Abstract:
The invention relates to a process for the preparation of L-amino acids. The process involves fermenting an L-amino acid producing coryneform bacteria in a culture medium, concentrating L-amino acid produced by the fermenting in the culture medium or in the cells of the bacteria, and isolating the L-amino acid produced. The bacteria has an overexpressed gene encoding 6-phosphogluconate dehydrogenase and a decreased or switched off gene encoding pyruvate oxidase. The L-amino acid may be L-lysine, L-threonine, L-isoleucine or L-tryptophan. `

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    The present application is a continuation-in-part of U.S. application Ser. No. 09/531,265, filed on Mar. 20, 2000, the contents of which are incorporated by reference herein in their entirety. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The invention relates to a process for the fermentative preparation of L-amino acids, in particular L-lysine, L-threonine, L-isoleucine and L-tryptophan, using coryneform bacteria in which at least the enzyme 6-phosphogluconate dehydrogenase encoded by the gnd gene is amplified.  
         BACKGROUND  
         [0003]    L-Amino acids are used in animal nutrition, in human medicine and in the pharmaceuticals industry and are prepared by fermentation from strains of coryneform bacteria, in particular  Corynebacterium glutamicum . Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements may relate to fermentation measures, e.g., stirring and supply of oxygen; the composition of the nutrient media, e.g., the sugar concentration during the fermentation; the working up to the product form, e.g., by ion exchange chromatography; or the intrinsic output properties of the microorganism itself.  
           [0004]    Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites (e.g., the threonine analogue α-amino-β-hydroxyvaleric acid (AHV), and the lysine analogue S-(2-aminoethyl)-L-cystein (AEC)) or which are auxotrophic for metabolites of regulatory importance and produce L-amino acids such as threonine `or lysine are obtained in this manner.  
           [0005]    Methods utilizing recombinant DNA techniques have also been employed for some years for improving  Corynebacterium glutamicum  strains which produce L-amino acids.  
         SUMMARY OF THE INVENTION  
         [0006]    L-Amino acids are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and especially in animal nutrition. There is therefore a general interest in providing improved processes for their preparation.  
           [0007]    In general, the present invention is directed to improved processes for the fermentative preparation of L-amino acids by coryneform bacteria. More specifically, the invention provides a process for the fermentative preparation of L-amino acids (particularly L-lysine, L-threonine, L-isoleucine and L-tryptophan) using coryneform bacteria in which the nucleotide sequence which codes for the enzyme 6-phosphogluconate dehydrogenase (EC number 1.1.1.44) (gnd gene) is amplified, in particular over-expressed. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0008]    Embodiments of the present invention will be described with reference to the following Figures, in which:  
         [0009]    [0009]FIG. 1 is a map of the plasmid pEC-T18mob2;  
         [0010]    [0010]FIG. 2 is a map of the plasmid pECgnd;  
         [0011]    [0011]FIG. 3 is a map of the plasmid pBGNA; and  
         [0012]    [0012]FIG. 4 is a map of the plasmid pCR2.1poxBint. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0013]    The strains of bacteria employed in the present processes preferably already produce L-amino acids before amplification of the gnd gene. The term `“amplification” as used herein describes the increase in the intracellular activity of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA. This may be accomplished, 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, or by combining these measures.  
         [0014]    By amplification measures, in particular over-expression, the activity or concentration of the corresponding enzyme or 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%, compared to that of the wild-type enzyme or the activity or concentration of the enzyme in the starting microorganism.  
