EP1278862A2

EP1278862A2 - Nucleotide sequences coding for the cls gene

Info

Publication number: EP1278862A2
Application number: EP01933892A
Authority: EP
Inventors: Bettina Möckel; Walter Pfefferle; Madhavan Nampoothiri; Lothar Eggeling; Hermann Sahm
Original assignee: Forschungszentrum Juelich GmbH; Degussa GmbH
Current assignee: Forschungszentrum Juelich GmbH; Evonik Operations GmbH
Priority date: 2000-05-04
Filing date: 2001-04-26
Publication date: 2003-01-29
Also published as: AU2001260253A1; WO2001083767A3; KR20020097251A; DE10021826A1; WO2001083767A2

Abstract

This invention relates to a genetically modified coryneform bacterium, the cls gene of which is enhanced, and to an isolated polynucleotide, which codes for cardiolipin synthase from coryneform bacteria and to a process for the fermentative production of L-amino acids with enhancement of the cls gene in the bacteria and to the use of the polynucleotide as a primer or hybridization probe.

Description

Novel nucleotide sequences coding for the els gene

The invention provides genetically modified coryneform bacteria, nucleotide sequences coding for cardiolipin synthase and a process for the fermentative production of amino acids, in particular L-glutamate, using coryneform bacteria, in which the els gene, which codes for cardiolipin synthase, is enhanced.

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

It is known that amino acids are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up to yield the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.

The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection.

For some years, methods of recombinant DNA technology have moreover been used to improve strains of Corynebacterium which produce amino acids by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production. Review articles on this subject may be found inter alia in Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and

Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).

The object of the present invention was to provide novel auxiliaries for the improved fermentative production of amino acids, in particular L-glutamate.

Amino acids, in particular L-glutamate, are used in human medicine, in animal nutrition, in the pharmaceuticals industry, and in particular in the foodstuffs industry. There is accordingly general interest in providing novel improved processes for the production of amino acids, in particular L-glutamate.

Any subsequent mention of L-glutamate or glutamate should be taken to mean not only the base, but also the salts thereof.

The invention provides a genetically modified coryneform bacterium, in which the els gene of which, which codes for cardiolipin synthase, is enhanced.

In this connection, the term "enhancement" describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are coded by the corresponding DNA.

Enhancement may be achieved by means of various manipulations of the bacterial cell.

Enhancement, in particular overexpression, may be achieved by increasing the copy number of the corresponding genes, by using a strong promoter or by mutating the promoter and regulation region or the ribosome-binding site located upstream from the structural gene. Expression cassettes incorporated upstream from the structural gene act in the o cπ o cπ

^>α τs ι-3 Hi Ό O OJ 3 O o Ω 3 O ua ^•σ ι-3 Ω CL Φ rt Φ ιQ 3 φ CO o o 3^J o OJ β cr Φ Ό o o μ- rt H - o Φ 3 O I-¹ Φ φ IX OJ -" H φ H H μ- μ- 3 φ H H Ω OJ β o Φ 3 ιQ N Φ 3 OJ Ό 3

* •< rt ct rt Ω > ^ H H Ω α O" H ι Φ <! Φ 3 H φ

3 3 Ό φ μ- OJ μ- μ- μ- 3 3 o Ω O β 3 μ- OJ j3 J O Φ c β H X Ω σ rt o φ Φ Φ o 3^* CO Ω μ- 3 α Φ r+ rt ≤ CO -3

Ω Ω Φ OJ β H ^ 3 CD tr Hh H Φ φ Ω φ OJ Φ Φ 3" O CO OJ

CO 3 Φ Φ 0J o ιQ *_* H α rt J 3 α μ- μ- 3

Φ Φ Φ Ό OJ rt CL Ω l-i OJ Ω tr¹ o μ- Ω Q. Ω o 3

O O 3 μ^j H CD o o rt 3 3 Φ O 1 o OJ o rt OJ 3^* μ- 3 Φ rt ft rt Dd ω to O O O o φ rt N, Φ μ- H β OJ H rt 3 μ- rt Ω 3 4 μ- H- H H n O O O O O o H T3 s: ^ H σ o H" Ω 3 ιQ <J 3' rt Ω P- • α H- Φ φ Φ H H n H l-i rt H) OJ H 3' b μ- OJ 3 β H μ- OJ * o μ- Φ μ- O β φ Φ 3 < <! <! *< > ^ • •« 3^* μ- o μ- φ β Ω CO O 3 3 μ- Hi rt <l α Ω H H

<! H- H- μ- 3 3 3 3 3 Φ rt 3 Ω tr 3 rt O CO o μ- >< μ- μ- φ μ- μ- rt

CO Mi Φ cr σ cr Φ Φ Φ Φ Φ 3- O β 3' O Φ 3 CO Φ ω H¹ rt μ- rt CO σ 3 φ l-i 3 OJ OJ OJ σ cr tr cr σ ? Φ Ω 0 H J Φ ^ OJ 3 • ^ μ- Hi ^ ιQ μ-

