WO2002034775A2 - Nucleotide sequences encoding the hemd and hemb genes - Google Patents

Abstract

The invention relates to an isolated polynucleotide containing a polynucleotide sequence, selected from the group a) polynucleotide that is at least 70 % identical with a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID No. 2, b) polynucleotide that is at least 70 % identical with a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID No. 3, c) polynucleotide encoding a polypeptide that contains an amino acid sequence that is at least 70 % identical with the amino acid sequence of SEQ ID No. 2, d) polynucleotide encoding a polypeptide that contains an amino acid sequence that is at least 70 % identical with the amino acid sequence of SEQ ID No. 3, e) polynucleotide that is complementary to the polynucleotides of a), b), c) and d), and f) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b), c), d) and e), and a method for the fermentative production of L-amino acids using coryneform bacteria, in which at least the hemD-gene and/or the hemB-gene is/are amplified, and the use, as Hybridization probes, of polynucleotides that contain the sequences according to the invention.

Description

Nucleotide Sequences Encoding the hemD and hemB Genes

Field of the Invention

The invention relates to nucleotide sequences encoding the hemD and hemB genes from corynefor bacteria and a method for the fermentative production of amino acids using bacteria, in which the endogenous hemD-gene and/or the endogenous hemB-gene is amplified.

Prior Art

L-amino acids, in particular L-lysine, have applications in human medicine and in the pharmaceutical industry, in the food industry and very particularly in animal feeding.

It is known that amino acids are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of the great importance thereof, work is constantly being done to improve the production methods . Production improvements can relate to fermentation technology measures, such as, for example, agitating and supplying with oxygen, or to the composition of the nutrient media, such as, for example, the sugar concentration during ermentation, or to processing into a product form by, for example, ion exchange chromatography, or to the intrinsic performance properties of the microorganism itself.

To improve the performance properties of said microorganisms, methods of mutagenesis, selection and mutant selection are used. In this way, strains are obtained, which are resistant to antimetabolites or auxotrophic for metabolites that are important as regulators and produce amino acids .

For some years, methods of recombinant DNA-technology have also been used for strain-improvement of L-amino acid producing strains of Corynebacterium by amplifying individual amino acid-biosynthesis genes and studying the effect on amino acid production.

Object of the Invention

The inventors have set themselves the object of providing novel methods for the improved fermentative production of amino acids .

Summary of the Invention

Where L-amino acids or amino acids are mentioned below, reference is made thereby to one or more amino acids including the salts thereof, 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. Particularly preferred is L-lysine.

Where L-lysine or lysine are mentioned below, reference is made thereby not only to the bases, but also to the salts, such as, for example, lysine monohydrochloride or lysine sulfate.

The invention relates to isolated polynucleotides from coryneform bacteria, containing a polynucleotide sequence encoding the hemD-gene and/or the hemB-gene, selected from the group

a) polynucleotide that is at least 70% identical with a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID No . 2,

b) polynucleotide that is at least 70% identical with a polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID No . 3, c) polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical with the amino acid sequence of SEQ ID No . 2,

d) polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical with the amino acid sequence of SEQ ID No . 3,

e) polynucleotide that is complementary to the polynucleotides of a) , b) , c) or d) ,

the polypeptides preferably having the activity of uroporphyrinogene-III-synthase and/or of delta- aminolevulinic acid dehydratase.

The invention also relates to the above-mentioned polynucleotides, preferably with replicable DNA containing:

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

(ii) at least one sequence corresponding to the sequence (i) within the degenerateness of the genetic code, or

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

(iv) functionally neutral sense mutations in (i) that do not alter 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 243, b) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 244 and 2322,

c) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 2323 and 2858,

d) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 2859 and 3875,

e) polynucleotides containing at least 15 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No. 1 between positions 3876 and 4070.

The invention further relates to

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

polynucleotides encoding polypeptides containing the amino acid sequence, as shown in SEQ ID No . 2 and SEQ ID No. 3,;

a vector, containing one or both of the polynucleotides according to the invention, in particular a shuttle vector or plasmid vector, and

coryneform bacteria that contain the vector or in which the endogenous hemD-gene and/or the endogenous hemB-gene is/are amplified.

The invention also relates to polynucleotides that consist essentially of a polynucleotide sequence, that are obtainable by screening by means of hybridization of a corresponding gene bank of a coryneform bacterium, which bank contains the complete gene or parts thereof, using a probe that contains the sequence of the polynucleotides according to the invention according to SEQ ID No . 1 or a fragment thereof, and isolation of the above-mentioned polynucleotide sequence.

