WO2002059329A1 - Novel nucleotide sequences that encode the cite gene - Google Patents

Abstract

The invention relates to an isolated polynucleotide that contains a polynucleotide sequence selected from the group comprising a) a polynucleotide that has 70 % identity with a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID No. 2, b) a polynucleotide encoding a polypeptide that contains an amino acid sequence that has 70 % identity with the amino acid sequence of SEQ ID No. 2, c) a polynucleotide that is complementary to the polynucleotides of a) or b), and d) a polynucleotide that contains at least 15 subsequent nucleotides of the polynucleotide sequence of a), b) or c). The invention further relates to a method for the fermentative production of L amino acids using coryneform bacteria in which at least the citE gene is present in an attenuated form. The invention also relates to the use as hybridization probes of polynucleotides that contain the inventive sequences.

Description

 New nucleotide sequences coding for the citE gene

The invention relates to nucleotide sequences coding for the citE gene from coryneform bacteria and a method for the fermentative production of amino acids using bacteria in which the citE gene is weakened.

State of the art

L-amino acids, in particular L-lysine, are used in human medicine and in the pharmaceutical industry, in the food industry and very particularly in animal nutrition.

It is known that amino acids are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of the great importance, work is constantly being carried out to improve the manufacturing processes. Process improvements can relate to fermentation-related measures such as stirring and supply of oxygen, or the composition of the nutrient media, such as the sugar concentration during fermentation, or the processing into the product form by, for example, ion exchange chromatography or the intrinsic performance properties of the microorganism itself.

To improve the performance characteristics of this

Microorganisms use mutagenesis, selection and mutant selection methods. In this way, strains are obtained which are resistant to antimetabolites or auxotrophic for regulatory-important metabolites and which produce amino acids.

For some years now, methods of recombinant DNA technology for strain improvement of L- Strains of Corynebacterium producing amino acids were used by amplifying individual amino acid biosynthesis genes and examining the effect on amino acid production.

Object of the invention

The inventors have set themselves the task of providing new measures for improved fermentative production of amino acids.

Description of the invention

If L-amino acids or amino acids are mentioned below, one or more amino acids including their salts are selected from the group 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 are meant. L-lysine is particularly preferred.

If 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 is meant.

The invention relates to an isolated polynucleotide from coryneform bacteria, containing a polynucleotide sequence coding for the citE gene, selected from the group

a) Polynucleotide that is at least 70% identical to a polynucleotide that codes for a polypeptide that contains the amino acid sequence of SEQ ID No. 2 contains

b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID No. c) polynucleotide which is complementary to the polynucleotides of a) or b),

d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c),

wherein the polypeptide preferably has the activity of citrate lyase E.

The invention also relates to the above-mentioned polynucleotide, which is preferably a replicable DNA containing:

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

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

(iii) at least one sequence that corresponds to that of the

Sequences (i) or (ii) complementary sequences hybridized, and optionally

(iv) functionally neutral sense mutations in (i) which do not change the activity of the protein / polypeptide.

Finally, the invention further relates to polynucleotides selected from the group

a) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 1 and 601

b) polynucleotides containing at least 15 consecutive nucleotides selected from the Nucleotide sequence of SEQ ID No. 1 between positions 602 and 1423

c) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 1424 and 1964

Other items include:

a replicable polynucleotide, in particular DNA, containing the nucleotide sequence as shown in SEQ ID No. 1;

a polynucleotide encoding a polypeptide having the amino acid sequence as described in SEQ ID No. 2 includes;

a vector containing parts of the polynucleotide according to the invention, but at least 15 consecutive nucleotides of the claimed sequence,

and coryneform bacteria in which the citE gene is weakened, in particular by an insertion or deletion.

The invention also relates to polynucleotides which essentially consist of a polynucleotide sequence which can be obtained by screening by means of hybridization of a corresponding library of a coryneform bacterium which contains the complete gene or parts thereof, with a probe which has the sequence of the invention

Contains polynucleotide according to SEQ ID No. 1 or a fragment thereof and isolation of said polynucleotide sequence.

Polynucleotides containing the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA to nucleic acids respectively

Isolate polynucleotides or full-length genes that code for the citrate lyase E, or to isolate those nucleic acids or polynucleotides or genes which are very similar to the sequence of the citE gene. They are also suitable for installation in so-called "arrays", "micro arrays" or "DNA chips" in order to detect and determine the corresponding polynucleotides.

Polynucleotides which contain the sequences according to the invention are furthermore suitable as primers, with the aid of which polymerase chain reaction (PCR) can be used to produce DNA from genes which code for citrate lyase E.

Such oligonucleotides serving as probes or primers contain at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, very particularly preferably at least 15, 16, 17, 18 or 19 successive nucleotides. Oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides are also suitable , If applicable, too

Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are suitable.

"Isolated" means separated from its natural environment.

"Polynucleotide" generally refers to

Polyribonucleotides and polydeoxyribonucleotides, which can be unmodified 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 produced therefrom and also one which is at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90% and entirely are particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No. 1 or a fragment made from it.

“Polypeptides” are understood to mean peptides or proteins which contain two or more amino acids linked 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 citrate lyase E 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 too at least 91%, 93%, 95%, 97% or 99% are identical to the polypeptide according to SEQ ID No. 2 and have said activity.

The invention further relates to a process for the fermentative production of amino acids, selected from the group 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 those for the nucleotide sequences encoding the citE gene are weakened, in particular switched off or expressed at a low level.

The term "weakening" describes in this

Context, the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which is responsible for a corresponding enzyme or protein encodes a low activity or inactivates the corresponding gene or enzyme (protein) and, if appropriate, combines these measures. The microorganisms which are the subject of the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be representative of coryneform bacteria, in particular of the genus Corynebacterium. In the genus Corynebacterium, the species Corynebacterium glutamicum should be mentioned in particular, which is known in the art for its ability to produce L-amino acids.

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

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavumvATCC1408bacterium Brev

and mutants or strains producing L-amino acids produced therefrom, such as the L-lysine producing strains

Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 Corynebacterium glutamicebum DM58-1M5numebutumum DM58-1 C5 The new citE gene from C. glutamicum coding for the enzyme citrate lyase E (EC No. 4.1.3.6) was isolated.