         [0015]    The microorganisms which the present invention provide can prepare L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They are representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, the most preferred species is  Corynebacterium glutamicum , which is known among experts for its ability to produce L-amino acids. Suitable strains include the wild-type strains:  
         [0016]    [0016] Corynebacterium glutamicum  ATCC13032;  
         [0017]    [0017] Corynebacterium acetoglutamicum  ATCC 15806;  
         [0018]    [0018] Corynebacterium acetoacidophilum  ATCC13870;  
         [0019]    [0019] Corynebacterium thermoaminogenes  FERM BP-1539;  
         [0020]    [0020] Brevibacterium flavum  ATCC14067;  
         [0021]    [0021] Brevibacterium lactofermentum  ATCC13869;  
         [0022]    Brevibacterium divaricatum ATCC14020;  
         [0023]    L-amino acid-producing mutants prepared from the strains above may also be used. Such strains include: the L-threonine-producing strains:  
         [0024]    [0024] Corynebacterium glutamicum  ATCC21649;  
         [0025]    [0025] Brevibacterium flavum  BB69;  
         [0026]    [0026] Brevibacterium flavum  DSM5399;  
         [0027]    [0027] Brevibacterium lactofermentum  FERM-BP 269;  
         [0028]    [0028] Brevibacterium lactofermentum  TBB- 10; `   
         [0029]    the L-isoleucine-producing strains:  
         [0030]    [0030] Corynebacterium glutamicum  ATCC 14309;  
         [0031]    [0031] Corynebacterium glutamicum  ATCC 14310;  
         [0032]    [0032] Corynebacterium glutamicum  ATCC 14311;  
         [0033]    [0033] Corynebacterium glutamicum  ATCC 15168;  
         [0034]    [0034] Corynebacterium ammoniagenes  ATCC 6871;  
         [0035]    the L-tryptophan-producing strains:  
         [0036]    [0036] Corynebacterium glutamicum  ATCC21850;  
         [0037]    [0037] Corynebacterium glutamicum  KY9218(pKW9901);  
         [0038]    and the L-lysine-producing strains:  
         [0039]    [0039] Corynebacterium glutamicum  FERM-P 1709;  
         [0040]    [0040] Brevibacterium flavum  FERM-P 1708;  
         [0041]    [0041] Brevibacterium lactofermentum  FERM-P 1712;  
         [0042]    [0042] Corynebacterium glutamicum  FERM-P 6463;  
         [0043]    [0043] Corynebacterium glutamicum  FERM-P 6464;  
         [0044]    [0044] Corynebacterium glutamicum  DSM5715;  
         [0045]    [0045] Corynebacterium glutamicum  DM58-1; and  
         [0046]    [0046] Corynebacterium glutamicum  DSM12866.  
         [0047]    It has been found that coryneform bacteria produce L-amino acids, in particular L-lysine, L-threonine, L-isoleucine and L-tryptophan, in an improved manner after over-expression of the gnd gene. The gnd gene codes for the enzyme 6-phosphogluconate dehydrogenase (EC number 1.1.1.44) which catalyses the oxidative decarboxylation of 6-phosphogluconic acid to ribulose 5-phosphate. The nucleotide sequence of the gnd gene is disclosed in JP-A-9-224662. Alleles of the gnd gene which result from the degeneracy of the genetic code or which are due to sense mutations of neutral function can furthermore be used. Genes encoding proteins with 6-phosphogluconate dehydrogenase activity from Gram-negative bacteria, e.g.  Escherichia coli , or other Gram-positive bacteria, e.g., Streptomyces or Bacillus, may optionally be used.  
         [0048]    The use of endogenous, genes in particular endogenous genes from `coryneform bacteria, is preferred. The terms “endogenous genes” or “endogenous nucleotide sequences” refer to genes or nucleotide sequences which are available in the population of a species.  
         [0049]    To achieve an amplification (e.g., over-expression) of a protein, the number of copies of the corresponding gene is increased, or the promoter and regulation region or the ribosome binding site upstream of the structural gene are mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same way. Using inducible promoters, it is additionally possible to increase the expression in the course of fermentative L-amino acid formation. Expression may also be improved by measures to prolong the life of the m-RNA. Enzyme activity may be increased by preventing the degradation of the enzyme protein.  
         [0050]    Genes or gene constructs may either be provided in plasmids with a varying number of copies, or may be integrated and amplified in the chromosome. Alternatively, an over-expression of the genes in question can 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 European Patent Specification EPS 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler (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 Patent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in Japanese Laid-Open Specification JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and in known textbooks of genetics and molecular biology.  
         [0051]    By way of example, 6-phosphogluconate dehydrogenase was over-expressed with the aid of a plasmid. The  E. coli - C. glutamicum  shuttle vector pEC-T18mob2 shown in FIG. 1 was used for this. After incorporation of the gnd gene into the EcoRI cleavage site of pEC-T18mob2, the plasmid pECgnd shown in FIG. 2 was `formed. Other plasmid vectors which are capable of replication in  C. glutamicum , such as pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pZ8-1 (EP-B-0 375 889), can be used in the same way.  
         [0052]    In addition, it may be advantageous for the production of L-amino acids to amplify one or more enzymes of the relevant biosynthesis pathway, of glycolysis, of anaplerosis, of the pentose phosphate pathway or of amino acid export, in addition to amplification of the gnd gene. For example, for the preparation of L-threonine, one or more of the following genes can be amplified (over-expressed):  
         [0053]    the hom gene which codes for homoserine dehydrogenase (Peoples et al., Molecular Microbiology 2, 63-72 (1988)) or the hom dr  allele which codes for a “feed back resistant” homoserine dehydrogenase (Archer et al., Gene 107, 53-59 (1991),  
         [0054]    the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns et al., Journal of Bacteriology 174: 6076-6086 (1992)),  
         [0055]    the pyc gene which codes for pyruvate carboxylase (Peters-Wendisch et al., Microbiology 144: 915-927 (1998)),  
         [0056]    the mqo gene which codes for malate:quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),  
         [0057]    the tkt gene which codes for transketolase (accession number AB023377 of the databank of European Molecular Biology Laboratories (EMBL, Heidelberg, Germany)),  
         [0058]    the zwf gene which codes for glucose 6-phosphate dehydrogenase (JP-A-09224661),  
         [0059]    the thrE gene which codes for threonine export (DE 199 41 478.5; DSM 12840),  
         [0060]    the zwa1 gene (DE 199 59 328.0; DSM 13115),  
         [0061]    the eno gene which codes for enolase (DE: 199 41 478.5).  