Detailed Description of the Invention

Polynucleotides that contain the sequences according to the invention are suitable as hybridization-probes for RNA, cDNA and DNA in order to isolate in their full length nucleic acids or polynucleotides or genes encoding uroporphyrinogene-III-synthase and/or delta-aminolevulinic acid dehydratase or in order to isolate such nucleic acids or polynucleotides or genes that have a high similarity to the sequence with that of the hemD-gene and/or of the hemB- gene. They can also be applied as a probe on so-called 'arrays', 'micro arrays' or 'DNA chips', in order to detect corresponding polynucleotides or sequences derived therefrom, such as, for example, RNA or cDNA.

Polynucleotides that contain the sequences according to the invention are, furthermore, suitable as primers, with the aid of which DNA can be produced by means of the polymerase-chain reaction (PCR) from genes that encode uroporphyrinogene-III-synthase and/or delta-aminolevulinic acid dehydratase.

Such oligonucleotides that serve as probes or primers contain at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24 and particularly preferably at least 15, 16, 17, 18 or 19 consecutive nucleotides. Also suitable are likewise oligonucleotides having 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. Optionally, oligonucleotides having a length of at least 100, 150, 200, 250 or 300 consecutive nucleotides are suitable.

'Isolated¹ means removed from its natural environment. 'Polynucleotide' relates in general to polyribonucleotides and polydeoxyribonucleotides, involving 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 produced therefrom and also such polynucleotides that are at least 70% to 80%, preferably at least 81% to 85%, especially preferably at least 86% to 90% and particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No . 1 or a fragment produced therefrom.

' Polypeptides ' are interpreted as peptides or proteins that contain two or more by peptide-bonded amino acids .

The polypeptides according to the invention include the polypeptides according to SEQ ID No . 2, and SEQ ID No . 3, in particular such polypeptides having the biological activity of uroporphyrinogene-III-synthase and of delta- aminolevulinic acid dehydratase and also such polypeptides that are at least 70% to 80%, preferably at least 81% to 85% and especially at least 86% to 90% and particularly preferably at least 91%, 93%, 95%, 97% or 99% identical with the polypeptides according to SEQ ID No . 2 and SEQ ID No. 3 and have the above-mentioned activities.

The invention further relates to a method 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 the nucleotide sequences encoding the hemD-Gene and/or the hemB-Gene are amplified and in particular overexpressed. The term 'amplification' means in this context the increase in the intracellular activity of one or more enzymes in a microorganism that are encoded by the corresponding DNA, by, for example, increasing the copy number of the gene or genes, using a strong promoter or a gene that encodes a corresponding enzyme having a high activity and optionally combines said measures.

By the measures of amplification, in particular of overexpression, the activity or concentration of the corresponding protein is generally increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, by a maximum of up to 1000% or 2000% with regard to that of the wild type protein or of the activity or concentration of the protein in the starter microorganism.

The microorganisms that form the subject matter of the present invention can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. These microorganisms can be coryneform bacteria, in particular of the genus Corynebacterium. In the genus Corynebacterium, particular mention is made of the species Corynebacterium glutamicum, which is known to 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 elassecola ATCC17965 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and L-amino acid-producing mutants or strains produced therefrom.

The novel hemD and hemB genes of C. glutamicum encoding the enzymes uroporphyrinogene-III-synthase (EC 4.2.1.75) and delta-aminolevulinic acid dehydratase (EC 4.2.1.24) were isolated.

To isolate the hemD-gene, the hemB-gene or also other genes of C. glutamicum, a gene bank of said microorganism is first constructed in Escherichia coli (E. coli) . The construction of gene banks is described in generally known textbooks and handbooks. As examples, one. could mention the textbook by Winnacker: Gene und Klone, Eine Einfuhrung in die Genetechnologie [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) . A very well known gene bank is that of E. coli K-12 strain W3110, which was constructed by Kohara et al. in λ-vectors (Cell 50, 495-508 (1987)). Bathe et al . (Molecular and General Genetics, 252:255-265, 1996) describe a gene bank of C. glutamicum ATCC13032, which was constructed 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 E. coli K-12 strain NM554 (Raleigh et al . , 1988, Nucleic Acids Research 16:1563- 1575) .

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

For the production of a gene bank of C. glutamicum in E. coli, plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al . , 1982, Gene 19:259- 268) can also be used. Particularly suitable as hosts are such E. coli strains that are restriction- and recombination-defective. An example thereof is the strain DH5 mcr, 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 can subsequently be subcloned again into common vectors suitable for sequencing and subsequently sequenced, as described, for example, in Sanger et al . (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).

The DNA-sequences obtained can then be studied using known algorithms or sequence analysis-programs , such as, for example, the one described by Staden (Nucleic Acids Research 14, 217-232(1986)), the one described by Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG- program described by Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).

