WO2002038598A1

WO2002038598A1 - Nucleotide sequences which code for the nada gene

Info

Publication number: WO2002038598A1
Application number: PCT/EP2001/012042
Authority: WO
Inventors: Christine Bastuck; Brigitte Bathe; Nicole Dusch; Bettina Möckel; Georg Thierbach
Original assignee: Degussa Ag
Priority date: 2000-11-10
Filing date: 2001-10-12
Publication date: 2002-05-16
Also published as: AU2002223623A1

Abstract

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

Description

Nucleotide Sequences which Code for the nadA Gene

The invention provides nucleotide sequences from coryneform bacteria which code for the nadA gene and a process for the fermentative preparation of nicotinic acid or nicotinic acid derivatives using coryneform bacteria in which the endogenous nadA gene is enhanced.

Prior Art

Nicotinic acid and nicotinic acid derivatives are used in human medicine, in the pharmaceuticals industry, in the foodstuffs industry and in animal nutrition.

It is known that -amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. No processes for the preparation of nicotinic acid or nicotinic acid derivatives usirig coryneform bacteria are known.

Object of the Invention

The inventors had the object of providing processes for the fermentative preparation of nicotinic acid and nicotinic acid derivatives .

Description of the Invention

If nicotinic acid or nicotinic acid derivatives are mentioned in the following, this means one or more compounds, including their salts, chosen from the group consisting of nicotinic acid, nicotinamide, uinolinic acid (quinolinate) , nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) . Nicotinic acid is particularly preferred.

The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the nadA gene chosen from the group consisting of

a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,

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

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

the polypeptide preferably having the activity of quinolinic acid synthetase A (quinolinate synthetase A) .

The invention also provides the above-mentioned polynucleotide, this preferably being a DNA which is capable of replication, comprising:

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

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

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

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

Finally, the invention also provides polynucleotides chosen from the group consisting of a) polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 1 and 742;

b) polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 743 and 2029; and

c) polynucleotides comprising at least 15 successive nucleotides chosen from the nucleotide sequence of SEQ ID No. 1 between positions 2030 and 2730.

The invention also provides

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

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

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

coryneform bacteria which contain the vector or in which the endogenous nadA gene is enhanced.

_-The -invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library of a coryneform bacterium, which comprises the complete gene or parts thereof, with a probe which comprises the sequence of the polynucleotide according to the invention according to SEQ ID No.l or a fragment thereof, and isolation of the polynucleotide sequence mentioned. Polynucleotides which comprise the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids or polynucleotides or genes which code for quinolinate synthetase or to isolate those nucleic acids or polynucleotides or genes which have a high similarity with the sequence of the nadA gene. They are also suitable for incorporation into so-called "arrays", micro arrays" or DNA chips" in order to detect and determine the corresponding polynucleotides .

Polynucleotides which comprise the sequences according to the invention are furthermore suitable as primers with the aid of which DNA of genes which code for quinolinate synthetase A can be prepared by the polymerase chain reaction (PCR) .

Such polynucleotides or oligonucleotides which serve as probes or primers comprise at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, most particularly preferably at least 15, 16, 17, 18 or 19 successive nucleotides. Oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides are also suitable. Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are optionally also suitable.

"Isolated" means separated out of its natural environment.

"Polynucleotide" in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.

The polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90% , and most particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No. 1 or a fragment prepared therefrom.

"Polypeptides " are understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds .

The polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those with the biological activity of quinolinate synthetase A, and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90%, and most particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polypeptide according to SEQ ID No. 2 and have the activity mentioned.

The invention also relates to a process for the fermentative preparation of nicotinic acid or nicotinic acid derivatives chosen from the group consisting of nicotinic acid, nicotinamide, quinolinic acid (quinolinate) , nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) using coryneform bacteria in which the nucleotide sequences which code for the nadA gene are enhanced, in particular over-expressed.

The term "enhancement" in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or using a gene which codes for a corresponding enzyme having a high activity, and optionally combining these measures. By enhancement measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on the starting microorganism.

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

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

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870

Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC17965 Brevibacterium flavu ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020.

The new nadA gene from C. glutamicum which codes for the enzyme quinolinate synthetase A has been isolated.

To isolate the nadA gene or also other genes of C. glutamicum, a gene library of this microorganism is first set up in Escherichia coli (E. coli) . The setting up of gene libraries is described in generally known textbooks and handbooks. The textbook by innacker: Gene und Klone, Eine Einfϋhrung in die Gentechnologie [Genes and Clones, An Introduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany, 1990) , or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may be mentioned as an example. A well-known gene library is that of the E. coli K-12 strain W3110 set up in λ vectors by Kohara et al . (Cell 50, 495 -508 (1987)). Bathe et al . (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was set up with the aid of the cos id vector SuperCos I (Wahl et al . , 1987, Proceedings of the National Academy of Sciences, USA, 84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575).