To isolate the citE gene or other genes from C. glutamicum, a gene bank of this microorganism is first created in Escherichia coli (E. coli). The creation of gene banks is recorded in well-known textbooks and manuals. Examples include the textbook by Winnacker: Genes and Clones, An Introduction to Genetic Technology (Verlag Chemie, Weinheim, Germany, 1990), or the manual by Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well-known gene bank is that of the E. coli K-12 strain W3110, which was developed 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 bank of C. glutamicum ATCC13032 which can be generated 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).

Börmann et al. (Molecular Microbiology 6 (3), 317-326 (1992)) in turn describe a gene bank of C. glutamicum ATCC13032 using the cosmide pHC79 (Hohn and Collins, 1980, Gene 11, 291-298).

Plasmids such as pBR322 (Bolivar, 1979, Life Sciences, 25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) can also be used to produce a C. glutamicum gene bank in E. coli become. Particularly suitable hosts are E. coli strains which are defective in terms of restriction and recombination, such as the strain

DH5αmcr, which was described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned with the aid of cosmids or other λ vectors can then in turn be used in conventional ones suitable for DNA sequencing <-υ CO r) μ ¹

(-π 0 n 0 π σ cn

£ d tr ≤ Hi ιQ "> £ ^ 1 a σ Φ C W υ S σ DJ S <! X iQ σ S CΛ> σ O <

Φ ^ Φ Φ d = μ CΛ CQ H- DJ 0 2 σ H- O σ φ Φ £ EP | tr Φ 0 Φ μ- 3 Φ DJ rt y- ³ μ- 3 -? a

DJ O Φ to CL N μ 3 'd H- ts Φ Ω • Φ Φ α CQ r + μ- ιQ rt μ l-S d φ DJ 3 μ DJ ιQ Φ Φ rt? V

Φ Φ H 3 3 DJ p- 1 3 μ- Ό tr DJ 3 Φ 3 ^" rt Ω O μ μ- rt

3 0 H- Φ Φ 3 HN £ h- ¹ ω Hi σ Φ DJ μ- 3 0> O μ- <J DJ 3 ^ 0? Φ μ Φ μ- O μ- O rt α Ω 3 3 CQ 3 DJ Φ φ ⁾ Φ H 0 3 CQ φ α φ 0 3 Φ CQ 0 3 μ- μ Ω 3 φ μ

Φ 3 "* _* DJ: d ιQ 03 H 3" iQ (- ^■ ιQ Φ r + 3 rt DJ: μ- Φ ιQ μ μ- d μ- 0 _^ - rt tr DJ DJ Φ

H- μ 3 d rt rt H- • r + • <: d r- ¹ Φ 3 Φ μ- d r + 3 φ H "Ω Φ CQ 1 a _^ - tr DJ H φ 3

0 Φ 3 0-, N D> 3 tr φ CO 3 3 3 li φ 3 μ- d ^f U da 3 μ ^j or 0 r + et • (- ^■ r + CO> O K. 3 Φ Φ 0- Φ r + α φ 3 Hi) μ Ω d Φ rt ~ J> co

1 Φ 3 ^J H- H- DJ: d H- H- N DO H? er DJ CO • μ- Φ ιQ d: I) O Ω 3 φ! t- »Ω N d tr t-3 μ Ω O d ω 3 α Φ Φ H- σ O: N tP Φ Φ 3 Φ s: μ _^ - ιQ Φ μ- 3 •• DJ • tr

Φ Φ S! tr 3 t r + H- 3 CO Φ .2 3 s: xQ H 3 d μ μ- φ tr Φ (-π C ΪV

Φ μ Φ DJ Hi Φ Φ Φ DJ to r + l-i> 3 • d 3?> Σ CL μ -J DJ Ω μ- N 3 Φ dd

H- 3 H- 3 d 3 13 d s: H- DJ r + 1 Φ μ- φ 3 3 Φ DJ J-- 3 Ω € CTι 3 • 0

3 H- rt 3 ^ ιQ CQ Φ Φ 3 - CΛ 3 ^ H 3 CΛ μ-> 3 Φ O 1 §> • σ ω ^ 3 rt 3 Φ Φ <J Φ Ω H- H H- 0. Φ Φ » 0 r + NM 3 1 <£> Ω> a 1 tr μ- d μ rt Φ, -. ιQ er r + φ Φ r + H- Λ H co lO 0 CΛ Cd ιQ Ω -J <! μ- Ω ω <-π 0 Φ Φ

D ⁾ : ω t Φ H- HO Φ Φ 3 Φ rt d 3 Φ α CQ Φ H Φ μ. O 0- μ- H 1 Hl μ- μ

O H- 3 O {D: φ 3 H _^ 3 H- Φ ^• <μ- φ H DJ: Λ Hi Hi rt -— - 3 co 0- & CΛ CTι rt "φ 3 3 3 3 0 p- H- (- ^■ α 3 iQ CQ σ dd μ- d M ¹ CO d Φ -J CΛ ω rr P- DJ co ω -π 3 H- ω r + Φ N 3 Φ li φ 3 3 1 Ω Dd PO Φ Λ Ω DJ d

H- 3 H- d 3 Φ φ (- • P Φ α CO Φ Φ 3 Ω a φ 3 0- Q od φ PO 3 d μ- 3 3 ιQ Φ co 0 φ H d cn CQ ω φ 3 rr Φ μ - 0 CO N d Φ Φ 00 rt CO Φ N φ Φ ιQ α

Φ co rt ^* dd 3 r +? T f + MH ιQ • »3 ¹ rr. φ 3 3 3 - φ CQ DJ 3 VT) 3 Φ

3 rt 3 d DJ •: O DJ Φ μ- MM ώ 3 iQ _^ • - Φ DJ Φ 3 N - Ω μ DJ

• X) tr DJ μ ιQ rt 3 Ω 3 3 M 3 O- O 3 1 N> d μ-? μ μ DJ DJ Φ -J Φ 3

O μ φ H DJ DJ H- H- CQ α HK Φ μ- 3 NO Φ rt μ- 0 d Ω μ ^> 3 - CQ φ CQ α O \ CQ - r + 3 P Φ r + σ Hi r + φ μ- ^ Φ μ- 3 CO μ- 3, -. 3 ^J Ω ^> rt Ω

