WO2002022827A1

WO2002022827A1 - Isolation and sequences of the ptsi gene of c. glutamicum

Info

Publication number: WO2002022827A1
Application number: PCT/EP2001/010072
Authority: WO
Inventors: Bettina Möckel; Stephan Hans; Natalie Schischka; Walter Pfefferle
Original assignee: Degussa Ag
Priority date: 2000-09-13
Filing date: 2001-08-31
Publication date: 2002-03-21
Also published as: DE60132341D1; US20030198991A1; DE60132341T2; DE10045496A1; AU2001289858A1; US6680187B2; US7160703B2; EP1317549A1; ATE383428T1; US20020132323A1; EP1317549B1

Abstract

The invention relates to the phosphotransferase system enzyme I of C. glutamicum and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the ptsI gene is present in enhanced form, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

Description

ISOLATION AND SEQUENCING OF THE PTSI GENE OF C . GLUTAMICUM

Field of the Invention

The invention provides nucleotide sequences from coryneform bacteria which code for the ptsl gene and a process for the fermentative preparation of amino acids using bacteria in which the endogenous ptsl gene is enhanced.

Prior Art

L-Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.

It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and produce amino acids are obtained in this manner.

Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.

Object of the Invention

The inventors had the object of providing new measures for improved fermentative preparation of amino acids.

Summary of the Invention

Where L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L- asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L- isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L- histidine, L-lysine, L-tryptophan and L-arginine. L-Lysine is particularly preferred.

When L-lysine or lysine are mentioned in the following, not only the bases but also the salts, such as e.g. lysine monohydrochloride or lysine sulfate, are meant by this.

The invention provides an isolated polynucleotide from coryneform bacteria, comprising .a polynucleotide sequence which codes for the ptsl 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 phosphotransferase system enzyme I.

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. 1, or

(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or

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

(iv) sense mutations of neutral function in (i) .

The invention also provides

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

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

a vector containing the polynucleotide according to the invention, in particular a shuttle vector or plasmid vector, and

coryneform bacteria which contain the vector or in which the endogenous ptsl gene is enhanced. C M M h-^r o cπ o Cπ o cπ

._ O α-» ω O H" Ω CO 13 T3 Cu rt TJ _ CU O 3 rt 3 Ω rt TJ CO Hi Cu s: Ω Ω o O H3

H cπ <S\ en Φ Φ O C- o tr μ- - o Φ 3^" H μ- e 3" d α " O Φ l-S Ω 3^ O o σ 3 3^J co o H- *_* H- CU Cu 9 Ω (-^■ o Φ rt μ- CO 3 Ω Φ Ω ^•z Φ 0-1 Cu Ω μ- 3 l-S rt cr Φ

O ιQ ιQ CO O Ό '≤ CO ^ Φ Ω o μ- H > ; C- ^■Q O Ω TJ H Cu Cfl

H O O iH- O o rt rt H 3 ^■o o μ- 3 H H Φ TJ Φ μ- 3 Φ 3 l-S 3* H Φ μ- rt μ-

H 3 -J σ> 3 H- o φ t Hi 3 C- 3 O O u μ- 3^ μ- cu 3 C- 3 φ r μ- CO 3 cυ 3 rt 3 3 ¹ ro CO H n o <1 Ω μ- C- H Ω 0 Ω 3 <J Ω Ω 3 μ- Ω co TJ Cu 3 <!

(D OJ o O cπ o Φ H- Cu rt ^■ Φ 3 Cu p. μ- CO Φ H Φ rt 3 O φ o cr rt φ o_ o H .C- CO H _^ iQ O H ¹ 3 ro φ l-S li- rt u ^•υ u 3 φ ! 3 O 3 H μ- 3 o φ CD ~J Φ CU O φ CU μ- ct o Cu * Ω 3^J Ω σ rt 0 3 rt TJ Or φ Cu rt o _^ o M rt H 3 Ω μ- rt eCu σ μ- o μ- s μ-- rt φ 3^* rt H rt μ- μ-

3 0 rt rt cn H> C- Ω CO t o μ- s' o α rt o μ- 3 Φ 0 μ- 3^J 3 cr 0 ω 3 H- ri=- CO H- _^ •*_* H^J Ω tr H) 3 Or Φ CO Φ ri CO H CO " 3 rt l-S CO Φ iQ •<: •<: 3 cu Ω α IT> CO Φ H u φ D Φ 3 CU Φ μ- Φ rt φ

3 M Φ *_* Φ μ> M Cu Φ H- H 2! Cu CO Ω Hi CO O 3 o μ- Cu CO o o 3^J CO Ω i-3 CO Ω Cu ω Φ CO O O -j r CO O 3 Cu H O O o φ l-S CO h 3 H 3 Hi Φ O Φ o 0 H

O H O •_* rt rt O Φ s: H H f-S tQ Hi Φ ≤: Φ _^ rt 3 3 H 3 O to rt s: <£> H- H φ O 3* H C- TJ Φ TJ o 3^J Or μ- 3^J TJ Φ Φ TJ o

CD H- H- Cπ H- rO ) α Φ Hi Hi μ- Φ -3 μ- φ o l-S o H CO μ- • CU 3 Φ Φ l-S

TJ rt o o piCO CO Cπ Φ CU CO C- Ω O o 3 3 μ^j Cu H Ω 3 <i Φ H 3 μ- TJ

CU Φ 3^J l-S s' *» O Ω ^< ιQ H 3* ^■υ s Ω Ω ^ CO ^ Φ μ- 3^J cx Φ CO rt μ- μ- o H ι-s O 3 o O rt CO Φ rt o 0 Φ 3 Φ 3 H rt 3 Φ Φ cr 3 Φ O

0J Cu 3 ιC- u H H N3 s: H- rt 3 3^J Ω 3 l-S C- C- Cu Ω μ- rt ι-3 H W <! rt DJ o o n - o Φ Φ Φ o a Ω Ό s: Ω CO Ω rt a- O O μ- d ιQ Cu u μ-

Φ H H *_* H » O μ- 3 3 CO H 3 μ- 3^* o μ- H ^ H o -~ 3 o O Φ Φ H cr Or

Φ Φ Φ Φ <£> *- Ω 3 ^■υ 3 μ- H rt Φ CO Φ φ TJ M 3 3 3 ^ ^ TJ Φ

3 o o 3 *_* NJ -r φ ^■ζ O H >--! T3 Cu O rt o μ- K u Ω Φ o CO

O o -Q rt H ιQ O -J T) 3 3^J l-S μ- CO rt rt Φ rt CO Cu μ- rt CU Φ o 3

3 TJ rt H- rt 3 <i _^ CO o N μ- Φ CO ^•σ - μ- rt μ- 3 μ- O CO ω H- Ω O Hi φ ^■υ rt rt 3" Cu 3* o Φ Φ ?α "≤ Ω φ O 0 3^J (-" φ O Ω O H Cu 3 0

H- Φ rt o H rO h 3 3^J CO μ- 3 Cu Φ Φ Φ Cu 3 O Hi CU 3 3 H

O O O Cfl o φ ^■<! CO • rt ■ 3 rt O 3 CO rt rt h TJ O Ω ^!