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

To prepare a gene library of C. glutamicum in E. coli it is also possible to use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al . , 1982, Gene, 19:259-268). Suitable hosts are, in particular, those E. coli strains which are restriction- and recombination- defective. An example of these is the strain DH5 mcr, which has been described by Grant et al . (Proceedings of the National Academy of Sciences, USA, 87 (1990) 4645-4649) . The long DNA fragments cloned with the aid of cos ids can in turn be subcloned in the usual vectors suitable for sequencing and then sequenced, as is described e.g. by Sanger et al . (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977) .

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

The new DNA sequence of C. glutamicum which codes for the nadA gene and which, as SEQ ID No. 1, is a constituent of the present invention has been found. The amino acid sequence of the corresponding protein has furthermore been derived from the present DNA sequence by the methods described above. The resulting amino acid sequence of the nadA gene product is shown in SEQ ID No. 2. It is known that, during or after translation, enzymes endogenous in the host can split off the N-terminal amino acid methionine or for ylmethionine from proteins formed.

Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy of the genetic code are also a constituent of the invention. In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Conservative amino acid exchanges, such as e.g. exchange of glycine for alanine or of aspartic acid for glutamic acid in proteins, are furthermore known among experts as "sense mutations" which do not lead to a fundamental change in the activity of the protein, i.e. are of neutral function. Such mutations are also called, inter alia, neutral substitutions. It is furthermore known that changes on the N and/or C terminus of a protein cannot substantially impair or can even stabilize the function thereof. Information in this context can be found by the expert, inter alia, in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al . (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences which result in a corresponding manner from SEQ ID No. 2 are also a constituent of the invention. In the same way, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are a constituent of the invention. Finally, DNA sequences which are prepared by the polymerase chain reaction (PCR) using primers which result from SEQ ID No. 1 are a constituent of the invention. Such oligonucleotides typically have a length of at least 15 nucleotides .

Instructions for identifying DNA sequences by means of hybridization can be found by the expert, inter alia, in the handbook "The DIG System Users Guide for Filter

Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al . (International Journal of Systematic Bacteriology (1991) 41: 255-260). The hybridization takes place under stringent conditions, that is to say only hybrids in which the probe and target sequence, i. e. the polynucleotides treated with the probe, are at least 70% identical are formed. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration.

The hybridization reaction is preferably carried out under a relatively low stringency compared with the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996) .

A 5x SSC buffer at a temperature of approx. 50²C - 68^SC, for ^"example^", ^~can^~be employed for the hybridization reaction. Probes can also hybridize here with polynucleotides which are less than 70% identical to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2x SSC and optionally subsequently 0.5x SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approx. 50^SC - 68^aC being established. It is optionally possible to lower the salt concentration to O.lx SSC. Polynucleotide fragments which are, for example, at least 70% or at least 80% or at least 90% to 95% or at least 96% to 99% identical to the sequence of the probe employed can be isolated by increasing the hybridization temperature stepwise from 50^SC to 68^eC in steps of approx. l^aC - 2⁹C. It is also possible to isolate polynucleotide fragments which are completely identical to the sequence of the probe employed. Further instructions on hybridization are obtainable on the market in the form of so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558) .

instructions for amplification of DNA sequences with the aid of the polymerase chain reaction (PCR) can be found by the expert, inter alia, in the handbook by Gait:

Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) .

It has been found that coryneform bacteria produce nicotinic acid in an improved manner after over-expression of the nadA gene.

To achieve an over-expression, the number of copies of the corresponding genes can be increased, or the promoter and regulation region or the ribosome binding site upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same way. By inducible promoters, it is additionally possible to increase the expression in the course of fermentative nicotinic acid production. The expression is likewise improved by measures to prolong the life of the - RNA. Furthermore, the enzyme activity is also increased by preventing the degradation of the enzyme protein. The genes or gene constructs can either be present in plasmids with a. varying number of copies, or can be integrated and amplified in the chromosome. Alternatively, an over- expression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.

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

By way of example, for enhancement the nadA gene according to the invention was over-expressed with the aid of episomal plasmids. Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as e.g. pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al . , Gene 102:93-98 (1991)) or pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHMl519, pBLl or pGAl . Other plasmid vectors, such as e.g. those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119- 124 (1990)), or pAGl (US-A 5,158,891), can be used in the same manner.