Λ o rt Ω Ω Ω 0⁾ OJ 0J 0J 3 o Φ μ- rt S μ- * Ω CO Φ co Φ Hi Φ ω β 3 H- rt rt rt Ω Ω Ω Ω Ω o O H) co φ μ- QJ O |__. μ- ^ rt O Hi

Φ o Φ Φ φ rt rr rt rt rt s: ^•υ P H rt Ω H OJ CL Φ 3 μ- M H •73 Φ OJ

3 Ω 3 l-i l-i H φ Φ φ Φ φ 3 φ rt 1 ? μ. 3^* μ- O Ω CO T3 3 o 3 tx H H

Ω o P- H- μ- H l-i H H H Ω 3" OJ 3 C μ- 3 3 Hi rt H N H φ T3 J O 3 α

Φ l-i 0⁾ β β β μ- μ- μ- μ- μ- s: μ- Φ 3 o 3 3 • H O o ^ Φ H 3 Φ μ-

» - 3 3 3 β β β 6 β μ- Φ μ- ≤ T3 H O CO μ- <! |3 o o Φ Ω o 3 rt

CO 3 CO 3 3 3 3 3 CO ιQ 3 3 ^Q rr OJ μ- 3 Φ 3 μ- Φ <! Hi ω O rr rt μ-

Φ Φ o H" H) Φ o (-. 3^* H ω ^ . • Φ O H Φ J 0 l-¹ Hi H- 0⁾ -¹ 3 rt 0J OJ ιQ 3 μ- c Φ rt Φ iQ ^•υ Φ H rt μ- H H rt 3 φ o Tf <! Ω OJ φ Ω Ω μ-¹ rt 0 β 0J 3 rt μ- I-¹ Hi OJ D. ^3 - o Φ CO H- OJ

Ω H H OJ rt ! r-^> Φ Φ φ β •< CO o 0) vQ Ω H •<: H H 3¹ OJ Φ 3 CO • < H rt 3 o H O β OJ ι-i rt rt rt Ό ^ μ- O 3 Φ β φ Ω β rt σ Φ Ω 13 Φ

Φ H- l-h 3 D 3 O 0 OJ φ a n α Ό μ. 3 - ^•σ φ Ω μ- CO 3^* 3 μ- O H • α σ H- Ω Φ en O OJ iQ 3 (D o CO 0 β OJ H l-i rt Ω Φ μ- » co 3 rt H"

OJ α 0⁾ l-i φ OJ Ω μ- CD tr H . Ω c CO H φ O 0 β rt φ a CL 1 •ϋ l-h Ω Φ rt 3 ι-3 Ω 3 μ- β Ω rt 0) ^< μ- 3 CO CO - 3 <! OJ μ- μ- iQ o

H rt CO β Φ O O μ- rt β l-i o 3 OJ O o φ Φ 0⁾ φ φ φ l-¹ 3 co H ω

O Φ 3 3 O i-¹ 3 O OJ 3 OJ rt φ μ- 3 o Hi 3 OJ ^ H OJ α. O ιQ β CO

3 H OJ rt μ» OJ o * 3 μ- Φ σ μ- OJ l-i rt 3 3 CO 0 J rt μ-

H- 3 > β vQ t μ- ! 3 OJ co

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O o ι-3 O CO 3 I-¹ H μ- OJ iQ φ 3^* O β 3 H Φ H 3 Ό Φ rt iQ 3 - >fc> O I-¹ > ^Q μ- ι-i T( Ω Φ O OJ CO rt rt 3 Φ 0 Φ o o

H rt 0⁾ o Ω -J •Tl > 1-3 o β Ω J rt 3 3 Φ OJ J <J J <! CO o ⁽ ) M H π CO Ό

OJ rt > I-¹ c 3 M H Φ β O O ^ 3 μ- •< Φ < Φ 3' O H μ- β H- φ o ω CTi !Λ o M rt φ rt H CO Hi 0⁾.

00 CJI 3 3 ¹ D 2 o OJ μ- o 3

• 3 a 0J CO μ- μ- • σ> H> CP 3 rt <! 0 rt < σ α Ω

H- 3 μ- Ω β rt o 03 CO o Φ Φ Ό μ- μ- σ μ- H β H

3 μ. 3 β ^fτ) 00 O 3 ^{^} ι-3 *_* 3 rt 3 3 • 3 Φ Ω Φ lΩ Ω H¹ Φ 3^" OJ rt μ- vQ Ω ιQ rt OJ

0⁾ 1 -J σ> o c l-i 0J Φ O Hi μ- o H 3 rt μ- O p⁾ 3 o l-i rt O H o 3 φ Φ J

Oi o o Φ μ- 3^* φ ω o 3 OJ OJ J 3 3 rt σ Φ CO 3 CO CO β ft CO φ φ β CO cr 3 H Φ > Φ 0⁾ μ- φ

•< σ 3 CO OJ Φ

a) polynucleotide which is at least 70% homologous to a polynucleotide which codes for a polypeptide containing the amino acid sequence of SEQ ID no. 2,

b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% homologous 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 containing at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c) .