Hi 3 Hi Hi O _^ Φ Ω μ- 3¹ 3 μ- N Φ cr 3" O O. rt Cu Ω μ-¹ 3

Cu Cu φ O TJ H O rt Φ c o 3 O o ~< 0 "*_* H Φ Hi μ- " H O 0 φ 3

H- (-' CU H C Cu H- Cu ro U 0- U Ω H rt Hi H J3 l-S μ- 3 Φ rt H Hi o Ω rt H rt φ cn rt <! H CO Ω Φ σ CO H α o Φ μ- Or O rt iQ CO ^ rt O ■< rt Φ rt Cu H φ Φ Φ rt iQ ιQ 3 μ- φ 3^J TJ 3 3¹ μ- Φ

\-^> Cu H H- O ^■υ 3 Hi Φ -Q O H O ^* Φ H Φ N iJQ Φ rt O rt Φ •< x O

3 Cu Φ (-^■ j-» Φ 3 Ω H H O C- rt O Φ 3 _^ 3 rt Cu d O 3^ Hi cr φ rt

CU H Cu CO Cu 3 3 O σ l-S Cu Φ μ- rt 1 Φ Φ 3¹ rt φ TJ ^ Φ O l-S μ- rt CO O o " O Ω M CO ty Φ CO 3 o Ω ^■υ O O CO Φ μ- 3 O CΛ 3 H H μ- O 0- a O rt rt CU o Φ ft o φ CU rt H o Ω ^ M 3 Φ 3 φ φ l-S O Φ H _^ CO *σ 3^J Ό Φ O H CO ^■z s: Hi 3 Φ ^ lO Ω O μ- -3 O

Cu O μ> 3 K. CO O H Φ H CO Φ H H r rt 3^{^} C- CO 3 μ- Hi cr N 3

H 3 o H- •_^ H> rt • Ό o Φ μ- rt Φ μ- O μ- TJ 3 H Φ *_* cu CU Φ

H- o rt H- H n ^•τs 3 CU Φ ιQ Ω Ω H H C Ω D 0 Ω rt 3

Φ rt "> u J-» α TJ φ Cu φ Ω Ω Φ 3^J μ- 3" O Ω H rt S3 rt μ- Ω S⁴

3 u cr co CO H Hi Ό H l-S Ω rt 3 3 co I-" σ Ω Φ a H- μ- Φ o Φ μ-

<J σ *_* M O Φ Φ H Φ CO O Cu Φ 3' o Ω Φ Φ O O o a ell-S 3 _"* Ω

H- UI Φ • Hi H H- C H Cu H . Cu O 3 O H rt • Φ s' μ- 3^*

4 Φ O • d-» Φ CU 3 ≤ Or 3 H <l CU iQ Or μ- 3 0 -3

O • tP-. CO H σ φ σ μ- μ- α CU m Φ rt Φ rt Hi μ- P- CU 3 Hi 3^*

3 O CU r-¹ M > rt 3 ^ " Φ 3' O 3 Φ o _^ μ-

3 rv) cr •< CO 3¹ iQ CO Φ Cu Hi l-S ^■Q H TJ CU Ω

Φ O it-. M rt -! •< rt o l-S s: 3'

3 O cπ ⁾ ^•< CU t-f rt rt 3^J 3^* H CI rt CU o rt ^ rt

*» φ 3^" O Cu μ- O 23 O μ- Cu

CU Φ H ω rt φ 3" φ > Φ Ω H Φ

"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 with the biological activity of the phosphotransferase system enzyme I, and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polypeptide according to SEQ ID No. 2 and have the activity mentioned.

The invention furthermore relates to a process for the fermentative preparation of amino acids chosen from the group consisting of L-asparagine, L-threonine, L-serine, L- glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan and L- arginine using coryneform bacteria which in particular already produce amino acids and in which the nucleotide sequences which code for the ptsl gene are enhanced, in particular over-expressed. The term "enhancement" in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene which codes for a corresponding enzyme having a high activity, and optionally combining these measures.

By enhancement measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism.

The microorganisms which the present invention provides can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among experts for its ability to produce L-amino acids .

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-amino acid-producing mutants or strains prepared therefrom.

The new ptsl gene from C. glutamicum which codes for the enzyme phosphotransferase system enzyme I (EC 2.7.1.69) has been isolated.

To isolate the ptsl 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 Einfuhrung in die Gentechnologie [Genes and Clones, An Introduction to Genetic Engineering] (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 Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination- defective. 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 ptsl gene and which, as SEQ ID No. 1, is a constituent of the present invention has been found. The amino acid sequence of the corresponding protein has furthermore been derived from the' present DNA sequence by the methods described above. The resulting amino acid sequence of the ptsl gene product is shown in SEQ ID No. 2.

Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also a constituent of the invention. In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Conservative amino acid exchanges, such as e.g. exchange of glycine for alanine or of aspartic acid,; for glutamic acid in proteins, are furthermore known among experts as "sense mutations" which do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. It is furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID No. 2 are also a constituent of the invention.

In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID No. 1 are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 nucleotides.

Instructions for identifying DNA sequences by means of hybridization can be found, by the expert, inter alia, in 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°C - 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 CO CO > IV) μ> μ»

Cπ o cπ o cπ o Cπ

pi μ- Hi TJ μ- s: d^¬ H Ω μ. TJ Cu H O O rt Cu H o Cfl 3^* CO 3¹ J Hi Cπ S CO rt Cu H cn

2, μ- Φ o 3 3^* s' Φ o o rt Ω rt ?v X ¹ 3^J μ- 3 O rt ^■ l-S o l-S O σ Cu "< O Ω Φ Φ

?r H fl μ- Φ ιQ H CO μ- Cu Hi μ- Φ CO rj¹ 1 σ Φ cr o o\o Cu s: 3 Cfl 3" 3 -Q φ 3 Cfl rt Ω 3 H Cu H \ r o U rt ffi Ω H TJ H cr Φ o 3 rt rO μ- o 3

^■t φ μ- 3' 3" CO Φ Ω co Cu φ l-S O Φ O H ^ CU μ- O μ- Φ 0 3 H 3^" Φ X! Φ <i Φ

> 1 μ- 3 cr Φ rt Cu Cfl 3" U3 CO 3 Or 3 X ^' Hi 3 α α H φ 1 Φ 3 <i φ 3 d co rt μ^j Cu H rt TJ μ- Φ μ- μ- •_" 3 J Ω S M μ- O μ- Φ 3 el^¬ μ- CΛ Φ Or Ω

H Φ Cu φ CO i-s 3 μ- O Φ 3 3 cr O Ω Φ rt rt Cu Φ N Hi N 3 Cu er s' cn 3 CΛ Φ el^¬ rt Cu ro Ω 0 3 <l Φ Φ Ω G H l-S 3- μ- 3 0- Cu u TJ rt co Φ fl n cr s' μ- μ- rt 3 rt 3 . φ • Cu Φ 3- pt Φ rt Φ o 3 rt Cu rt H Φ ^ O