An example of such a plasmid is the plasmid pZ-nadAex shown in Figure 1. Plasmid vectors which are furthermore suitable are also those with the aid of which the process of gene amplification by integration into the chromosome can be used, as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994) ) for duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned in a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum. Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKlδmob or pKl9mob (Schafer et al . , Gene 145, 69- 73 (1994)), pGEM-T (Promega corporation, Madison, I, USA), PCR2.1-T0P0 (Shuman (1994). Journal of Biological Chemistry 269:32678-84; US-A 5,487,993), pCR®Blunt (Invitrogen, Groningen, Holland; Bernard et al . , Journal of Molecular Biology, 234: 534-541 (1993)), pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGSδ (Spratt et al.,1986, Gene 41: 337-342). The plasmid vector which contains the gene to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or. transformation. The method of conjugation is described, . for example, by Schafer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362

(1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al . (FEMS Microbiological Letters- 123, 343-347 (1994)). After homologous recombination by means of a "cross over" event, . the resulting strain contains at least two copies of the gene in question.

In addition, it may be advantageous for the production of nicotinic acid or nicotinic acid derivatives to enhance, in particular over-express, one or more enzymes of glycolysis, of anaplerosis and optionally regulatory proteins, in addition to the nadA gene. Thus, for the preparation of nicotinic acid or nicotinic acid derivatives, in addition to enhancement of the nadA gene, one or more genes endogenous chosen from the group consisting of

* the pyc gene which codes for pyruvate carboxylase (DE-A-198 31 609) ,

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

* the prs gene which codes for phosphoribosyl pyrophosphate synthetase (ACCESSION No. : U76387)

can be enhanced, in particular over-expressed.

It may furthermore be advantageous for the production of nicotinic acid or nicotinic acid derivatives, in addition to enhancement of the nadA gene, for one or more genes chosen from the group consisting of

• the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1; DSM 13047),

• the poxB gene which codes for pyruvate oxidase (DE: 199 51 975.7; DSM 13114),

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

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

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

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

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

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

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

Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) ) .

The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General

Bacteriology" of the American Society for Bacteriology (Washington D.C. , USA, 1981). Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substance can be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya, bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture.

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

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid, or sulfuric acid, can be employed in a suitable manner to control the pH. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 20²C to 45²C, and preferably 25^aC to 40^aC. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours.

Methods for the determination of nicotinic acid or nicotinic acid derivatives are known from the prior art. The concentration of nicotinic acid or nicotinic acid derivatives formed can be determined with microbiological methods, such as, for example, the Lactobacillus plantarum test (DIFCO MANUAL, 10^th Edition, p. 1100-1102; Michigan, USA) .

A pure culture of the C. glutamicum strain DSMl2455/pZ- nadAex was deposited on 25th October 2000 at the Deutsche Sammlung fϋr Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty as DSM13794.

The process according to the invention is used for fermentative preparation of nicotinic acid and nicotinic acid derivatives .

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

The isolation of plasmid DNA from Escherichia coli and all techniques of restriction, Klenow and alkaline phosphatase treatment are carried out by the method of Sambrook et al. (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA) . Methods for transformation of Escherichia coli are also described in this handbook. The composition of the usual nutrient media, such as LB or TY medium, can also be found in the handbook by Sambrook et al.

The strain ATCC13032ΔilvA is deposited as DSM12455 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen in Braunschweig (Germany) in accordance with the Budapest Treaty and is described in EP-A-1006189.

Example 1

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

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 is isolated as described by Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). The DNA fragments are dephosphorylated 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 Stratagene (La Jolla, USA, Product Description SuperCosl Cosmid Vektor Kit, Code no. 251301) is cleaved with the restriction enzyme Xba (Amersham Pharmacia, Freiburg, Germany, Product Description Xbal, Code no. 27- 0948-02) and likewise 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 manner is mixed with the treated ATCC13032 DNA and the batch 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 with the aid of 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. The infection and titering of the cosmid library are carried out as described by 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^SC, recombinant individual clones are selected.

Example 2

Isolation and sequencing of the nadA gene

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

The DNA of the sequencing vector pZero-1, obtained from Invitrogen (Groningen, Holland, Product Description Zero Background Cloning Kit, Product No. K2500-01) , is cleaved with the restriction enzyme BamHI (Amersham Pharmacia, • • Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04) . The ligation of the cosmid fragments in the sequencing vector pZero-1 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) . This ligation mixture is then electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences, USA, 87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 zeocin.

