H- H- H- H- H- H- 0 Ω H- 3 3 3 d d d d d 3 ω Hi H- H- Ω 3 3 3 3 3 TJ P- co d h- 1 Hi TJ φ ft 03 h- 1 H- J H rt 3 0J 0> ιQ OJ Ω t • ? 0J <! Ω OJ tr CD Ω Ω H hi H- Φ D hi QJ et Φ H Φ φ c ft φ 0 i-i O G hi OJ et r+ rt H- Cfl LQ s: ft H- Hi 3 3 co O o OJ Ω Φ 3 " o CD o Cfl OJ iQ 3 d o 3 Φ OJ hi > OJ CD Ω - 1 H- O d 0 ) rt 3 H3 3 Ω H- d Ω 0 ) hi O 3 O d φ O H- O r+ d hi ^< o T 3 3 O 3 - o 0> 3 3 o 3 Φ et I- 1 o PU Ό 3 rt φ O iQ Ω ! d J-» Q 3 * H- > 3 J tr hi H- ι-3 3 o CD > H- Ω H3 Φ OJ ^ Φ Φ O σ> 3 ι-3 H d O Ω 3 •xj CO o -j CD O d 3 π pr rt Φ TJ h-» >-3 CO O 3 3 Φ σ Φ O J-. n H 1 ω o H 0 ) hi O o o **] -J > ι-3 o s H- Ω rt t-j tV> P. <£> 3 o ω 3 d rt O O ω ^ Sd σ. Ω π IV) 3 Φ 3 CO S Cπ O H» s: hi Hi Φ cn Cn H- ιQ H- o U3 Gd OJ CO σ H 1 H d hi 0J j CO o d 3 Ω OJ 1 ^1 σ, 1 rt H- ft 3 O rt OJ rt Φ Q. ( l ■ 3 P- CO hi ω TJ H- ti P- D Φ Ω ) d d 3 CO 3 H- rt H hi . — . H- 0 ) o rt W- • • < 3 cn ft O and L-amino acid-producing mutants or strains prepared therefrom. The new menE gene from C. glutainicum which codes for the enzyme O-succinylbenzoic acid CoA ligase (EC 6.2.1.26) has been isolated. To isolate the menE gene or also other genes of C. glutamicum, a gene library of this microorganism is first set up in Escherichia coli (E. coli) . The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Eine Einfϋhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990), or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known gene library is that of the E. coli K-12 strain W3110 set up in λ vectors by Kohara et al . (Cell 50, 495 -508 (1987)). Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bόrmann et al. (Molecular Microbiology 6(3), 317-326)) (1992)) in turn describe a gene library of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, 1980, Gene 11, 291-298) . To prepare a gene library of C. glutamicum in E. coli it is also possible to use plasmids such as pBR322 (Bolivar, 1979, Life Sciences, 25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination-defective, such as, for example, the strain DH5αmcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) . The long DNA fragments cloned with the aid of cosmids or other λ vectors can then in turn be subcloned and subsequently sequenced in the usual vectors which are suitable for DNA sequencing, such as is described e. g. by Sanger et al . (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977) . The resulting DNA sequences can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217- 232(1986)), that of Marck (Nucleic Acids Research 16, 1829- 1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)). The new DNA sequence of C. glutamicum which codes for the menE gene and which, as SEQ ID No. 1, is a constituent of the present invention has been found. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the menE gene product is shown in SEQ ID No. 2. Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also a constituent of the invention. In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Conservative amino acid exchanges, such as e.g. exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, are furthermore known among experts as "sense mutations" which do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. It is furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of CO o IV) IV) cπ o Cπ o Cπ O cπ CO IV) IV) h- n o cπ o cπ o cπ OJ sQ Cfl Ω ι-3 o Ω 3 Hi sQ OJ H > o o rt J H o co tr O 3 " T CO H cπ S CΛ rt OJ hi CO Ω Φ μ- d 3 " TJ OJ φ O Φ Ω PI ¬ pr 3 " μ- 3 3 O ^ rt • hi o hi O o OJ ■ O Ω Φ Φ rt 3 Q H Φ rt 3 3 s μ- OJ HI μ- Φ α. fl σ 1 tr Φ tr O o\° OJ it, 0 3 CO 3 " 3 Λ μ- Φ 3 rt μ- M OJ Φ S ' O ιQ ft in Ω ii TJ hi tr iQ Φ O 3 ft IV) μ- O d <! J d hi o tr Ω Cfl J Φ hi o Φ o hi » OJ μ- Cfl μ- Φ o 3 hi 3- Φ X Φ <! Φ OJ Φ H hi φ 3 Φ sQ tr Cfl 3 3 ? Hi d h- 1 Qπ Qπ hi Φ 1 Φ 3 <! Φ 3 rt X μ- On OJ Φ μ- μ- μ- • d TJ Ω s h- 1 μ- o μ- Φ 3 PI ¬ μ- CΛ Φ α Ω 0 TJ Cfl 3 d h- 1 hi 3 φ 3 tr cn Ω Φ PI ¬ rt J Φ N Hi N 3 OJ . rt S' CΛ 3 CΛ Qπ φ hi hi rt iQ Ω Φ Φ <! φ Ω h- 1 hi S' μ- 3 C OJ OJ TJ rt Cfl Φ CO CΛ O tr Φ hi rt ^ φ OJ φ 3' PI φ rt Φ o 3 rt OJ rt H o Φ ■ <: o sQ cn d O μ- d o 3 3 φ o 3 3" pr μ- T μ- O h- 1 S CO n S hi J Hi Hi Φ cn Ω hi O tr Ω hi OJ ii rt TJ Cfl Φ μ- O TJ 0 • Φ tr OJ J - 3 o 3 μ- rt 3 Φ Φ 3 μ- Hi μ 1 μ- μ- O μ- rt 3 hi 3 φ J μ- H tr 3 Cfl π hi OJ PI ¬ Φ O d tr π PI ¬ 3 o < SD π 3 - Hi 3 co O Qπ Cfl Ω rt Φ 3 cn S ' Cfl 3 hi ^ μ- Ω S' OJ TJ d Φ CO Φ et ^ o • ^ J X rt rt tr μ- tr Ω O Φ 3 " Φ ^ Φ 3 o O Φ rt hj 3 hi J-> Φ 3 hi . — . hi • Φ a Ω 3 φ d TJ P μ- OJ CO iQ 3 hi r o Qπ H — cn ii φ Q φ Φ 3 0 ) o OJ O iQ μ- μ- rt OJ 3 TJ O hi Φ iQ tr Ω Φ <! OJ ■ ii OJ Φ h- 1 TJ 3 o ϋ 3 3 μ- iQ li TJ Φ O 3 Φ μ- φ OJ 3 Φ et Q OJ 3 OJ J 3 hi sQ O Φ o\° φ Ω Φ Φ o TJ o φ ^» Hi Φ 3 3 rt d Qπ tr " * 3 PI ¬ μ- 1 Cfl TJ 3 . tr 1 hi tr Φ CO 3 - 1 d tr hi rt Φ Φ μ- J OJ OJ CL S' μ- Φ H OJ rt OJ Φ rt 3 rt Q Hi OJ Φ 3 Φ OJ Hi sQ μ- On 3 rt 3 rt tc Φ OJ μ- 3 α J IV) rt O Hi rt OJ Φ o CL • rt O Φ Ω Φ • μ- •< μ- OJ Φ μ- cn Ω h H μ- o d μ- O hi