Φ 3 <! o φ μ- 3 μ- 3 3 φ O 3 S⁴ W μ- TJ μ- 0 s; CO O S l-S cu Hi Hi

H TJ φ 3 i-S i-s 3 Cu H rt TJ O Φ μ- o TJ o << φ 3' Cu Cu - 3 O s;

3 i-s μ- ^■ Ω Cu Φ i-3 3 μ- Hi » μ- μ- 0 μ- rt 3 l-S 3 Φ Cu μ- H^J cr 3 CO P- l-S Cu el^¬ o 0 CU 3 Cu 0 ¹ ιQ 3 O < l£> 3 " Hi 3 O 0 CO Ω rt φ 3 Cfl s'

H <! 3 Ω ^■ H μ- v-3 O TJ 3 φ CO Φ rt ~< O • CU X rt rt .s^* μ- 3^* Q o Φ 3^J Φ

Φ Φ μ- l-S TJ i-3 o Φ i-s 3 Φ 3 H ,— , H • Φ Ω Ω 3 Φ 3 TJ X μ-

3 φ O Φ 3 3 Φ o α — CQ l-S Φ Q Φ Φ 3 u (Ω Cu O μ- μ- rt Cu 3 J

O Cu Dd H 3 Φ l-S <! Cu ^ i-s C Φ • > TJ 3 o H 3 3 μ- 3 0 l-S er cr CO ^ Cu Φ O Cfl 1 Φ rt ιQ Cu 3 Cu cu 3 l-S i-3 o Φ o\o Φ Ω φ _" φ o TJ O

3" • : Cu φ rt K φ α 3^J 3 rt H CO TJ 3 • cr 1 H cr ^ Φ Cfl 3 μ^j 3 cr

Φ Ω μ- Φ Ω Ω X Cu Or 3- μ- φ Cϋ rt Cu Φ rt 3 rt Q Hi Cu Φ 3 Φ

3 μ- el3 P. Cu rt CU J 3 rt ffi Φ u μ- 3 D CD ) rt O Hi er Cu Φ o *_* •

Φ Φ α s' Or 3 S^J 3 l-S Cu φ μ- CD ^ Ω Hi • H μ- 3 μ- O H σ l-S H H φ

3 u Φ 3 3 Φ φ 3 Ω μ- 3 μ- 3- μ- Q 3 O H Cfl <£> H Cu M 3 ^■<: cr H CΛ

N CO TJ Ω TJ cr cr CO 3 O α Cfl μ-^' Cu Ω Cu Φ o cπ rt μ- Cu ^■<: 3

-<j 3 H Φ μ- CO φ l-S Φ CO Φ H Φ S • • 3 μ- cu Ω .73 cr o\o Φ μ- CO 3 μ- CO CO Ω

3 H o X cr rt μ- μ- l-S • : μ- Φ 3 rt Cu CU M •n CO Cu X O 3^* •<: μ- 3 rt 3" φ φ 0- J H H 3 cr μ- O 3 cr £ rt μ- rt CO Φ 3 rt ef μ- CU 3 φ rt cr O H O d l-S Φ Φ 3 o 3 3 Cu Φ Φ rt 3^* H 0 Cu • H Φ Φ 3 Or Φ CO μ- 3^*

Cu Ω φ Cu rt CO Ω Hi Hi h O τ3 Φ Φ 3 o el^¬ TJ Or Φ TJ rt H φ Φ 3 •<!

Ω rt rt CO TJ 3 CU o H rt o i-3 3 l-S Cu O W 3 s' € 3 O u> μQ H U3 cr rt O μ- Cfl l-S rt 3 Φ el^¬ Φ H *_* Cu 3" Ω O ιQ ^ Φ μ- σ rt Φ H <X⁾ K 3 μ- Φ H μ- o μ- o O Φ φ Cu s' l-S 3 Cu Ω Cu rt Hi 3 σ el^¬ l-S fl ^< μ- _"* o rt cπ •< Φ 3 3 μ-

< TJ 3 o 3 Hi α CO Φ o 3 rt 3 μ- Φ s' φ Ω . ~— - cr 3 i-3 rt Or μ- H • 3 o • cr Φ O cr φ μ- o α Hi Φ μ- μ- Cu Cu μ> μ- l-S rt CO rt O rt el^¬ μ- 3 <J Cu l-S Ω σ 3 a 2, H 3 Hi 3 rt X Cfl Cu μ- " el^¬ Ω

^ * H3 μ- Φ s' M 3 *_* 3 Φ Ω 3 Q CU O 0 O 3 Cfl K Ω s' o Cu

O 3" 3 H Φ |x! Or 3 H rt Cu H O . — . • 3 Cu rt o H er H CΛ o rt μ- Φ 3 H μ- 3 Φ CO J μ- O r 1 Φ 3 Cu ?v <τ) O H H 3 Φ O Φ ω TJ Φ O o 0- Φ

Cfl iQ rt " CO l-S 3 H Φ φ H ^■ : 3 O φ ϊσ ΪV 3 Cu Cu a rt 3 u Cfl μ-

Φ 3^* rt Φ vQ H ' X μ- ^ CU TJ σ 2 1-3- cn 0 Φ Ω π CO el^¬ Cfl • μ- TJ rt Cπ Cu rt

Cu el^¬ X Φ μ- i-S CO rt TJ Cu 3 J ^■< 3 o Ω rt rt o μ- s' rt 0 Φ μ- X H μ- Φ s' TJ rt 3 Cfl CO 3^J o H μ> l-S Φ CO 3^* μ- 3 Φ TJ 3 H o rt o Cfl

CO Φ l-S Ω Ω μ- μ- Φ Hi Φ TJ O o Ω 3 Cπ Φ μ- O O iQ o Cu Cu 3 O 3 Cfl

0 Φ O μ- rt o rt Cfl l-S - TI CU Cu Cu Ω Cπ 3 3 Cfl o rt ω Ω CO

H Cfl d n 3 3 Φ TJ Ω CO o d > O Ω μ- 3 Φ CO D Cfl rt rt Φ o\° H 3 O O . Cfl μ- μ- fl i-s H l-S O μ- α P3 3^* rt CO — μ- el^¬ o 3^* ι-3 3 ^ H Dd 3 rt

3 Hi μ- CO u Cu Ω 3 o TJ o 3 • • σ • cu s' o Φ 3 0 3 Φ o _^^ Ω ι-3 cu

Ω Φ o Φ Cu TJ 3 μ- 3 Ω — . _*~_* Φ Ξ ιQ Φ 3 cn Φ l-S Ω TJ φ ι-3 Φ 3' cr

H 3 Or CO Cfl o φ φ CΛ H μ- 3 3 H o O 3^* r 3 μ- H φ o o μ- ιQ CO rt rt CO O TJ J. Hi rt o Hi Ω CU Φ Cfl Hi H Φ rt Cfl Φ