         [0062]    For the preparation of L-lysine, one or more of the following genes can be amplified, in particular over-expressed, at the same time as gnd.  
         [0063]    the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0 197 335),  
         [0064]    a lysC gene which codes for a feed back resistant aspartate kinase (Kalinowski et al. (1990), Molecular and General Genetics 224: 317-324),  
         [0065]    the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns `(1992), Journal of Bacteriology 174:6076-6086),  
         [0066]    the pyc gene which codes for pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),  
         [0067]    the mqo gene which codes for malate-quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),  
         [0068]    the tkt gene which codes for transketolase (accession number AB023377 of the databank of European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany)),  
         [0069]    the zwf gene which codes for glucose 6-phosphate dehydrogenase (JP-A-09224661),  
         [0070]    the lysE gene which codes for lysine export  
         [0071]     (DE-A-195 48 222),  
         [0072]    the zwa1 gene (DE 199 59 328.0; DSM 13115),  
         [0073]    the eno gene which codes for enolase (DE 199 47 791.4).  
         [0074]    The use of endogenous genes is preferred.  
         [0075]    It may furthermore be advantageous for the production of L-amino acids to attenuate one or more of the following genes while at the same time amplifying gnd:  
         [0076]    the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1; DSM 13047),  
         [0077]    the pgi gene which codes for glucose 6-phosphate isomerase (U.S. Ser. No. 09/396,478, DSM 12969),  
         [0078]    the poxB gene which codes for pyruvate oxidase  
         [0079]     (DE 199 51 975.7; DSM 13114),  
         [0080]    the zwa2 gene (DE: 199 59 327.2; DSM 13113).  
         [0081]    In this connection, the term “attenuation” means reducing or suppressing the intracellular activity or concentration of one or more enzymes or proteins in a microorganism. This may be accomplished using the genes which encode the proteins, for example by using a weak promoter or a gene or allele which codes for a corresponding protein which has a low activity or inactivates the corresponding enzyme and optionally by combining these measures. By attenuation measures, the activity or concentration of the corresponding enzyme or protein is in general reduced to 0 to 75%, `0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type enzyme or of the activity or concentration of the enzyme in the starting microorganism.  
         [0082]    In addition to over-expression of 6-phosphogluconate dehydrogenase, it may furthermore be advantageous for the production of L-amino acids to eliminate undesirable side reactions (see, Nakayama: “Breeding of Amino Acid Producing Microorganisms,” in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).  
         [0083]    The microorganisms prepared according to the invention can be cultured continuously or discontinuously in a batch process (batch culture) or in a fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of L-amino acid production. A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einführung 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 microorganisms 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 substances 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. 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.  
         [0084]    Basic compounds, such as sodium hydroxide, potassium hydroxide, 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 fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing is continued until a maximum of L-amino acid has formed. This target is usually reached within 10 hours to 160 hours.  
         [0085]    The analysis of L-amino acids can be carried out by anion exchange chromatography with subsequent ninhydrin derivation, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190), or it can take place by reversed phase HPLC as described by Lindroth et al. (Analytical Chemistry (1979) 51:. 1167-1174).  
         [0086]    The following microorganism has been deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty:  Escherichia coli  K-12 DH5α/pEC-T18mob2 as DSM 13244.  