σ ii li h- 1 φ O hi 3 Ps 3 rt o 3 Ω μ- 3 μ- 3 * μ- ^« Q 3 n ii Cfl CD hi J - > 3 > tr H CΛ hj Φ 3 TJ OJ μ- 3 3 O C cn μ- OJ Ω J • Φ o cπ rt μ- OJ ^ ^ d cn Φ O hi rt Ω ^ Φ ii φ 3 • • 3 μ- OJ O IS σ μ- 1 o\° Φ μ- co 3 μ- CO cn Ω . J>^ Φ C Φ Φ hi •< r- φ 3 rt J OJ * CO OJ X O S' • d l- 1 rt S' OJ X μ- Cfl On TJ Φ 3 tr s: > rt μ- rt CO Φ d rt rt μ- OJ 3 φ ^« rt tr o hi TJ 3 TJ H) CO hi μ- OJ Φ φ rt tr hi o OJ ■ <: hi Φ Φ Qπ 3 CL Φ cn s: μ- tr hi TJ hj μ- μ- o rt Hi Hi hj o <v φ Φ 3 r- o pi ¬ T π φ T rt • - 1 ii Φ Φ 3 ><: O Φ Ω O h3 T 3- rt O iQ 3 hi OJ O tr 3 s' s: 3 O SD iQ hi iQ tr 3 Φ Cfl OJ 3 3' φ Φ Φ hi OJ tr Ω o iQ « < Φ μ- tr rt φ H CD tc d μ- Φ hi o ^ CO rt Φ hi H hi 3 OJ Ω J rt Hi d σ PI ¬ hi co ■ < μ- ^ * o rt Cπ ■ <: Φ 3 3 μ- rt μ- μ- Ω rt Ω 3 rt 3 μ- φ S' φ Ω • — - tr 3 sQ rt CL Φ hj o O J rt μ- rt OJ tr φ CL μ- o o Hi Φ μ- μ- J OJ μ- ϋ rt CO hi Φ 3 3 3 φ tr rt OJ hi Ω σ 3 3 3 hi 3 hh 3 rt X co J μ- rt Ω cn TJ • o Cfl φ rt Ω 3 Ω OJ o > O O 3 CO Ii Ω CL " tr O OJ hi . — . rt Φ rt J hi H o ^ -. 3 OJ rt 0 li rt h- > CΛ 0 rt μ- φ 3 hi Φ CΛ 3 OJ 3 0 Φ 3 Φ 3 OJ pr *υ Cfl hi hi 3 Φ O Φ CΛ TJ Φ CO o CL Φ OJ O μ- d p φ Hi X d H *< tr > o φ PJ p d OJ J O rt 3 OJ • O μ- rr O ιQ rt Φ J TJ OJ μ- OJ TJ tr 2 μQ C o Φ Ω cπ CO PI ¬ CO μ- TJ rt cn OJ rt - 1 rr O 3 J O rt h rt OJ 3 TJ • d O Ω et rt ' o μ- S ' r o Φ μ- X h- 1 μ- Φ Φ hi J rt TJ d 3" Φ μ- H 1 H Φ CO 3 1 μ- 0 3 Φ TJ 3 hi 0 rt O CO 3 H μ- H hi Φ CO O TJ CO O n Ω 3 Cn Φ μ- O O u3 ~J o OJ OJ 3 CΛ 3 CO d ιQ o OJ φ cn 3 hi SD πj J 0 ) 0J Ω Cπ 3 3 Cfl o H h- 1 rt CΛ Ω CΛ OJ Φ CO 3 Ω O Φ μ- O o Ω μ- 3 Φ CO α fl rt PI ¬ Φ o\° • <; d o O . CO rt 3 rt — Φ 3 0 O to 3" rt cn μ- PI ¬ 0 S' »Q 3 hj Cd 3 rt o Φ hi Ω N 3 h • tr • OJ S' O Φ d O d Φ O ^ - ^ Ω ι-3 OJ hi * * Cfl d O tr OJ *< O ^ -» ^ - ^ Φ cn s: Q φ 3 CΛ φ hi Ω TJ φ 1-3 Φ 3' tr Ω Hi *<; 3 g O pi ¬ CO H μ- 3 CO 3 o O 3 * tr 3 μ- h- > rr φ Hi s' TJ |X) Hi rt O Hi 0 Ω OJ φ CO Hi li φ rt co Φ d PI ¬ CO Φ OJ Φ IT 1 o tr CO o O Φ rt o O μ- hj hi S' d TJ PI ¬ 3 r d rt hi rt μ- OJ 3 o OJ Ω OJ Φ Φ μ- hi S' 3 μ- et hd 3 et μ- 3 μ- 0 μ- tr TJ sQ H rt OJ 3 CO rt 0 Φ Φ 3 hi ii CL tr 0 3 Hi Φ CL TJ Φ Ω μ- 3 Qπ J rt 3 0 d φ Φ CO o OJ φ Φ li i 0 O tr Φ M 3 cn σ Hi rt CO O 3 tr OJ Hi μ- cn > 3" et rt X φ H Φ 3 Φ O Φ ribosome binding sites, the start codon and terminators. The expert can find information on this e.g. in the patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al . (Microbiology 142: 1297 (1996)), Vasicova et al. (Journal of Bacteriology 181: 6188 (1999)) and in known textbooks of genetics and molecular biology, such as e.g. the textbook by Knippers ("Molekulare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that by Winnacker ("Gene und Klone ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) . Mutations which lead to a change or reduction in the catalytic properties of enzyme proteins are known from the prior art; examples which may be mentioned are the works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611- 8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Mδckel ("Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms", Reports from the Jϋlich Research Center, Jϋl-2906, ISSN09442952, Jϋlich, Germany, 1994). Summarizing descriptions can be found in known textbooks of genetics and molecular biology, such as e.g. that by Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) . Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, "missense mutations" or "nonsense mutations" are referred to. Insertions or deletions of at least one base pair (bp) in a gene lead to frame shift mutations, as a consequence of which incorrect amino acids are incorporated or translation is interrupted prematurely. Deletions of several codons co CO IV) IV) μ> cπ O cπ o π O Cπ ro Φ o O O <i - n to Ω CO | -J Φ Ω TJ Ω H ^ CL 3 < O Pi CΛ _ 3 OJ H rt μ- I- 1 > Hi • Hi Φ CO μ- hi J^ CΛ TJ O O o ■ o 3 h- n φ Φ Φ li h- > ft 3 o 3 3 * O OJ O TJ Ω CD o o • tr li d CD OJ h- 1 OJ CL co co PI ¬ Ω li o d 2 -n Qπ cn TJ rt 3 CD TJ Ω iQ rt r μ- 1 3 C 0 li — . CO 3 μ- Cfl μ- piCD Ω S' o -n rt 3 rt O φ rt μ- Φ TJ >I h- 1 0 h- 1 3 * O 0 μ- σ 3 3 3 μ> O TJ ~— 3 3 s' μ> H o OJ 3- Φ rt h- 1 Ω a hi Ω Ω h- 1 - — μ- 3 Cπ Φ li sQ 3 CΛ OJ φ OJ CD — μ- μ- iQ Φ ■ — μ- Qπ iQ OJ 3 iQ Φ d ØJ d OJ tr Φ •—* φ CJ- rt | "< iQ 3 iQ h- 1 CD — Φ Qn — tr 0 ti ¬ ^ OJ pr I- 1 3 Ω - 3 • π d J Q s: o Φ TJ OJ J-. σ H 3 Φ O 3 hi d OJ rt h- 1 0 iQ 3 Φ S 1 c 3 OJ ,— * 0 — - d < Φ Φ CL Hi CΛ r < rt i- 1 ϋ tr μ- < < μ- tr S OJ OJ μ- 3 μ- hi M •* * rt Ω Hi — TJ TJ rt Φ iQ PI ¬ 3 rt ^ Ω OJ φ 0 O ^ φ 3 rt Ω Φ Ω 3 O Φ CD O pi ω Ω μ- S' tr 3 φ d tc li tr 3 iQ PI ¬ CL μ- C 3- OJ *. t 3 CD li ω h- n G 3 rt O O ^ iQ PI ¬ rt tc Ω Φ μ- Hi CD φ ^< H S ' o 3 μ- h- 1 .. o rt »1 TJ OJ TJ CO TJ O O 3 CL Φ S' et OJ < Φ o o OJ 3- o M 3 ω Ω h- 1 — O Ω Pi 3 O rt hi CΛ 3 o iQ iQ Φ hi Ω •_. d o CL tv> μ- CL 3 σ O o cn cπ • hi rt μ> o o O O Ω Φ Qπ OJ Φ hi 3 Φ o 3 3 CD φ cn <! μ- • <; pi ¬ 3 Hi O OJ OJ Φ CO tr h- n μ- $ Hi Hi 3' H 3 h- 1 OJ 3 iQ π ft •« * hi μ- CO s' rt .J 3 J-. -l rt hi 3 3 tr s: CL O rt OJ OJ 3 Φ ^ iQ o tr Hi hi Ω φ OJ tO 1 On CO o μ- μ- o o . — ^ μ- rt iQ OJ μ- Hi 3 iQ * rt μ- Φ co OJ O o CL o 3 μ- OJ cπ "* « ^1 d O o tr li CΛ n Ω 3' Φ hi co 3 cn μ- 3 3 OJ cπ Ω hi 3 φ 3 3 Ω d-* hi 3 CO μ- . 