Cu Hi μ- Hi rt Φ φ h Φ Hi Cu φ t-i o CO O O Φ rt o Cfl μ- i O Cfl μ- 3 rt Φ H 0 3 3 er h rt μ- CU S α Cu o Cu φ rt 0 Φ rt CU Hi rt μ- rt TJ^' 3 el^¬ μ- 3 μ- O H μ- cr TJ iQ H rt Cu 3

S 3 Φ 3 Cu 3" 3 H H r s' Ω 3 Hi Φ H TJ Φ Q μ- 3 α φ Cu Cu CO 3 el^¬ Φ o 3 Φ φ CO o Cu Φ Φ l-S H O cr H Ω o s' 3 O cr Hi el^¬ CO o 3 cr U

>< 3 el^¬ Hi Φ CO ^■< rt rt X Φ H

1 s' Φ O φ

preventing the degradation of the enzyme protein. The genes or gene constructs can either be present in plasmids with a varying number of copies, or can be integrated and amplified in the chromosome. Alternatively, an over- expression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.

Instructions in this context can be found by the expert, 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, in US 4,601,893, in Schwarzer and Puhler (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, for enhancement the ptsl 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 ρNG2 (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. CO CO IV) M I-¹ μ>

Cπ σ cπ O cπ o Cπ

J μ* Cu CO el^¬ H μ-¹ CU CO _ U rt S Φ er el^¬ Ω Cu ro Ω I\) TJ φ Ω TJ O _ CU 3 u el^¬ TJ

3¹ tV> 3 φ s' el^¬ φ μ> μ> μ- X H s' O H O μ- H ( i Ω CO μ¹ X O μ- TJ CO s'

0 o α -3 Φ cu co ^• > • Cu Ω Cu CU Φ 3 • 3 o 0 Ω Pi D Cu Cu Φ Φ TJ 0 CU O \ 3 s' O Ω 1 00 3 i-s 3 3 rt ^ H μ^j 3 • • M ^-~. 3 μ- to i-s VD μ^j CO O

TJ Cu o φ CO Cu rt l-S CO sD . — . CO o TJ to X Cu μ> 3 o μ- CO • CO TJ • — 3 O _^ μ- φ 3

3- 3 H 3 φ CO o — — Hi cr - Hi Φ μ- <£) Cu iQ 3 ro l£> ' — μ^j _^ μ- 3 — Hi μ-

0 Or Ω Ω et cn ~J — - — TJ 0 μ- ro O to s CO H ^ i-3 C t 1 > > - — Φ Or • - — TJ Cu μ- Q- et CU Φ rt Hi s: to > J h 0 _" l-S μ- Cfl cn Φ ! HI ιfc» ^ σ TJ CO Ω μ-

K 3 μ- 3 0 —. CU H 3 H 3 l-S 0 3 CO O — '. 3 H Hi - u er u IV) μ- o o l-S o 3 ϋ μ- Cu o cr Cu φ el^¬ Hi M •*_* 1 .TJ — ' TJ TJ rt Φ 3 o μ- tr rt 3' Φ

3 -J 3 Hi 3 rt Ω <! t£> 3 Φ et ιQ ^■ rt Or s' Ω CO O Pi O a H Φ cu μ- Ω

Cfl o 3' O Φ <£> 3 0- μ- •<: μ- Φ Φ dd *- X *=> μ> G 3 rt el^¬ to o el^¬ ef-

H 0 Φ cr Φ TJ l-S it-. μ_ μ- 0 CO 0 Cfl 3 u • • o _^^ T CO TJ 0 0 s' Or 3 s' 0

Φ Hi Cu 3 Φ H μ- u Ω S 3 cn Ω 3 el- K3 Φ Ω ω Ω 3 CO cr H μ- 3 u cr Φ l-S l-S Ω N rt 3 φ — 3 CU μ- σ S- • H Φ er Cπ α 3" t? o o Cfl TJ 3 Φ cr CO cu el^¬ μ- ^ Ξ o CO φ • Ω 3 Ω Cu Cu: Cu 3 Φ CO CU O 3^' S cr μ- s: Or Φ *< cu

CO s' 3 Φ i-s <! 3" H H Hi ι-3 μ- Φ μ» l-S .£- 3 1 3 1 3 3" 3 μ- 3 μ-

Φ Φ φ Φ φ o Φ > Cu o Φ Φ 3^* 3 .. μ- 1 > Cu H o ~_^ μ- Φ Ω M μ- α 3'

Φ 3 Hi 3 Hi φ 3 σ cπ l-S Φ rt o Cπ _• 3 H O Ω Ω elCU 3 3 μ-

CO Cu X H σ rt et rt α μ- P- cn 0 O CO J-* Cπ .. — . μ- • 3- s' rt <! Φ rt o Ω

^■ 3 Ω TJ Φ el^¬ * Φ o Φ 1 Φ 3 Hi CO o μ¹ to ,-, TJ TJ 3 O μ- μ- CD Φ Hi 3'

Cfl μ- 3- O O Φ s' H Cu w CD rt Φ cr -J i-3 Φ H « o ιQ Ω Or o l-S Ω ιQ et 3 Cu Hi Cfl 3 Φ el^¬ 3- O Ω cπ rt o Φ 1 ^< _*-_*. l-S 00 ^£) O ¹ 3 H Cu 3 o H H ≤ CU φ o 3 μ- s' 3" • μ- i-3 i-s Cu 3' . O μ> 3 -J υ? 3 VD 3 3 3 μ- Cu 3" H

3 iQ el^¬ rt Hi Φ O <1 •< μ- \-> O u > Ω Cu > it-- Φ 3 Φ rt rt o 3 cr er μ- Φ

_^ Cu Φ s' μ- o Φ 3 . — 3 cr . — . • 0- iQ 3 ro <£> l-S vo — ιQ o rt Cu H 3' H φ Φ μ- Ω

Ω CO Φ o 3 3 H O -. Cu CU Φ TJ o CO Or D • Cu cr 3 Φ Φ 3 Or O tr Hi

CO μ- 3 3 Φ Φ Cd 3 3 a. O ,— _^ 0 3 • • ¹ — CO Cu μ- TJ Cu rt % 3 3 rr 0- μ- TJ Or CO O S o. *_* < > Hi rt μ- it-. — Φ — C-I Ω _^-_^ Ω Ω 3 Cu el^¬ l-S o 3 3' μ> μ- 3 CO . — J-» J Cu Hi 1-3 cπ s rt o O ω • 3 μ- O ^■υ Hi μ- s' et s: cn O CO rt 3 < O W DO Hi — TJ Ω 3 μ- 3' μ> 3 H o -> 3 Ω 3 μ- o 3 φ 3'

3 φ rt CO di3" rt 3 S μ- μ- o — -> O μ- φ Φ o TJ Cu J H J 3" u TJ μ- S i-s rt Φ

Λ 3- J Cu iQ μ- to μ- o o i-s • μ- 3 Ω Or 1 a μ^> o 3 O Cu: 03 rt -^> Hi μ- O J i-s μ- 3 Φ S' et er 3 3 Ω \ et Φ 3 TJ s: PJ Cu H Hi μ- J Φ Φ μ- Ω Φ HS 3