         [0087]    In the accompanying Figures, the base pair numbers stated are approx. values obtained in the context of reproducibility. The abbreviations used in the Figures have the following meaning: ` 
                                       In FIG. 1:           Tet:   Resistance gene for tetracycline       oriV:   Plasmid-coded replication origin of  E. coli         RP4mob:   mob region for mobilizing the plasmid       rep:   Plasmid-coded replication origin from  C. glutamicum             plasmid pGA1       per:   Gene for controlling the number of copies from pGA1       lacZ-alpha:   lacZα gene fragment (N-terminus) of the β-Galactosidase           gene.       In FIG. 2:       Tet:   Resistance gene for tetracycline       rep:   Plasmid-coded replication origin from  C. glutamicum             plasmid pGA1       per:   Gene for controlling the number of copies from PGA1       lacZ   Cloning relict of the lacZα gene fragment from           pEC-T18mob2       gnd:   6-Phosphogluconate dehydrogenase gene.       In FIG. 3:       LacP:   Promoter of the  E. coli  lactose operon       CMV:   Promoter of cytomegalovirus       ColE1:   Replication origin of the plasmid ColE1       TkpolyA:   Polyadenylation site       Kan r:   Kanamycin resistance gene       SV40ori:   Replication origin of Simian virus 40       gnd:   6-Phosphogluconate dehydrogenase gene.       In FIG. 4:       ColE1 ori:   Replication origin of the plasmid ColE1       lacZ:   Cloning relict of the lacZα gene fragment       fl ori:   Replication origin of phage f1       KmR:   Kanamycin resistance       ApR:   Ampicillin resistance       poxBint:   internal fragment of the poxB gene                  
 
         [0088]    The following abbreviations have also been used herein:  
                                                       AccI:   Cleavage site of the restriction enzyme AccI           BamHI:   Cleavage site of the restriction enzyme BamHI           EcoRI:   Cleavage site of the restriction enzyme EcoRI           HindIII:   Cleavage site of the restriction enzyme HindIII           KpnI:   Cleavage site of the restriction enzyme KpnI           PstI:   Cleavage site of the restriction enzyme PstI           PvuI:   Cleavage site of the restriction enzyme PvuI           SalI:   Cleavage site of the restriction enzyme SalI           SacI:   Cleavage site of the restriction enzyme SacI           SmaI:   Cleavage site of the restriction enzyme SmaI           SphI:   Cleavage site of the restriction enzyme SphI           XbaI:   Cleavage site of the restriction enzyme XbaI           XhoI:   Cleavage site of the restriction enzyme XhoI                      
 
         [0089]    The following examples will further illustrate this invention. The molecular biology techniques, e.g. plasmid DNA isolation, restriction enzyme treatment, ligations, standard transformations of  Escherichia coli  etc. used are, (unless stated otherwise), are described by Sambrook et al., (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratories, USA).  
       EXAMPLE 1  
     Construction of a Gene Library of  Corynebacterium glutamicum  Strain AS019  
       [0090]    A DNA library of  Corynebacterium glutamicum  strain AS019 (Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985)) was constructed using λ Zap Express™ system, (Short et al., (1988) Nucleic Acids Research 16: 7583-7600), as described by O&#39;Donohue (O&#39;Donohue, M. (1997). The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from  Corynebacterium glutamicum.  Ph.D. Thesis, National University of Ireland, Galway). λ Zap Express™ kit was purchased from Stratagene (Stratagene, 11011 North Torrey Pines Rd., La Jolla, Calif. 92037) and used according to the manufacturer&#39;s instructions. AS019-DNA was digested with restriction enzyme Sau3A and ligated to `BamHI treated and dephosphorylated λ Zap Express™ arms.  
       EXAMPLE 2  
     Cloning and Sequencing of the gnd Gene  
       [0091]    2.1 Construction of a gnd Probe  
         [0092]    A radio-labeled oligonucleotide, internal to the gnd gene, was used to probe the AS019 λ Zap Express™ library described above. The oligonucleotide was produced using degenerate PCR primers internal to the gnd gene. The degenerate nucleotide primers designed for the PCR amplification of gnd DNA fragments were as follows:  
         [0093]    gnd1: 5′ ATG GTK CAC ACY GGY ATY GAR TA 3′ (SEQ ID NO 7)  
         [0094]    gnd2: 5′ RGT CCA YTT RCC RGT RCC YTT 3′ (SEQ ID NO 8)  
         [0095]    with R=A+G; Y=C+T; K=T+G.  
         [0096]    The estimated size of the resulting PCR product was 252 bp approximately. Optimal PCR conditions were determined to be as follows:  
         [0097]    35 cycles  
         [0098]    94° C. for 1 minute  
         [0099]    55° C. for 1 minute  
         [0100]    72° C. for 30 seconds  
         [0101]    2.5-3.5 mM MgCl 2    
         [0102]    100-150 ng AS019 genomic DNA.  
         [0103]    Sequence analysis of the resulting PCR product confirmed the product to be an internal portion of a gnd gene. Sequence analysis was carried out using the universal forward and reverse primers, and T7 sequencing kit from Pharmacia Biotech, (St. Albans, Herts, UK). The sequence of the PCR product is shown in SEQ ID No. 1.  
