EP1313758A1 - Nucleotide sequences which code for the oxyr gene - Google Patents

Abstract

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

Description

PC i / tP 01/08388

A. CLASSIFICATION OF SUBJECT MATTER

IPC 7 C07K14/34 C12P13/04 C12P13/08

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 7 C07K C12P

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

BIOSIS, EPO-Internal, EMBL, WPI Data, PAJ, MEDLINE, CHEM ABS Data, EMBASE, SCISEARCH, LIFESCIENCES, SEQUENCE SEARCH

c. DOCUMENTS CONSIDERED TO BE RELEVANT

Category ⁰ Citation of document, with indication, where appropπate, of the relevant passages Relevant to claim No

DATABASE EMBL 'Online! 1-6

EBI;

Acc.Nr.: U18263, 3 June 1995 (1995-06-03)

SHERMAN DR ET AL : "Mycobacteπ^'um avi u al kyl hydroxperoxidase C (ahpC) gene, and

OxyR homol og gene"

XP002185667 abstract

DATABASE EMBL Online ! 1-6

EBI;

Acc.Nr.: AF186371,

9 November 1999 (1999-11-09)

HAHN JS AND ROE JH: "Strepto yces coelicolor A3(2) ahpD, ahpC, and oxyR genes"

XP002185668 abstract

-/- xl Further documents are listed in the continuation of box C Patent family members are listed in annex

⁰ Special categories of cited documents

"T" later document published after the international filing date or pπonty date and not in conflict with the application but

"A" document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance Invention

"E" earlier document but published on or after the international "X" document of particular relevance the claimed Invention filing date cannot be considered novel or cannot be considered to "L" document which may throw doubts on pπonty claιm(s) or involve an Inventive step when the document Is taken alone which is cited to establish the publication date of another "Y" document of particular relevance, the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the "O" document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docuother means ments, such combination being obvious to a person skilled "P" document published prior to the international filing date but in the art later than the priority date claimed '&^■ document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

14 December 2001 14/01/2002

Mame and mailing address of the ISA Authorized officer

European Patent Office, P B 5818 Patentlaan 2 NL- 2280 H Ri]swi)k Tel (+31-70) 340-2040, Tx 31 651 epo nl, Fax (+31-70) 340-3016 Bassi as , I

Form PCT/ISA/210 (second sheet) (July 1992)

C.(Contιnuatιon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category ° Citation of document, with indication.where appropriate, of the relevant passages Relevant to claim No

DATABASE EMBL ' Onl i ne ! 1-6

EBI ;

Acc . Nr . : AF034861 ,

28 September 1998 (1998-09-28)

PAGAN-RAMOS E ET AL: "Mycobacterium marinum ahpC and oxyR genes"

XP002185669 abstract

KRAMER R: "Genetic and physiological approaches for the production of amino acids"

JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE

PUBLISHERS, AMSTERDAM, NL, vol. 45, no. 1,

12 February 1996 (1996-02-12), pages 1-21,

XP004036833

ISSN: 0168-1656 the whole document

P ,X EP 1 108790 A (KYOWA HAKKO KOGYO KK) 1-6 20 June 2001 (2001-06-20) sequences 7065, 5614, 2113, 2114

P ,A L00S ANDREA ET AL: "Development and 19-21 validation of Corynebacterium DNA microarrays."

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 67, no. 5, May 2001 (2001-05), pages

2310-2318, XP002185618

ISSN: 0099-2240 the whole document

Form PCT/ISA/210 (continuation ot second sheet) (July 1992) International Application No. PCT/EP 01 /08388

FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210

Continuation of Box 1.2 Claims Nos. : la)

Present claim la) relates to a polynucleotide having at least 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

However, whilst such a claim is formally clear, the complexity and scope of the subject-matter encompassed by a claim of this type results in serious problems with the assessment of novelty and inventive step since there is no method (computer-based or otherwise) currently available that can compare the almost infinite number of hypothetical sequences claimed with all the known sequences of the prior art. Accordingly, the search can only be carried out on the "real" sequences which are disclosed in the application but not on all the other hypothetical sequences also encoding the polypeptide of SEQ ID N0:2.

Consequently, the search for claim la) has been carried out only for the polynucleotide containing the nucleotide sequence as shown in SEQ ID NO: 1.

The applicant's attention is drawn to the fact that claims, or parts of claims, relating to inventions in respect of which no international search report has been established need not be the subject of an international preliminary examination (Rule 66.1(e) PCT). The applicant is advised that the EPO policy when acting as an International Preliminary Examining Authority is normally not to carry out a preliminary examination on matter which has not been searched. This is the case irrespective of whether or not the claims are amended following receipt of the search report or during any Chapter II procedure.

Drmation on patent family members

PC / tP 01/08388

Patent document Publication Patent family Publication cited in search report date member(s) date

EP 1108790 20-06-2001 EP 1108790 A2 20-06-2001

Form PCT/ISA/210 (patent family annex) (July 1992) Nucleotide sequences which code for the oxyR gene

The invention provides nucleotide sequences from coryneform bacteria which code for the oxyR gene and a process for the fermentative preparation of amino acids, in particular L- lysine, using bacteria in which the oxyR gene is enhanced. The oxyR gene codes for the transcription regulator OxyR, which belongs to the LysR family.

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 utagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to anti etabolites, such as e.g. the lysine analogue S-(2- aminoethyl) -cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-lysine 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, in particular L-lysine.

Description 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.

If L-lysine or lysine are mentioned in the following, this also means the salts, such as e.g. lysine monohydrochloride or lysine sulfate.

The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the oxyR 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 the transcription regulator OxyR.

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 sequence complementary to sequence (i) or (ii) , and optionally

(iv) sense mutations of neutral function in (i) which do not modify the activity of the protein/polypeptide

The invention also provides

a) polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 1 and 490;

b) polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 491 and 1471; and c) polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 1472 and 1675.

The invention also provides

5 a polynucleotide, in particular DNA, which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No. 1;

a polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID 10 No. 2;

a vector containing the DNA sequence of C. glutamicum which codes for the oxyR gene, deposited in Corynebacterium glutamicum as pT-oxyRexp under DSM 13457, and

coryneform bacteria serving as the host cell, which contain 15. the vector or in which the oxyR gene is enhanced.

The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library of a coryneform bacterium, which 20 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.

25 Polynucleotide 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 the transcription regulator OxyR, or to isolate those nucleic acids or

30 polynucleotides or genes which have a high similarity of sequence with that of the oxyR 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.

Polynucleotide sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for the transcription regulator OxyR can be prepared with the polymerase chain reaction (PCR) .

Such oligonucleotides which serve as probes or primers comprise at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, very particularly preferably at least 15, 16, 17, 18 or 19 successive nucleotides.

Oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 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 in particular 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 o IV) ) I-¹ H¹ o cπ O π o n

Hi O rt rt K CO ιQ Ό H-3 rt φ ø Ω Ω Φ μ- Hi * CO Cυ t-¹ tr¹ t-i t¹ iQ Hi m Cu SO H tJ O €

O O tr - Φ rt M H 3^J tr 3 CO 0 0 3 3 3^J Cu φ 1 1 1 1 H Φ tr Ω Φ i-s μ-

X~i H Φ Φ ^•ϋ Cu 3 Φ Φ Φ N μ- 3 H N Ω Φ l-S Λ H Cυ n 3 iQ O H Φ Ω o\o Cu φ * rt

* H r-i H Ω Ό¹ CO *<; 3 μ- H > * H rt ø Φ H tr Φ H" C- 3 O ^_* CO Hi ?o tr

H- 3 Φ ιQ Φ Ω O Cu 3 Φ 3 ιQ Φ Φ 3 Φ rt μ- Φ Cu ιQ φ rt μ- H

H- c Ό φ rt φ rt CD Φ O 3^{^} CQ r-i Φ CO CO Φ Cu Φ Ω 3 μ- 3 tr rt 3 3 0_ SO rt

CO tr 3 3 Φ » Φ Φ O 3 CU ^■n CO CO l-S 0 Ω ^ 3 ^■< μ- CU Ω rt <! μ- ω 00 Cu Cυ tr