3 Φ O o Φ U3 H i-s t-3 ro Φ S α 3 3 μ- Cπ ^ ® Cu Φ o o rt Ω l-S X rt o o

3 CO rt u CO Φ o Φ Ω X φ i-3 Cfl Cu > Dd rt l-S \ CO μ- Φ CU o CU 3' Ω ts

CΛ Ω Φ H 3 rt D Ω σ Ω S Cu el^¬ u Cu cr CO cn — . -i l—i O μ- j CO 3 rt cr 3 Φ Φ φ

M Φ Ω Cu μ- μ- rt O μ- 3" 3 3 s' 3 rt ^ rt 3 — CO O 3 Hi o Φ Φ μ- iQ μ- μ- TJ CO rt 3 O 3 o H 3 o 3 o TJ O α μ- 3" μ- Ω 3 3 3 rt Ω cr Cu Φ o 0 H Ω Cfl CD

O μ- cn o 3 Cu o H O \-> Or o Ω Φ o S¹ hi ct dd » 3" H 3 3 H Φ 3' 3

H Hi O Hi o • μ- μ- O o φ CO K 3 O 3 H f-S 3 μ- u 3 Φ r Φ O H o μ- α 3 Φ Hi Cu 3 3 i-3 0 i-3 *_* 3 3 <1 3 Cu o S O 0 O 0 O Hi rt

Φ i-S Ω Cu μ- iQ Hi <! μ- rt Φ TJ 3 H (—1 - Cu ¹ to μ- Hi ^< cr 3 cu s 3 cr CU el^¬ μ- Ω et Ω "< cr 0 μ- Cfl 3 i-S Ω Dd Ό tV> 3 0 O Φ rt o 0 U σ

O N φ Cfl s' Or O μ- CU * H H v-3 Cu rt Ω HI O

— O <! ιQ μ- i£3 Ω el^¬ cn O Φ H

• et φ φ 3 O -J ⁽X⁾ r CU 3 O CO Hi μ- μ- to Ω rt _^~» Ω s' o 5 o 3 Φ

3 ^■t Φ et 3 _"» H 3 Φ rt to l-S Φ c- Ω j o Φ O rt μ-¹ Φ _"« φ 3 Φ

IV) φ φ Cfl Cu [x Cu t-¹ S⁴ 3 to μ- Hi rt S cr _^— H Cu 3 3 μ> H ^ o μ- Φ Cu

_^ φ "< 3 Ω μ- Φ μ> CO μ- Φ Ω o Φ O

3 ^ et o cπ φ 3 l-S 3 H 3^* CΛ CU o H" Φ ^ Cfl TJ 3 3' > 3 l-S

H 3 O μ- 3' μ- Φ O M CO Ω Φ μ- et" 3 Cu CO rt cn cn H. rt μ- l-S μ- TJ Φ Φ Ω s: μ* cu Ω r 3 cn Ω CU

3 o TJ Φ o 3 3 Φ 1 tr Cυ cr o φ Ω H % Ω 3 3" H CO l-S Cu 1 1 3- 3 Cu

TJ Hi o 3 iQ. Cu Φ l-S 1 CO u μ¹ Φ l-S α. 3' Cu 3 Φ -« cπ Φ μ- el^¬ μ Φ

H CO Cu Φ rt Cu CO n -o α rt 3 »» 1 3 s' CO μ- cr CO

0 rt μ- Ω 3 O r 3- •_^ μ- el- *£> CU μ- G ~J ^ l-S IV) α Φ o

< 3' rt μ- to μ» 1 3 W3 H to CO en <£i Hi Cu Dd

Φ Φ μ- μ- tV> o Φ Hi er Φ μ> rt t O M Φ o 3 O O rt O O rt H 1 H rt

3 Hi H

O IV) IV) μ» o Cπ o cπ o cπ

3 X cr Ω > M o t"¹ M μ- rt 3 3 to Ω Ω H Ω o D Cu μ- Cu to j TJ CU TJ Cu O J J el¬

3 Φ * 3' ?r μ- CU CO 3 H 3 μ- 3 O O α O O a Ω to μ^> rt S' o Ω rt H l-S rt l-S H s' rt μ- CU u 3 μ- cr Ω ro rt rt cr 3 3 3 3 μ- H ts ro CD μ- o S- φ O Φ

Cu 23 μ- α to ιQ 0 3" Dd Cu Cu H CO <! rt 3 rt rt α to φ Cu μ- TJ rt μ- Φ Hi Or ua Φ Φ 3 ro rt O H Φ Cu rt iQ O et Φ Cu o Cu Cu CO t μ- cr rt •_^ O Φ 3 i-S Φ 3 Cu

Φ H € 3 Φ 3 Cu l-S Ω 3 φ to Cu 3 μ- • μ- μ- φ Cu o Φ 3 Cu μ- CD μ- μ- l-S Ω 3

3 σ rt H μ- μ- 3 rt μ- et ro 3 O 3 rt 3 3 3 Λ rt € to o μ- μ- 3 3 J Cu ft μ-

Φ Φ 0 ro Cfl U Ω 0 Ω ro 3 Φ ιQ Ω μ- Φ Φ CO 3 3 Ω D Φ 3 3 rt O φ H cr μ- 3 cn h 3 CU Ω Φ H l-S 3- i-s rt to 3 Φ O Or • — Or Φ TJ 0 S μ- v-3 O H 0 o μ- ιQ Ω 3- i-s Φ μ- μ- μ- Cu CO 3 et 3 0 μ- Or μ> CD μ» TJ o Φ Cu rt ^ 3 fl *_* Cu rt Φ O u u ^■£, CO 3 _^» Cu Cu U Cu 3^" Ω CO 3 φ cπ Φ CO Cu 3 3 rt ro *> CU

. 3 μ- l-S rt J μ- • μ- M et 3 et Φ Φ μ- CD *- Q H μ- μ- Ω μ» Or o Ω μ- H Ω rt CD α rt CO 3 _"» rt Ω J 3 tr¹ O μ- D 3 < Φ Or ro o Ω 3' H μ- 3 3 J ^■υ cr 0 μ- t? Hi Φ μ- IV⁾ O o 1 Hi

<£> Ω Φ 3 φ cn M ro 3 3 Ω 3 O rt 0 Cu Hi o o μ- 3 Φ Ω CO Cu CO iQ

H —. l-S Φ Cfl CO μ- 3 3' ro tr rt to 3' CD ω 3 l-S to Ω <: 3 3 μ- φ ^ Ω J

- — Cu J rt φ ^ <! Cu μ- Φ μ- φ rt H Φ Ω μ- rt 3 CD 0 H

--. 3^* Ω CU μ- ,—, u rt μ- o u i-3 rt rt S⁴ μ> σ Cu CO 3^* CU φ 3 μ- μ- μ- H O

Cu 2 iQ. Ω μ_ Ω 3 μ- l-S rt i-s to Φ μ- σ μ- ιQ Φ IV) 3^* O Cu H X o 0 Ω 3 ^• : Or