         [0104]    [0104] 2 . 2  Cloning  
         [0105]    Screening of the AS019 λ Zap Express™ library was carried out according to the λ Zap Express™ system protocol, (Stratagene, 11011 North Torrey Pines Rd., La Jolla, Calif. 92037). Southern Blot analysis was then carried out on isolated clones. Southern transfer of DNA was as described in the Schleicher and Schuell protocols manual employing Nytran™ as membrane (,,Nytran, Modified Nylon `66  Membrane Filters” (March 1987), Schleicher and Schuell, Dassel, Germany). Double stranded DNA fragments, generated using the same primers and optimal PCR conditions as described above, were radio-labeled with α- 32 P-dCTP using the Multiprime™ DNA labeling kit from Amersham Life Science (Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK) according to the manufacturers instructions. Prehybridization, hybridization and washing conditions were as described in the Schleicher and Schuell protocols manual. Autoradiography was carried out according to the procedure outlined in the handbook of Sambrook et al. using AgFa Curix RPIL film. Thus several gnd clones were identified. Plasmid DNA was isolated from one of the clones, designated pBGNA (FIG. 3) and chosen for further analysis.  
         [0106]    2.3 Sequencing  
         [0107]    The Sanger Dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences USA 74, 5463-5467 (1977)) was used to sequence the cloned insert of pBGNA. The method was applied using the T7 sequencing kit and α- 35 S-dCTP from Pharmacia Biotech (St. Albans, Herts, UK). Samples were electrophoresed for 3-8 hours on 6% polyacrylamide/urea gels in TBE buffer at a constant current of 50 mA, according to the Pharmacia cloning and sequencing instructions manual (,, T7  Sequencing™ Kit”,ref.XY-010-00-19, Pharmacia Biotech, 1994). Sequence analysis was carried out using internal primers designed from the sequence known of the internal gnd PCR product (SEQ ID NO 1) allowing the entire gnd gene sequence to be deduced. The sequences of the internal primers were as follows:  
                                           Internal primer 1:                   5′ GGT GGA TGC TGA AAC CG 3′   (SEQ ID NO 9)                       Internal primer 2:           5′ GCT GCA TGC CTG CTG CG 3′   (SEQ ID NO 10)                       Internal primer 3:           5′ TTG TTG CTT ACG CAC AG 3′   (SEQ ID NO 11)                       Internal primer 4:           5′ TCG TAG GAC TTT GCT GG 3′   (SEQ ID NO 12)          
 
         [0108]    Sequences obtained were analyzed using the DNA Strider program, (Marck (1988), Nucleic Acids Research 16: 1829-1836), version 1.0 on an Apple Macintosh computer. This program allowed for analyses such as restriction site usage, open reading frame analysis and codon usage determination. Searches between DNA sequences obtained and those in EMBL and Genbank databases were performed using the BLAST program (Altschul et al., (1997), Nucleic Acids Research 25: 3389-3402). DNA and protein sequences were aligned using the Clustal V and Clustal W programs (Higgins and Sharp, 1988 Gene 73: 237-244).  
         [0109]    The sequence thus obtained is shown in SEQ ID NO 2. The analysis of the nucleotide sequence obtained revealed an open reading frame of 1377 base pairs which was designated as gnd gene. It codes for a protein of 459 amino acids shown in SEQ ID NO 3.  
       EXAMPLE 3  
     Preparation of the Shuttle Vector pEC-T18mob2  
       [0110]    The  E. coli - C. glutamicum  shuttle vector pEC-T18mob2 was constructed according to the prior art. The vector contains the replication region, rep, of the plasmid pGA1 including the replication effector, per (U.S. Pat. No. 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline resistance-imparting tetA(Z) gene of the plasmid, pAG1 (U.S. Pat. No. 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA) with accession number AF121000), the replication region, oriV, of the plasmid pMB1 (Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979)), the lacZ gene fragment including the lac promoter and a multiple cloning site (mcs) (Norrander et al. Gene 26, 101-106 (1983)) and the mob region of the plasmid RP4 (Simon et al.,(1983) Bio/Technology 1:784-791).  
         [0111]    The vector constructed was transformed in the  E. coli  strain DH5α (Hanahan, In: DNA cloning. A practical approach. Vol. I. IRL-Press, Oxford, Washington D.C., USA, 1985). Selection for plasmid-carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 nd  Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA, 1989), which had been supplemented with 5 mg/l tetracycline. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and HindIII subsequent agarose gel electrophoresis (0.8%). The plasmid was called pEC-T18mob2 and is shown in FIG. 1. It is deposited in the form of the strain  Escherichia coli  K-12 strain DH5α/pEC-T18mob2 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) as DSM 13244.  