Cu Cυ 3 3 "» H Φ tr o O φ 3 H* Φ μ- o 3 O CU φ 3 o\o en σ 3 Φ

Cυ Ω ^< CO rt Ω l→ O Cu C iQ H, 3 μ- CU CO Ό 3 CU 3 Cu 3 rt 3 ιQ _^ o\o !-^■ tr ri" Cu Φ O 1 O CO <J Φ 3 μ- _z l-S H φ M μ- rt CO μ- rt •< tr

H- Φ tr O rt H 3 CU H P- 3 rt μ- 3 φ s: 0 Cu 3 Φ μ- <! μ- rt S rt cu μ- h-¹ H Φ O μ- H¹ Ω 3 lΩ. l-S 3 Φ 3^{^} 3 Cu 3 O 3^* c 3 Φ *_* CO Φ O O Cπ O Cυ μ^j o

H- H- H <i 3 H H- C φ U3 Φ iQ rt 3^* <! μ- c O μ- rt 3 o\o rt CO r 3 3 • Φ O 3 3 O o 3 t Cu Φ Ω Ω μ- 3 t^"1 μ- 13 O _^ so o o

^ 3 Φ 3 CO o CO o H- • C H Q D μ- Φ 3 h " Φ 3 Φ ir¹ 1 3 H Hi M o X-¹ iQ

3 Φ O Φ co Φ a Ω Ω 1 sQ •*« 1 sQ iQ Φ C O < ⁾ o\° Φ rt μ- rt iQ rt tr O φ CU 3 3" CU 3 > H μ- Φ φ Ω Cυ μ- Ό H -J _^ Cυ ^* Ω

O M H- Cu Hi Ω to H- Φ o 3 3 !x! O 3 Ω t-¹ co "< O Cυ rt H o\o CO O Cu

3 O Ω o H- ιQ H> O rt Φ Ό μ- o 1 o Ω Hi H tr D Cυ rt CO

T3 rr 3 rt Ω H CU s: 3^J Φ o Hi l-S H 3 H Φ 3 H 3" μ- Cυ φ O 3 Φ ri a) Φ Φ O Ω CO 3" H H o ιQ Cu rt Φ o ^ μ- Φ 3 H" rt H -2 H c» Cυ

O 3 O. l-J i-s Hi rt H- Cu Φ H CU Ω CQ Hi 3 co φ 1 μ- 3 O H¹ X Ω

H- H- H O Ω Ω U3 3 Φ CO 0 cυ Φ rt Ω *_* Cu o O SO <J o\o tr rt

3 Ω H- 3 3 O CO H- 3^* rt X Φ Φ μ- H¹ μ- Φ K Ω Hi μ- μ- CO 3 H <JD Φ μ- μ-

Ω 3 3 Φ 3 Φ 3 H- t 3 co H 3 o_ μ- 0 3 H¹ T3 Φ rυ o\o i-s rt Ω <!

Φ 3 Hi • rt μ- Φ C 3 0 rt α H μ- Φ 1 CU o • O tr μ-

Ό O vQ J 3^* H- Ω CO 3 H rt 3^{^} CO 3 3 _^ CU H Hi H υ μ- rt tr" Cu o H Hi Cu Φ rt ¹ > ^•o s: Cu ^{^} Φ φ Cu Φ 3 Ό fJO cu ^ i s: H l-h 3 •< l-S H •< 3" t-J μ- Cυ tr • tr" CU ιQ Cu h-¹ 0_ Φ Cυ Cπ H

CU tr rt Ω c rt Ό _^ Ω C Φ μ- ω O 3 cu 1 3 μ- 3 υ 3 H o\° Φ O

3 μ- μ- rt tr Φ Ω H- H O CO Ω Cu C Ω tr" H μ- 3 μ- rt tr rt rt Hi

H- Ω Ω r u l-j rt Ω Φ CU μ- σ 3^J Ω Ω *< (ϊ- 1 Φ 3 φ 3 Φ υ μ- μ- υ

3 tr 3 Φ Ω O O 3 CO 3 Φ 3 • rt O W μ- φ μ^j φ *_* o CO <! Ω Ω *a rt rt

O H rt H CO Φ 0- CO ιQ Cu μ- 3 3 l-S ^ Ω •» Φ Cu 3 Cυ tr

H- Cυ ιQ Φ Φ CU 3 μ- l-S <! 3 ιQ μ- CO μ- H" Cυ rt H H Φ

CU O i Φ H CU *% H rt O Hi CU 3 φ μ- Φ Φ ≤ u μ- 3 t-f 1 Ω O rt Cυ rt φ

Ω 3 μ- 3 Ό o Ω rt Ω 3 3" 3 . Φ 1 rt μ- tr rt H μ- Cu rt

H- *- rt C Cu 3 tr¹ H- rt M ^•ϋ l-S Ω ^ rt Φ μ- s: φ *_* Ω t Cυ Φ O Ω CO H α a 3^* CO _^ Cu 1 3 H- O Φ O μ- Ω tr ^■ i-s O •<: C rt U

CO o Φ <! O u rt u . O O υ 3^J μ- tr¹ co Φ Ό u rt 3

• s: O H- rt rt *< Φ 3 Φ CQ φ l-h 3 H Ω ir¹ 1 rt o Ω l-S Ω - Ό Cυ ~J CO

3 O O 3 tr O CO 3 Cu Ω 3 μ- α Φ rt 3' 1 rt Φ 3 V O rt Φ H H o Ω

Ό ri Cu O H- rt M O rt 3 O 3^J rt *< μ- μ- o Ω μ- Φ o\° l-S cu Φ Ό 3 Φ 3 H- H¹ H ιQ cr 3 φ Φ Φ μ- i-S l-S 3 3 O Φ <! T3 Hi >< μ-

3 Ω 3 u O Φ O ^•<; H Ό • Φ CO 3 3 ^■ o Φ φ φ CO μ- O Φ rt O

0 H- Φ H l-¹ »_* 3 Φ H rt Ω 3¹ 3 Ό CQ *» 3 CO rt |_. H t> Q rt

3 Φ σ rt • 3 Ω CO O 3" rt O i Cυ C Ό rt μ- •<: ^ CU H μ- iQ CO (D H- o H, Ό o Ό 3 Φ 3^J H μ- 3 Ω cυ o 3 t-¹ HI Hi Hi J tr Φ OD O

Ω Ω Hi H H g O o Φ tr Ω h-¹ i-s 13 φ 1 1 l-S o 3 Φ Hi O 3

Φ rt 3 3¹ Cu o O ΓJ¹ 3 rt 3 3 Φ Φ Φ rt tr <J O o l-S Φ O • Φ o\o

X Φ H Φ CO 3 <! H- α Φ C- O O α 0 μ- Cυ Cu φ 3 3 rt H i-S

^•α H Cu ^< CO H- 3 μ- t-J 3 H rt Ω 3 H¹ t-s rt rt μ- CU Cu Φ

Φ H- l-j φ μ- 3 & Φ rt μ- C μ- μ- rt tr μ- rt tr sQ

H 3 O CO Φ 3 uQ O Φ 3¹ μ- o. X-- u 3 3 tr φ O Φ C r 3 O Cu ^•* CO v-3 H l-S Φ 3 φ υ 3 φ Φ Φ 3 (-^{■ >}

CO Hi 3 H *_{* "}« Φ Φ CU

O o 0_ Cυ Cu rt tr Cu Hi CO rt O Φ 3 rt H

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

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806

Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC17965 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaricatum ATCC14020

and L-lysine-producing mutants or strains prepared therefrom, such as, for example

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

Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM5715.

The inventors have succeeded in isolating the new oxyR gene of C. glutamicum which codes for the transcription regulator OxyR.

To isolate the oxyR gene or also other genes of C. glutamicum, a gene library of this microorganism is first set up in Escherichia coli (E. coli) . The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by Winnacker: Gene und Klone, Eine Einfϋhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known gene library is that of the E. coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50, 495 -508 (1987)). Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA,

84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575).