Ω 3 _"* • o α Ω o H *_* 3 O U o 3 CO s TJ o Hi 3 Ω 3 Φ 3 3

Cu M CO x O 3 Φ 3 CO 3 u TJ o l-S iQ t"¹ 3" Cu Cu ro Ω

3 „ — , cn EG 3 > i • ^>< * Hi CD ro 3 rt Cu o 3 rt Φ I Cu Ω 3 cu Hi et J 3 Φ i-3 Φ H 3 t O μ- Cπ Cπ μ- to 3 et 3' iQ 3 μ- O μ- rt O μ-

Hi Ω Ω μ- μ- CO > cu Φ i-i H to cn et f 3 H Or Φ μ- ro Hi μ^j iQ Or -J 3 H O

3 Pi 3^* α 3 Dd J u 3 rt Cu IV) 3" O Φ 1 • μ- 3 O 3 Φ o TJ 3 3

H * Φ φ i-s H rt tr¹ μ- ET <! Φ TJ • -<: μ» t"¹ 3 cu H rt Φ μ- o et cu * φ O μ- ro Cu 3 O μ- X! l-S ^ μ- μ- cπ 1 μ. CO h-" Cu Hi X Cfl u O cr

3' CO Cfl cr i-s 3 α cr ro Or o Cu o μ- 3 3 Ω S¹ o tfl 3¹ 3 0 Ω Ω μ- u μ-

Φ TJ ro μ- 3 3 o to 3 Ω 3 CD ^ Φ Hi 0 φ 1 μ- l-S Φ μ- rt Ω 3

H φ μ- l-S 3 • • ω l-S . — . ro TJ ro ro rt et CO Ω Ω 3 ><! μ- et

3 w Cfl ιQ ιQ EC Cu Cu S et H CO 3^* Φ Cu rt TJ -3 H Φ ^< t-ι Ω o Φ TJ

O rt *_* Ω CU Ω et μ- CO Φ CO μ- Φ Cu Φ l-S 3" O CD cu 1 μ- 3" Φ 3 l-S Cu

H n Or Hi Φ H rt o μ^j 3 *% ro 3 r μ- o μ- J rt Ω i-3 3 Cu X μ- H

Φ 3 ro μ> O 3 cr Φ i-s H Ω μ- to CD TJ Φ 3 et Ω 3 rt Φ μ- iQ 3 0 μ¹ Cu rt

3 ω U3 H rt o H *< φ ET ιQ 2, rt rt O μ- Φ Φ 3' O to μ- P- 3 iQ H IV) μ- cr Ω V-D Φ H μ- l-S 3" 1 3 Φ CO Hi φ μ- . H 3 rt tr¹ Φ 3 CO Ω

Φ U" H CO ttf Ω ^ Cu s Cu rt 3 CD Cu H Cu 3 et 1 ro Cu 1 α 0- 3

?sr μ- — ' ro 3^* CU to φ μ- Or rt ,—. et 3^* CO μ-¹ μ- 3 Cu μ- μ- H

3 cu cr v<3 3 μ> 3 C-I Ω el^¬ 3 ^■ 3^* CO Ω Φ . Cπ μ- co μ- Hi 3 co Cu

CO Φ o μ- O U3 Ω 3 Cu s' i-3 o μ- Φ M TJ O J-- to Ω O iQ H

Φ Φ l-S 3 <£> o Cu Hi 3 ^< Hi lO O 3 Cu j -J TJ μ- H φ

3 3 O IV⁾ μ^j H EC O H 3 σ CD rt 3 3^* TJ ts Cu 3 tr¹ X rt μ- μ- rt ro iQ — α H σ 1 3 et 1 Cu H μ- Cu μ- Φ Ω H φ Ω μ- CU 1 0 3^"

Hi CD 3 3^* i-s μ- CU • • Φ z rt 3' rt to σ rt μ- 3 0 φ Hi μ- 3 3 ¹ r Φ o Ω Φ CO Φ o CO 3 ro - Cu Φ Φ μ- 3 o TJ 3 Hi φ Or Φ •< • cu l-S S⁴ rt ' ' H TJ > X 3 1 U3 J 3 O CO rt rt Φ i-s . to 3 H

Φ 3^* TJ -» Hi Cu CD 3 Φ cr Φ Cu O 3 Cu CD u H H o O μ- iQ ^ et H Φ O 3: 3 μ- EC co 3 Φ μ- 3 Q) r tr¹ Ω H μ- H φ i-s et 3 φ to

3^" CO l-S 3 3 Cu 3' TJ P- rt μ- CD U3 ro 1 μ- 1 3 Φ 3" Φ C μ-

Φ < 3' > TJ rt ιQ 3 o H" H Ω φ Φ 3 CO CO i-3 α μ¹ φ 0- t-¹ Φ 3

Φ Cu 3 ro Cu 0- H ro Hi O 3 μ- — -J cπ 1 l-S u Hi ro

H 3 Φ fV iQ x σ rt • o 1 Ω Φ 3 • 3 to it-- > Cu 3 o

H 0- H et Φ Cu o l-S S •<; Φ et Φ -J μ- rt CO i-s

CU cr Cu H 3 t o Ω CU 1 et . — . •-3 Cu Λ 3 TJ o to 3 μ-

•*_* σ - O rt O CΛ 3^* 3 3 Cu o Cu o Φ 3 Ω o 3 3" Φ μ- Φ l-S H 3 l-S Hi H Φ μ- !θ Ω 3 et ^•< o to 3 Ω μ- IT¹

H Φ Φ Φ Ω 1

The corresponding alleles or mutations are sequenced and introduced into the chromosome by the method of gene replacement such as is described, for example, by Peters- Wendisch et al. (Microbiology 144, 915 - 927 (1998)) for the pyc gene of C. glutamicum, by Schafer et al. (Gene 145: 69-73 (1994)) for the hom-thrB gene region of C. glutamicum or by Schafer et al. (Journal of Bacteriology 176: 7309- 7319 (1994)) for the cgl gene region of C. glutamicum. The corresponding alleles or the associated proteins can optionally be enhanced in turn.

In addition, it may be advantageous for the production of L-amino acids to enhance, in particular over-express one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and optionally regulatory proteins, in addition to the ptsl gene.