       EXAMPLE 4  
     Cloning of the gnd Gene into the  E. coli - C. glutamicum  Shuttle Vector pEC-T18mob2  
       [0112]    PCR was used to amplify DNA fragments containing the entire gnd gene of  C. glutamicum  and flanking upstream and downstream regions using pBGNA as template. PCR reactions were carried out using oligonucleotide primers designed from SEQ ID NO 2. The primers used were:  
                               gnd fwd. primer:               5′ ACT CTA GTC GGC CTA AAA TGG 3′   (SEQ ID NO 13)               gnd rev. primer:       5′ CAC ACA GGA AAC AGA TAT GAC 3′.   (SEQ ID NO 14)          
 
         [0113]    PCR parameters were as follows:  
         [0114]    35 cycles  
         [0115]    95° C. for 6 minutes  
         [0116]    94° C. for 1 minute  
         [0117]    50° C. for 1 minute  
         [0118]    72° C. for 45 seconds  
         [0119]    1 mM MgCl 2    
         [0120]    approx. 150-200 ng pBGNA-DNA as template.  
         [0121]    The PCR product obtained was cloned into the commercially available pGEM-T vector purchased from Promega Corp. (pGEM-T Easy Vector System 1, cat. no. A1360, Promega UK, Southampton) using  E. coli  strain JM109 (Yanisch-Perron et al. Gene, 33: 103-119 (1985)) as a host. The entire gnd gene was subsequently isolated from the pGEM T-vector on an EcoRI fragment and cloned into the lacZ EcoRI site of the  E. coli - C. glutamicum  shuttle vector pEC-T18mob2 (FIG. 1), and designated pECgnd (FIG. 2). Restriction enzyme analysis with AccI (Boehringer Mannheim GmbH, Germany) revealed the correct orientation (i.e., downstream the lac-Promotor) of the gnd gene in the lacZα gene of pEC-T18mob2.  
       EXAMPLE 5  
     Preparation of Amino Acid Producers with Amplified 6-phosphogluconate Dehydrogenase  
       [0122]    Plasmid pECgnd from Example 3 was electroporated by the electroporation method of Tauch et al. (FEMS Microbiological Letters, 123:343-347 (1994)) in the strains  Corynebacterium glutamicum  DSM 5399 and DSM 5714. The strain DSM 5399 is a threonine producer described in EP-B-0358940. The strain DSM 5714 is a lysine producer described in EP-B-0435132. Selection of transformants was carried out by plating out the electroporation batch on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 nd  Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 25 mg/l kanamycin. The strains DSM5399/pECgnd and DSM5714/pECgnd were formed in this manner.  
       EXAMPLE 6  
     Preparation of Threonine  
       [0123]    The  C. glutamicum  strain DSM5399/pECgnd obtained in Example 5 was cultured in a nutrient medium suitable for the production of threonine and the threonine content in the culture supernatant was determined. For this, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with tetracycline (5 mg/l)) for 24 hours at 33° C. Starting from this agar plate culture, a preculture was seeded (10 ml medium in a 100 ml conical flask). Brain-heart broth (Merck, Darmstadt, Germany) was used as the medium for the preculture. Tetracycline (5 mg/l) was added to this medium. The preculture was incubated for 24 hours at 33° C. at 240 rpm on a shaking machine. A main culture was seeded from this preculture such that the initial OD (660 nm) of the main culture was 0.1. The medium MM-threonine was used for the main culture.  
                                                 Medium MM-threonine                                    CSL   5   g/l           MOPS   20   g/l           Glucose(autoclaved separately)   50   g/l           Salts:           (NH 4 ) 2 SO 4     25   g/l           KH 2 PO 4     0.1   g/l           MgSO 4  * 7 H 2 O   1.0   g/l           CaCl 2  * 2 H 2 O   10   mg/l           FeSO 4  * 7 H 2 O   10   mg/l           MnSO 4  * H 2 O   5.0   mg/l           Biotin (sterile-filtered)   0.3   mg/l           Thiamine * HCl (sterile-filtered)   0.2   mg/l           CaCO 3     25   g/l                      
 
         [0124]    The CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions were then added, as well as the CaCO 3  autoclaved in the dry state. Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Tetracycline (5 mg/l) was added. Culturing was carried out at 33° C. and 80% atmospheric humidity. After 48 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The concentration of threonine formed was determined with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection. The result of the experiment is shown in Table 1.  
                               TABLE 1                                       OD   L-Threonin           Strain   (660 nm)   g/l                           DSM5399/pECgnd   11.9   1.29           DSM5399   11.8   0.33                      
 
       EXAMPLE 7  
     Preparation of Lysine  
       [0125]    The  C. glutamicum  strain DSM5714/pECgnd obtained in Example 5 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined. For this, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with tetracycline (5 mg/l)) for 24 hours at 33° C. Starting from this agar plate culture, a preculture was seeded (10 ml medium in a 100 ml conical flask). The complete medium Cg III was used as the medium for the preculture.  