Φ et DJ Φ H- CO H φ H- μ- 3 3 3 CQ N α rt φ μ- rt S tr CQ - tr 0 tr ι-i φ 3 3 CQ H 0 DJ: iQ <! H- -2 3 co Q Φ Ό rt Φ. Φ Φ Φ μ> φ O: Φ Hi DJ

H- 3 Φ H- 3 d Φ DJ - ¹ 0 O. Ω μ- H Q- 3 JHH rt CTι μ> 1 3 CQ - μ-

W 3 rt d 3 CQ l-i ιQ r + • d 3- Ω Φ 0- 0 0, φ S! Φ CO tr TJ 3 Ω rt. Φ

O co r + p-? R - - Φ Φ μ. α 3 Φ 3 "co μ- α μ- CQ 0 Φ CQ 3 d 0 ^. μ Φ tr 3- &> Q ι-i • rr P <! Φ H ¹ ιQ 3 φ d Φ σ μ- 0. Ω O- 0 3 μ Φ. - φ

0 ⁾ Q- DJ H- tr H- Φ • D ⁾ ιQ Φ Φ rt CΛ Φ 3 ^J CQ CO M ιQ μ- TJ 3

H Φ DJ f- ¹ α <! Φ 3> 0 ^ d Φ 2 r + Q 3 3 φ C rt NJ μ α Φ α μ co Φ Φ H-; M t? M α HO CQ σ 1 Φ μ- r + μ 10 ö 0 <X ⁾ - ~ J DJ DJ tr 3 O

CO co N. r + DJ ^ DJ 3 H Φ 3 α μ- r + CQ Ω Q tr 3 ^J 2. Hi 1 1 3 3 Φ μ- Ω Q rt φ: CΛ n> tυ: 3 li P μ. Hi H Φ ω φ 3 ^{J •} o Φ μ- μ> ro 3 3 3 rt φ φ

JD 3 3 d H rt 3 O H- 3 H- ιQ co H α ι-i CO 3 ö 1 μ ω co ω Φ Φ φ Λ tr α tT r + r + r + 3 O 3 H l- ¹ lO Φ 3 DJ φ Φ Ω CΛ μ- OJ KJ 3 3 μ- α α d

H- <- Φ ω H- α _^ Φ CO α Φ φ Φ r + μ- li Ü Ω 3 ^J ≤ a Φ Φ 0 CTi _^ -. μ- Q μ- φ d left ιQ Φ μ. O DJ: d H- μ. HH Φ 3 ιQ ιQ 3 ^J μ d 0 -Q 3 Ω> £ rt rt Λ 3 3

H- 3 d H- Φ Q α d α d 3 h- ¹ Ω vQ O 3 DJ Φ φ Φ μ- ι-i. d 3 ^J -—, ⁽ -0 μ- • rt iQ N co? 3 rt H- Φ Φ N iQ φ ^* φ H CO tr 3 Φ Φ Φ μ »CO φ DJ CO μ- μ. tr r + ιQ d S TJ φ 3 H Φ • 3 Φ er s <V Φ r + φ tr Φ »3 3 I ⁾ CTi φ rt Φ

Φ μ- Φ rt d H JD H Φ 0 H Φ 3 Φ φ N μ- CO - N? Φ O μ li 0 3 H- rt OND ⁾ <! d <! 3 • O y - 0- 3 3 0 ⁾ DO Ω CO -. J CQ Hi rt φ 3 O 0 ⁾ r + d 0 CO 3 0 -3 ^ «r + 3 μ ^> Φ φ d φ <! J - _^ 03 3

3 tu 3 rt Φ CO 3 c + 3 Φ (- ¹ Φ μ- r + T) 3 co O 0 - - • 3 O et

3 3 φ H- P-? R DJ 0, H- CO μ- 3 • h rt 3 0- rt Hi tr Φ

; r H- 3 O 3 Φ d φ CΛ CQ μ- α 3 0 0 0- DJ 0 Φ 0- Φ Φ μ

O: 0 2 3 CO H- CQ H M Φ 3 CO CQ π rt Φ 3 O 3 φ 3 0-

3 3 ^J 1 Φ P Ω D 0- H DJ: CQ Φ μ α Φ 3 Φ

3 r + 3 φ 3 ¹ O Ω d μ- rt μ <! 3 φ H Φ H H- 3 ^" h μ- 3 Φ O <

3 D 3 Φ Q ω μ- 3 O

• α r + 3

The person skilled in the art can find information on this from 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 well-known textbooks of genetics and molecular biology. Amino acid sequences which can be derived from SEQ ID No. 2 result, are also part of the invention.

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

The person skilled in the art can find instructions for identifying DNA sequences by means of hybridization in the manual "The DIG System Users Guide for Filter Hybridization" by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). The hybridization takes place under stringent conditions, ie only hybrids are formed in which the probe and target sequence, ie the polynucleotides treated with the probe, are at least 70% identical. 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 at a relatively low stringency compared to the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996). For example, a 5x SSC buffer at a temperature of approx. 50 ° C - 68 ° C can be used for the hybridization reaction. Probes can also hybridize with polynucleotides that have less than 70% identity to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be done, for example, by lowering the salt concentration to 2x SSC and possibly subsequently 0.5x SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim,

Mannheim, Germany, 1995) can be reached, with a temperature of about 50 ° C - 68 ° C is set. It may be possible to reduce the salt concentration to 0.1x SSC. By gradually increasing the hybridization temperature in steps of approx. 1-2 ° C from 50 ° C to 68 ° C, polynucleotide fragments can be isolated which, for example, contain at least 70% or at least 80% or at least 90% to 95% or at least 96% to 99 % Identity to the sequence of the probe used. It is also possible to isolate polynucleotide fragments that are completely identical to the sequence of the probe used. Further instructions for hybridization are available on the market in the form of so-called kits (e.g. DIG Easy Hyb from Röche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).