3 " Φ 3 N li 3 iQ pr CΛ Φ CΛ tr 3 CO C_l. r CO rt h- n x ) D 3 " « o OJ TJ 3 Φ Φ d d O ,— Φ -s 3 d ? li Ω 1 rt φ Ω d hj Φ Φ O OJ sQ Ω P^ o uq Ω iQ hi TJ rt CO 3 Cfl O Ω 3 OJ O O φ ii 3 li iQ OJ rt li . — . li O S *< DO h- 1 3 H OJ Φ α rt øJ CΛ Φ CO 3' ό rt 3 CΛ rt OJ rt μ- OJ 3 tr μ- s 3 — OJ CD d 3 3 μ- OJ μ- rt •— ' H M cn OΛ s: μ- TJ IV) 3 OJ tr rt O Φ O CD OJ 0 h- 1 o 3 Φ rt Φ Cfl 3 o μ- • h- 1 - — PI ¬ 0J 3 o H OJ cn Φ μ- Hi > CD li Hi μ- -J li o rt 0 ) li ii α 3 3 iQ cn S' O 3 Φ .— -. (- ■ Hi CL o Φ Ω o O π TJ co 4-. tr 3 Φ O d 5 iQ Φ Ω rt ft h- 1 • O S •«« 3 hi Φ iQ — - n ttJ O TJ OJ μ- TJ Hi TJ TJ 3 3 * pi ¬ Φ φ Φ o Φ CD li μ- ii 3 ■ <: — Φ J μ- 3 Pi -— - Ω h- 1 rt d= OJ iQ Φ OJ s' CL • J>^ Hi CO -~ ^ 3 Ω Hi o φ rt rt ® 0 ^ Cπ t- 1 CΛ • d μ- μ- μ- S' 3 Φ μ- Hi OJ μ- iQ rt - 1 CO ≥ OJ li O H Qπ hj o w H J^ CD Ω -. 3 Ω 3 0 h- 1 Q. 3 3 rt rt rt tr Cfl O — TJ et o H OJ -J H OJ j o S! CΛ 3 S' OJ rt 3 φ Φ φ • μ- o d Cfl — TJ .. μ- tr rt μ- H O d iQ H ) o OJ: ω rt Φ hi 5 Cfl tr o rt o Ω CΛ h- 1 o μ- Φ H 3 • • TJ • s μ- 1 tr HI μ- TJ Φ hi OJ iQ Ω S! ■ <: 3 tr pr tr μ- 3 o X OJ rt TJ >i M ^ rr Ω CΛ Cfl Φ o O Φ ^ — ^ Φ O Φ Φ φ cn o α φ μ- OJ 3 0 OJ Cn 3 OJ G cπ OJ hi ^ CO μ- CO Ω t 3 Hi 3 μ- 3 Hi d C OJ o M O li - n -> 00 Ω ι-3 O 3 rt φ μ- Φ Φ 3 μ- Ω rt o d 3 hi iQ TJ Hi PI ¬ rt o O M ,--» to Φ Φ μ- 3 o Ω rt 3 * 3 φ Φ Hi rt PI ¬ μ- s Φ "< h- 1 o S' 1 ^ - ^ d 3 o μ 1 rt Ω σ OJ μ- rt \ hi μ- Φ 3 o ? ØJ S' Ω μ- φ H Φ O J-> CΛ hi s- CD . — . 3 * H CA li m φ ? μ- fu li rt iQ rt Φ OJ Ω Qπ CΛ " 3 Hi Cπ Ω 3 μ- φ O C-l QJ 3 Φ 3' OJ Φ TJ iQ li Φ O OJ 3 Ω iQ tr φ μ- 3 CL 3- O rt 3 TJ IV) OJ: h- 1 o O O o h- 1 M pr o 3 0 Φ O Cfl " * tr rt Φ PI ¬ CΛ H h- 1 j μ- n — iQ • μ- <! CO H tr OJ d Φ m o Φ 3 3 OJ tr Ω > < μ- Cfl S' — ' d o CΛ pϋ — Φ ^ o Φ rt O T 3 Ω rt Ω Ω li 3' pr rt CO N 3 μ- hi -J o μ- Φ M o IQ rt IV) h{ iQ Ω 3 OJ o φ øJ d Φ μ- μ- CL o μ- Cπ CΛ 3 hi TJ Hi Φ hi Ω ^ rt ^^ Φ hi H 3 3 Cfl ii . Φ tr Ω OJ Sj h- 1 tr CΛ Ω " Φ Ω H Hi 3 • H 1 TJ Φ Φ O rt CL rt o φ μ- rt 3 3 3 •< Cfl hi Φ CΛ O Φ 1 3' CL O tr S 1 Ω rt 3 μ> hi ^ sQ 3 Ω OJ ø Ω φ Φ tr iQ Qπ ~J OJ: ι-3 CL Φ Φ o IV) 1-3 n Φ " ^ Cfl TJ μ- o * pi- d 3 Φ Pi ØJ OJ μ- ^ " " cn Hi 3 " CO μ- rt CΛ O S DJ μ- 3 Hi 3 3 3 TJ Ω <! D 3 Φ >< 3 C < Φ ft 3 TJ Φ CD TJ 1 - ~j OJ Ω < ) **• Φ Φ μ- CL ii rt 3 OJ hh hi li Q μ- OJ Ω o ι-3 • its. CO hi OJ J pi ¬ ^ μ- μ- H TJ μ- μ- OJ 3 0 3 OJ OJ h- 1 d O Cπ ^. Φ H s' CΛ Cfl -n C TJ 0 <! 3 C hi Φ φ μ- hi CO h- 1 IV) 1 H Φ CO Ω CO 3 OJ Φ hi μ- rt rt 3 Φ 3 OJ CΛ ^ rt 3" CD CL iQ rt tr sQ μ- hi CΛ CD Hi 1 3' . Φ O cn OJ OJ o O μ- CO CO μ 1 O M CO Φ ii ^^ ϋ C Hi o 1 I li ^ rt 3 (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 coding region of the gene in question is interrupted by the vector sequence and two incomplete alleles are obtained, one lacking the 3' end and one lacking the 5' end. This method has been used, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994) ) to eliminate the recA gene of C. glutamicum. In the method of "gene replacement", a mutation, such as e.g. a deletion, insertion or base exchange, is established in vitro in the gene of interest. The allele prepared is in turn cloned in a vector which is not replicative for C. glutamicum and this is then transferred into the desired host of C. glutamicum by trans ormation or conjugation. After homologous recombination by means of a first "crossover" event which effects integration and a suitable second "cross-over" event which effects excision in the target gene or in the target sequence, the incorporation of the mutation or of the allele is achieved. This method was used, for example, by Peters-Wendisch et al. (Microbiology 144, 915 - 927 (1998)) to eliminate the pyc gene of C. glutamicum by a deletion. A deletion, insertion or a base exchange can be incorporated into the menE gene in this manner. In addition, it may be advantageous for the production of L-amino acids to enhance, in particular over-express, one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and optionally regulatory proteins, in addition to the attenuation of the menE gene. The term "enhancement" in this connection describes the increase in the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene or allele which codes for a corresponding enzyme (protein) having a high activity, and optionally combining these measures. By enhancement measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on the starting microorganism. Thus, for the preparation of L-amino acids, in addition to the attenuation of the menE gene at the same time one or more of the genes chosen from the group consisting of • the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0 197 335), • the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) , • the