Bδrmann et al. (Molecular Microbiology 6(3), 317-326) (1992)) in turn describe_ a gene library of C. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). To prepare a gene library of C. glutamicum in E. coli it is also possible to use plasmids such as pBR322 (Bolivar, Life Science^'s, 25, 807-818 (1979)) or pϋC9 (Viera et al., 1982, Gene, 19:259-268). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination-defective. An example of these is the strain DH5αmcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments _ cloned with the aid of cosmids can in turn be subcloned in the usual vectors suitable for sequencing and then sequenced, as is described e.g. by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).

The resulting DNA sequences can then be investigated with known algorithms or sequence analysis programs, such as e.g. that of Staden (Nucleic Acids Research 14, 217-232 (1986)), that of Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG program of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).

• The new DNA sequence of C. glutamicum which codes for the oxyR gene and which, as SEQ ID No. 1, is a constituent of the present invention has been found in this manner. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by O CO ro ro H> cπ o cπ - o Cπ O Cπ tr Hi 3 o Hi •d μ- a tr Hi Ω ? Ό α Hi o Φ Ω tr rt α O Hi o co rt

<< 3 3 H o 3 o • 3 Cυ 3 I-S O 3 Hi X O >< tr Φ O O Hi rt φ tr tr CO Ω μ- o X-¹ <! • 3 3 o O H Ω 3 tr Φ ιQ H Hi Λ φ

H rt H¹ i-2 3 Φ rt Ω 3 X rt 3 rt CU tr CO H Φ μ- 3 μ- 3 μ- H Φ o 3 tr rt O 3 Φ O tr CO CU rt μ- μ- 3 3 ^■ rt o Φ 3

3 O 3 CO φ rt Φ μ- rt μ- rt Φ Ό 3 μ- o_ 3 φ iQ H¹ tr 3 φ μ- Ω ct 3 M H μ- o o rt 3 H cυ A rt H- < i-s 3 φ ? Ω rt

N rt P- Ω Cu O i-s Φ 3 CO tr 3 H Φ 3 tM Φ Cu σ φ 3 Φ tr

Cυ μ- CO 3 X 3 Cυ Φ o rt CO Φ Φ 3 Ω a rt a o o rt o Φ Φ H Φ Ό O rt tr rt μ- Cu H μ- 3 rt *< tr 1 X O a.

H- 3 CO 0 D Cu Φ tr CO φ Ω rt S μ- μ- rt 3 Hi O

0 O rt Ω ro H S Φ rt Ω φ O μ- 0 0 ω 0 Φ

3 μ- 2 tr μ- rt rt l-S CU tr rt ? Cυ 3 O rt 3 Hi φ 3 i-s rt rt a

Hi o_ O Cυ 3 co *_* φ 3 Cυ 0 3 Ω Ω l-h tr • Λ μ- 3 tr tr Φ

Ω O φ μ- Cυ o rt 3 Cυ o μ- tr ft 3 3 μ- Cυ Φ O

Cυ H CQ 3 H O μ- Hi μ- «£) H Cυ O- rt O H tr Φ φ 3 rt Ω

3 H» H¹ Hi 3 • cυ Φ Φ 3 Cυ tr ro 3 Φ 3 Cυ o H μ- rt H ^< rt H¹ CO Hi Hi O Φ ιO Ω o H¹ Φ X μ. tr ^> Cυ φ co Φ H O 3 Cυ O rt ιQ Φ Hi 3 ^<< σ

Φ φ Ό H Cυ H H 3 O Hi 3 3 H Φ μ- H tr φ O Cu N to Φ

3 μ- Φ Ω α Hi 3 o 3 D Φ 3 rt 3 D-

Hi ft Ω rt a Cυ o μ- 3 Cυ 3 ιQ sQ <! Φ X tr μ- 3 iQ o μ- Cυ Cu μ- HI i-s 3 rt Φ 3 ιQ M • Φ a CO rt tr Φ 3 Φ φ CU

3 Hl o α μ- 3 • 3^* 3 Φ 3 3 o cu μ- μ- 0 CO 3 tr

3 •<: I-¹ Ω 3 CD Cυ Cυ u Φ cϊ- 3 Φ rt Φ <-r . 3 Ω Ω ts Φ o o- μ- ^ O Φ a rt μ- H rt X Cυ X μ- φ tr H Cu Φ <!

3 3 _*—. Λ o μ- l-S a Cυ Cυ Ό 3 Ω o Ω o Ω 3 t3 Φ tr A tr CQ ro 3 . μ- o I-¹ Φ μ- tr 3 X O H rt μ- C H •

* Cυ ft O Φ 3 3 o Cu l-S Ω Cυ o Cυ Φ Φ O O

D <1 μ- •5 3 H¹ H 3 Hi Ω rt 3 i-s ^< Φ O μ- ιQ H rt a φ rt Ω ω μ- α 3 tr co Cυ tQ o 3 3 3 Φ 3 tr tr 3 Φ Cu Φ 3 Ω \ 3 cu Ω Φ o TJ Cυ -¹ φ 3 Ω Φ φ Cυ Φ 3 CO H 3 Cu o Ω 3 Cυ μ- 3 Cu α H rt Hi rt O rt O 3 CO Φ 1 rt 3 h rt ιQ CO 0_ O O H a Φ o tr 3 H φ φ rt μ- X ω tr μ- Φ Hi φ rt ^ Hi H μ- CO μ- Φ

X Λ Φ 3 tr Cu Cυ μ- Φ o O z μ- H O Cυ H 3 o CQ CO

Ό 3 3 O iQ μ- CO co < 3 μ- . CO 3 ιQ < O o Φ 3 μ- 3

Φ Φ iQ Hi Ω Ω CO Φ rt • 3 Φ H Cυ O φ O 3 μ- 3 ώ H"

H 3 rt TJ tr 0 Cυ Ω 3 Φ 3 Ό ^> rt Hi Λ O • 3 tr rt rt Ω tr rt H 3 rt o H H rt CO H Ω μ- 3 co φ rt 0 μ-

-» Φ tr μ- Cυ O 3 O 3 rt tr Φ O μ- <! O Φ υ ro tr X 3

CO o Φ 3 l-S rt Φ rt rt μ- φ rt 3 φ ro 3. to o Φ 3 ιQ μ- Hi φ Φ μ- rt Φ Cu 3 μ- 3 Φ φ ιO Ω Ω H

3 tr μ- i rt X tr 3 o Cυ 3 μ- cυ Φ O tr μ- Cυ rt *< Cu 3 CO Ό 3 Cυ rt μ- O Ω rt 3 H, 3 H O 3 D o 3 3

Φ rt <! ι-s Φ H¹ X-" Hi rt υ CO O μ- σ O CQ μ- l-S 3 Φ X φ 3 • Ω μ- O 3 μ- rt •• H 3 ≤ rt a rt CO 3 φ 3 t *σ rt Cυ N Hi H <! μ- o a tr μ- o M o

Cυ Cυ Φ rt μ- Cυ . — . 3 Φ rt μ- o Cυ CU o μ- rt Ω

H 3 Cu μ- Ω H O Cυ tr rt 3 H Cυ • Ω 3 Cυ Cυ μ- CQ O o tr φ Hi O σ rt φ •<; CO Φ Cυ Ω tr Φ (-> 3 H Ω u rt 3 3 Φ tr O H s 3 μ- H¹ 3 D μ-

O • H rt H φ l-S 3 o μ- rt tr CO α

Hi Φ tr tr 3 H) O O Hi X 3 Cυ ^• T3 a μ- π CO CO ^ Φ Cυ o rt H tr Φ l-S O o

3 3 3 3 Φ Φ ft μ- Φ Hi rt μ- Ω rt 3 μ- tr Ω O tr rt tr rt 3" O 0_ 3 φ tr H Cυ Φ ro Φ Hi

10

the handbook "The DIG System Users Guide for Filter Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260) . The hybridization takes place under stringent conditions, that is to say only hybrids in which the probe and target sequence, i.e. the polynucleotides treated with the probe, are at least 70 % identical are formed. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out under a relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996) .