The novel DNA sequences of C. glutamicum encoding the hemD and hemB genes, which sequences are a component of the present invention as SEQ ID No . 1, were found. Furthermore, the amino acid sequence of the corresponding proteins was derived from the available DNA sequences using the methods described above. In SEQ ID No . 2 and SEQ ID No . 3, the resulting amino acid sequences of the hemD- and hemB-gene products are shown. It is known that host-specific enzymes can split the N-terminal amino acid methionine or formyl methionine of the protein formed.

Coding DNA sequences that result from SEQ ID No . 1 as a result of the degenerateness of the genetic code, are likewise a component of the invention. In the same way,

DNA-sequences that hybridize with SEQ ID No . 1 or parts of SEQ ID No . 1 are a component of the invention. Furthermore, amongst experts, conservative amino acid exchanges, such as, for example, the exchange of glycine for alanine or of aspartic acid for glutamic acid are known in proteins as 'sense mutations', which do not lead to any basic change in the activity of the proteins i.e. which are functionally neutral. Such mutations are also described inter alia as neutral substitutions. Furthermore, it is known that alterations to the N- and/or C-terminal of a protein do not essentially impair the function thereof or can even stabilize it. A person skilled in the art can find information about this 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 and SEQ ID No . 3, are likewise a component of the invention.

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

Guidance on the identification of DNA- sequences by means of hybridization can be found by a person skilled in the art inter alia in the handbook 'The DIG System Users Guide for Filter Hybridization' published by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al . (International Journal of Systematic Bacteriology (1991) 41: 255-260). Hybridization is carried out under stringent conditions, that is, only hybrids are formed, in which the probe and target sequence, i.e. the polynucleotides treated with the probe, are at least 70% identical. It is known that the stringency of hybridization, including that of the washing stages, is influenced or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is preferably carried out with a relatively low stringency compared with the washing stages (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996) .

For the Hybridization reaction, a 5x SSC-buffer can be used, for example, at a temperature of approx. 50°C - 68 °C. In said reaction, probes can also hybridize with polynucleotides, that are less than 70% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2x SSC and, optionally, subsequently to 0.5x SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) , a temperature of approx. 50°C - 68°C being set. It is optionally possible to reduce the salt concentration to 0. Ix SSC. By gradually increasing the Hybridization temperature in stages of approx. 1°C - 2°C from 50°C to 68°C, polynucleotide fragments can be isolated, which, for example, are at least 70% or at least 80% or at least 90% to 95% or at least 96% to 99% identical to the sequence of the probe used. It is likewise possible to isolate polynucleotide fragments that are completely identical to the sequence of the probe used. Further guidance on

Hybridization is obtainable on the market in the form of so-called kits (e.g. DIG Easy Hyb from the company Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558) .

A person skilled in the art can find guidance on the amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) inter alia in Gait's handbook: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) . It was found that, after overexpression of the hemD-gene and/or of the hemB-gene, coryneform bacteria produce amino acids in an improved manner .

To obtain an overexpression, the copy number of the corresponding genes can be increased, or the promoter- and regulation region or the ribosome bonding point, which is located upstream of the structural gene, can be mutated. Expression cassettes that are inserted upstream of the structural gene work in a similar way. It is additionally possible, using inducible promoters, to increase expression in the course of fermentative amino acid production. Expression is likewise improved by measures to lengthen the life of the m-RNA. Furthermore, enzyme activity is likewise amplified by preventing the breakdown of the enzyme protein. The genes or gene constructs can either be present in plasmids that have a different copy number or be integrated and amplified in the chromosome. Alternatively, an overexpression of the relevant genes can furthermore be achieved by altering the composition of the media and culturing method.

A person skilled in the art can find guidance on this point in inter alia 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 Piihler

(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. For the amplification, the hemD and hemB genes according to the invention were overexpressed, for example, with the aid of episomal plasmids . Suitable plasmids are those that are replicated in coryneform bacteria. Numerous known plasmid vectors such as, for example, pZl (Menkel et al . , Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl

(Eikmanns et al . , Gene 102:93-98 (1991)) or pHS2-l (Sonnen et al . , Gene 107:69-74 (1991)) are based on the cryptic plasmids pHMl519, pBLl or pGAl . Other plasmid vectors such as, for example, those that are based on pCG4

(US-A 4,489,160), or pNG2 (Serwold-Davis et al . , FEMS Microbiology Letters 66, 119-124 (1990)), or pAGl

(US-A 5,158,891) can be used in a similar manner.