For the purposes of the present application, a polynucleotide sequence is "homologous" to the sequence according to the invention if the base composition and sequence thereof at least 70%, preferably at least 80%, particularly preferably at least 90% matches the sequence according to the invention. According to the present invention, a "homologous protein" should be taken to mean proteins which have an amino acid sequence which at least 70%, preferably at least 80%, particularly preferably at least 90% matches the amino acid sequence which is coded by the els gene (SEQ ID no. 1), wherein "matching" should be taken to mean that the corresponding amino acids are either identical or comprise mutually homologous amino acids. "Homologous amino acids" are those having corresponding properties, in particular with regard to charge, hydrophobicity, steric properties etc..

The invention moreover provides a polynucleotide as described above, wherein it preferably comprises replicable DNA containing:

(i) the nucleotide sequence shown in SEQ ID no. 1, or (ii) at least one sequence which corresponds to the sequence (i) within the degeneration range of the genetic code, or

(iii) at least one sequence which hybridizes with the complementary sequence to sequence (i) or (ii) and optionally

(iv) functionally neutral mutations in (i) which give rise to the same or a homologous amino acid.

The invention also provides

a replicable polynucleotide which comprises or consists of the nucleotide sequence SEQ ID no. 1,

a polynucleotide which codes for a polypeptide which comprises or consists of the amino acid sequence SEQ ID no. 2,

a vector containing the DNA sequence of C. glutamicum which codes for the els gene, contained in the vector (plas id) pJClcls, deposited in Corynebacterium glutamicum under the number DSM 13250,

and coryneform bacteria acting as host cell which contain the vector or in which the els gene is enhanced.

The invention also provides polynucleotides which contain the complete gene with the polynucleotide sequence according to SEQ ID no. 1 or fragments thereof and which are obtainable by screening by means of hybridization of a suitable gene library with a probe which contains the sequence of the stated polynucleotide according to SEQ ID no. 1 or a fragment thereof and isolation of the stated DNA sequence.

Polynucleotide sequences according to the invention are also suitable as hybridization probes for RNA, cDNA and DNA in order to isolate full length cDNA which code for cardiolipin synthase and to isolate such cDNA or genes, which exhibit a high level of similarity with the sequence of the cardiolipin synthase gene.

Polynucleotide sequences according to the invention are furthermore suitable as primers for the polymerase chain reaction (PCR) for the production of DNA which codes for cardiolipin synthase.

Such oligonucleotides acting as probes or primers may contain more than 30, preferably up to 30, particularly preferably up to 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides having a length of at least 40 or 50 nucleotides are also suitable.

"Isolated" means separated from its natural environment.

"Polynucleotide" generally relates to polyribonucleotides and polydeoxyribonueleotides, wherein the RNA or DNA may be unmodified or modified.

"Polypeptides" are taken to mean peptides or proteins which contain two or more amino acids connected by peptide bonds.

The polypeptides according to the invention include a polypeptide according to SEQ ID no. 2, in particular those having the biological activity of cardiolipin synthase and also those, which are at least 70%, preferably at least 80%, homologous to the polypeptide according to SEQ ID no. 2 and in particular which exhibit 90% to 95% homology to the polypeptide according to SEQ ID no. 2 and exhibit the stated activity.

The invention moreover relates to a process for the fermentative production of amino acids, in particular L-glutamate, using coryneform bacteria, which in particular already produce an amino acid and in which the nucleotide sequences which code for the els gene are enhanced, in particular overexpressed. The present invention presents for the first time the els gene of C. glutamicum which codes for cardiolipin synthase.

The els gene or also other genes from C. glutamicum are isolated by initially constructing a gene library of this microorganism in E. coli . The construction of gene libraries is described in generally known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker, Gene und Klone, Eine Einfϋhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al., Molecular Cloning, A

Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) . One very well known gene library is that of E. coli K-12 strain W3110, which was constructed by Kohara et al. (Cell 50, 495-508 (1987)) in λ-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was constructed using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bόrmann et al. (Molecular Microbiology 6(3), 317-326, 1992)) also describe a gene library of C. glutamicum ATCC 13032, using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). A gene library of C. glutamicum in E. coli may also be produced using plasmids such as pBR322 (Bolivar, Life

Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are in particular those E. coli strains with restriction and recombination defects. One example of such a strain 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 assistance of cosmids may then in turn be sub-cloned in usual vectors suitable for sequencing and then be sequenced, as described, for example, in Sanger et al. (Proceedings of the National ω t t

Cn o n σ cn o cn

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Ω OJ O co ιQ o φ o h CO 3 Ω 3 rt φ 13 φ Φ μ- Ω β O Cn α

H rt H H fl μ- Φ \ _*—. _^-> H Φ rt iQ Hi 0⁾ μ- μ- Φ 3 OJ 3 3 3 H Ω Φ Ω 3 tj-» Φ

O μ- D Φ 13 3 3 ι-3 Tl OJ C 3 o OJ Φ β 3 OJ 3 μ- rt H rt Φ 13 O μ- φ CO 3 H 0 < ι -3