tpi gene which codes for triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), • the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), • the zwf gene which codes for glucose 6-phosphate dehydrogenase (JP-A-09224661) , • the pyc gene which codes for pyruvate carboxylase (DE-A- 198 31 609) , • the mqo gene which codes for malate-quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), • the lysC gene which codes for a feed-back resistant aspartate kinase (Accession No.P26512; EP-B-0387527; EP- A-0699759; WO 00/63388), • the lysE gene which codes for lysine export (DE-A-195 48 222) , • the horn gene which codes for homoserine dehydrogenase (EP-A 0131171) , • the ilvA gene which codes for threonine dehydratase (Mδckel et al . , Journal of Bacteriology (1992) 8065- 8072)) or the ilvA(Fbr) allele which codes for a "feed back resistant" threonine dehydratase (Mδckel et al., (1994) Molecular Microbiology 13: 833-842), • the ilvBN gene which codes for acetohydroxy-acid synthase (EP-B 0356739) , • the ilvD gene which codes for dihydroxy-acid dehydratase (Sahm and Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979), • the zwal gene which codes for the Zwal protein (DE: 19959328.0, DSM 13115) can be enhanced, in particular over-expressed. It may furthermore be advantageous for the production of amino acids, in addition to the attenuation of the menE gene, at the same time for one or more of the genes chosen from the group consisting of • the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047), • the pgi gene which codes for glucose 6-phosphate isomerase (US 09/396,478, DSM 12969), • the poxB gene which codes for pyruvate oxidase (DE:1995 1975.7, DSM 13114), • the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2, DSM 13113) to be attenuated, in particular for the expression thereof to be reduced. In addition to the attenuation of the menE gene it may furthermore be advantageous for the production of amino acids to eliminate 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 invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of L-amino acids. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfϋhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) ) . The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols, such as, for example, glycerol and ethanol, and organic acids, such as, for example, acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture. Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the above-mentioned substances . Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner. Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture. Antifoams, such as, for example, fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as, for example, antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as, for example, air, are introduced into the culture. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours. Methods for the determination of L-amino acids are known from the prior art. The analysis can thus be carried out, for example, as described by Spackman et al . (Analytical Chemistry, 30, (1958), 1190) by anion exchange chromatography with subsequent ninhydrin derivation, or it can be carried out by reversed phase HPLC, for example as described by Lindroth et al . (Analytical Chemistry (1979) 51: 1167-1174) . The process according to the invention is used for fermentative preparation of amino acids. The following microorganism was deposited on 23.02.2001 as a pure culture at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty: • Escherichia coli ToplO/pCR2. lmenEint as DSM 14080. The present invention is explained in more detail in the following with the aid of embodiment examples. The isolation of plasmid DNA from Escherichia coli and all techniques of restriction, Klenow and alkaline phosphatase treatment were carried out by the method of Sambrook et al. (Molecular Cloning. A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA) . Methods for transformation of Escherichia coli are also described in this handbook. The composition of the usual nutrient media, such as LB or TY medium, can also be found in the handbook by Sambrook et al. Example 1 Preparation of a genomic cosmid gene library from C. glutamicum ATCC 13032 Chromosomal DNA from C. glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02) . The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250) . The DNA of the cosmid vector SuperCosl (Wahl et al. (1987), Proceedings of the National Academy of Sciences, USA 84:2160-2164), obtained from Stratagene (La Jolla, USA, Product Description SuperCosl Cosmid Vector Kit, Code no. 251301) was cleaved with the restriction enzyme Xbal (Amersham Pharmacia, Freiburg, Germany, Product Description Xbal, Code no. 27- 0948-02) and likewise dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04) . The cosmid DNA treated in this manner was mixed with the treated ATCC13032 DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA- Ligase, Code