Furthermore those plasmid vectors, with the aid of which the method of gene amplification by integration into the chromosome can be used, are also suitable, as described, for example, by Reinscheid et al . (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB-operon. In said method, the complete gene is cloned into a plasmid vector that can replicate in a host (typically E. coli), but not in C. glutamicum. Possible vectors are, for example, pSUP301 (Simon et al . , Bio/Technology 1, 784-791 (1983)), pKlδmob or pKl9mob (Schafer et al . , Gene 145, 69- 73 (1994)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5 , 487 , 993 ) , pCR®Blunt (Invitrogen company, Groningen, Netherlands; Bernard et al . , Journal of Molecular Biology, 234: 534-541 (1993)), pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al.,1986, Gene 41: 337-342). The plasmid vector that contains the gene to be amplified is subsequently transferred by conjugation or transformation into the desired strain of C. glutamicum. The conjugation method is described for example in Schafer et al . (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods are described, for example, in Thierbach et al . (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al . (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a 'cross over '-event, the resulting strain receives at least two copies of the relevant gene.

Additionally, it can be advantageous for the production of L-amino acids, to amplify, in particular to overexpress, in addition to the hemD-gene and/or the hemB-gene, one or a plurality of enzymes of each biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and, optionally, regulatory proteins.

Thus, for the production of L-amino acids, in addition to the amplification of the hemD-gene and/or of the hemB-gene, one or several endogenous genes can be amplified, in particular overexpressed, said genes being selected from the group

» the dapA gene encoding dihydrodipicolinate synthase (EP-B 0 197 335) ,

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

the tpi gene encoding triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

® the pgk gene encoding 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

• the zwf gene encoding glucose-6-phosphate dehydrogenase (JP-A-09224661) , » the pyc gene encoding pyruvate carboxylase (DE-A-198 31 609),

* the mqo gene encoding malate quinone oxidoreductase (Molenaar et al . , European Journal of Biochemistry 254, 395-403 (1998)),

• the lysC gene encoding a feed-back resistant aspartate kinase (Accession No . P26512 ; EP-B-0387527 ; EP-A-0699759 ) ,

® the lysE gene encoding lysine-export (DE-A-195 48 222),

^β the horn gene encoding homoserine-dehydrogenase (EP-A 0131171),

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

» the ilvBN gene encoding acetohydroxy acid synthase (EP-B 0356739) ,

» the ilvD gene encoding dihydroxy acid dehydratase (Sahm and Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979),

β the zwa 1 gene encoding Zwa 1 protein (DE: 19959328.0, DSM 13115) .

Furthermore, it can be advantageous for the production of L-amino acids, in addition to the amplification of the hemD-gene and/or of the hemB-gene, to attenuate, in particular to reduce the expression of one or more genes, selected from the group

® the pck gene encoding phosphoenolpyruvate carboxykinase (DE 199 50 409.1; DSM 13047), © the pgi gene encoding glucose-6-phosphate isomerase (US 09/396,478; DSM 12969) ,

the poxB gene encoding pyruvate oxidase (DE: 1995 1975.7; DSM 13114) ,

the zwa 2 gene encoding the Zwa 2 protein (DE: 19959327.2, DSM 13113)

The term 'attenuation' describes in this context the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism, which are encoded by the corresponding DNA, by using, for example, a weak promoter or a gene or allele that encodes a corresponding enzyme having a low activity or inactivates the corresponding gene or enzyme (protein) and optionally combines said measures .

By means of attenuation measures, the activity or concentration of the corresponding protein is generally 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 starter microorganism.

Furthermore it can be advantageous for the production of amino acids, to eliminate, in addition to the overexpression of the hemD-gene and/or of the hemB-gene, unwanted secondary reactions (Nakayama: 'Breeding of Amino Acid Producing Microorganisms', in: Overproduction of Microbial Products, Krumphanzl, sikyta, Vanek (eds.), Academic Press, London, UK, 1982) .

The microorganisms produced according to the invention are likewise the subject matter of the invention and can be cultured continuously or discontinuously by the batch method or by the fed batch method or by the repeated fed batch method for the production of amino acids . A summary of known culturing methods is given in Chmiel ' s textbook (Bioprozeβtechnik 1. Einfϋhrung in die Bioverf hrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991) ) or in Storhas ' textbook (Bioreaktoren und periphere Einrichtungen [bioreactors and peripheral installations] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) ) .

The culture medium that is to be used must satisfy the requirements of each of the strains in an appropriate manner. Descriptions of culture media for various microorganisms are contained in the American Society for Bacteriology's handbook 'Manual of Methods for General Bacteriology' (Washington D.C . , USA, 1981).

As a carbon source, sugar and carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol and ethanol and organic acids such as, for example, acetic acid can be used. Said substances can be used individually or as a mixture. As a nitrogen source, organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used. The nitrogen sources can be used individually or as a mixture.

As a phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used. The culture medium must furthermore contain metal salts such as, for example, magnesium sulfate or ferrous sulfate, which are required for growth. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above-mentioned substances. Moreover, suitable preliminary stages can be added to the culture medium. The above-mentioned ingredients can be added to the culture in the form of a single mixture or fed to it in an appropriate manner during culturing.