A 5x SSC buffer at a temperature of approx. 50 - 68°C, for example, can be employed for the hybridization reaction. Probes can also hybridize' here with polynucleotides which are less than 70 % identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2x SSC and subsequently 0.5x SSC (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approx. 50 - 68°C being established. It is optionally possible to lower the salt concentration to O.lx SSC. Polynucleotide fragments which are, for example, at least 70 % or at least 80 % or at least 90 % to 95 % identical to the sequence of the probe employed can be isolated by increasing the hybridization temperature stepwise in steps of approx. 1 - 2°C. Further instructions on hybridization are obtainable on the market in the form of so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558). CO CO ro ro

Cπ o Cπ o Cπ O cπ en ro O ro μ- m μ- H Ω Cu Φ cu X ιQ tr 3 M Hi TJ μ- X rt Ω Hi o Cu H O o rt Cυ o Φ Ω Cυ 3 CQ H¹ 3 3 3 Ω X 3 μ- Φ *< 1 μ- Φ 0 3 tr tr Φ O o <J Ω rt Λ" X H* tr μ- 3 μ- tr rt 3 Ό rt O H tr 13 13 rt 3 ro Λ" i-s CO μ- Φ ιQ H Φ μ- Cu Hi μ- Φ CO

3 X Φ ro Ω 00 Φ rt rt μ- H H tr Φ 13 a φ 3 O rt Ω 3 H Cυ l-S tr O ιQ rt l-> O Cυ 3 μ- tr -J H H 3 φ φ μ- CO l-S > s: φ μ- tr tr CO Φ Ω 1 CO cυ Φ H o Φ o x-i ro Ω l-S rt μ- ^— 3 t-s <! O Hi Cυ Φ μ- 3 σ Φ rt Cυ CO tr φ O 3 α 3 X Hi 3

C tr N $ "< — ' Cυ Ω Φ φ CO μ- O <! O rt H Cu i-s rt TJ μ- X μ- 3 TJ Ω

1 Φ Φ o Cυ Cυ . H^J rt P_ μ- Φ <i H Φ ro Φ Cυ Φ CO l-S 3 μ- O Φ TJ μ- tr CO Ω Φ rt rt μ- l-S TJ 3 H- μ- TJ o x Cu 3 3 rt u φ Ω O 3 <i i-s 3 φ Ω K tr μ-

CO Φ 3 Cυ μ- Cu o H tr 3 H ιQ rt H μ- μ- rt 3 rt 3 Φ Φ φ & « Φ rt Φ o ro Cυ Ω O 3 3 3 0 *< μ- ^ Φ μ- rt 3 < o Φ μ- 3 μ- O TJ 3 φ o 3

Φ 3 μ- O Ω 0 3 μ- 3 3 3^* 13 Φ 3 H H 3 u CO Cυ ι-s rt TJ O

. — . rt P_ Hi Φ μ- Φ Ω Hi 3 Φ vQ Φ l-S μ- s: Ω Cυ Φ iQ 3 μ- H Hi H¹ μ- μ- o h-¹ μ- rt s 3 3 μ- tr rt ιQ r. O tr¹ 3 Cυ O h-> ιQ o rt O < D 3 Hi so Cυ ro Ω o Φ 3 3 cu rt tr Ω rt 3 <! 1 Ω •<; H μ- Q o 3 μ- 3 Φ 00 Φ rt o so 3= Cυ cυ l-S H S H 3 tr Φ 3 O tr O Φ H l-S TJ ιQ o Φ <1 Ω 3 H J^. Φ 3 li

J-- • tr rt H" μ- H Cυ rt Φ ιQ φ 3 3 φ H α •<: φ O Φ 3 3 φ O 3 α H — CO H Φ

— μ- • 3 Φ l-S tr • μ- Ω 3 CO Φ CO Cυ ω H 3 Φ l-S Hi H¹ Cυ ^ l-S Cυ

— _^— _* φ o Cu H rt μ- 3 ιQ ¹ o rt α tr μ- CO > Cu Φ O O 1 Cυ rt ιQ Cυ 3 Cu CU

*. 3

H 3 ,—> iQ O μ- O ιQ Φ H Φ H Φ ^ 3 Φ rt i-s φ rt l-S tr •^ 3 rt -¹ CQ TJ

TJ O Cυ 3 3 o H 3 ιQ ft φ μ- Φ Ω Ω X tr u α 3' μ- φ H μ- TJ . — . o Φ Φ Ω rt Φ 3 Ω l-S tr 3 rt 3 x Cυ rt CU TJ φ Hi rt tc Φ Cυ μ-

3 ω 3 -— . rt Φ O 3" CO o O rt cυ Φ Φ T tr 3 tr 3 N 1 Φ μ- CO Ω Hi μ- μ- α-> Φ ω rt 3 Φ CO Hi CO P_ Cu H Φ 3 3 Φ φ o H" Ω μ- 3 μ- tr μ-

H" Φ o μ- Cυ rt μ- o Cu Φ CQ O Ω TJ σ tr O X ^ O Ω

\ ro α co μ- cυ

Cυ H> o H" Cu Φ Ω 3 3 Ω Ω rt 3 3 Φ μ- CO Φ H φ CO • CO H Φ a • • 3 μ- cυ ω ι-3 o \ • H psj O φ O Cu μ- N H 3 X σ rt μ- μ- ro μ- " H" φ 3 rt

Cu u Φ 00 ro H3 • rt 3 Λ • 13 3 o Φ Ω TJ l-¹ H 3 tr μ- o 3 3 tr s: > rt μ- ii 3 Ω en * φ TJ 3 μ- 3 3 CQ rt H Φ Φ 3 o 3 3 iQ φ Φ Φ rt tr ri o i-i tr <o Ω Q —. Ω o Φ > φ Φ Φ μ- Φ υ rt O Ω ^ Φ Hi i-s o ro φ Φ 3

Φ 3 -> O t Φ ω Cu O CO }-• O μ- 0 rt 0 CO TJ 3 Cυ 0 H 3 O U3 3 i-i cυ ro o CO 3 3 μ- 3 μ- rt rv rt Hi Cυ 0 3 CO l-S rt 3 Φ rt Φ μ- f-S ^ Cυ tr Ω o

Φ 3 μ- 1 O Φ 0 rt μ- φ tr Ω • μ- O O Φ φ Cυ tr • 3 3 Cu Ω Cυ rt Hi rt <! o 3 SO \ tr μ- o H O Φ rt rt TJ o 3 Hi O φ Q 3 rt 3 μ- μ- ιQ 00 o H¹ m φ o 3 3 H H tr μ- H HI 3 o tr Φ Cu σ Φ P- μ- o O

Cυ l-S ^■< α <-α CO Φ 3 CU φ <! O ^* rt rt μ- 3 3 Cυ H Ω tr 3 a o O -— ^ 00 Ω Hi Ω rt Ω σ μ- H Φ μ- Φ tr . H 3 3 Ω 3 CD Cυ o

. >

3 kO H tr O o Cu H- Cu Φ Φ rt O 3 H Φ X g μ- rt cu H O ^" .—»

3 ^ ro SO en 3 3 Hi 3 < 3 3 "< 3 Φ O TJ μ- o - 3 Φ 3 cυ ? >τ) O

_^-, Φ u SO CO o 3 Φ 13 N iQ X rt ^ CO H 3 H φ TJ l-S • tr Ω φ

C-| 3 00 rt H¹ cπ H" CL. rt Hi tr H 1 μ- TJ tr rt Φ ιQ H H μ- ^ Cu TJ tr ro Λ

O rt .£-. Φ — ' 4i» 1 o 3" 3 ^< Φ φ o rt N Φ μ- H CO rt O cυ 3 TJ •<: 3

3 Cυ 1 3 — ro >fe 1-3 tr Φ l-S *« CO φ ^* Φ rt 3 CO O 3^* O H Φ

H H 00 rt _^ CO H¹ •<: ^ rt μ- φ Cυ Φ CO Ω Ω μ- μ- Φ Hi Φ TJ U3 o o Ω 3

3 -J 1 3 tr Cυ 3 3 13 CO O μ- rt o rt P- i-s li⁾ ro CU Cu cu Ω

Cυ S μ- —_* Cπ rt Φ φ 3 rt rt l-S O H μ- 3 o 3 3 Φ TJ Ω O 0-» o Ω μ- 3 Φ μ- —- 3 CO ^•* tr P_ H Φ 0 O μ- 0 h H H O 3 ro tr rt O