                                                 Medium Cg III                                    NaCl   2.5   g/l           Bacto-Peptone   10   g/l           Bacto-Yeast extract   10   g/l           Glucose (autoclaved separately)   2%   (w/v)                                  
 
         [0126]    Tetracycline (5 mg/l) was added to this medium. The preculture was incubated for 24 hours at 33° C. at 240 rpm on a shaking machine. A main culture was seeded from this preculture such that the initial OD (660 nm) of the main culture was 0.05. Medium MM was used for the main culture.  
                                                 Medium MM                                    CSL (corn steep liquor)   5   g/l           MOPS (morpholinopropanesulfonic acid)   20   g/l           Glucose (autoclaved separately)   50   g/l           (NH 4 ) 2 SO 4             KH 2 PO 4     25   g/l           MgSO 4  * 7 H 2 O   0.1   g/l           CaCl 2  * 2 H 2 O   1.0   g/l           FeSO 4  * 7 H 2 O   10   mg/l           MnSO 4  * H 2 O   10   mg/l           Biotin (sterile-filtered)   0.3   mg/l           Thiamine * HCl (sterile-filtered)   0.2   mg/l           L-Leucine (sterile-filtered)   0.1   g/l           CaCO 3     25   g/l                      
 
         [0127]    The CSL, MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions were then added, as well as the CaCO 3  autoclaved in the dry state. Culturing was carried out in a 10 ml volume in a 100 ml conical flask with baffles. Tetracycline (5 mg/l) was added. Culturing was carried out at 33° C. and 80% atmospheric humidity.  
         [0128]    After 48 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, München). The amount of lysine formed was determined with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection. The result of the experiment is shown in Table 2.  
                               TABLE 2                                       OD   Lysine HCl           Strain   (660 nm)   g/l                           DSM5715/pECgnd   7.7   14.7           DSM5715   7.1   13.7                      
 
       EXAMPLE 8  
     Preparation of a Genomic Cosmid Gene Library from  Corynebacterium glutamicum  ATCC 13032  
       [0129]    Chromosomal DNA from  Corynebacterium glutamicum  ATCC 13032 was isolated as described by Tauch et al., (1995, Plasmid 33:168-179), and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-O 2 ). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164), obtained from Stratagene (La Jolla, USA, Product Description SuperCos1 Cosmid Vektor Kit, Code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-O 2 ) and likewise dephosphorylated with shrimp alkaline phosphatase.  
         [0130]    The cosmid DNA was 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 was mixed with the treated ATCC13032 DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04). The ligation mixture was then packed in phages with the aid of Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). For infection of the  E. coli  strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575) the cells were taken up in 10 mM MgSO 4  and mixed with an aliquot of the phage suspension. The infection and titering of the cosmid library were 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)+100 μg/ml ampicillin. After incubation overnight at 37° C., recombinant individual clones were selected.  
       EXAMPLE 9  
     Isolation and Sequencing of the poxB Gene  
       [0131]    The cosmid DNA of an individual colony (Example 8) was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer&#39;s instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-O 2 ). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250). After separation by gel electrophoresis, the cosmid fragments in the size range of 1500 to 2000 bp were isolated with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1, obtained from Invitrogen (Groningen, Holland, Product Description Zero Background Cloning Kit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04).  
         [0132]    The ligation of the cosmid fragments in the sequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), the DNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) into the  E. coli  strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin. The plasmid preparation of the recombinant clones was carried out with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out by the dideoxy chain-stopping method of Sanger et al. (1977, Proceedings of the National Academies of Sciences U.S.A., 74:5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067). The “RR dRhodamin Terminator Cycle Sequencing Kit” from PE Applied Biosystems(Product No. 403044, Weiterstadt, Germany) was used. The separation by gel electrophoresis and analysis of the sequencing reaction were carried out in a “Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE Applied Biosystems (Weiterstadt, Germany).  
         [0133]    The raw sequence data obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) version 97-0. The individual sequences of the pZero1 derivatives were assembled to a continuous contig. The computer-assisted coding region analysis were prepared with the XNIP program (Staden, 1986, Nucleic Acids Research 14:217-231). Further analyses were carried out with the “BLAST search program” (Altschul et al., 1997, Nucleic Acids Research 25:3389-3402), against the non-redundant databank of the “National Center for Biotechnology Information” (NCBI, Bethesda, Md., USA).  
         [0134]    The resulting nucleotide sequence is shown in SEQ ID No. 4. Analysis of the nucleotide sequence showed an open reading frame of 1737 base pairs, which was called the poxB gene. The poxB gene codes for a polypeptide of 579 amino acids (SEQ ID NO. 5).  