The person skilled in the art can find instructions for amplifying DNA sequences with the aid of the polymerase chain reaction (PCR) in the Gait handbook, inter alia: Oligonucleotide synthesis: A Practical Approach (IRL Press, Oxford, UK

1984) and with Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

It has been found that corynefor e bacteria produce amino acids in an improved manner after weakening the citE gene. co CO to NJ P>P>

Cn o cn O cn o cn σ μ. ^■ Pö ω α «σ Ω Cd K Cd N

Φ Φ φ et Φ μ- Φ d P- Φ 3 DJ X d d μ μ rt T3 ιQ μ Φ ιQ N rt Ό μ rt 3 Φ μ 3 rt Φ * DJ μ

CQ J μ- 3 Φ DJ CΛ d <tr 3 Φ Cd

Ω DJ 3 CQ P ¹ μ- μ Φ φ Ό * co μ tr rt DJ DJ CO CO ιQ Hl μ 3 μ rt CQ N

H Φ rt rt o rt 3 d: μ Φ O μ- μ- μ-

SU 3 o O μ μ DJ 3 ^J μ- 3 rt CO o Φ

3 rt μ μ iQ d H μ 3 Hi Φ Ω 3 H α DJ φ Φ Φ? CQ d ιQ DJ μ- tr d

3 3 3 3 rt rt 3 Φ H 3 φ Q, 3

3 • * _* Φ d μ ιO μ H CQ 3 Φ iQ p> Φ _"* μ dd CO \ CQ

<-0 H PC φ Ϊ o 3 tr μ Φ

<> α 3 μ-> 3 rt α ιQ T Φ Φ Ω P- d ιQ tr d Φ O: μ u3 μ- 3

- 3 DJ o et μ μ α 3 DJ d rt Φ ιQ tr CQ μ- φ Φ Φ 3 tr H Cd μ iQ Φ o <μ 3 μ Φ U-5 DJ 1

Φ s: 3 3 D ⁾ d 3 Φ rt Ω

3 o φ rt Ω μ Ω co 0 Φ tr

DJ - 3 0 Φ Φ Ω Φ tr φ μ 3 CO

3 υs μ- tr μ 3 μ tr 3 Φ rt μ- CQ Ω

3 σ> Φ μ- lO. Φ Φ P- N co tr et \ μ 3 Φ Ω ιQ C rt Ω O s;

, P ¹ N α- 3 Ό Φ φ O φ tr α DJ: ndd Φ μ 3 3 μ O φ Φ Ω

N NJ 3 Φ Φ Φ φ 2 α 3 μ "d J-- Hi ιQ CQ X! Rt co DJ Φ d

CQ CD μ- CQ o co Tf P- co 5o μ M α- 3

DJ 3 ω Ό μ- μ CO μ- 3 μ- μ- iQ

3 α rt φ 0 Φ Ω 0 DJ DJ ιQ Φ

§ tr Φ φ μ 3 CQ tr 3 tr d Φ? T φ Φ rt p <DJ co Φ 3 co 3 O:

3 P- rt co μ-? R Φ iQ co 3

Hi α Φ O μ- o <DJ 3 Φ Ω 3

DJ 00 Φ 3 μ 3 3 φ 3 CO 3 ^J Φ

CQ o μ Φ μ 3 ^* Ω DJ 3 co * 3 Φ DJ: O 3 "Hi φ α ^ 0- tr μ 3 0- 3 DJ rt Φ

3 DJ DJ Φ Hl α d tr Φ α 3 d Ω CO J P- 0 φ μ P- rt 3 rt

Φ 3 3 "μ CO P" μ Ω 3 Φ s:

0- 3 CΛ O • d iQ d tr P- rt Φ σ DJ rt Φ Φ

DJ 3 P- Φ 3 α

2 3 DJ o Φ 3 ιQ lΩ. μ S, o Φ μ d 3 μ rt P "Φ rt Φ μ o μ rt O CQ Φ μ rt T3 N μ € 2 μ- s: Q. 0,

Φ -. O Φ φ d U3 Φ Φ μ-

^ to 3 P- rt 3 μ 3 Φ

H o CQ DJ Φ d α

3 Φ rt rt Φ

3 μ- P- Φ 3 Q 3 d O

Φ 3 3

3

can read well-known textbooks in genetics and molecular biology such as that of Hagemann ("General Genetics", Gustav Fischer Verlag, Stuttgart, 1986) are taken.

Transitions, transversions,

Insertions and deletions are considered. Depending on the effect of the amino acid exchange on the enzyme activity, one speaks of missense mutations or nonsense mutations. Insertions or deletions of at least one base pair (bp) in a gene lead to

Frame shift mutations, as a result of which incorrect amino acids are incorporated or translation prematurely terminates. Deletions from several codons typically lead to a complete failure of the enzyme activity. Instructions for generating such mutations are state of the art and can be found in known textbooks of Genetics and molecular biology such as the textbook by Knippers ("Molecular Genetics", 6th edition, Georg Thieme Verlag,

Stuttgart, Germany, 1995), from Winnacker ("Gene and Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or from Hagemann ("General Genetics", Gustav Fischer Verlag, Stuttgart, 1986).

A common method of mutating C. glutamicum genes is the method of gene disruption ("gene disruption") and gene exchange described by Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)) (" gene replacement ").

In the gene disruption method, a central part of the coding region of the gene of interest is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum. Examples of vectors are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pKlδmob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pKlδmobsacB or pK19mobsacB (Jäger et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269: 32678-84; US Patent 5,487,993), pCR®Blunt (Invitrogen, Groningen, 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 central part of the coding region of the gene, is then converted into the desired strain of C. glutamicum by conjugation or transformation. The conjugation method is described, for example, by Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross-over" event, the coding region of the gene in question is interrupted by the vector sequence and two incomplete alleles are obtained, each of which lacks the 3 'and 5' ends. This method was, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) used to switch off the recA gene from C. glutamicum.

In the method of gene replacement, a mutation such as a deletion, insertion or base exchange in the gene of interest is produced in vitro. The allele produced is in turn cloned into a vector which is not replicative for C. glutamicum and then cloned by Transformation or conjugation transferred to the desired host by C. glutamicum after homologous recombination by means of a first, integration-causing "cross-over" event and a suitable one second excision-causing "cross-over" event in the target gene or in the target sequence is achieved by incorporating the mutation or the alley. This method was, for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) used to switch off the pyc gene from C. glutamicum by deletion.