O * Λ H ιQ Φ Φ H 3 O OJ Hi 3 O 3 Φ H Φ 3 μ- rt μ- H φ cr α φ rt O < CO ^<

3 3 *_* 3 β O rt Ω O β rt • rt Ω 3 μ- CO o CO O 0⁾ D iQ Φ Λ φ 1

O o Φ <! μ- μ- 3^* rt φ H μ- μ- rt rt rt O O _^ 3 3 Ω a β CL Hi β μ- iQ H Cn o

O • 3 μ- 3 Ω 3 Φ rt 3 o ι-3 OJ O μ- OJ rt l-i . 0 • *< Φ H Φ 3 Φ it-. Hi

T3 a Ω O o μ- OJ 3 3^* O I-¹ Hi Hi 3 OJ o 3 <! 3 D cn μ» O μ» Φ Φ OJ 3 OJ I-¹ Φ rt 3 Ω O O o σ O CO Ω cr 3 Ω Φ Φ a CΛ

JO CO a OJ o μ- *< 3¹ OJ Ω μ- Hi H o CQ a Hi φ Φ o Φ 3 _^ Ω

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H φ h s; σ O a Ω Hi Φ μ- OJ Ω OJ Φ CO lO rt β O Φ V o μ- 3 O μ> Φ

Φ β Λ φ f > H μ- rt H 3 a *< 3 fl X φ CO - Φ Hi • Φ Hi O φ D 3 O β μ- H 3 σi φ o Cd 3- CO 13 ιQ Φ 13 OJ H φ Φ 3 3 iJQ Ω

OJ μ- Φ CO Ω rt Φ O •• 3 φ 0⁾ μ- o OJ OJ 3 Φ O OJ 3 <! D iQ Ω rt CΛ J rt ^ β Φ

H 3 3 μ- t r ω μ- H» Ω 3 Ω co 3 μ- 3 Φ H 13 OJ β vQ Φ 3^J H σ 3¹ OJ Φ — CO ω ιQ Ω 3 Φ 13 Φ ω φ Φ rt H α β μ- OJ rt " rt 3 Φ Φ CO Φ o Φ s: CO 3 o Φ μ- £T O Ω IV) CO Φ Ω CO ^_^ rt 3 O μ- φ μ- 0 3 3 <! OJ Ω o

O O • μ- 3 β -J H o ?*r o O H Ω <! • Ω Φ OJ 13 μt φ Ω CO CΛ Φ Hi

T) OJ σ 3 H 1 ω ^I μ- 3 μ- H H OJ OJ rt Φ Φ rt H H D 0 pd

H μ- TS l-i μ- ft⁾ I-¹ • • • • 0 3 μ^j M Ω O OJ μ¹ ω μ- μ- O D H 0 O Hi rt o ιQ H l-¹ μ- Φ 3 H J > M ¹ Φ r-¹ 3 O • rt φ Ω Φ CO Ω co CL. 3 a l-i cr l-i 3^{^}

<i 0 o *< 3 iQ N) 4-» O o Ω Φ OJ μ- 3 μ- β OJ s: μ- β 0 > Φ rt H O Φ μ- 3 μ- rt cr Cπ O ιQ rt • rt <! CO CD cr H 3^* Ω 3 Ω • CO OJ α 3 β β Ό N μ- 3 μ- 1 1 *< μ- Φ rt μ- φ β CO φ μ- O iQ rt CD 13 μ- σ

Φ Ω Ω H Φ O OJ O ΓΌ N> o μ- Φ H rt Hi cr rr Ω CL ro Φ O 3 3 o 3

CL M Φ o 3 3 H μ> ■£-. n 3 3 < 3 μ- •< 3 O CD μ- CO 3^* Φ Hi 0 vQ 3 φ O μ-

Φ α <S € • 3 O VD ι < ι Φ μ- CQ β H rt rt μ- l-i Hi CO β CL rt cr O μ- μ- Φ ιQ 00 CD μ- rt 3 3 o rt μ- β 3 t OJ O 3- φ μ- 'Q Φ

•< rt cr rt H ^ 00 _^^ _^~~ • • 3 " β Hi Hi OJ ιQ rt rt μ- "< H 3 rt O 3 3 μ- -. α μ- *< Φ 3- • •—' μ» μ> -J rt Φ CO ω β rt t-¹ β μ- ^j cr Φ 3^{^} s: Ω iQ 3 _^ c r α Hi — CD U3 cn Φ rt l-i rt μ- β rt O OJ H CΛ Φ CO Φ rt Λ

3^* Φ rt CΛ H t_? <o 00 H OJ OJ o rt 3" O rt μ- 3 H μ- OJ M 13 rt μ- KU rt

Φ 3" σ M O 3 OJ *. 1 i σ Hi 3' Φ 3 OJ 0 O μ-¹ CL M lO Ω rt β H 3¹ CO B OJ

13 φ • 3 μ- 3 — _" ^^ OJ rt μ- Φ CO 3 3 ^ μ- O H 3^J CO O μ- μ- rt μ- l-i 3 a — — _' cn M OJ H 13 μ- o N O H CO Φ μ- rt CO 13 o Φ