The plasmid preparation of the recombinant clones is carried out with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) . The sequencing is carried out by the dideoxy chain-stopping method of Sanger et al . (1977, Proceedings of the National Academy of Sciences, USA, 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 by gel electrophoresis and analysis of the sequencing reaction are carried out in a "Rotiphoresis NF Acrylamide/Bisacrylamide" Gel (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377" sequencer from PE Applied Biosystems (Weiterstadt, Germany) .

The raw sequence data obtained 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 assembled to a continuous contig. The computer-assisted coding region analysis is prepared with the XNIP program (Staden, 1986, Nucleic Acids Research 14:217-231) .

The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis of the nucleotide sequence shows an open reading frame of 1287 base pairs, which was called the nadA gene. The nadA gene codes for a protein of 428 amino acids.

Example 3

Preparation of the shuttle vector pZ-nadAex for enhancement of the nadA gene in C. glutamicum

3.1. Cloning of the nadA gene

From the strain ATCC 13032, chromosomal DNA is isolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) . On the basis of the sequence of the nadA gene known for C. glutamicum from example 2, the following oligonucleotides are chosen for the polymerase chain reaction. In addition, suitable restriction cleavage sites which allow cloning into the target vector are inserted:

nadA-exl shown in SEQ ID No. 3 5'GAT CTA GTC GAC ATG ACC ACC TCA ATC ACC 3

nadA-ex2 shown in SEQ ID No. 4

5^VAAG TCT GTC GAC ACG ATG CGG TCA ATA TGG 3'

The primers shown were synthesized by ARK Scientific GmbH Biosystems (Darmstadt, Germany) . The primers nadA-exl and nadA-ex2 contain the sequence for the cleavage site of the restriction endonuclease Sail, which are marked by underlining in the nucleotide sequences shown above. The PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim, Germany) . With the aid of the polymerase chain reaction, the primers allow amplification of a DNA fragment 1287 bp in size, which carries the nadA gene from Corynebacterium glutamicum without a potential promoter region. The fragment amplified in this way is tested electrophoretically in a 0.8% agarose gel and checked by sequencing. The PCR fragment obtained in this manner is cleaved completely with the restriction enzy Sail and, after separation in a 0.8% agarose gel, isolated from the gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .

3.2. Cloning of nadA in the vector pZ8-l

The E. coli - C. glutamicum shuttle expression vector pZ8-l (EP 0 375 889) is employed as the base vector for expression both in C. glutamicum and in E. coli. DNA of this plasmid is cleaved completely with the restriction enzyme Sail and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 17582.50) . The nadA fragment isolated from the agarose gel in example 3.1 is mixed with the vector pZ8-l prepared in this way and the batch is treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04) .

The ligation batch is transformed in the E. coli strain DH5 αmcr (Hanahan, In: DNA cloning. A Practical Approach, Vol. I, IRL-Press, Oxford, Washington DC, USA) . Selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 kana ycin. After incubation overnight at 37 ^aC, recombinant individual clones are selected. Plasmid DNA is isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and checked by restriction cleavage. The resulting plasmid is called pZ-nadAex. It is shown in Figure 1. Example 4

Transformation of the strain ATCCl3032ΔilvA with the plasmid pZ-nadAex

The strain ATCCl3032ΔilvA is transformed with the plasmid pZ-nadAex using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989)). Selection of the transformants takes place on LBHIS agar comprising 18.5 g/1 brain-heart infusion broth, 0.5 M sorbitol, - 5-. g/1 Bacto-tryptone, 2.5 g/1 Bacto-yeast extract, 5 g/1 NaCl and 18 g/1' Bacto-agar, which has been supplemented with 25 mg/1 kanamycin. Incubation is carried out for 2 days at 33 ^SC.

Plasmid DNA is isolated from a transformant by conventional methods (Peters-Wendisch et al . , 1998, Microbiology, 144, 915-927) , cleaved with the restriction endonuclease Sail, and the plasmid is checked by subsequent agarose gel electrophoresis .

The resulting strain is called C. glutamicum ATCC13032Δ ilvA/pZ-nadAex or DSMl2455/pZnadAex.

Example 5

Preparation of Nicotinic Acid

The formation of nicotinic acid by the C. glutamicum strains ATCC13032ΔilvA/pZ8-l and ATCCl3032ΔilvA/pZ-nadAex is tested in medium CGXII (Keilhauer et al., 1993, Journal of Bacteriology, 175:5595-5603), which was supplemented with 25 μg/ml kanamycin and 1 mM L-isoleucine.