In this way, a deletion, insertion or base exchange can be incorporated into the citE gene.

In addition, it can be advantageous for the production of L-amino acids, in addition to the weakening of the citE gene, one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, the amino acid export and, if appropriate to reinforce regulatory proteins, in particular to overexpress them.

In this context, the term “amplification” describes the increase in the intracellular activity of one or more enzymes (proteins) in a microorganism which are encoded by the corresponding DNA, for example by increasing the copy number of the gene or genes, using a strong promoter or uses a gene or allele which codes for a corresponding enzyme (protein) with a high activity and, if appropriate, combines these measures.

So for the production of L-lysine in addition to

Attenuation of the citE gene simultaneously one or more of the genes selected from the group

The gene dapA coding for the dihydrodipicolinate synthase (EP-B 0 197 335),

The gene gap coding for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), The gene tpi coding for the triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),

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

Gene zwf coding for glucose-6-phosphate dehydrogenase (JP-A-09224661),

The pyc gene coding for pyruvate carboxylase (DE-A-198 31 609),

The mqo gene coding for the malate quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry

254, 395-403 (1998)),

The lysC gene coding for a feed-back resistant aspartate kinase (Accession No.P26512; EP-B-0387527; EP-A-0699759; WO 00/63388), • the lysE gene coding for lysine export (DE-A -195 48 222),

The gene zwal coding for the Zwal protein (DE: 19959328.0, DSM 13115)

amplified, especially overexpressed.

Furthermore, it can be advantageous for the production of L-lysine, in addition to the attenuation of the citE gene, at the same time one or more of the genes selected from the group

The gene pck coding for the phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047),

The gene pgi coding for glucose-6-phosphate isomerase (US 09 / 396,478, DSM 12969), The gene poxB coding for the pyruvate oxidase (DE: 1995 1975.7, DSM 13114),

The gene coding for the Zwa2 protein zwa2 (DE: 19959327.2, DSM 13113),

The gene hom coding for homoserine dehydrogenase (EP-A-0131171),

The gene coding for the homoserine kinase thrB (Peoples, O.W., et al., Molecular Microbiology 2 (1988): 63-72), and

The gene coding for the aspartate decarboxylase panD (EP-A-1006192)

attenuate, especially to decrease expression.

The weakening of the homoserine dehydrogenase can also be caused, inter alia, by amino acid exchanges, such as, for example, by exchanging L-valine for L-alanine, L-glycine or L-leucine at position 59 of the enzyme protein, by exchanging L-valine for L- Isoleucine, L-valine or L-leucine at position 104 of the enzyme protein and / or by replacing L-asparagine with L-threonine or L-serine at position 118 of the enzyme protein.

The weakening of the homoserine kinase can also be caused, inter alia, by amino acid exchanges, such as, for example, by exchanging L-alanine for L-valine, L-glycine or L-leucine at position 133 of the enzyme protein and / or by exchanging L-proline for L-threonine, L-isoleucine or L-serine can be achieved at position 138 of the enzyme protein.

The attenuation of the aspartate decarboxylase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by replacing L-alanine with L-glycine, L-valine or L-isoleucine at position 36 of the enzyme protein. o IV) NJ P> o Cπ o Cn σ cπ

2 <! Cd d T! S μ- Φ rt 3 φ Φ o μ tr 0- rt μ-

Ω x DJ rt rt tr φ 3 tr ¹ co Φ d 3 O P-DJ: μ

3 or P ^J 3 d tr Q Φ 0 μ μ- rt d H Φ 3

<! 3 co 3

Φ s: Q- D: * _* r μ Φ d DJ

X μ O μ x 3

Φ α μ φ μ- 3

3 φ ιQ -. Φ α. 3 DJ Φ

Φ • 3> N co rt μ- d co? 3 St.

X σ Ω o d:

Φ μ- tr tr to μ μ Φ φ o Φ α o μ- 0- φ Φ CΛ Φ co P-

3 DJ: • d φ

• Λ d s: μ- rt μ P- Φ TJ o φ Φ P * μ

St. 3 or

Hi "> N TJ α

DJ d DJ d s: 3 Ϊ

? μ- 3 rt

O: φ to P- μ-

3 Φ et o

3 N P- P- 3

Φ d co 3

3 3 Ό co

P-DJ: o

Φ to φ d 3 μ- Φ P- μ

3 μ- φ

N CO t?

Ω § φ * d μ- P-

P-> CΛ 3

3 φ Ω rt O

H Φ Φ CQ

O μ DJ DJ:

P- c μ- μ d

Φ CO 3 μ- μ μ co 3 φ p- d o 3

DJ ιQ 3 DJ: co d ω DJ: μ d φ μ φ

o CO IV) rv> P ¹ P ¹

(-n o cn o Cπ o cπ

CO tv>

P- n P-

Ω o Φ

3 Ω ιΩ et

Φ tr

P- µ- 3

3 C0 o μ

2> t-. 3

DJ o DJ o P ¹

P- Ω φ

3 • μ d X

3 O Φ μ- μ-

CL φ CQ

Φ φ

CQ d tr ιΩ Φ

Μ rt μ-

X d d: μ r

3 O

CO X o

Ω μ- Ω

3 "μ rt CL tr

Φ μ-

3 co co o

TJ P ¹ μ DJ cn

O 3 o α iQ Ω d Φ d et Hl 3

Φ O P. co μ rt <: ι Ω o

Φ Φ μ tr CD N μ- Φ d * rt ιΩ α N co

Φ rt ≤ rt φ μ-

3 "tr CQ

DJ μ- φ rt CO tr Φ

P-

This goal is usually achieved within 10 hours to 160 hours.

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

A pure culture of the Escherichia coli strain Top 10 / pCR2. lcitEint was deposited on January 12, 2001 with the German Collection for Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) as DSM13981 in accordance with the Budapest Treaty.

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

The present invention is explained in more detail below on the basis of exemplary embodiments.