Ω (-> σi o <£> φ μ- 3 o ιQ μ- r Hi 3 O cυ Cυ Ω 3 o TJ cυ 3 . .. tr

O i-S ιχ> o ro υa ΪD o < H 3 Φ μ- Φ φ H" Cυ TJ 3 μ- 3 Ω , — _* — Φ X

Hi o <£> H¹ 3 — .£-. CO Φ l-S Φ Cυ H- 3 μ- P- O O o Φ 3 Φ O M μ- tr H» *•_* H H» " — -J X Cu Φ H rt Ό 3 Ω O o O μ- vQ CO rt rt CO Φ TJ ro Hi rt μ- — 00 0 <£> - — 1 TJ 3 1 Φ H • ι-s Hi Hi rt Φ Φ H Φ υ φ H" o tr o ^ SO TJ 00 H» Φ 0_ tr Ω- Cυ φ μ- 3 rt Φ h O 3 3

CO φ 00 i-s Φ CO Hi Cυ ft o Φ rt Cu Hi CU μ- rt ro 3 rt o μ- •> Cυ ^•~-^ en rt rt Cυ 3 tr O 3^* 3 Φ 3 Cυ Hi 3 l-S H t iQ 3 3 — tr 3 μ- φ Φ Φ cu Cu CO 3 rt rt O 3 Φ Φ

•< μ- Φ CL α X-¹ Ω o 0_ tr Φ 3 CO tr

3 CO rt Hi φ l-S CO • :

^•<:

12

Bacteriology 175, 1001-1007 (1993)), in Patent Application WO 96/15246, in Malumbres et al. (Gene 134, 15 - 24 (1993) ) , in Japanese Laid-Open Specification JP-A-10- 229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides

(Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.

By way of example, for enhancement the oxyR gene- according to the invention was over-expressed with the aid of episomal plasmids. Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as e.g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl . (Eikmanns et al., Gene 102:93-98 (1991)) or^' pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBLl or pGAl . Other plasmid vectors, such as e.g. those based on pCG4 (US-A 4,489,160) or pNG2. (Serwold-Davis et al., FEMS Microbiology Letters 66, 119- 124 (1990)) or pAGl (US-A 5,158,891), can be used in the same manner.

An example of a plasmid with the aid of which the oxyR gene can be over-expressed is the E. coli-C. glutamicum shuttle vector pT-oxyRexp. It contains the replication region rep of the plasmid pGAl including the replication effector per (US-A- 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline resistance- imparting tetA(Z) gene of the plasmid pAGl (US-A- 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with accession number AF121000, the replication origin oriV of the plasmid pMBl (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979)), the lacZα gene fragment including the lac promoter and a "multiple cloning site" (mcs) (Norrander, J.M. et al . Gene 26, 101-106 13

(1983)) and the mob region of the plasmid RP4 (Simon et al.,(1983) Bio/Technology 1:784-791).

The plasmid pT-oxyRexp is shown in figure 2.

Plasmid vectors which are furthermore suitable are also those with the aid of which the process of gene amplification by integration into the chromosome can be used, as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994) ) for duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum. Possible vectors are, for example, pSUP301. (Simon et al., Bio/Technology 1, 784-791 (1983)), pKlδmob or pK19mob (Schafer et al.., Gene 145, 69- 73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), PCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5,487,993), pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al,,1986, Gene 41: 337-342). The plasmid vector which contains the gene to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described, for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross over" event, the resulting strain contains at least two copies of the gene in question.

In addition, it may be advantageous for the production of amino acids, in particular L-lysine, to enhance one or more 14

enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the pentose phosphate cycle, of the citric acid cycle or of amino acid export and optionally regulatory proteins, in addition to the oxyR gene.

Thus, for example, for the preparation of amino acids, in particular L-lysine, one or more genes chosen from the group consisting of

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

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

• the tpi gene which codes for triose phosphate isomerase ^' (Eikmanns (1992). Journal of Bacteriology 174:6076-6086),

• the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns (1992). Journal of Bacteriology 174:6076-6086),

• the pyc gene which codes for pyruvate carboxylase (Peters-Wendisch et al. (Microbiology 144, 915 - 927 (1998)),

• the lysC gene which codes for a feed back resistant aspartate kinase (EP-B-0387527; EP-A-0699759;

WO 00/63388)

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

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

• the zwf gene which codes for glucose 6-phosphate dehydrogenase (JP-A-09224661) , 15

• the gnd gene which codes for 6-phosphogluconate dehydrogenase (US: 09/531,265),

• the sod gene which codes for superoxide dismutase (US: 09/373,731),

• the zwal gene which codes for the Zwal protein (DE: 199 59 328.0, DSM 13115)

can be enhanced, in particular over-expressed.

It may furthermore be advantageous for the production of amino acids, in particular L-lysine, in addition to the enhancement of the oxyR gene, for one or more 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: 199 51,975.7, DSM 13114) ,

• the zwa2 gene which codes for the Zwa2 protein (DE: 199 59,327.2, DSM 13113)

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

The term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme (protein) , and optionally combining these measures. 16

In addition to over-expression of the oxyR gene it may furthermore be advantageous, for the production of amino acids, in particular L-lysine, to eliminate undesirable side reactions, (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds . ) , Academic Press, London, UK, 1982) .

The microorganisms prepared according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of amino acids, in particular L-lysine. A summary of known culture methods is described in the textbook by Ch iel (Bioprozesstechnik 1. Einfiihrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) ) .

The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981).

Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty ^'acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substance can be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep CO CO ro ro cπ o cπ o cπ O cπ

O Hi Hi s 3 3 Ω ιQ 3 rt Cυ a Φ μ- CO tr Cd α rt 3 CO Φ CO s: 3 Hi Ω ex ro tr 3 TJ CO H" tr O H φ CQ 3 cπ 3 υ υ O o • 3 3 >< cυ 3 tr Φ 3 3 3 tr Φ tr O μ- tr φ O tr 3 μ- φ H O rt 3 rt o CO μ- CO 3 iQ Ω O ti Φ o_ μ- TJ σ μ- liΦ 3 TJ o Φ o Ω Λ

3 3 tr Cυ μ- O rt 3 rt Φ rt Cu tr H μ- μ- μ- rt CO Ω eu rt o CO 3 ex O tr 3 μ- Φ o X~^> *» 3 3 rt tr H o Ω 3 Ω 3 Cu o rt tr H¹ Ω Cu rt TJ CO TJ o co X rt PL H li μ- cu Φ Φ O Hi CQ cu X ιQ 3 3 tr ^>< Cυ CO 3 μ- Cu tr Φ Cυ tr cu H ct (D tr O •< u υ Φ X μ- Ω μ^j CU 3 O μ- Ω μ^j H" φ 3 cυ "_* 3 CO o ex CO Cυ CO *_* ii 3 φ 3 • rt 3 3 rt rt μ- α 0 rt rt Φ ex Ω ii rt μ- CO li rt

^■<: J Hi ii 3 ex 3 φ μ- rt rt rt TJ Φ 3 tr 3 Hi Φ φ CO 3 3 μ- μ- μ- rt Φ Cu CO

*_* H J o Φ Cu m H Cυ P_ <J tr • : Cυ tr TJ φ li tr J μ- CO 3 li ιQ 3 Ω 3 tr 3 0

Φ ri i Cυ X TJ tr Φ Φ μ- Φ Φ tr o o Φ Φ H 3 *_* 3 Ω Φ 3 ex φ ^<<

CO H- Ω μ- H Φ CO li 3 CU H CO cυ 3 a Φ φ tr CO cυ μ- CU o cυ o O rt tr 3 φ O 3 Cυ P_ Ω Φ TJ g 3 3 μ- CO Ω cυ CO Ω 3 Cu tr Ω <i CO 3 3