Thus, for the preparation of L-amino acids, in addition to enhancement of the ptsl gene, one or more endogenous genes chosen from the group consisting of

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

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

• the tpi gene which codes for triose phosphate isomerase

(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

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

• the zwf gene which codes for glucose 6-phosphate dehydrogenase .(JP-A-09224661) , • the pyc gene which codes for pyruvate carboxylase (DE-A- 198 31 609),

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

• the lysC gene which codes for a feed-back resistant aspartate kinase (Accession No.P26512; EP-B-0387527; EP-

. A-0699759),

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

• the horn gene which codes for homoserine dehydrogenase (EP-A 0131171) ,

• the ilvA gene which codes for threonine dehydratase (Mδckel et al., Journal of Bacteriology (1992) 8065- 8072)) or the ilvA(Fbr) allele which codes for a "feed back resistant" threonine dehydratase (Mδckel et al., (1994) Molecular Microbiology.13: 833-842),

• the ilvBN gene which codes for acetohydroxy-acid synthase (EP-B 0356739) ,

• the ilvD gene which codes for dihydroxy-acid dehydratase (Sahm and Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979),

• the zwal gene which codes for the Zwal protein (DE: 19959328.0, DSM 13115) ,

can be enhanced, in particular over-expressed.

It may furthermore be advantageous .for .the production of L- amino acids, in addition to the enhancement of the ptsl 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: 1995 1975.7; DSM 13114) ,

• the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2, DSM 13113)

to be attenuated, in particular reduced in expression.

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.

By attenuation measures, the activity or concentration of the corresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the starting microorganism.

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

The invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of amino acids. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfϋhrung in die

Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral

Equipment] (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 liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e. g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the above-mentioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40 °C. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours.

Methods for the determination of L-amino acids are known from the prior art. The analysis can thus be carried out, for example, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by ion exchange chromatography with subsequent ninhydrin derivation, or it can be carried out by reversed phase HPLC, for example as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167- 1174) .

A pure culture of the Corynebacterium glutamicum strain DM1547 was deposited on 16th January 2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) as DSM 13994 in accordance with the Budapest Treaty.

A pure culture of the Escherichia coli strain DH5alphamcr/pEC-K18mob2ptsIexp (= DH5 mcr/pEC- K18mob2ptsIexp) was deposited on 2nd May 2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) as DSM 14278 in accordance with the Budapest Treaty.

The process according to the invention is used for fermentative preparation of amino acids .

The present invention is explained in more detail in the following with the aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques of restriction, Klenow and alkaline phosphatase treatment were carried out by the method of Sambrook et al. (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA) . Methods for transformation of Escherichia coli are also described in this handbook.

The composition of the usual nutrient media, such as LB or TY medium, can also be found in the handbook by Sambrook et al.

Example 1

Preparation of a genomic cosmid gene library from Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid

33:168-179) and partly cleaved with the restriction enzyme CO IV) IV) μ* o cπ o Cπ o π

^■s. ro l-S ro to ro

H

Φ

Ω rt

Φ

Or

Example 2 ^{• ■}

Isolation and sequencing of the ptsl 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., 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 the 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 Biosystems (Product No. 403044, Weiterstadt, Germany) was used. The separation by gel electrophoresis and analysis of the sequencing reaction were carried out in a "Rotiphoresis NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI^' Prism 377" sequencer from PE Applied Biosystems (Weiterstadt, Germany) .

The raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, ^' 14:217-231) version 97-0. The individual sequences of the pZerol derivatives were assembled to a continuous contig. The computer-assisted coding region 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 1707 base pairs, which was called the ptsl gene. The ptsl gene codes for a protein of 568 amino acids.

The DNA section lying upstream of SEQ ID No. 1 was identified in the same way, this section being shown in SEQ ID No. 5. The ptsl. gene region extended by SEQ ID No. 5 is shown in SEQ ID No. 6. Example 3

Preparation of a shuttle vector pEC-Kl8mob2ptsIexp for enhancement of the ptsl gene in C. glutamicum

3.1 Cloning of the ptsl gene in the vector pCR®Blunt II

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 ptsl gene known for C. glutamicum from example 2, the following oligonucleotides were chosen for the polymerase chain reaction (see also SEQ ID No. 8 and SEQ ID No. 9) :

ptsI2expl:

5^{^}-GGC TGA CCA TGC TTA ACC TC-3' ptsI2exp2 :

5 -AGA CTG CTG CGT CGA TCA 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. 2121 bp in size, which carries the ptsl gene with the potential promoter region. The DNA sequence of the amplified DNA fragment was checked by sequencing.

The amplified DNA fragment 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 E. coli strain TOP10 was then electroporated with the ligation batch (Hanahan, In: DNA cloning. A Practical Approach. Vol. I, IRL-Press, Oxford, Washington DC, USA, 1985) . Selection for plasmid-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 and checked by restriction with the restriction enzyme EcoRI and subsequent agarose gel electrophoresis (0.8%). The plasmid was called pCRBl-ptsIexp.

3.2 Preparation of the E. coli - C. glutamicum shuttle vector pEC-Kl8mob2

The E. coli - C. glutamicum shuttle vector was constructed according to the prior art. The vector 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 kanamycin resistance-imparting aph(3')-IIa gene of the transposon Tn5 (Beck et al., Gene 19, 327-336 (1982)), the replication region oriV of the plasmid pMBl (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979) ) , the lacZ 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., Bio/Technology 1:784-791 (1983)).

The vector pEC-K18mob2 constructed was transferred into C. glutamicum DSM5715 by means of electroporation (Liebl et al., 1989, FEMS Microbiology Letters, 53:299-303). 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 25 mg/1 kanamycin. 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 Hindlll, and the plasmid was checked by subsequent agarose gel electrophoresis .

The plasmid construction thus obtained was called pEC- K18mob2 and is shown in figure 1. The strain obtained by electroporation of the plasmid pEC-K18mob2 in the C. glutamicum strain DSM5715 was called DSM5715/pEC-Kl8mob2 and deposited on 20th January 2000 as DSM 13245 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.

3.3 Cloning of ptsl in the E. coli-C. glutamicum shuttle vector pEC-K18mob2

For cloning of the .ptsl gene in the E. coli - C. glutamicum shuttle vector pEC-K18mob2 described in example 3.2, plasmid DNA of pEC-K18mob2 was cleaved completely with the restriction endonuclease EcoRI and treated with alkaline ■ phosphatase (Alkaline Phosphatase, Roche Diagnostics GmbH, Mannheim, Germany) .

The vector pCRBl-ptsIexp was isolated from Escherichia coli ToplO and cleaved completely with the restriction endonuclease EcoRI and the fragment approx. 2140 bp in size with the ptsl gene was purified from a 0.8% agarose gel (QIAquick Gel Extraction Kit der Firma Qiagen, Hilden, Germany) . The fragment with the ptsl gene was then ligated with the vector pEC-K18mob2 (T4-Ligase, Roche Diagnostics GmbH, Mannheim; Germany) . The ligation batch was transformed in the E. coli strain DH5αmcr (Hanahan, In: DNA cloning. A Practical Approach. Vol.. I, IRL-Press, Oxford, Washington DC, USA) . Selection for plasmid-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 Pstl with subsequent agarose gel electrophoresis. The plasmid was called pEC-Kl8mob2ptsIexp and is shown in figure 2.