no.27-0870-04) . The ligation mixture was then packed in phages with the aid of Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). For infection of the E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Res. 16:1563-1575) the cells were taken up in 10 mM MgS0 and mixed with an aliquot of the phage suspension. The infection and titering of the cosmid library were carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) , the cells being plated out on LB agar (Lennox, 1955, Virology, 1:190) + 100 μg/ l ampicillin. After incubation overnight at 37°C, recombinant individual clones were selected. Example 2 Isolation and sequencing of the menE gene The cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02) . The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250) . After separation by gel electrophoresis, the cosmid fragments in the size range of 1500 to 2000 bp were isolated with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) . The DNA of the sequencing vector pZero-1, obtained from Invitrogen (Groningen, The Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04). The ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) , the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany) . This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol. Letters, 123:343-7) into the E. coli strain DH5txmcr (Grant, 1990, Proceedings of the National Academy of Sciences, U.S.A., 87:4645-4649). Letters, 123:343-7) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin. The plasmid preparation of the recombinant clones was carried out with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) . The sequencing was carried out by the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academies of Sciences, U.S.A., 74:5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067). The "RR dRhodamin Terminator Cycle Sequencing Kit" from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) was used. The separation by gel electrophoresis and analysis of the sequencing reaction were carried out in a "Rotiphoresis NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377" sequencer from PE Applied Biosystems (Weiterstadt, Germany) . The raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0. The individual sequences of the pZerol derivatives were assembled to a continuous contig. The computer-assisted coding region analyses were prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyses were carried out with the "BLAST search program" (Altschul et al . , 1997, Nucleic Acids Research, 25:3389-3402) against the non- redundant databank of the "National Center for Biotechnology Information" (NCBI, Bethesda, MD, USA) . The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed an open reading frame of 1131 bp, which was called the menE gene. The menE gene codes for a polypeptide of 376 amino acids. Example 3 Preparation of an integration vector for integration mutagenesis of the menE gene From the strain ATCC 13032, chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817 - 1828 (1994)). On the basis of the sequence of the menE gene known for C. glutamicum from example 2, the following oligonucleotides were chosen for the polymerase chain reaction (see SEQ ID No. 3 and SEQ ID No. 4) : menE-intl : 5 CTC ACT CCG TTG AAT TTG G 3 menE-int2 : 5 CAG GTG CAT TTC TGT AGC C 3 The primers shown were synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction was carried out by the standard PCR method of Innis et al . (PCR protocols. A guide to methods and applications, 1990, Academic Press) with the Taq-polymerase from Boehringer Mannheim (Germany, Product Description Taq DNA polymerase, Product No. 1 146 165) . With the aid of the polymerase chain reaction, the primers allow amplification of an internal fragment of the menE gene 520 bp in size. The product amplified in this way was tested electrophoretically in a 0.8% agarose gel. The amplified DNA fragment was ligated with the TOPO TA Cloning Kit from Invitrogen Corporation (Carlsbad, CA, USA; Catalogue Number K4500-01) in the vector pCR2.1-T0PO (Mead at al. (1991) Bio/Technology 9:657-663). The E. coli strain TOP10 was then electroporated with the ligation batch (Hanahan, In: DNA cloning. A practical approach. Vol. I. IRL-Press, Oxford, Washington DC, USA, 1985) . Selection of plasmid-carrying cells was carried out by plating out the transformation batch on LB Agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 50 mg/1 kanamycin. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and subsequent agarose gel electrophoresis (0.8%). The plasmid was called pCR2. ImenEint and is shown in figure 1. Example 4 Integration mutagenesis of the menE gene in the strain DSM 5715 The vector pCR2. ImenEint mentioned in example 3 was electroporated by the electroporation method of Tauch et al. (FEMS Microbiological Letters, 123:343-347 (1994)) in Corynebacterium glutamicum DSM 5715. The strain DSM 5715 is an AEC-resistant lysine producer. The vector pCR2. ImenEint cannot replicate independently in DSM5715 and is retained in the cell only if it has integrated into the chromosome of DSM 5715. Selection of clones with pCR2. ImenEint integrated into the chromosome was carried out by plating out the electroporation batch on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which had been supplemented with 15 mg/1 kanamycin. For detection of the integration, the menEint fragment was labelled with the Dig hybridization kit from Boehringer by the method of "The DIG System Users Guide for Filter Hybridization" of Boehringer Mannheim GmbH (Mannheim, Germany, 1993) . Chromosomal DNA of a potential integrant was isolated by the method of Eikmanns et al . (Microbiology 140: 1817 - 1828 (1994)) and in each case cleaved with the restriction enzymes EcoRI and Pstl. The fragments formed were separated by means of agarose gel electrophoresis and hybridized at 68°C with the Dig hybridization kit from Boehringer. The plasmid pCR2. ImenEint mentioned in example 3 had been inserted into the chromosome of DSM5715 within the chromosomal menE gene. The strain was called DSM5715 : : pCR2. ImenEint . Example 5 Preparation of lysine The C. glutamicum strain DSM5715: :pCR2. ImenEint obtained in example 4 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined. For this, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg/1) for 24 hours at 33°C. Starting from this agar plate culture, a preculture was seeded (10 ml medium in a 100 ml conical flask) . The complete medium Cglll was used as the medium for the preculture. Medium Cg III NaCl 2.5 g/1 Bacto-Peptone 10 g/1 Bacto-Yeast extract 10 g/1 Glucose (autoclaved separately) 2% (w/v) The pH was brought to pH 7.4 Kanamycin (25 mg/1) was added to this. The preculture was incubated for 16 hours at 33°C at 240 rpm on a shaking machine. A main culture was seeded from this preculture such that the initial OD (660 nm) of the main culture was 0.1 OD. Medium MM was used for the main culture. Medium MM CSL (corn steep liquor) 5 g/1 MOPS (morpholinopropanesulfonic acid) 20 g/1 Glucose (autoclaved separately) 50g/l Salts: (NH 4 ) 2 S0 4 25 g/1 KH 2 P0 4 0.1 g/1 MgS0 4 * 7 H 2 0 1.0 g/1 CaCl 2 * 2 H 2 0 10 mg/1 FeS0 4 * 7 H 2 0 10 mg/1 MnS0 4 * H 2 0 5.0mg/l Biotin (sterile-filtered) 0.3 mg/1 Thiamine * HC1 (sterile-filtered) 0.2 mg/1 Leucine (sterile-filtered) 0.1 g/1 CaC0 3 25 g/1 The CSL, MOPS and the salt solution are brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions are then added, and the CaC0 3 autoclaved in the dry state is added. Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Kanamycin (25 mg/1) was added. Culturing was carried out at 33°C and 80% atmospheric humidity. After 72 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich) . The amount of lysine formed was determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection. The result of the experiment is shown in Table 1. Table 1 Brief Description of the Figure: Figure 1: Map of the plasmid pCR2. ImenEint The abbreviations and designations used have the following meaning. KmR: Kanamycin resistance gene EcoRI: Cleavage site of the restriction enzyme EcoRI Pstl: Cleavage site of the restriction enzyme Pstl menEint : Internal fragment of the menE gene ColEl: Replication origin of the plasmid ColEl SEQUENCE PROTOCOL <110> Degussa AG <120> Nucleotide sequences which code for the menE gene <130> 000551 BT <140> <141> <160> 4 <170> Patentln Ver. 2.1 <210> 1 <211> 1570 <212> DNA <213> Corynebacterium glutamicum <220> <221> CDS <222> (230) .. (1357) <223> menE gene <400> 1 ttcgttgcca tagacatgct cttcgcagca ctgtttgcgc acgtctcctc cggcatcttt 60 gtcaccaaca atggttggga actcaccggc gcaatcggcg ctggcgcgct gcttctcatc 120 gcagttggcg caggtgcatg gagcatcgac ggggttctgg caaaacgcaa ggcctaaatc 180 tagcgccaca actccgaatt ctgaaccatc ggcactagaa tctcggaat atg aat act 238 Met Asn Thr 1 cgc gtc etc gaa gca eta cct gtt gat ctt gca gat ccc ace gca att 286 Arg Val Leu Glu Ala Leu Pro Val Asp Leu Ala Asp Pro Thr Ala lie 5 10 15 ctg gga gat etc gag gac gca ate tct ggg aag aaa act ttc etc ccc 334 Leu Gly Asp Leu Glu Asp Ala lie Ser Gly Lys Lys Thr Phe Leu Pro 20 25 30 35 ate cct gta caa gat aaa ace cgt gca cag ttg ctg cgc gat tct caa 382 lie Pro Val Gin Asp Lys Thr Arg Ala Gin Leu Leu Arg Asp Ser Gin 40 45 50 cga gtt ggc etc gcc ate gat cct teg ate get ttg gtg atg gee act 430 Arg Val Gly Leu Ala lie Asp Pro Ser lie Ala Leu Val Met Ala Thr 55 60 65 tct ggt tct aca ggt ace ccg aag ggc get cag etc act ccg ttg aat 478 Ser Gly Ser Thr Gly Thr Pro Lys Gly Ala Gin Leu Thr Pro Leu Asn 70 75 80 ttg gtg agt tec gee gat get acg cat cag ttt tta ggt ggc gaa ggc 526 Leu Val Ser Ser Ala Asp Ala Thr His Gin Phe Leu Gly Gly Glu Gly 85 90 95 cag tgg ttg ctt gcc atg cca gca cac cac att gca ggc atg cag gtg 574 Gin Trp Leu Leu Ala Met Pro Ala His His He Ala Gly Met Gin Val 100 105 110 115 ctt ctt cga age etc att get gga gtt gag cca eta get att gat etc 622 Leu Leu Arg Ser Leu He