Cu ii tr Φ 3 Hi rt tr Ω Φ μ- tr H 3 rt 3 ex 3 φ Cu φ *< μ- μ- o 3 μ- tr

CO Φ ex 3 Φ Φ O μ- rt rt < o_ 3 0 o CO rt Cu H ex Ω CO CO ex rt ex ex 3 o 3 Φ

_~. Cυ l-S 3 3 Ω O μ- Φ Cu H 3 _"•. 3 rt li O μ- tr CO μ- CO H 3 li 3 3 cu

H» ex H ex s o Cυ TJ Ω O TJ 3 μ- μ- H tr rt O rt . Cυ φ 3 o Cu Ω μ- 3

SO Φ rt φ μ- Hi σ Φ tr Ω 3 3 O 0 3 Ω cu CQ Φ φ μ- li μ- Cu li • 3 Ω ιQ TJ Φ 3 CO

Cπ CO • rt rt li o cu *» TJ Φ 3 3 CO o CO ^ ιQ Cυ φ o H 3 3 Hi oo Ω Φ tr H¹ *< Cu Cυ 3 μ- 3 ^ H Cυ o Ω μ- Hi iQ 3 . 3 3 3 rt • 0 H¹ li m H μ- • rt O ex 3 CQ Φ iQ Ω H tr 3 O Ω Cυ Hi 3 cu Hi Ω Hi

_^ o μ- tr 3 3 O ro 3 μ- rt 3 3 H" rt μ- H CO Cυ rt - 3 o 3 O tr TJ co o CU CU 3 tr φ H- μ- Cπ H Φ rt Cυ Ω rt ^<< O P_ Cυ cu CU 3 3 3 o μ- H cυ rt Φ tr CO H 3 H rt li

H¹ Φ 3 3 Φ • μ- μ- tr Ω Λ CO tr 3 iQ o μ- μ- tr Φ

H» cx cυ cυ o Φ π iQ o 3 O O Ω o 3 CO o CQ 3 rt O ιQ 3 Hi 3 CO 3 Cυ rt o * Cu

VD 3 rt O • 3 u Hi H O H Φ CO 3 Hi liO tr ti Φ 3 CO TJ 3 CO H 3 3

O tr Cυ μ- " tr rt Hi CO rt CQ 3 o O μ- eu li φ cυ O CO li Φ tr O CU Cu ex

— ^ H" O O cυ O Cυ •» tr Hi Φ rt CO 3 rt Cu 3 Φ Ω X μ- rt ex cυ ex Cu sQ rt 3

><: 3 3 O rt μ- Φ φ O CQ H 3 O μ- cυ Ω O Cu μ- rt 3 tr rt μ- Φ Φ 3 tr CO co H 4-» 3" H o • Cu rt O H¹ 3 tr CO Φ <! tr cx tr 3 Φ eυ φ tr 3 3 O H

*< TJ μ- O CO Hi o Φ X CO ιQ 3 Φ H Hi Cu 3 M H- CO Φ o CO . H CO >< μ- Cυ 3 Φ

CU Cfl Hi o * rt • H 3 g Φ 3 H <! CO 3 o ex X 3 μ- CU μ- Ω rt ii O Ω Cu iQ CU CO rt H 3 tr iQ 3 Φ Cu ro 3 O rt i H rt m ex 3 _" o _ Ω H" O 3 • 3 H Φ tr υ O tr μ- 0 " 3 r-¹ φ tr 1 3 μ- ti tr 0 3 tr 3

3 3 cu 1 Φ H Φ 3 μ- 3 3 Φ Ω 3 cu Φ 3 Φ 3 ex 3 Hi Φ cp rt iQ H Φ Cυ o

CU 3 Cυ H> ex o rt rt μ- Ω H- li 3 rt φ cu CU tr φ Φ 3 Ω H

Φ 3 3 en • 3 3 μ- O H- μ- rt Cu TJ cυ Cυ o 3 tr μ- Cυ 3 <! rt Ω CO Φ 3 . . ω 3 tr

X rt μ- o H 3 H rt tr Cu 3 tc Ω *< X Φ Cu O a rt μ- Φ o o o O X-¹ μ-

Ω Φ tr 3 m rt Φ rt *< μ- σ • μ- μ- H rt 3 CX H- rt ^ 3 3 Ω TJ 3 3 o 3 tr rt 3 O tr tr 3 li O 0 H tr o α Ω Φ φ o Cυ O TJ H o tr ii μ- H 0 cυ CO o μ- fi μ- O X O rt Φ Φ > _^ ii Φ tr Q. CX 3 3 H li Ω H o Ω 3 μ- H

3 Cυ cυ 3 co μ- o ex Hi μ- 3 _^ Φ μ- Φ μ- φ ri O Φ 3 α iQ iQ H tr Ω H 3 3 iQ Ω CO Φ rt Ω Cυ Ω rt 3 CO H- co Φ TJ CO Φ CU φ Φ μ- CO rt ιQ 3 Ω Φ TJ CO 3 3 μ- Cυ Ω TJ o Cu O CO CO H Φ 0 CO tr 3 3 ex • cu CO Φ 3 H _^ tr TJ Hi 3 μ- o ii 3 CO φ O Hi J cυ O μ- μ-

Ω — . Ω CO H μ- 3 CX 1 CU CO h-* O P_ rt rt 3 3 3 CO o rt Hi rt CU Ω tr Cυ ι3 co u Ω CO Ω rt o Cυ tr Cυ Ω tr tr tr Ω rt Cυ TJ 3 φ H 9

H 3 H cυ Φ μ- O 3 CU Cυ ^•<: 3 Φ Ω CO CU Φ φ O cυ μ- CO H" tr ex 3 Cυ 3 Ω o Cυ H H rt Ω 3 3 μ- 3 3 Φ CO O CO 3 rt 3 cυ 3 rt o μ- o μ- rt O O

3 μ- Φ o ^> rt rt ex Ω Φ 3 μ- cu Ω Cυ co O 3 H rt Φ 3

CU μ- 3

^■< Φ 3 O CU CO tr Φ 3 TJ 3 tr a. 3 3 tr Hi TJ "3 li *> μ- TJ rt rt ex Ϊ co rt ro μ- • φ CO rt CO TJ o 3 Φ o_ Ω φ ιQ Cυ O 3 O 3 o

O μ- 3 μ- o rt 3 0 3 μ^j 3 Φ rt Φ H ft Hi o O iQ Ω 3 3 sQ Ω o O 3 o tr μ- Hi Cυ 3^* Ω O 3 Hi a. 3 CO O Φ H o Φ Cu 3

H Cu 3 s: 3 O Φ 3 o Q. Cυ tr * Φ ii . X _^ 3 ex 3 3 ex

Cu H¹ rt 3 Φ iQ a. <i Φ CQ rt Φ rt CO μ- O J •»» CX rt φ μ- Cυ o. o tr . 3 Ω tr *_* tr 0 o_ 3 CO μ- 3 Cu Φ t< »Q 3 1 3

18

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) .

A pure culture of the strain Corynebacterium glutamicum

DSM5715/pT-oxyRexp was deposited on 17th April 2000 as DSM 13457 at the Deutsche Sammlung fiir Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.

A pure culture of the strain Escherichia coli DH5α/pEC- T18mob2 was deposited on 20th January 2000 as DSM 13244 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.