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

The composition of common nutrient media such as LB or TY medium can also be found in the manual by Sambrook et al. be removed. example 1

Production of a genomic cosmid library from C. glutamicum ATCC 13032

Chromosomal DNA from C. glutamicum ATCC 13032 is as described by Tauch et al. (1995, plasmid 33: 168-179) described isolated and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, code no. 27-0913-02). The DNA fragments are dephosphorylated with shrip alkaline phosphatase (Röche Molecular Biochemicals, Mannheim, Germany, product description SAP, code no. 1758250). The DNA of the cosmid vector SuperCosl (Wahl et al. (1987), Proceedings of the National Academy of Sciences, USA 84: 2160-2164), obtained from Stratagene (La Jolla, USA, product description SuperCosl Cosmid Vector Kit, Code no. 251301) is cleaved with the restriction enzyme Xbal (Amersham Pharmacia, Freiburg, Germany, product description Xbal, code no. 27-0948-02) and also dephosphorylated with shrimp alkaline phosphatase.

The cosmid DNA is then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04). The cosmid DNA treated in this way is mixed with the treated ATCC13032 DNA and the mixture is treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code no.27-0870-04) , The ligation mixture is then packed in phages using the Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, product description Gigapack II XL Packing Extract, Code no. 200217).

For infection of the E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Res. 16: 1563-1575), the cells are taken up in 10 mM MgSO ₄ and with an aliquot of the Phage suspension mixed. Infection and titering of the cosmid bank are, as in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), wherein the cells are plated on LB agar (Lennox, 1955, Virology, 1: 190) + 100 mg / 1 ampicillin. After overnight incubation at 37 ° C., recombinant individual clones are selected.

Example 2

Isolation and sequencing of the citE gene

The cosmid DNA of a single colony is isolated using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, Product No. 27-0913 -02) partially split. The DNA fragments are dephosphorylated with shrimp alkaline phosphatase (Röche Molecular Biochemicals, Mannheim, Germany, product description SAP, product No. 1758250). After gel electrophoretic separation, the cosmid fragments in the size range from 1500 to 2000 bp are isolated using the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The DNA of the sequencing vector pZero-1 obtained from Invitrogen (Groningen, Netherlands, product description Zero Background Cloning Kit, Product No. K2500-01) is mixed with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, Product No. 27 - 0868-04) split. The ligation of the cosmid fragments in the sequencing vector pZero-1 is carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), wherein the DNA mixture is incubated with T4 ligase (Pharmacia Biotech, Freiburg, Germany) overnight. This ligation mixture is then converted into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences, USA, 87: 4645-4649) electroporated (Tauch et al. 1994, FEMS Microbiol. Letters, 123: 343-7) and on LB agar (Lennox, 1955, Virology, 1: 190 ) plated with 50 mg / 1 Zeocin.

The plasmid preparation of the recombinant clones is carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing is carried out according to the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academies of Sciences, U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990,

Nucleic Acids Research, 18: 1067). The "RR dRhodamin Terminator Cycle Sequencing Kit" from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) is used. The gel electrophoretic separation and analysis of the sequencing reaction takes place 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 are then processed using the Staden program package (1986, Nucleic Acids Research, 14: 217-231) version 97-0. The individual sequences of the pZerol derivatives are blended into a coherent contig asse. The computer-aided coding area analysis is carried out with the program XNIP (Staden, 1986, Nucleic Acids Research, 14: 217-231).

The nucleotide sequence obtained is shown in SEQ ID No. 1 shown. Analysis of the nucleotide sequence reveals an open reading frame of 821 bp, which is referred to as the citE gene. The citE gene codes for a polypeptide of 273 amino acids. Example 3

Generation of an integration vector for the integration mutagenesis of the citE gene

The ATCC 13032 strain is used according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) chromosomal DNA isolated. Based on the sequence of the citE gene known from Example 2 for C. glutamicum, the following oligonucleotides are selected for the polymerase chain reaction (see also SEQ ID No. 3 and SEQ ID No. 4):

citE-intl

5 "GAC GTG CTG AGA TCA TTC C 3 ^V citE-int2

5 TAA GCC TCA TGG TGT CTC G 3 ^N

The primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and after

Standard PCR method by Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) carried out the PCR reaction with the Taq polymerase from Boehringer Mannheim (Germany, product description Taq DNA Polymerase, Product No. 1 146 165). With the help of the polymerase chain reaction, the primers enable the amplification of a 450 bp internal fragment of the citE gene. The product amplified in this way is tested electrophoretically in a 0.8% agarose gel.

The amplified DNA fragment is inserted into the vector pCR2.1-TOPO (Mead at al. (1991) Bio / Technology 9: 657) using the TOPO TA cloning kit from Invitrogen Corporation (Carlsbad, CA, USA; catalog number K4500-01) -663).

The E. coli strain TOP10 is then electroporated using the ligation approach (Hanahan, In: DNA cloning. A practical approach. Vol.I. IRL-Press, Oxford, Washington DC, USA, 1985). Plasmid-bearing cells are selected by plating out the Transformation approach on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 ^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), which was supplemented with 50 mg / 1 kanamycin. Plasmid DNA is isolated from a transformant using 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 becomes pCR2. Called lcitEint and is shown in Figure 1.

Example 4

Integration mutagenesis of the citE gene in the strain DSM 5715

The vector pCR2 mentioned in Example 3. lcitEint is used according to the electroporation method of Tauch et.al. (FEMS Microbiological Letters, 123: 343-347 (1994)) in

Corynebacterium glutamicum DSM 5715 electroporated. The strain DSM 5715 is an AEC-resistant lysine producer, which is described in EP-B-04351342. The vector pCR2. lcitEint cannot replicate independently in DSM5715 and only remains in the cell if it has integrated into the chromosome of DSM 5715. Selection of clones with pCR2 integrated in the chromosome. lcitEint is carried out by plating the electroporation batch onto LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2nd ^and Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), which has been supplemented with 15 mg / 1 kanamycin.