3 μ- 13 rt CΛ o _^ ~J μ- € μ- 3 H _^ Ω O Hi Ό φ D 3 Φ H c

< 3 o 3- a M μ- 0⁾ μ- N 13 O O Hi H 13 iQ H iQ μ- 3 o 3 φ φ I-¹ Φ OJ 3 μ- μ- . — . " Φ H H rt s: OJ OJ o s: H 3 Φ Φ 3 0⁾ < O

3 H 3 Ω 3 3 μ» μ- o Φ Φ 3^* Ω ιQ 3 <! μ- O O 3 CO rt 3 μ- s: Hi rt CO j3 μ- o H μ- ? Ω 3 rt rt μ- μ- μ- μ- μ- rt <J . Φ β 3^* S 3 3" μ- φ 3 σ a 3 ffi CΛ CD Hi 3^* Φ ?T 3 Ω • 3 3" μ- . Φ OJ Φ Φ μ- to

O o H <! o o OJ ω μ- Φ μ- 3 _^ - ^_* Ω O Φ CL μ» rt CO H L Ω g

3 σ OJ Φ » 3 co Ω 3¹ — Φ 3 3 O μ- . CΛ φ OJ 3 . 3^* Φ

• rt CO 3 o φ 3 3^* μ- — 3 a Hi s: μ- H OJ 3 OJ M 3 Φ Hi cr H

OJ Φ rt o . Λ β 3 _^ 1 β CL 3 . Φ H Φ o σ O β rt rt β ι-3 ^ Ω μ-

CΛ μ- μ- H β rt 1 CO 3 O φ CO Φ μ- •< σ φ rr H 3" O Ω β 3 Ω 0 N) φ Φ μ- t-3 μ- 0⁾ Ω rt • β Hi H OJ o rt Φ rt CL OJ

Ω Φ 3^* 3 13 3 X O 3 CO rt 3 3" μ- Ϊ 0 CO rt o rt 3 CL 3- ^* Φ

3^* α OJ • OJ OJ Ω cf φ rt CO μ- O OJ rt rt 3 H ^* rt O μ- O Φ OJ Φ CO μ- H H Φ σ rt 3" OJ o rt rt 3- 0 μ- Φ 3 3- 3 l-i

Hi 3

3 rt Φ CO o rt 3 Φ μ- s 3 O φ rt 0 3 μ- Hi

H CO 0 OJ Φ : 3 3 • " O 3 O o - μ-- rt Φ Φ H O H

3 O CD " rt 3 μ- μ» Φ

Hi 0 3

oligonucleotides typically have a length of at least 15 nucleotides .

The person skilled in the art may find instructions for identifying DNA sequences by means of hybridization inter alia in the manual "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: 255-26Q) . The person skilled in the art may find instructions for amplifying DNA sequences using the polymerase chain reaction (PCR) inter alia in the manual by Gait, Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton & Graham, PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) .

During work on the present invention, it proved possible to establish that coryneform bacteria produce amino acids, in particular L-glutamate, in an improved manner once the els gene has been enhanced.

The genes or gene constructs under consideration may either be present in plasmids in a variable copy number or be integrated into the chromosome and enhanced. Alternatively, overexpression of the genes concerned may also be achieved by modifying the composition of the nutrient media and culture conditions.

The person skilled in the art will find guidance in this connection inter alia in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP 0 472 869, US patent 4,601,893, in Schwarzer and Pϋhler (Bio/Technology 9, 84-87 (1991)), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996) ) and in known textbooks of genetics and molecular biology.

By way of example, the els gene according to the invention was overexpressed with the assistance of plasmids.

Suitable plasmids are those which are replicated and expressed in coryneform bacteria. Numerous known plasmid vectors, such as for example 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 for example 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) may be used in the same manner.

One example of a plasmid by means of which the els gene may be overexpressed is pJClcls (Fig. 1), which is based on the E. coli-C. glutamicum shuttle vector pJCl (Cremer et al., 1990, Molecular and General Genetics 220: 478-480) and contains the DNA sequence of C. glutamicum which codes for the els gene. It is contained in the strain DSM5715/pJClcls.

Further suitable plasmid vectors are those with the assistance of which gene enhancement may be performed by integration into the chromosome, as has for example been described by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication or enhancement of the hom-thrB operon. In this method, the complete gene is cloned into a plasmid vector which can replicate in a host (typically E. coli) , but not in C. glutamicum. Vectors which may be considered are, for example, pSUP301 (Simon et al., Bio/Technology 1,. 784-791 (1983)), pK18mob or pK19mob (Schafer et al., Gene 145, 69- 73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5, 87, 993) , pCR®Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516). The plasmid vector which contains the gene to be enhanced is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The conjugation method is described, for example, in Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods are described, for example, in Thierbaeh 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 "crossing over", the resultant strain contains at least two copies of the gene in question.

It may additionally be advantageous for the production of amino acids, in particular L-glutamate, to amplify or overexpress not only the els gene, but also one or more enzymes of the particular biosynthetic pathway, of glycolysis, of anaplerotic metabolism, of the citric acid cycle or of amino acid export.