The process according to the invention is used for the fermentative preparation of amino acids, in particular L-lysine ._

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. CO ro ro H> H» o Cπ o Cπ O cπ

H TJ li- H¹ ro o ro TJ tr¹ ro D rt α ro m TJ o ro X CO Hi a < D X D CO co X n o ro ro

H- tr Cυ <£> o μ- X Cυ μ- tr a H φ CU tr 3" <o li μ- 3 H Cυ Φ Φ ro Φ Φ eυ CO eυ tr o H X tr cu K¹ 00 H sΩ. rt Ω iQ cυ > Φ O 3 Φ o >fc. φ rt TJ o rt Ω CO o H CO 3 • • CO H ii Φ Cu

H iQ Φ 00 cυ H ΪV cu H Cυ Ω ffi CO CO μ- tr Φ 3 μ- rt Ω Ω Φ Ω CO o •< J

Cυ 3 φ 3 μ- TJ cu Φ CO 3 cu rt ii H Ω TJ 1 tr H o O H tr H to cn μ- 3 3 cυ TJ li 3 cu Ω ex φ cυ 3 Φ μ- 0 tr o 3 rt O CO 3 li μ- Φ ex μ- H CO co o Φ H

^ O 3 a Hi Ω rt Ω ex ex J _*— . O Cu ro ii tr o rt Cυ TJ φ TJ 1 o O σ Cu φ

3 TJ 3 Φ μ- μ- rt to 3 rt- - — iQ φ CO h CO rt o TJ rt _^^ H> o Cu ft s: CO Ω Ω -— . 3 o Cυ rt μ- μ- 3 μ- Cu ^ Cυ 3 μ- μ- tr μ- to -J cυ 3 Ω μ- H¹ φ TJ μ- rt X-A O o 3" 3 o Φ ex CO cυ H rt > TJ o cu o o 3 so rt Cυ ft o

H Φ 3 φ H- H rt TJ ex Φ 3 H Φ 3 Q φ O CU Ω Φ 3 iQ CQ 3 Φ — Φ H¹ φ 3 φ 3 μ- O H tr Φ ro rt CO D ex Φ Q o ^Q Cυ H 3 TJ H ii

CQ I-¹ Ω 3 X CU cυ H tr tr to tr a h rt CQ Φ α o CO o tr co CO cu D μ- O

Ω μ- o Hi rt iQ 3 Φ cu μ- Cu cu 3 ii 3 3 φ 0 CO o eυ tr 3 Cυ a 3 Hi

CU o > O Cυ Φ o μ- rt CO 3 3 μ- Cυ μ- μ- Φ 3 CO ro rt H 3 CU ex CO 3 li 3 3 Ω Hi n_) iQ CO . tr Ω tα X ? 3 Ω ^ μ- *< CO 3 CU

H • § μ- cu Φ ro 3 ^r 3 H ro Cu φ > rt Ω I-¹ > TJ ex Hi iQ μ- ex rt Ω 3 X -J li cu tr CO X ^ μ- < t-ι o , — . o CO Cυ HI ro Cu φ ti iQ

Φ m 2 tr ?r Φ μ- 1 iQ X 3 Cυ μ- o Φ Cυ Hi S! 0 rt ^ tr H CO o 3 Φ

CX tr iQ ro Φ μ- rt o ^ Cυ 3 o H rt co ro 3 Ω cu ex Q Φ cυ rt Ω 3 li3

Φ CO Φ 3 tr 00 CO Φ o 3 tr φ H rt C- CO tr Φ ex o ii H eu O

O O CO ro iQ tr⁴ -J Q li ex Cυ φ o Φ o O Ω Q¹ o 3 ^ p. O 3 3

3 μ- Φ • Cυ rt o Φ rt Φ Ω 3 α 3 ti H μ- 3 tc X cu tr o μ- μ- rt 3 Cυ ro tr 1 H H X μ- Φ 3 N φ φ o •_"* μ- Φ Ω Ω Φ h Ω Ω

Hi Cυ H Ω X -l φ o Ω TJ Ω "<; 3 3 Φ eυ 3 eυ « CU 3 rt . rt μ- H¹ ex •< 3

Cu Φ Ω O rt o d-» CU Cυ CO 0 H tr rt 3 μ- Ω ex s tr 3 Cυ Φ 3 3 Ω to Ω tr H H H CU — 3 rt • Φ 0 Φ rt φ Cυ H> eυ o ^• cυ σ φ O rt μ- cu H μ- .

3 • Φ 3 ro Cυ CO D G CO -J 3 CO . <i ^ tr CQ ex μ- H¹ Ω cυ ex *_* ex μ- ro li <! TJ Φ X CO . Cn 3 tr ro φ CU m 3

Φ O μ- cn O rt _^ Hi X -j Φ Φ tr CO X σ n > σ CO tr H ro H m Ω o μ- O 3 • • rt «_* o tr ro X Φ 1 μ- ex o Ω Cυ o •_" ω o Φ μ- -~ Φ cu rt o α

Ω φ H> li α Hi Φ H μ- ex o σ li H H to H> cπ μ- 3 1 μ- X 3 Φ li CU ex π cu O CO 0 rt 00 3 s: *< μ- Φ ro V£⁾ o

.— _* 3 TJ o tr μ- Ω H H> U3 μ- 3 en μ- o s: > cn ex tr X en H μ- TJ H 00 CO — •^ SO 3 rt tr μ- CO φ tr ex CO 3 ex •^ μ- μ- 3 μ- CO iQ rt Cu rt to 3 O J-> -J . Cu H¹ H tr 3 O 3

Φ μ- 1 Φ iQ sΩ, Ω m rt 1 •_* tr rt μ- o ex .. — Q H* CO iQ Φ 3 CO φ ex rt rt μ¹ a ro Cu CU rt ^ tr o Φ o Φ . 3 ro HI Φ f 1 _* rt rt- ro μ- tr cπ s ^' 3 ti TJ rt Λ. o rt CX 3 ti Ω H> T3 tr . ti Cυ O tr iQ \-> tr rt -j Cπ o o Cυ p. α σ rt — φ tr O ro rt cn li Φ 3 to o Φ cυ μ-

Φ cυ Cπ cπ . ex Ω o Φ li φ X X tr cπ o O CU μ- • — φ H 3 3 a tr • tr i — J-. 3 rT 3 CO Φ 3 p. tr eυ H¹ α 1 Ω- D 3 3 • li H . rt- H

CO μ- ro o Ω m Cυ rt Cυ 3 CO Φ ro Φ a ^ Φ 3 φ cυ Cυ

Cu 3 Cu rt _^-~. o rt H 3 li H rt tr 3 Φ tr H o CO H¹ Φ > ,_"• Hi Cu CO ,— 3 ri

3 sQ tr ro o HI μ- μ- p. H φ •< CO ^ ro H¹ Ω cn ex TJ tr 3 rt H¹ μ- ^ ^* μ- Φ Cυ ro σ X TJ A Φ rt O tr — - li „-- μ- o ro tr φ • H lO Ω

H O H¹ Φ X rt ft CU CU Ω li tr 3 Hi

0 Hi 3 n cu μ- — ^• 3 Hi H o ^ μ- O

Ω Φ ~j CO HI 3 μ- CO rt o μ- H 0 o r o H X TJ 2 o CO D Ω π 3 H

O φ μ- — Ω H o φ Φ O H Ω μ- rt ex 3 Cυ rt CO rt CX TJ a ro rt 0 - ft H ιQ H ro Φ 3 ex 3 0 rt 3 Φ cu CO μ- o tr 3 tr > ii μ- 3 tr I-¹ tr p. CU . — μ- ex μ- TJ Ω o tr o Φ Ω Cυ 0 O ro m

Φ φ O CQ TJ Ω X Hi t? ex 3 o 3 μ- Ω 3 rt Hi rt rt Hi ex 3 rt Hi φ rt Cυ .t H Ω 3 Cu o cυ H Cυ Ω Cυ K 3 cu o

Ω - μ- CO 1 Φ rt ft s: rt μ o σ rt x-> . Φ μ- o CO eυ Ω Φ CO

Cu O Φ tr o 3 o H o a Φ ? Cυ 3 tr CO Φ iQ ft 3

CO iQ 3 H¹ li eυ 3 rt Φ a O H¹ > 3 cu ro <! Φ φ 3 3 N μ- CO

• 3 Φ \-> tr > 3" CO N -j Φ

• ex μ- Φ ^ ex O μ- Φ 1 Cυ o "<| p. 1 ex ex 3 3 CO ex 3 3 CO 3 rt^¬ Φ to ro φ Φ

20

(1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , the cells being plated out on LB agar (Lennox, 1955, Virology, 1:190) with 100 mg/1 ampicillin. After incubation overnight at 37°C, recombinant individual clones were selected.

Example 2

Isolation and sequencing of the oxyR gene

The cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02) . The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) . After separation by gel electrophoresis, the cosmid fragments in the size range of 1500 to 2000 bp were isolated with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .

The DNA of the sequencing vector pZero-1, obtained from Invitrogen (Groningen, Holland, Product Description Zero Background Cloning Kit, Product No. K2500-01) , was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04) . The ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany) . This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 21

87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 zeocin.