Ala Gly Val Glu Pro Leu Ala He Asp Leu 120 125 130 age aca ggt ttt cac att gac get ttc gca ggc gcc gcg gca gaa ctg 670 Ser Thr Gly Phe His He Asp Ala Phe Ala Gly Ala Ala Ala Glu Leu 135 140 145 aaa aat ace ggc gac cgc gtc tat aca tec ttg act cca atg cag tta 718 Lys Asn Thr Gly Asp Arg Val Tyr Thr Ser Leu Thr Pro Met Gin Leu 150 155 160 ctt aaa gca atg gac tec ttg caa ggc att gaa gcc ctg aaa ctt ttt 766 Leu Lys Ala Met Asp Ser Leu Gin Gly He Glu Ala Leu Lys Leu Phe 165 170 175 gat gtc att ctt gtt ggc ggt get gca ttg tct aag cag gcc cga att 814 Asp Val He Leu Val Gly Gly Ala Ala Leu Ser Lys Gin Ala Arg He 180 185 190 195 tct gcg gag cag eta gac ate aac att gtc ace ace tac ggc tec tea 862 Ser Ala Glu Gin Leu Asp He Asn He Val Thr Thr Tyr Gly Ser Ser 200 205 210 gag act tea ggt ggc tgc gtt tat gat ggc aag ccc att ccc ggc gcg 910 Glu Thr Ser Gly Gly Cys Val Tyr Asp Gly Lys Pro He Pro Gly Ala 215 220 225 aaa gtc cgt att teg gat gag cgc att gag ttg ggt ggc ccg atg att 958 Lys Val Arg He Ser Asp Glu Arg He Glu Leu Gly Gly Pro Met He 230 235 240 gcg cag ggc tac aga aat gca cct gaa cat ccg gat ttc gcc aac gag 1006 Ala Gin Gly Tyr Arg Asn Ala Pro Glu His Pro Asp Phe Ala Asn Glu 245 250 255 ggt tgg ttt ace ace tct gat tea ggt gaa etc cac gac ggg att etc 1054 Gly Trp Phe Thr Thr Ser Asp Ser Gly Glu Leu His Asp Gly He Leu 260 265 270 275 ace gtg act ggt cgc gtg gat ace gtc att gat tec ggt gga ttg aag 1102 Thr Val Thr Gly Arg Val Asp Thr Val He Asp Ser Gly Gly Leu Lys 280 285 290 ttg cac cca gag gta ctg gaa cgt gcc ate gca gat att aaa ggt gtc 1150 Leu His Pro Glu Val Leu Glu Arg Ala He Ala Asp He Lys Gly Val 295 300 305 ace gcg gcg tgt gtt gtg ggt att ccc gat ccc cga tta ggc caa gca 1198 Thr Ala Ala Cys Val Val Gly He Pro Asp Pro Arg Leu Gly Gin Ala 310 315 320 att gtg gcc gcg tac tec gga teg ate agt ccg tct gaa gtt att gaa 1246 He Val Ala Ala Tyr Ser Gly Ser He Ser Pro Ser Glu Val He Glu 325 330 335 ggc etc gac gat eta cct cgt tgg cag ctt ccc aaa egg ctg aag cat 1294 Gly Leu Asp Asp Leu Pro Arg Trp Gin Leu Pro Lys Arg Leu Lys His 340 345 350 355 ctg gaa tct ttg ccc age att ggt cct gga aaa get gat cga cgt get 1342 Leu Glu Ser Leu Pro Ser He Gly Pro Gly Lys Ala Asp Arg Arg Ala 360 365 370 ate gcg aag ctg ttt tagtcttcat tcttgctggc tgcaactagt tttgccacat 1397 He Ala Lys Leu Phe 375 ettcatcggt gtacactttg gegatetget eateatttcc acceatgagg gtgttgccaa 1457 caactagtgc tcccaettgg gtggtgggca cgacagcgaa gtgteggggc tgagcgtaga 1517 cctggcgaat agggtgatca gagcgcagtg cgcaggcatg cagccatacg tea 1570 <210> 2 <211> 376 <212> PRT <213> Corynebacterium glutamicum <400> 2 Met Asn Thr Arg Val Leu Glu Ala Leu Pro Val Asp Leu Ala Asp Pro 1 5 10 15 Thr Ala He Leu Gly Asp Leu Glu Asp Ala He Ser Gly Lys Lys Thr 20 25 30 Phe Leu Pro He Pro Val Gin Asp Lys Thr Arg Ala Gin Leu Leu Arg 35 40 45 Asp Ser Gin Arg Val Gly Leu Ala He Asp Pro Ser He Ala Leu Val 50 55 60 Met Ala Thr Ser Gly Ser Thr Gly Thr Pro Lys Gly Ala Gin Leu Thr 65 70 75 80 Pro Leu Asn Leu Val Ser Ser Ala Asp Ala Thr His Gin Phe Leu Gly 85 90 95 Gly Glu Gly Gin Trp Leu Leu Ala Met Pro Ala His His He Ala Gly 100 105 110 Met Gin Val Leu Leu Arg Ser Leu He Ala Gly Val Glu Pro Leu Ala 115 120 125 He Asp Leu Ser Thr Gly Phe His He Asp Ala Phe Ala Gly Ala Ala 130 135 140 Ala Glu Leu Lys Asn Thr Gly Asp Arg Val Tyr Thr Ser Leu Thr Pro 145 150 155 160 Met Gin Leu Leu Lys Ala Met Asp Ser Leu Gin Gly He Glu Ala Leu 165 170 175 Lys Leu Phe Asp Val He Leu Val Gly Gly Ala Ala Leu Ser Lys Gin 180 185 190 Ala Arg He Ser Ala Glu Gin Leu Asp He Asn He Val Thr Thr Tyr 195 200 205 Gly Ser Ser Glu Thr Ser Gly Gly Cys Val Tyr Asp Gly Lys Pro He 210 215 220 Pro Gly Ala Lys Val Arg He Ser Asp Glu Arg He Glu Leu Gly Gly 225 230 235 240 Pro Met He Ala Gin Gly Tyr Arg Asn Ala Pro Glu His Pro Asp Phe 245 250 255 Ala Asn Glu Gly Trp Phe Thr Thr Ser Asp Ser Gly Glu Leu His Asp 260 265 270 Gly He Leu Thr Val Thr Gly Arg Val Asp Thr Val He Asp Ser Gly 275 280 285 Gly Leu Lys Leu His Pro Glu Val Leu Glu Arg Ala He Ala Asp He 290 295 300 Lys Gly Val Thr Ala Ala Cys Val Val Gly He Pro Asp Pro Arg Leu 305 310 315 320 Gly Gin Ala He Val Ala Ala Tyr Ser Gly Ser He Ser Pro Ser Glu 325 330 335 Val He Glu Gly Leu Asp Asp Leu Pro Arg Trp Gin Leu Pro Lys Arg 340 345 350 Leu Lys His Leu Glu Ser Leu Pro Ser He Gly Pro Gly Lys Ala Asp 355 360 365 Arg Arg Ala He Ala Lys Leu Phe 370 375 <210> 3 <211> 19 <212> DNA <213> Corynebacterium glutamicum <220> <223> Primer enE-intl <400> 3 ctcactccgt tgaatttgg 19 <210> 4 <211> 19 <212> DNA <213> Corynebacterium glutamicum <220> <223> Primer menE-int2 <400> 4 caggtgcatt tctgtagcc 19