This medium is called C. glutamicum test medium in the following. In each case 50 ml of freshly prepared C. glutamicum test medium are inoculated with a 16 hours old preculture of the same medium such that the optical density of the culture suspension (OD₅₈₀) at the start of incubation is 0.1. The cultures are incubated at 30^aC and 130 rp . After incubation for 48 hours the optical density (OD5₈₀) of the culture is determined and the cells are then removed by centrifugation at 5000 g for 10 minutes and the supernatant subjected to sterile filtration.

A Novaspec II photometer from Pharmacia (Freiburg, Germany) is employed at a measurement wavelength of 580 n for determination of the optical density.

The nicotinic acid in the culture supernatant is quantified by means of Lactobacillus plantarum ATCC 8014 in accordance with the instructions in the handbook of DIFCO (DIFCO MANUAL, 10^th Edition, p. 1100-1102; Michigan, USA). Nicotinic acid from Sigma (Deisenhofen, Germany) is used for the calibration.

Table 1

Brief Description of the Figure:

Figure 1: Map of the plasmid pZ-nadAex

The abbreviations and designations used have the following meaning. The base pair numbers stated are approximate values obtained in the context of reproducibility of measurements .

Ptac Ptac promoter rep: plasmid-coded replication region from glutamicum plasmid pGAl rrnB: terminator T1T2 of the rrnB gene of E.coli

Kan: resistance gene for kanamycin nadA-ex:- nadA gene of C. glutamicum without promoter region Sail: cleavage site of the restriction enzyme Sail

Claims

What is claimed is:

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

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

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

d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a) , b) or c)

the polypeptide preferably having the activity of quinolinate synthetase A.

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

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

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

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

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

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

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

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

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

9. Corynebacterium glutamicum DSMl2455/pZ-nadAex deposited on 25th October 2000 as DSM13794 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures) .

10. Process for the fermentative preparation of nicotinic acid or nicotinic acid derivatives, wherein the following steps are carried out:

a) fermentation of the coryneform bacteria which produce the desired nicotinic acid or nicotinic acid derivative and in which at least the endogenous nadA gene or nucleotide sequences which code for it is or are enhanced, in particular over- expressed;

b) concentration of the nicotinic acid or nicotinic acid derivative in the medium or in the cells of the bacteria, and

c) isolation of the nicotinic acid or nicotinic acid derivative.

11. Process according to claim 10, wherein bacteria in which further genes of the biosynthesis pathway of the desired nicotinic acid or nicotinic acid derivative are additionally enhanced are employed.

12. Process according to claim 10, wherein bacteria in which the metabolic pathways which reduce the formation of the desired nicotinic acid or nicotinic acid derivative are at least partly eliminated are employed.

13. Process according to claim 10, wherein a strain transformed with a plasmid vector is employed, and the plasmid vector carries the nucleotide sequence which codes for the nadA gene.

14. Process according to claim 10, wherein the expression of the polynucleotide (s) which code(s) for the nadA gene is enhanced, in particular over-expressed.

15. Process according to claim 10, wherein the regulatory/catalytic properties of the polypeptide (enzyme protein) for which the polynucleotide nadA codes are increased.

16. Process according to claim 10, wherein, for the preparation of nicotinic acid or nicotinic acid derivatives, coryneform microorganisms in which one or more of the endogenous genes chosen from the group consisting of

16.1 the pyc gene which codes for pyruvate carboxylase,

16.2 the zwal gene which codes for the Zwal protein,

16.3 the prs gene which codes for phosphoribosyl pyrophosphate synthetase

is or are amplified, in particular over-expressed, at the same time are fermented.

17. Process according to claim 10, wherein, for the preparation of nicotinic acid or nicotinic acid derivatives, coryneform microorganisms in which one or more of the genes chosen from the group consisting of

17.1 the pck gene which codes for phosphoenol pyruvate carboxykinase,

17.2 the poxB gene which codes for pyruvate oxidase

17.3 the zwa2 gene which codes for the Zwa2 protein

is or are attenuated at the same time are fermented.

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

19. Process according to one or more of the claims 10-17, wherein microorganisms of the genus Corynebacterium glutamicum are employed.

20. Process for discovering RNA, cDNA and DNA in order to isolate nucleic acids or polynucleotides or genes which code for quinolinate synthetase A or have a high similarity with the sequence of the nadA gene, wherein the polynucleotide comprising the polynucleotide sequences according to claims 1, 2, 3 or 4 is employed as hybridization probes.

1. Process according to claim 20, wherein arrays, micro arrays or DNA chips are employed.