The plasmid preparation of the recombinant clones was carried out with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) . The sequencing was carried out by the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067). The "RR dRhodamin Terminator Cycle Sequencing Kit" from PE Applied Biosyst^'ems (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 analysis was prepared with the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231).

The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence showed an open reading frame of 981 base pairs, which was called the oxyR gene. The oxyR gene codes for a protein of 327 amino acids. 22

Example 3

Preparation of a shuttle vector pT-oxyRexp for enhancement of the oxyR gene in C. glutamicum

3.1. Cloning of the oxyR 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 oxyR 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) .

OxyR (oxy-exp) :

5 GAT CGA GAA TTC AAA GGA AGA TCA GCT TAG 3^V

OxyR (oxy R2) :

5^V GGA AAA CCT CTA GAA AAA CT 3 The primers shown were synthesized by ARK Scientific GmbH Biosystems (Darmstadt, Germany), and the PCR reaction was carried out by the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) with Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim, Germany) . With the aid of the polymerase chain reaction, the primers allow amplification of a DNA fragment approx. 1.43 kb in size, which carries the oxyR gene. Furthermore, the primer OxyR (oxy-exp) contains the sequence for the cleavage site of the restriction endonuclease EcoRI, and the primer OxyR (oxy R2) the cleavage site of the restriction endonuclease Xbal, which are marked by underlining in the nucleotide sequence shown above .

The amplified DNA fragment of approx. 1.43 kb which carries the oxyR gene was ligated with the Zero Blunt™ Kit of

Invitrogen Corporation (Carlsbad, CA, USA; Catalogue Number K2700-20) in den vector pCR®Blunt II (Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)). The 23'

E. coli strain- ToplO (Grant et al., Proceedings of the National^' Academy of Sciences USA, 87 (1990) 4645-4649) was then transformed with the ligation batch in accordance with the instructions of the manufacturer . of the kit (Invitrogen Corporation, Carlsbad, CA, USA) . Selection for plas id- carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al., Molecular cloning: A Laboratory Manual. 2^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 25 mg/1 kanamycin. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany) and checked by treatment with the restriction enzyme Xbal and EcoRI with subsequent agarose gel electrophoresis (0.8 %) . The DNA sequence of the amplified DNA fragment was checked by sequencing. The plasmid was called pCR-oxyRexp. The strain was called E. coli ToplO / pCR-oxyRexp.

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

The E. coli - C. glutamicum shuttle vector was constructed according to the prior art. The vector contains the replication region reg of the plasmid pGAl including the replication effector per (US-A- 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline resistance-imparting tetA(Z) gene of the plasmid pAGl (US-A- 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with the accession number AF121000), the replication region oriV of the plasmid pMBl (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979)), the lacZα gene fragment including, the lac... promoter and a multiple cloning site'(mcs) (Norrander, J.M. et al. Gene 26, 101-106 (1983)) and the mob region of the plasmid RP4 (Simon et al.,(1983) Bio/Technology 1:784-791). The vector constructed was transformed in the E. coli 24

strain DH5α (Hanahan, In: DNA cloning. A Practical Approach. Vol. I. IRL-Press, Oxford, Washington DC, USA). Selection of plasmid-carrying cells was carried out by plating out the transformation batch on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which had been supplemented with 5 mg/1 tetracycline. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and Hindlll and subsequent agarose gel electrophoresis (0.8 %) . The plasmid was called pEC-T18mob2 and is shown in figure 1.

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

The E. coli - C. glutamicum shuttle vector pEC-Tl8mob2 described in example 3.2 was used as the vector. DNA of this plasmid was cleaved completely with the restriction enzymes EcoRI and Xbal and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH,

Mannheim, Germany, Product Description SAP, Product No. 1758250) .

The oxyR gene was isolated from the plasmid pCR-oxyRexp described in example 3.1. by complete cleavage with the enzymes EcoRI and Xbal. The oxyR fragment approx. 1400bp in size was isolated from the agarose gel with the QiaExII.Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .

The oxyR fragment obtained in this manner was mixed with the prepared vector pEC-T18mob2 and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no. 27-0870-04) . The ligation batch was transformed in the E. coli strain DH5α (Hanahan, In: DNA cloning. A Practical 25

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

Example 4

Transformation of the strain DSM5715 with the plasmid pT- oxyRexp

The strain DSM5715 was transformed with the plasmid pT- oxyRexp using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989)). Selection of the transformants took place on LBHIS agar comprising 18.5 g/1 brain-heart infusion broth, 0.5 M sorbitol, 5 g/1 Bacto-tryptone, 2.5 g/1 Bacto-yeast extract, 5 g/1 NaCl and 18 g/1 Bacto-agar, which had been supplemented with 5 mg/1 tetracycline. Incubation was carried out for 2 days at 33°C.

Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915 -927), cleaved with the restriction endonucleases EcoRI and Xbal, and the plasmid was checked by subsequent agarose gel electrophoresis. The resulting strain was called DSM5715/pT-oxyRexp.

Claims

26Example 5Preparation of lysineThe C. glutamicum strain DSM5715/pT-oxyRexp obtained in example 4 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined.For this, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with tetracycline (5 mg/1)) for 24 hours a't 33°C. Starting from this agar plate culture, a pre-culture was seeded (10 ml medium in a 100 ml conical flask) . The complete medium Cglll was used as the medium for the pre-culture.Medium Cg IIINaCl 2.5 g/1Bacto-Peptone 10 g/1 ιBacto-Yeast extract 10 g/1Glucose (autoclaved separately) 2 % (w/v)The pH was brought to pH 7.4Tetracycline (5 mg/1) was added to this. The pre-culture was incubated for 16 hours at 33°C at 240 rp on a shaking machine. A main culture was seeded from this pre-culture such that the initial OD (660 nm) of the main culture was 0.05. Medium MM was used for the main culture. 27Medium MMCSL (corn steep liquor) 5 g/1MOPS (morpholinopropanesulfonic 20 g/1 acid)Glucose (autoclaved separately) 50 g/1(NH4)2S04 25 g/1KH2P04 σ.l g/1MgS0 * 7 H20 1.0 g/1CaCl2 * -2 H20 10 mg/1FeS04 * 7 H20 10 mg/1MnS04 * H20 5.0mg/lBiotin (sterile-filtered) 0.3 mg/1Thiamine * HCl (sterile-filtered) 0.2 mg/1L-Leucine (sterile-filtered) 0.1 g/1CaC03 25 g/1The CSL, MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions were then added, as well as the CaC03 autoclaved in the dry state.Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Tetracycline (5 mg/1) was added. Culturing was carried out at 33°C and 80 % atmospheric humidity. 28After 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 129Brief Description of the Figures:Figure 1: Map of the plasmid pEC-Tlδmob2Figure 2 : Map of the plasmid pT-oxyRexpThe abbreviations and designations used have the following meaning:per: Gene for controlling the number of copies fromPGAl oriV: ColEl-similar origin from pMBl rep: Plasmid-coded replication region from C. glutamicum plasmid pGAlRP4mob: RP4 mobilization site lacZ-alpha: lacZ gene fragment from E. coliTet: Resistance gene for tetracycline oxyR: oxyR gene of C. glutamicumEcoRI : Cleavage site of the restriction enzyme EcoRIEcll36II: Cleavage site of the restriction enzymeEcll36IIHindlll: Cleavage site of the restriction enzyme HindlllKpnl: Cleavage site of the restriction enzyme KpnlSail: Cleavage site of the restriction enzyme SailSmal: Cleavage site of the restriction enzyme SmalPstl: Cleavage site of the restriction enzyme PstlBamHI : Cleavage site of the restriction enzyme BamHIXbal: Cleavage site of the restriction enzyme XbalXmal : Cleavage site of the restriction enzyme XmalXhol: Cleavage site of the restriction enzyme XholPstl: Cleavage site of the restriction enzyme Pstl 30What is claimed is :

1. An isolated polynucleotide from coryneform bacteria, 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) ,

the polypeptide preferably having the activity of the transcription regulator OxyR.

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

5. DNA as claimed in claim 2 which is capable of replication, comprising

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