For pH-control of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammoniacal water or acid compounds such as phosphoric acid or sulfuric acid are used in an appropriate manner. To control the development of foam, anti-foaming agents such as, for example, fatty acid polyglycol esters can be used. To maintain the stability of plasmids, appropriate selectively acting substances, e.g. antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as air, for example, are inserted into the culture. The temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C. The culture is maintained until a maximum of the desired product has formed. The above objective is normally achieved within 10 hours to 160 hours.

Methods for determining L-amino acids are known from the prior art. Analysis can be carried out, for example, as described, in Spackman et al . (Analytical Chemistry, 30, (1958) , 1190) or by ion exchange chromatography with subsequent ninhydrin-derivation, or it can be carried out by reversed phase HPLC, as described in Lindroth et al .

(Analytical Chemistry (1979) 51: 1167-1174).

The method according to the invention is for the fermentative production of amino acids.

The following microorganism was deposited on July 18 2001 in the Deutsche Sammlung fur Mikroorganismen und

Zellkulturen [German Collection for Microorganisms and Cell Cultures (DSMZ, Brunswick, Germany) in accordance with the Treaty of Budapest: » Escherichia coli DH5alphamcr/pEC-XK99EhemBa2ex ( = DH5otmcr/pEC-XK99EhemBa2ex) as DSM 14408.

The present invention is described in more detail below in the form of embodiments .

The isolation of plasmid-DNA from Escherichia coli and all techniques for restriction, Klenow treatment 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 the transformation of

Escherichia coli are likewise described in this handbook.

The composition of common nutrient media such as LB- or TY- medium can likewise be found in the handbook by Sambrook et al.

Example 1

Production of genomic cosmid gene bank from Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 is isolated as described in Tauch et al . (1995, Plasmid 33:168-179) and partially split with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, Code no. 27-0913-02) . The DNA fragments are dephosphorylized with shrimp alkaline phosphatase (Roche Diagnostics GmbH, 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 the Stratagene company (La Jolla, USA, product description SuperCosl Cosmid Vector Kit, Code no. 251301) is split with the restriction enzyme Xbal (Amersham

Pharmacia, Freiburg, Germany, product description Xbal, Code no. 27-0948-02) and likewise dephosphorylized using shrimp alkaline phosphatase. The cosmid-DNA is subsequently split with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, Code no. 27-0868-04) . The cosmid-DNA treated in this manner is mixed with the treated ATCC13032-DNA and the mixture treated with T4-DNA-ligase

(Amersham Pharmacia, Freiburg, Germany, product description T4-DNA-ligase, Code no .27-0870-04 ) . The ligation mixture is subsequently packed in phages with the aid of 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 Acids Research 16:1563-1575), the cells are taken up in 10 mM MgS0₄ and mixed with an aliquot of the phage suspension. Infection and titration of the cosmid bank are carried out as described in Sambrook et al . (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) , the cells being plated out on LB-Agar (Lennox, 1955, Virology, 1:190) with 100 mg/1 ampicillin. After incubation overnight at 37°C, recombinant individual clones are selected.

Example 2

Isolation and sequencing of the hemD-gene and of the hemB- Gene

The cosmid DNA of an individual colony is isolated using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagene, Hilden, Germany) according to the manufacturer's instructions and partially split using the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, product No. 27-0913-02). The DNA-fragments are dephosphorylized using shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, Product No. 1758250) . After separation using gel electrophoresis, isolation of the cosmid fragments in the size range of 1500 to 2000 bp is carried out, using the QiaExII Gel Extraction Kit (Product No. 20021, Qiagene, Hilden, Germany).

The DNA of the sequencing vector pZero-1, obtained from the Invitrogen company (Groningen, Netherlands, Product description Zero Background Cloning Kit, Product No. K2500- 01), is split using the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, product No. 27-0868-04). Ligation of the cosmid fragments into the sequencing vector pZero-1 is 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) . Said ligation mixture is subsequently electroporated into the E. coli strain DH5c.MCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) (Tauch et al . 1994, FEMS Microbiol Letters, 123:343-7) and plated out on LB-Agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 zeocin.

The plasmid preparation of the recombinant clones is carried out using Biorobot 9600 (Product No. 900200, Qiagene, Hilden, Germany) . Sequencing is carried out according to Sanger et al . ' s dideoxy chain-termination method (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) is used. The separation and analysis of the sequencing reaction using gel electrophoresis is carried out in a ' Rotiphoresis NF acrylamide/bisacrylamide ' gel (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany) using the ' ABI Prism 377' sequencing device from PE Applied Biosystems (Weiterstadt, Germany) . Raw sequence data are subsequently processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) Version 97-0. The individual sequences of the pZerol-derivatives are assembled into a continuous contig. The computer-assisted coding region analysis was prepared using the XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231).