For the production of L-glutamate, for example, it is thus possible simultaneously to amplify, in particular overexpress or amplify, one or more genes selected from the group

• the gdh gene which codes for glutamate dehydrogenase (DE: 19907347.3) and/or • the.pyc gene which codes for pyruvate carboxylase (Peters-Wendisch et al. (1998), Microbiology 144: 915- 927) .

It may furthermore be advantageous for the production of L- glutamate, in addition to amplifying the els gene, simultaneously to attenuate

• the odhA gene which codes for -ketoglutarate dehydrogenase (WO 9534672 Al 951221*), or

• the dtsRl gene which codes for the DtsRl protein (WO 952324 Al 950831*), or

• the dtsR2 gene which codes for the DtsR2 protein (WO 9902692A Al 990121*) .

It may furthermore be advantageous for the production of amino acids, in particular L-glutamate, in addition to overexpressing the els gene, to suppress unwanted secondary reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982) .

For the purposes of amino acid production, in particular of L-glutamate, the microorganisms produced according to the invention may be cultured continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process. A summary of known culture methods is given in the textbook by Chmiel

(Bioprozeβtechnik 1. Einfϋhrung in die Bioverfahrensteehnik (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 adequately satisfy the requirements of the particular strains. Culture media for various microorganisms are described¹ in "Manual of Methods for General Bacteriology" from the American Society for Bacteriology (Washington D.C., USA, 1981).

Carbon sources which may be used are sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose for example, oils and fats, such as soya oil, sunflower oil, peanut oil and coconut oil for example, fatty acids, such as palmitic acid, stearic acid and linoleic acid for example, alcohols, such as glycerol and ethanol for example, and organic acids, such as acetic acid for example. These substances may be used individually or as a mixture.

Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture.

Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The culture medium must furthermore contain metal salts, such as for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. Suitable precursors may furthermore be added to the culture medium. The stated feed substances may be added to the culture as a single batch or be fed appropriately during culturing.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture. Foaming may be controlled by using antifoaming agents such as fatty acid polyglycol esters for example. Plasmid stability may be maintained by the addition to the medium of suitable selectively acting substances, for example antibiotics. Oxygen or oxygen-containing gas mixtures, such as air for example, are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20°C to 45°C and preferably from 25 °C to 40°C. The culture is continued until the maximum quantity of glutamate has formed. This objective is normally achieved within 10 hours to 160 hours.

The following microorganism has been deposited with Deutsche Sa mlung fur Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty:

• Corynebacterium glutamicum strain DSM5715/pJClcls as DSM 13250

The purpose of the process according to the invention is the fermentative production of amino acids, in particular L-glutamate.

The present invention is illustrated in greater detail by the following practical Examples.

Example 1

Production of a genomic cosmid gene library from Corynebacteri um glutamicum ATCC13032

Chromosomal DNA from Corynebacterium glutamicum ATCC13032 was isolated as described in Tauch et al., (1995, Plasmid 33:168-179) and partially 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 cosmid vector SuperCosl (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164), purchased 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 also dephosphorylated with ≤hrimp 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) . Cosmid DNA treated in this manner was mixed with the treated ATCC 13032 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 using Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, product description Gigapack II XL Packing Extract, code no. 200217) . E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Res. 16:1563-1575) was infected by suspending the cells in 10 mM MgS0 and mixing them with an aliquot of the phage suspension. The cosmid library was infected and titred as described in Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , the cells being plated out on LB agar (Lennox,

1955, Virology, 1:190) with 100 mg/1 of ampicillin. After overnight incubation at 37 °C, individual recombinant clones were selected.

Example 2

Isolation and sequencing of the els gene

Cosmid DNA from an individual colony was isolated in accordance with the manufacturer's instructions using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) and partially 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) . Once separated by gel eleetrophoresis, the cosmid fragments of a size of 1500 to 2000 bp were isolated using the QiaExII Gel Extraction Kit (product no. 20021, Qiagen, Hilden, Germany) . The DNA of the sequencing vector pZero-1 purchased from Invitrogen (Groningen, 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) . Ligation of the cosmid fragments into the sequencing vector pZero-1 was performed 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 into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy. of Sciences

U.S.A., 87:4645-4649) (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) and plated out onto LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 of Zeocin. Plasmids of the recombinant clones were prepared using the Biorobot 9600 (product no. 900200, Qiagen, Hilden, Germany) . Sequencing was performed using the dideoxy chain termination method according to Sanger et al. (1977, Proceedings of the National Academy of Sciences U.S.A., 74:5463-5467) as modified by 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. Separation by gel eleetrophoresis and analysis of the sequencing reaction was performed in a "Rotiphorese NF" acrylamide/bisacrylamide gel (29:1) (product no. A124.1, Roth, Karlsruhe, Germany) using the "ABI Prism 377" sequencer from PE Applied Biosystems ' (Weiterstadt, Germany) .

The resultant raw sequence data were then processed using the Staden software package (1986, Nucleic Acids Research, 14:217-231), version 97-0.. The individual sequences of the pZero 1 derivatives were assembled into a cohesive contig. Computer-aided coding range analysis was performed using XNIP software (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analysis was performed using the "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402), against the non-redundant database of the "National Center for Biotechnology Information" (NCBI, Bethesda, MD, USA) .