For the verification of the integration, the citEint fragment is marked according to the method "The DIG System Users Guide for Filter Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) with the Dig Hybridization Kit from Boehringer. Chromosomal DNA of a potential integrant is extracted using the method of Eikmanns et al. (Microbiology 140: 1817 - 1828 (1994)) isolated and each with the

Restriction enzymes EcoRI, BamHI and Hindlll cut. The resulting fragments are separated by means of agarose gel electrophoresis and hybridized at 68 ° C. using the Boehringer dig-hybridization kit. The plasmid pCR2 mentioned in Example 3. lcitEint has inserted into the chromosome of DSM5715 within the chromosomal citE gene. The strain is called DSM5715:: pCR2. called lcitEint.

Example 5

Production of lysine

C. glutamicum strain DSM5715 obtained in Example 4:: pCR2. lcitEint is cultivated in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant is determined.

For this purpose, the strain is first incubated on agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg / 1) for 24 hours at 33 ° C. Starting from this agar plate culture, a preculture is inoculated (10 ml medium in 100 ml Erlenmeyer flask) The complete medium CgIII is used as the medium for the preculture.

Medium Cg III

NaCl 2.5 g / 1

Bacto peptone 10 g / 1

Bacto yeast extract 10 g / 1

Glucose (autoclaved separately) 2% (w / v)

The pH is adjusted to pH 7.4 Kanamycin (25 mg / 1) is added to this. The preculture is incubated on the shaker at 33 ° C. at 240 rpm for 48 hours. A main culture is inoculated from this preculture so that the initial OD (660 nm) of the main culture is 0.1 OD. The medium MM is used for the main culture

Medium MM

CSL (Corn Steep Liquor) 5 g / 1

MOPS (morpholinopropanesulfonic acid) 20 g / 1

Glucose (autoclaved separately) 50g / l

salts:

(NH ₄ ) ₂ S0 ₄ ) 25 g / 1

KH ₂ P0 ₄ 0.1 g / 1

MgS0 ₄ * 7 H ₂ 0 1.0 g / 1

CaCl ₂ * 2 H ₂ 0 10 mg / 1

FeS0 ₄ * 7 H ₂ 0 10 mg / 1

MnS0 ₄ * H ₂ 0 5.0 mg / l

Biotin (sterile filtered) 0.3 mg / 1

Thiamine * HC1 (sterile filtered) 0.2 mg / 1

Leucine (sterile filtered) 0.1 g / 1

CaC0 ₃ 25 g / 1

CSL, MOPS and the salt solution are adjusted to pH 7 with ammonia water and autoclaved. Then the sterile substrate and vitamin solutions are added, and the dry autoclaved CaC0 _{3 is} added. The cultivation is carried out in a volume of 10 ml in a 100 ml Erlenmeyer flask with baffles. Kanamycin (25 mg / 1) is added. The cultivation takes place at 33 ° C and 80% humidity.

After 72 hours, the OD is determined at a measuring wavelength of 660 nm using the Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine formed is determined using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.

Table 1 shows the result of the experiment,

Table 1

29

The following figure is attached:

Figure 1: Map of plasmid pCR2. lcitEint.

The abbreviations and designations used have the following meaning.

KmR: Kanamycin resistance gene

EcoRI: Interface of the restriction enzyme EcoRI

HindiII: interface of the restriction enzyme Hindlll

BamHI: interface of the restriction enzyme BamHI

citEint: internal fragment of the citE gene

ColEl: origin of replication of the plasmid ColEl

1APESTER TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Degussa Hüls AG

Kantstr. 2

33790 Halle / Künsebeck

FIRST DEPOSIT RECEIPT CONFIRMATION, issued in accordance with Rule 7 1 by the INTERNATIONAL DEPOSITOR below

I LABELING THE MICROORGANISM

Reference assigned by the DEPOSITER.2. IDENTIFICATION NUMBER assigned by the INTERNATIONAL DEPOSITORY

Top 10 / pCR2. lcitEint

DSM 13981

II SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC NAME

With the microorganism designated under I was

(X) a scientific description

(X) submitted a proposed taxonomic name (check where applicable)

HI INPUT AND ACCEPTANCE

This international depository accepts the microorganism referred to under I, which it received on 2 001 - 01 - 12 (date of first filing) ¹

IV RECEIPT OF CONVERSION APPLICATION

The microorganism referred to under I has been received by this International Agency for the filing of the application (date of first filing) and an application for the conversion of this first filing into a deposit under the Bndapester Treaty has been received on (date of receipt of the application for conversion)

V INTERNATIONAL DEPOSIT

Name DSMZ-DEUTSCHE SAMMLUNG VON Subsection (s) of the person (s) authorized to represent the international depositary MIKROORGANISMEN UND ZELLKULTUREN GmbH or the employee (s) authorized by them

Address Mascheroder Weg lb D- 81 ₂ Braunschweig <y, £ A- O

Date 2001 - 01 - 17

If Rule 64 (d) applies, this is the time when the status of an international depository was acquired Form DSMZ BP / 4 (single page) 0196 1APESTER TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURES

INTERNATIONAL FORM

Degussa Hüls AG

Kan str. 2

33790 Halle / Künsebeck

VITALITY CERTIFICATE issued in accordance with Rule 102 of the below

INTERNATIONAL DEPOSIT

I DEPOSIT II. LABELING OF THE MICROORGANISM

Name Degussa Hüls AG From the INTERNATIONAL DEPOSIT

Kantstr. 2 assigned INPUT NUMBER: address 33790 Halle / Künsebeck DSM 13 981

Date of filing or forwarding ¹ : 2 001 - 0 1 - 12

πi. LEBENSFAHIGKEITSBESCHEINIGUNG

The viability of the microorganism mentioned under II was checked on 20 01 - 01 - 15 ² at that time the microorganism was

(X) ¹ viable

() 'no longer viable

IV. CONDITIONS UNDER WHICH THE LIFE CHECK IS PERFORMED *

V INTERNATIONAL DEPOSIT

Surname. DSMZ-GERMAN COLLECTION OF signature (s) of the person (s) authorized to represent the international depositary MIKROORGANISMEN UND ZELLKULTUREN GmbH or the employee (s) authorized by them

Address: Mascheroder Weg lb D- ₃ S124 Braunschweig

Date. 2001-01-17

Indication of the date of the first filing If a new deposit or a transfer has been made, the date of the last renewed filing or value transfer.