The nucleotide sequence is shown in SEQ ID No. 1. The analysis of the nucleotide sequence produces two open grids of 2082 base pairs and 1020 base pairs, which are known as the hemD-gene and the hemB-gene. The hemD-Gene encodes a protein consisting of 693 amino acids and the hemB-Gene encodes a protein consisting of 339 amino acids.

Example 3

Production of a shuttle vector pEC-XK99EhemBa2ex for amplification of the hemB-gene in C. glutamicum.

3.1 Cloning of the hemB-gene

Chromosomal DNA was isolated from the strain ATCC 13032 according to the method of Eikmanns et al . (Microbiology 140: 1817-1828 (1994)). On the basis of the sequence of the hemB-gene known from Example 2 for C. glutamicum, the following oligonucleotides were selected for the polymerase chain reaction (see also SEQ ID No . 4 and SEQ ID No . 5) :

hemBexl : 5^s- ca tct aga-tgt cca ccg ctg act eta tg -3^'

hemBex2 :

5^s- tg tct aga-gca gtt cgc aga age aga ag -3^'

The primers shown were synthesized by the company MWG- Biotech AG (Ebersberg, Germany) and the PCR reaction was carried out according to Innis et al . ' s standard PCR method (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press), using Pwo-polymerase from the company Roche Diagnostics GmbH (Mannheim, Germany) . With the aid of the polymerase chain reaction, the primers allow the amplification of a 1201 bp long DNA fragment, which carries the hemB-gene. Furthermore, both primers contain the sequence for the site of the restriction endonuclease Xbal, which is underlined in the nucleotide sequence shown above.

The 1201 bp long hemB fragment was split with the restriction endonuclease Xbal and subsequently isolated from the agarose gel using the QiaExII Gel Extraction Kit (Product No. 20021, Qiagene, Hilden, Germany).

3.2 Construction of the shuttle-vectors pEC-XK99E

The E. coli - C. glutamicum shuttle-vector pEC-XK99E was constructed according to the prior art. The vector contains the replication region rep of the plasmid pGAl including that of the replication effector per (US-A-5 175 108; Nesvera et al . , Journal of Bacteriology 179, 1525-1532 (1997)), the kana ycin-resistance gene aph(3')-IIa of Escherichia coli (Beck et al . (1982), Gene 19: 327-336), the replication origin, the trc-promoter, the termination regions Tl and T2 , the Iacl^α-Gene (repressor of the lac- operon of E. coli) and a multiple cloning site (mcs ) (Norrander, J.M. et al . Gene 26, 101-106 (1983)) of the plasmid pTRC99A (Amann et al . (1988), Gene 69: 301-315).

The trc-promoter can be induced by addition of the lactose- derivative IPTG (isopropyl-β-D-thiogalactopyranoside) .

The constructed E. coli - C. glutamicum shuttle-vector pEC- XK99E was transferred by means of electroporation (Liebl et al., 1989, FEMS Microbiology Letters, 53:299-303) into C. glutamicum DSM5715. The selection of the transformants was carried out on LBHIS Agar consisting of 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 according to the usual methods (Peters-Wendisch et al . , 1998, Microbiology, 144, 915 - 927), cut with the restriction endonuclease Hindlll, and the plasmid subsequently examined by agarose gel electrophoresis.

The plasmid construct thus obtained was designated as pEC- XK99E (Figure 1) . The strain obtained by electroporation of the plasmid pEC-XK99E into the C. glutamicum strain DSM5715 was designated as DSM5715/pEC-XK99E and deposited as DSM13455 in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Brunswick, Germany) in accordance with the Treaty of Budapest.

3.3 Cloning of hemB into the E. coli-C. glutamicum shuttle vector pEC-XK99E

The E. coli-C. glutamicum shuttle vector pEC-XK99Em described in Example 3.2 was used as the vector. The DNA of said plasmid was completely split with the restriction enzyme Xbal and subsequently dephosphorylized with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, Product No. 1758250) .

The approx. 1190 bp long hemB-fragment described in Example 3.1, obtained by means of PCR and split using the restriction endonuclease Xbal, was mixed with the prepared vector pEC-XK99E and the mixture treated with T4-DNA-Ligase (Amersham Pharmacia, Freiburg, Germany, product description T4-DNA-Ligase, Code no .27-0870-04) . The ligation mixture was transformed into the E. coli strain DH5αmcr (Hanahan, In: DNA Cloning. A Practical Approach. Vol. I, IRL-Press,

Oxford, Washington DC, USA) . Selection of plasmid-bearing cells was carried out by plating out the transformation mixture on LB-Agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 kanamycin. After incubation overnight at 37°C, recombinant individual clones were selected. Plasmid DNA was isolated from a transformant using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagene, Hilden, Germany) according to the manufacturer's instructions and split once with each restriction enzyme Xbal and BamHI, in order to subsequently examine the plasmid by means of agarose gel electrophoresis. The plasmid obtained was designated as pEC-XK99EhemBa2ex. It is shown in Figure 2.