Brief Description of the Figures:

Figure 1: Map of the plasmid pEC-K18mob2

Figure 2: Map of the plasmid- pEC-K18mob2ptsIexp

The abbreviations and designations used have the following meaning:

Kan: Resistance gene for kanamycin

per: Gene for control of the number of copies from pGAl

oriV: ColEl-similar origin from pMBl

rep: Plasmid-coded replication region from C. glutamicum plasmid pGAl

RP4mob: RP4 mobilization site

ptsl ptsl gene from C. glutamicum

EcoRI : Cleavage site of the restriction enzyme EcoRI Hindlll: Cleavage site of the restriction enzyme

Hindlll

Pstl: Cleavage site of the restriction enzyme Pstl

Claims

What is claimed is :

1. Isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the ptsl gene, chosen from the group consisting of.

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 phosphotransferase system enzyme I.

2. Polynucleotide according to claim 1, wherein the polynucleotide is a preferably recombinant DNA which is capable of replication in coryneform bacteria.

3. Polynucleotide according to claim 1, wherein the polynucleotide is an RNA.

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

5. DNA according to claim 2 which is capable of replication, comprising (i) the nucleotide sequence shown in SEQ ID No. 1, or

(iv) sense mutations of neutral function in (i) .

6. DNA according to claim 5 which is capable of replication, characterized in that the hybridization is carried out under a stringency corresponding to at most 2x SSC.

7. Polynucleotide sequence according to claim 1, which codes for a polypeptide which comprises the amino acid sequence shown in SEQ ID No. 2.

8. Coryneform bacteria in which the ptsl gene is enhanced, in particular over-expressed.

9. Corynebacterium glutamicum DSM5715/pEC-Kl8mob2 deposited as DSM 13245 at the Deutsche Sammlung fur

Mikroorganismen und Zellkulturen [German Collection of Microorganisms and Cell Cultures] , DSMZ, Braunschweig, Germany.

10. Escherichia coli strain DH5alphamcr/pEC-Kl8mob2ptsIexp (= DH5αmcr/pEC-K18mob2ptsIexp) deposited as DSM 14278 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen [German Collection of Microorganisms and Cell Cultures] , Braunschweig, Germany.

11. Process for the fermentative preparation of L-amino acids, in particular L-lysine, characterized in that the following steps are carried out: a) fermentation of the coryneform bacteria which produce the desired L-amino acid and in which at least the ptsl gene or nucleotide sequences which code for it are enhanced, in particular over- expressed;

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

c) isolation of the L-amino acid.

12. Process according to claim 11, characterized in that bacteria in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally enhanced are employed.

13. Process according to claim 11, characterized in that bacteria in which the metabolic pathways which reduce the formation of the desired L-amino acid are' at least partly eliminated are employed.

14. Process according to claim 11, characterized in that a strain transformed with a plasmid vector is employed, and the plasmid vector carries the nucleotide sequence which codes for the ptsl gene.

15. Process according to claim 11, characterized in that the expression of the polynucleotide (s) which code(s) for the ptsl gene is enhanced, in particular over- expressed.

16. Process according to claim 11, characterized in that the catalytic properties of the polypeptide (enzyme protein) for which the polynucleotide ptsl codes are increased.

17. Process according to claim 11, characterized in that for the preparation of L-amino acids, coryneform microorganisms in which at the same time one or more of the endogenous genes chosen from the group consisting of,

17.1 the dapA gene which codes for dihydrodipicolinate synthase,

17.2 the gap gene which codes for glyceraldehyde 3- phosphate dehydrogenase,

17.3 the tpi gene which codes for triose phosphate isomerase,

17.4 the pgk gene which codes for 3-phosphoglycerate kinase,

17.5 the zwf gene which codes for glucose 6- phosphate dehydrogenase,

17.6 the pyc gene which codes for pyruvate carboxylase,

17.7 the mqo gene which codes for malate-quinone oxidoreductase,

17.8 the lysC gene which codes for a feed-back resistant aspartate kinase,

17.9 the lysE gene which codes for lysine export,

17.10 the horn gene which codes for homoserine dehydrogenase

17.11 the ilvA gene which codes for threonine . dehydratase or the ilvA(Fbr) allele which codes for a feed back resistant threonine dehydratase,

17.12 the ilvBN gene which codes for acetohydroxy- acid synthase,

17.13 the ilvD gene which codes for dihydroxy-acid dehydratase,

17.14 the zwal gene which codes for the Zwal protein

is or are enhanced or over-expressed are fermented.

18. Process according to claim 11, characterized in that for the preparation of L-amino acids, coryneform microorganisms in which at the same time one or more of the genes chosen from the group consisting of

18.1 the pck gene which codes for phosphoenol pyruvate carboxykinase,

18.2 the pgi gene which codes for glucose 6- phosphate isomerase,

18.3 the poxB gene which codes for pyruvate oxidase,

18.4 the zwa2 gene which codes for the Zwa2 protein,

is or are attenuated are fermented.

19. Coryneform bacteria which contain a vector which carries a polynucleotide according to claim 1.

20. Process according to one or more of claims 11-18, characterized in that microorganisms of the species Corynebacterium glutamicum are employed.

21. Process according to claim 20, characterized in that the Corynebacterium glutamicum strain DSM5715/pEC- K18mob2 is employed.

22. Process according to claim 20, characterized in that the Corynebacterium glutamicum strain DH5alphamcr/pEC-

K18mob2ptsIexp is employed.

23. Process for discovering RNA, cDNA and DNA in order to isolate nucleic acids or polynucleotides or genes which code for the phosphotransferase system enzyme I or have a high similarity with the sequence of the ptsl gene, characterized in that the polynucleotide comprising the polynucleotide sequences according to claims 1, 2, 3 or 4 is employed as hybridization probes.

24. Process according to claim 23, characterized in that arrays, micro arrays or DNA chips are employed.

25. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein ^' the associated amino acid sequences between positions 134 to 140 in SEQ ID No. 2 are altered by amino acid exchange.

26. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences between positions 120 to 127 in SEQ ID No. 2 are altered by amino acid exchange.

27. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences between positions 120 to 127 and 134 to 140 in SEQ ID No. 2 are altered by amino acid exchange.

28. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences at position 123 in SEQ ID No. 2 contain any other proteinogenic amino acid excluding L-lysine.

29. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences at position 123 in SEQ ID No. 2 contain L-glutamic acid or L-aspartic acid.

30. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences at position 123 in SEQ ID No. 2 contain L-glutamic acid.

31. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences at position 137 in SEQ ID No. 2 contain any other proteinogenic amino acid excluding L-arginine.

32. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences at position 137 in SEQ ID No. 2 contain L-cysteine.

33. DNA which originates from coryneform bacteria and codes for enzyme I of the phosphotransferase system, wherein the associated amino acid sequences at position 123 contains glutamic acid and at position 137 containes L- cysteine, shown in SEQ ID No. 4.

34. DNA according to claim 33, characterized in that this contains the nucleobase guanine at position 520 and the nucleobase thymine at position 562, shown in SEQ ID No 3.

35. Coryneform bacteria which contain a DNA according to one or more of claims 25 to 34.