The resultant nucleotide sequence is stated in SEQ ID no. 1. Analysis of the nucleotide sequence revealed an open reading frame of 1502 base pairs, which was designated the els gene. The els gene codes for a protein of 500 amino acids (SEQ ID no. 2) .

Example 3

Cloning of the els gene into vector pJCl

Chromosomal DNA from Corynebacterium glutamicum ATCC13032 was isolated as described in Tauch et al., (1995, Plasmid 33:168-179). A DNA fragment bearing the els gene was amplified with the assistance of the polymerase chain reaction. The following primers were used for this purpose:

5^S-TGC TCT AGA CGG TAA GTC GGT CCC TCT AAA AG-3 ^v

5 -TGC TCT AGA CAA CCG GCG CCT CTG ACC AC -3^*

Both oligonucleotides bear the sequence for the cleavage site of the restriction enzyme Xbal (underlined nucleotides) . The stated primers were synthesized by the company MWG Biotech (Ebersberg, Germany) and the PCR reaction was performed in accordance with the standard PCR method of Innis et al. (PCR protocol. A guide to methods and applications, 1990, Academic Press) . The primers allow the 1610 bp DNA fragment which bears the els gene from Corynebacterium glutamicum to be enhanced.

Once separated by gel eleetrophoresis, the PCR fragment was isolated from the agarose gel using the QiaExII Gel Extraction Kit (product no. 20021, Qiagen, Hilden, Germany) .

The PCR fragment obtained in this manner was completely cleaved with the restriction enzyme Xbal. The approx. 1600 bp els fragment was isolated from the agarose gel using the QiaExII Gel Extraction Kit (product no. 20021, Qiagen, Hilden, Germany) .

The vector used was the E. coli-C. glutamicum shuttle vector pJCl (Cremer et al., 1990, Molecular and General Genetics 220: 478 - 480). This plasmid was also completely cleaved with the restriction enzyme Xbal and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, product no. 1758250) .

The els fragment obtained in this manner was mixed with the prepared pJCl vector 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 then transformed into E. coli strain DH5α (Hanahan, in: DNA cloning. A practical approach. Vol. I. IRL-Press, Oxford, Washington DC, USA). Plasmid-bearing cells were selected by plating the transformation batch out onto LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 of kanamycin. After overnight incubation at 37 °C, individual recombinant clones were selected. Plasmid DNA was isolated from a transformant in accordance with the manufacturer's instructions using the Qiaprep Spin Miniprep Kit (product no. 27106, Qiagen, Hilden, Germany) and cleaved with the restriction enzyme Xbal in order to check the plasmid by subsequent agarose gel eleetrophoresis. The resultant plasmid was named pJClcls.

Example 4

Transformation of strain ATCC13032 with plasmid pJClcls

Strain ATCC 13032 was then transformed with plasmid pJClcls using the electroporation method described by Liebl et al. (FEMS Microbiology Letters, 53:299-303 (1989)) Transformant selection proceeded on LBHIS agar consisting of 18.5 g/1 of brain-heart infusion bouillon, 0.5 M sorbitol, 5 g/1 of Bacto tryptone, 2.5 g/1 of Bacto yeast extract, 5 g/1 of NaCl and 18 g/1 of Bacto agar, which had been supplemented with 25 mg/1 of kanamycin. Incubation was performed for 2 days at 33°C.

Plasmid DNA was isolated from a transformant using the conventional methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915 - 927), cut with the restriction endonuclease Xbal and the plasmid to be checked by subsequent agarose gel eleetrophoresis. The resultant strain was named ATCC13032/pJClcls .

The following microorganism has been deposited with Deutsche Sammlung fur Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty:

• Corynejacterium glutamicum strain DSM 5715/pJClcls as DSM 13250

Example 5

Production of glutamate

The C. glutamicum strain ATCC13032/pJClcls obtained in Example 5 was cultured in a nutrient medium suitable for the production of glutamate and the glutamate content of the culture supernatant was determined.

To this end, the strain was initially incubated for 24 hours at 33°C on an agar plate with the appropriate antibiotic (brain/heart agar with kanamycin (50 mg/1)). Starting from this agar plate culture, a preculture was inoculated (10 ml of medium in a 100 ml Erlenmeyer flask) . The medium used for the preculture was complete medium Cglll (2.5 g/1 of NaCl, 10 g/1 of Bacto peptone, 10 g/1 of Bacto yeast extract, 20 g/1 of glucose, pH 7.4). Kanamycin (25 mg/1) was added to this medium. The preculture was incubated for 16 hours at 33°C on a shaker at 240 rpm. A main culture was inoculated from this preculture, such that the initial OD (660 nm) of the main culture was 0.1.

After preculturing in medium Cglll (Keilhauer et al . 1993, Journal of Bacteriology 175:5595-5603), strain ATCCl3032/pJClcls was cultured for the main culture in production medium CgXII (Keilhauer et al. 1993, Journal of Bacteriology 175:5595-5603). 4% of glucose and 50 mg/1 of kanamycin sulfate were added.