Example 4

Transformation of strain DSM5715 using the plasmid pEC- XK99EhemBa2ex

Strain DSM5715 was transformed with the plasmid pEC- XK99EhemBa2ex using the electroporation method described by Liebl et al . , (FEMS Microbiology Letters, 53:299-303

(1989)). Selection of the transformants was carried out on LBHIS Agar consisting of 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 according to the usual methods (Peters-Wendisch et al . , 1998, Microbiology, 144, 915 - 927), cut with the restriction endonuclease BamHI, and the plasmid examined by subsequent agarose gel electrophoresis. The strain thus obtained was designated as DSM5715/pEC-XK99EhemBa2ex.

Example 5

Production of lysine

The C. glutamicum strain DSM5715/pEC-XK99EhemBa2ex obtained in Example 4 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined.

For this purpose, the strain was first incubated on an agar plate with the corresponding antibiotic (Brain-Heart Agar with kanamycin (25 mg/1)) for 24 hours at 33°C. Using said agar plate culture as a point of departure, a preculture was inoculated (10 ml medium in the 100 ml Erlenmeyer flask) . The medium used for the preculture was the complete medium Cg III.

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-value was adjusted to pH 7.4

Kanamycin (25 mg/1) was added thereto. The preculture was incubated for 16 hours at 33 °C and 240 rpm on the shaker. A main culture was inoculated from said preculture, with the result that the initial OD (660 rum) of the main culture was 0.05. The MM medium was used for the main culture.

MM Medium

CSL (Corn Steep Liquor) 5 g/1

MOPS (morpholinopropanesulfonic acid) 20 g/1

Glucose (autoclaved separately) 100 g/1

(NH₄)₂S0₄ 25 g/1

KH₂P0₄ 0.1 g/1

MgS0₄ * 7 H₂0 1.0 g/1

CaCl₂ * 2 H₂0 10 mg/1

FeS0₄ * 7 H₂0 10 mg/1

MnS0₄ * H₂0 5.0mg/l

Biotin (sterile-filtered) 0.3 mg/1

Thiamin * HCl (sterile-filtered) 0.2 mg/1

L-Leucine (sterile-filtered) 0.1 g/1

CaC0₃ 25 g/1

CSL, MOPS and the salt solution were adjusted to pH 7 with ammoniacal water and autoclaved. The sterile substrate- and vitamin solutions were subsequently added, likewise the dry autoclaved CaC0₃.

Culturing was carried out in 10 ml volume in a 100 ml Erlenmeyer flask with baffles. Kanamycin (25 mg/1) was added. Culturing was carried out at 33 °C and 80% air humidity. After 72 hours, the OD was determined at a measurement wavelength of 660 nm using the Biomek 1000 (Beckmann Instruments GmbH, Munchen) . The amount of lysine formed was determined using an amino acid analyzer from the Eppendorf- BioTronik company (Hamburg, Germany) by ion exchange chromatography and post-column derivation by ninhydrin detection.

Table 1 shows the result of the experiment.

Table 1

Brief Description of the Figures:

Figure 1: Map of plasmid pEC-XK99E

Figure 2: Map of plasmid pEC-XK99EhemBa2ex

The abbreviations and names used have the following meaning:

Kan: Kanamycin-resistance-gene aph(3^v)-Ha from Escherichia coli

BamHI interface of the restriction enzyme BamHI

HindiII interface of the restriction enzyme Hindlll

Xbal interface of the restriction enzyme Xbal

Ptrc trc-Promoter TI termination region Tl

T2 termination region T2

per replication effector per

rep replication region rep of the plasmid pGAl

laclq laclq-repressor of the lac-operon of Escherichia coli

hemB cloned hemB-gene

Claims

What is claimed is:

1. Isolated polynucleotide from coryneform bacteria, containing one polynucleotide sequence encoding the hemD-gene and/or the hemB-gene, selected from the group

a) polynucleotide that is at least 70% identical with a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID No . 2,

b) polynucleotide that is at least 70% identical with a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID No. 3,

c) polynucleotide encoding a polypeptide, that contains an amino acid sequence, that is at least 70% identical with the amino acid sequence of SEQ ID No. 2,

d) polynucleotide encoding a polypeptide that contains an amino acid sequence that is at least 70% identical with the amino acid sequence of SEQ ID No. 3,

e) polynucleotide that is complementary to the polynucleotides of a) , b) , c) or d) , and

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

the polypeptides preferably having the activity of uroporphyrinogene-III-synthase and/or delta- aminolevulinic acid dehydratase.