In the cases provided for in Rule 10 2 (a) (ii) and (m), state the last viability test.

Tick the appropriate

Complete if the information has been requested and if the results of the examinee were negative

Form DSMZ-BP / 9 (single page) 0196 BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNΓΠON OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

TERNTERNAΉONAL FORM

Degussa Hüls AG

Kantstr. 2

33790 Halle / Künsebeck

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this pagc

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITΥ:

Top 10 / pCR2. lcitEint

DSM 13981

II. SCIENΗHC DESCRIPTION AND / OR PROPOSED TAXONO C DESIGNATION

The microorganism identified under I. above was accompanied by:

(X) a scientific description

() a proposed taxonomic designation

(Mark with a cross where applicable).

III. RECEIPT AND ACCEPTANCE

This International Depositary Authorily accepts the microorganism identified under I. above, which was received by it on 20 01 - 0 1 - 12 (Date of the original deposit) '.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion)

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-GERMAN COLLECTION OF Signature (s) of person (s) having the power to represent the

MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized offιcial (s):

Address: Mascheroder Weg lb D- ₃ 81 ₂ Braunschweig

Date: 2001-01-17

¹ Where Rule 6. (d) applies, such date is the date on which the Status of international depositary authority was acquired. Form DSMZ-BP / 4 (sole page) 0196 BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Degussa Hüls AG

Kantstr. 2

33790 Halle / Künsebeck

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the

INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

¹ Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

² In the cases referred to in Rule 10 - (a) (ii) and (ϊ), refer to the most recent viability test Mark with a cross the applicable box.

⁴ Fill in if the information has been requested and if the results of the test were negative.

Form DSMZ-BP / 9 (sole page) 0196

Claims

claims

1. Isolated polynucleotide from coryneform bacteria, containing a polynucleotide sequence coding for the citE gene, selected from the group

a) Polynucleotide that is at least 70% identical to a polynucleotide that codes for a polypeptide that contains the amino acid sequence of SEQ ID No. 2 contains

b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least

70% identical 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 consecutive nucleotides of the polynucleotide sequence of a), b) or c), wherein

e) the polypeptide preferably has the activity of the citrate lyase E.

2. Polynucleotide according to claim 1, wherein the polynucleotide is a replicable in coryneform bacteria, preferably recombinant DNA.

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

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

5. Replicable DNA according to claim 2, containing

(i) the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence that corresponds to the sequence

(i) within the range of 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) functionally neutral mutations in (i).

6. Replicable DNA according to claim 5, so that the hybridization is carried out with a stringency corresponding to at most 2x SSC.

7. The polynucleotide sequence according to claim 1, which codes for a polypeptide which has the sequence shown in SEQ ID No. 2 contains amino acid sequence shown.

8. Coryneform bacteria in which the citE gene is weakened, in particular switched off.

9. A process for the fermentative production of L-amino acids, in particular L-lysine, characterized in that the following steps are carried out: ia) fermentation of the coryneform bacteria producing the desired L-amino acid, in which at least the citE gene or nucleotide sequences coding therefor are weakened , especially turns off;

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

c) isolating the L-amino acid.

10. The method according to claim 9, characterized in that bacteria uses, in which additional genes of the biosynthetic pathway of the desired L-amino acid are amplified.

11. The method according to claim 9, so that bacteria are used in which the metabolic pathways are at least partially switched off, which reduce the formation of the desired L-amino acid.

12. The method according to claim 9, d a d u r c h g e k e n n z e i c h n e t that the expression of the polynucleotide (s) which encode (s) for the citE gene (code) is attenuated, in particular switched off.

13. The method according to claim 9, so that the regulatory (or catalytic) properties of the polypeptide (enzyme protein) for which the polynucleotide citE codes are reduced.

14. The method according to claim 9, d a d u r c h g e k e n n z e i c h n e t that for the production of L-amino acids coryneform microorganisms in which one or more of the genes selected from the group are simultaneously fermented

14.1 the gene dapA coding for the dihydrodipicolinate synthase,

14.2 that for the glyceraldehyde-3-phosphate. Gene gap encoding dehydrogenase,

14.3 the gene tpi coding for the triose phosphate isomerase,

14.4 the coding for the 3-phosphoglycerate kinase

Gene pgk,

14.5 the gene coding for glucose-6-phosphate dehydrogenase zwf,

14.6 the pyc gene coding for the pyruvate carboxylase,

14.7 the mqo gene coding for the malate quinone oxidoreductase,

14.8 the gene lysC coding for a feedback-resistant aspartate kinase,

14.9 the gene lysE coding for lysine export,

14.10 the gene coding for the Zwal protein zwal

amplified or overexpressed.

15. The method according to claim 9, d a d u r c h g e k e n n z e i c h n e t that for the production of L-amino acids coryneform microorganisms are fermented, in which one or more of the genes selected from the group simultaneously

15.1 the gene pck coding for the phosphoenolpyruvate carboxykinase,

15.2 the pgi gene coding for glucose-6-phosphate isomerase,

15.3 the poxB gene coding for pyruvate oxidase

15.4 the zwa2 gene coding for the Zwa2 protein

15.5 the gene coding for homoserine dehydrogenase hom

15.6 the gene coding for the homoserine kinase thrB, and

15.7 the gene encoding the aspartate decarboxylase

attenuates, especially the expression decreases.

16. Coryneform bacteria which contain a vector which carries parts of the polynucleotide according to claim 1, but at least 15 consecutive nucleotides of the claimed sequence.

17. Isolated microorganism of the E. coli strain ToplO / pCR2. lcitEint, deposited with the German Collection for Microorganisms and Cell Cultures in Braunschweig under the number DSM 13981 in accordance with the Budapest Treaty.

18. The method according to one or more of the preceding claims, that the use of microorganisms of the type Corynebacterium glutamicum is used.

19. A method for finding RNA, cDNA and DNA in order to isolate nucleic acids or polynucleotides or genes which code for the citrate lyase E or are very similar to the sequence of the citE gene, characterized in that the polynucleotide, containing the polynucleotide sequences according to claims 1, 2, 3 or 4, used as hybridization probes.

20. The method according to claim 18, which also means that arrays, icro arrays or DNA chips are used.