Culturing is performed in a volume of 10 ml in a 100 ml Erlenmeyer flask with flow spoilers. Kanamycin (25 mg/1) was added. Culturing was performed at 33°C and 80% atmospheric humidity.

In order to induce glutamate formation, 20 g of Tween 60

(P-1629 Sigma-Aldrich, Deisenhofen, Germany) plus 80 ml of water were mixed and autoclaved. Some 4 hours after inoculation, 75 μl of this Tween solution were added to the culture and culturing was continued.

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

Table 1 shows the result of the test.

Table 1

Brief Description of the Figure:

Figure 1: Map of the plasmid pJClcls

The abbreviations and names are defined as follows:

Orf2,rep: plasmid-coded replication origin, C. glutamicum (from pHM1519)

lacZ-alpha^s part of the 5 'end of the β-galactosidase gene

els: els (cardiolipin synthase) gene from C. glutamicum ATCC13032

BamHI : restriction site of the restriction enzyme

BamHI

Xbal: restriction site of the restriction enzyme

Xbal

Pstl: restriction site of the restriction enzyme

Pstl Sail: restriction site of the restriction enzyme

Sail

EcoRI : restriction site of the restriction enzyme

EcoRI

Claims

What is claimed is:

1. A genetically modified coryneform bacterium, wherein the els gene thereof, which codes for cardiolipin synthase, is enhanced.

2. The genetically modified coryneform bacterium as claimed in claim 1, wherein the starting bacterium

(wild type) is selected from the group 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) .

3. The genetically modified coryneform bacterium as claimed in claim 1 or 2, wherein the els gene is enhanced by overexpressing the gene, in particular by increasing the copy number of the gene, by selecting a strong promoter or a regulation region upstream from the reading frame, by mutating the promoter, the regulation region or the ribosome-binding site, by incorporating a suitable expression cassette upstream from the structural gene or by incorporating inducible promoters, by extending the lifetime of the corresponding mRNA, by reducing degradation of the expressed proteins, or by combining two or more of these possibilities.

4. The genetically modified coryneform bacterium as claimed in one of claims 1 to 3, wherein the strain is transformed with a plasmid vector and the plasmid vector bears the nucleotide sequence which codes for the els gene.

. The genetically modified coryneform bacterium as claimed in one of claims 1 to 4, wherein it corresponds genotypically to the strain Corynebacterium glutamicum DSM 13250.

6. An isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence selected from the group

a) polynucleotide which is at least 70% homologous to a polynucleotide which codes for a polypeptide which comprises or consists of the amino acid sequence of SEQ ID no. 2,

b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is at least 70% homologous to the amino acid sequence of SEQ ID no. 2,

c) polynucleotide which is complementary to the polynucleotides of a) or b) and

7. The polynucleotide as claimed in claim 6, wherein the polynucleotide is a preferably recombinant DNA replicable in coryneform bacteria.

8. The polynucleotide as claimed in claim 6, wherein the polynucleotide is an RNA.

9. The replicable DNA as claimed in claim 7, containing

(iv) functionally neutral mutations in (i) which give rise to homologous amino acids .

10. A polynucleotide sequence as claimed in claim 7, 8 or 9 which codes for a polypeptide which comprises has the amino acid sequence SEQ ID no. 2.

11. A process for the fermentative production of L-amino acids, wherein the following steps are performed:

a) fermentation of L-amino acid producing coryneform bacteria in which at least the els gene or nucleotide sequences coding therefor is/are enhanced, in particular overexpressed,

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

c) isolation of the L-amino acid.

12. The process as claimed in claim 11, wherein a strain as claimed in one of claims 1 to 5 is used.

13. The process as claimed in claim 11 or 12, wherein further genes, which code for a protein of the biosynthetic pathway of the desired L-amino acid, are enhanced in the bacteria.

14. The process as claimed in one of claims 11 to 13, wherein metabolic pathways which reduce the formation of the desired amino acid are at least partially suppressed in the bacteria.

15. The process as claimed in one of claims 11 to 14, wherein the amino acid produced is L-glutamate.

16. The process as claimed in one of claims 11 to 15, wherein bacteria are fermented for the production of glutamate in which one or more of the genes selected from the group

a) the gdh gene which codes for glutamate dehydrogenase, or

b) the pyc gene which codes for pyruvate carboxylase

is/are simultaneously enhanced, in particular overexpressed or amplified.

17. The process as claimed in one of claims 11 to 16, wherein bacteria are fermented for the production of L-glutamate in which one or more of the genes selected from the group

a) the odhA gene which codes for α-ketoglutarate dehydrogenase,

b) the dtsRl gene which codes for the DtsRl protein, or

c) the dtsR2 gene which codes for the DtsR2 protein

is/are simultaneously attenuated.

18. A use of polynucleotide sequences or parts thereof as claimed in claim 6 as primers for the production of DNA of genes which code for cardiolipin synthase by the polymerase chain reaction.

19. A use of polynucleotide sequences as claimed in claim 6 as hybridization probes for the isolation of cDNA or genes which exhibit elevated homology with the sequence of the els gene.