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

3. Polynucleotide according to claim 1, the polynucleotide being RNA.

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

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 sequence (i) within the region of the degeneration of the genetic code, or

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

(iv) functionally neutral sense mutations in (i) .

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

7. Polynucleotide sequence according to claim 1, that encodes a polypeptide that contains the amino acid sequence shown in SEQ ID No. 2 and/or SEQ ID No. 3.

8. Coryneform bacteria, in which the hemD-Gene and/or the hemB-Gene is (are) amplified, in particular overexpressed.

9. Escherichia coli strain DH5alphamcr/pEC-XK99EhemBa2ex ( = DH5αmcr/pEC-XK99EhemBa2ex) deposited as DSM 14408 in the Deutsche Sammlung fur Mikroorganismen und Zellkulturen, DSMZ, Brunswick, Germany.

10. Method for the fermentative production of L-amino acids, in particular L-lysine, wherein the following steps are carried out:

a) fermentation of the coryneform bacteria producing the desired L-amino acid, in which at least the endogenous hemD-gene and/or the endogenous hemB- gene or nucleotide sequences encoding said gene is/are amplified, in particular overexpressed;

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

c) isolation of the L-amino acid.

11. Method according to claim 10, wherein bacteria are used, in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally amplified.

12. Method according to claim 10, wherein bacteria are used, in which the metabolic pathways that reduce the formation of the desired L-amino acid are at least partially eliminated.

13. Method according to claim 10, wherein a strain transformed with a plasmid vector is used, and the plasmid vector carries the nucleotide sequence encoding the hemD-gene and/or the hemB-gene.

14. Method according to claim 10, wherein the expression of the polynucleotide (s) that encode (s) the hemD-gene and/or the hemB-gene is amplified, in particular overexpressed .

15. Method according to claim 10, wherein the catalytic properties of the polypeptides (enzyme protein) encoded by the polynucleotides hemD and/or hemB are increased.

6. Method according to claim 10, wherein coryneform microorganisms used for the production of L-amino acids are fermented, in which one or more the endogenous genes is/are simultaneously amplified or overexpressed, said genes being selected from the group

16.1 the dapA gene encoding dihydrodipicolinate- synthase,

16.2 the gap gene encoding glyceraldehyde-3- phosphate dehydrogenase,

16.3 the tpi gene encoding triosephosphate isomerase,

16.4 the pgk gene encoding 3-phosphoglycerate kinase,

16.5 the zwf gene encoding glucose-6-phosphate dehydrogenase,

16.6 the pyc gene encoding pyruvate carboxylase,

16.7 the mqo gene encoding malate-quinone- oxidoreductase ,

16.8 the lysC gene encoding a feed-back resistant aspartate kinase,

16.9 the lysE gene encoding lysine-export,

16.10 the horn gene encoding ho oserine-dehydrogenase,

16.11 the ilvA gene encoding threonine-dehydratase or the ilvA(Fbr) allele encoding a feed-back resistant threonine-dehydratase,

16.12 the ilvBN gene encoding acetohydroxy acid synthase,

16.13 the ilvD gene encoding dihydroxy acid dehydratase,

16.14 the zwa 1 gene encoding Zwa 1 protein.

17. Method according to claim 10, wherein coryneform microorganisms used for the production of L-amino acids are fermented, wherein one or more the endogenous genes is/are simultaneously attenuated, said genes being selected from the group

17.1 the pck gene encoding phosphoenolpyruvate- carboxykinase,

17.2 the pgi gene encoding glucose-6-phosphate isomerase,

17.3 the poxB gene encoding pyruvate-oxidase,

17.4 the zwa 2 gene encoding Zwa 2-protein.

18. Coryneform bacteria that contain a vector that carries a polynucleotide according to claim 1.

19. Method according to one or more claims 10-17, wherein microorganisms of the species Corynebacterium glutamicum are used.

20. Method according to claim 19, wherein Corynebacterium glutamicum strain Escherichia coli DH5alphamcr/pEC- XK99EhemBa2ex ( = DH5αmcr/pEC-XK99EhemBa2ex) is used.

21. Method for the detection of RNA, cDNA and DNA, in order to isolate nucleic acids, or polynucleotides or genes that encode uroporphyrinogene-III-synthase and/or delta-aminolevulinic acid-dehydratase or have a high degree of similarity to the sequence of the hemD-gene and/or of the hemB-gene, characterized in that the polynucleotide containing the polynucleotide sequences according to claims 1, 2, 3 or 4 is used as hybridization probe.

22. Method according to claim 21, wherein arrays, micro arrays or DNA-chips are used.