Claims: What is claimed is: 1. An isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the menE gene, chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b) , and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c) , the polypeptide preferably having the activity of 0- succinylbenzoic acid CoA ligase. 2. A polynucleotide as claimed in claim 1, wherein the polynucleotide is a preferably recombinant DNA which is capable of replication in coryneform bacteria. 3. A polynucleotide as claimed in claim 1, wherein the polynucleotide is an RNA. 4. A polynucleotide as claimed in claim 2, comprising the nucleic acid sequence as shown in SEQ ID No. 1. 5. A DNA as claimed in claim 2 which is capable of replication, comprising (i) the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii) , and optionally (iv) sense mutations of neutral function in (i) . 6. A DNA as claimed in claim 5 which is capable of replication, wherein the hybridization is carried out under a stringency corresponding to at most 2x SSC. 7. A polynucleotide sequence as claimed in claim 1, which codes for a polypeptide which comprises the amino acid sequences shown in SEQ ID No. 2. 8. A coryneform bacterium in which the menE gene is attenuated, in particular eliminated. 9. The integration vector pCR2. ImenEint, which 9.1. carries an internal fragment of the menE gene 520 bp in size, 9.2. the restriction map of which is reproduced in figure 1, and 9.3. which is deposited in the E. coli strain ToplO/pCR2. ImenEint under no. DSM 14080 at the Deutsche Sammlung fur Mikroorganismen und Zellenkulturen] . 10. A process for the fermentative preparation of L-amino acids, in particular L-lysine, which comprises carrying out the following steps: a) fermentation of the coryneform bacteria which produce the desired L-amino acid and in which at least the menE gene or nucleotide sequences which code for it are attenuated, in particular eliminated; b) concentration of the L-amino acid in the medium or in the cells of the bacteria, and c) isolation of the L-amino acid. 11. A process as claimed in claim 10, wherein bacteria in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally enhanced are employed. 12. A process as claimed in claim 10, wherein bacteria in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated are employed. 13. A process as claimed in claim 10, wherein the expression of the polynucleotide (s) which code(s) for the menE gene is attenuated, in particular eliminated. 14. A process as claimed in claim 10, wherein the catalytic properties of the polypeptide (enzyme protein) for which the polynucleotide menE codes are reduced. 15. A process as claimed in claim 10, wherein for the preparation of L-amino acids, coryneform microorganisms in which at the same time one or more of the genes chosen from the group consisting of 15.1 the dapA gene which codes for dihydrodipicolinate synthase, 15.2 the gap gene which codes for glyceraldehyde 3- phosphate dehydrogenase, 15.3 the tpi gene which codes for triose phosphate isomerase, 15.4 the pgk gene which codes for 3-phosphoglycerate kinase, 15.5 the zwf gene which codes for glucose 6- phosphate dehydrogenase, 15.6 the pyc gene which codes for pyruvate carboxylase, 15.7 the mqo gene which codes for malate-quinone oxidoreductase, 15.8 the lysC gene which codes for a feed-back resistant aspartate kinase, 15.9 the lysE gene which codes for lysine export, 15.10 the horn gene which codes for homoserine dehydrogenase 15.11 the ilvA gene which codes for threonine dehydratase or the ilvA(Fbr) allele which codes for a feed back resistant threonine dehydratase, 15.12 the ilvBN gene which codes for acetohydroxy- acid synthase, 15.13 the ilvD gene which codes for dihydroxy-acid dehydratase, and 15.14 the zwal gene which codes for the Zwal protein is or are enhanced or over-expressed are fermented. 16. A process as claimed in claim 10, wherein for the preparation of L-amino acids, coryneform microorganisms in which at the same time one or more of the genes chosen from the group consisting of 16.1 the pck gene which codes for phosphoenol pyruvate carboxykinase, 16.2 the pgi gene which codes for glucose 6- phosphate isomerase, 16.3 the poxB gene which codes for pyruvate oxidase, and 16.4 the zwa2 gene which codes for the Zwa2 protein is or are attenuated are fermented. 17. A coryneform bacterium which contains a vector which carries parts of the polynucleotide as claimed in claim 1, but at least 15 successive nucleotides of the sequence claimed. 18. A process as claimed in one or more of the preceding claims, wherein microorganisms of the species Corynebacterium glutamicum are employed. 19. A process for discovering RNA, cDNA and DNA in order to isolate nucleic acids, or polynucleotides or genes which code for O-succinylbenzoic acid CoA ligase or have a high similarity with the sequence of the menE gene, which comprises employing the polynucleotide comprising the polynucleotide sequences as claimed in claims 1, 2, 3 or 4 as hybridization probes. 20. A process as claimed in claim 19, wherein arrays, micro arrays or DNA chips are employed.

Inventors: FARWICK MIKE, HUTHMACHER KLAUS, MARX ACHIM, PFEFFERLE WALTER, FARWICK, MIKE, HUTHMACHER, KLAUS, MARX, ACHIM, PFEFFERLE, WALTER
Applicants: DEGUSSA, DEGUSSA AG
Application year: 2001
Main classification: C12P13
IPC codes: C12N9, C12P13