       EXAMPLE 10  
     Preparation of an Integration Vector for Integration Mutagenesis of the poxB Gene  
       [0135]    From the strain ATCC 13032, chromosomal DNA was isolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis of the sequence of the poxB gene known for  C. glutamicum  from Example 9, the following oligonucleotides were chosen for the polymerase chain reaction:  
                               poxBint1:               5′ TGC GAG ATG GTG AAT GGT GG 3′   (SEQ ID NO 15)               poxBint2:       5′ GCA TGA GGC AAC GCA TTA GC 3′   (SEQ ID NO 16)          
 
         [0136]    The primers shown were synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction was carried out by the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) with Pwo-Polymerase from Boehringer. With the aid of the polymerase chain reaction, a DNA fragment approx. 0.9 kb in size was isolated, this carrying an internal fragment of the poxB gene and being shown in SEQ ID No:6.  
         [0137]    The amplified DNA fragment was ligated with the TOPO TA Cloning Kit from Invitrogen Corporation (Carlsbad, Calif., USA; Catalogue Number K4500-01) in the vector pCR2.1-TOPO (Mead at al. (1991) Bio/Technology 9:657-663). The  E. coli  Stamm DH5α was then electroporated with the ligation batch (Hanahan, In: DNA cloning. A practical approach. Vol. 1. IRL-Press, Oxford, Washington D.C., USA, 1985). Selection for plasmid-carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 nd  Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 25 mg/l kanamycin. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and subsequent agarose gel electrophoresis (0.8%). The plasmid was called pCR2.1poxBint (FIG. 4).  
         [0138]    Plasmid pCR2.1poxBint has been deposited in the form of the strain  Escherichia coli  DH5α/pCR2.1poxBint as DSM 13114 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.  
       EXAMPLE 11  
     Integration Mutagenesis of the poxB Gene in the Lysine Producer DSM 5715  
       [0139]    The vector pCR2.1poxBint mentioned in Example 10 was electroporated by the electroporation method of Tauch et al.(FEMS Microbiological Letters, 123:343-347 (1994)) in  Corynebacterium glutamicum  DSM 5715. Strain DSM 5715 is an AEC-resistant lysine producer. The vector pCR2.1poxBint cannot replicate independently in DSM5715 and is retained only if it has integrated into the cell&#39;s chromosome. Selection of clones with pCR2.1poxBint integrated into the chromosome was carried out by plating out the electroporation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd  ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which had been supplemented with 15 mg/l kanamycin. For detection of the integration, the poxBint fragment was labeled with the Dig hybridization kit from Boehringer by the method of “The DIG System Users Guide for Filter Hybridization” of Boehringer Mannheim GmbH (Mannheim, Germany, 1993). Chromosomal DNA of a potential integrant was isolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and in each case cleaved with the restriction enzymes SalI, SacI and HindIII. The fragments formed were separated by agarose gel electrophoresis and hybridized at 68° C. with the Dig hybridization kit from Boehringer. The plasmid pCR2.1poxBint mentioned in Example 9 had been inserted into the chromosome of DSM5715 within the chromosomal poxB gene. The strain was called DSM5715::pCR2.1poxBint.  
       EXAMPLE 12  
     Effect of Over-Expression of the gnd Gene with Simultaneous Elimination of the poxB Gene on the Preparation of Lysine  
       [0140]    12.1 Preparation of the Strain DSM5715::pCR2.1poxBint/pECgnd  
         [0141]    The strain DSM5715::pCR2.1poxBint was transformed with the plasmid pECgnd using the electroporation method described by Liebl et al., (FEMS Microbiology Letters, 53:299-303 (1989)). Selection of the transformants took place on LBHIS agar comprising 18.5 μl brain-heart infusion broth, 0.5 M sorbitol, 5 g/l Bacto-tryptone, 2.5 g/l Bacto-yeast extract, 5 g/l NaCl and 18 g/l Bacto-agar, which had been supplemented with 5 mg/l tetracycline and 25 mg/l kanamycin. Incubation was carried out for 2 days at 33° C.  
         [0142]    Plasmid DNA was isolated in each case from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology 144, 915-927), cleaved with the restriction endonuclease AccI, and the plasmid was checked by subsequent agarose gel electrophoresis. The strain obtained in this way was called DSM5715:pCR2.1poxBint/pECgnd.  
         [0143]    12.2 Preparation of L-lysine  
         [0144]    The  C. glutamicum  strain DSM5715::pCR2.1poxBint/pECgnd obtained in Example 12.1 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined. For this, the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with tetracycline (5 mg/l) and kanamycin (25 mg/l)) for 24 hours at 33° C. The cultures of the comparison strains were supplemented according to their resistance to antibiotics. Starting from this agar plate culture, a preculture was seeded (10 ml medium in a 100 ml conical flask). The complete medium CgIII was used as the medium for the preculture.  
                                                 Medium Cg III                                    NaCl   2.5   g/l           Bacto-Peptone   10   g/l           Bacto-Yeast extract   10   g/l           Glucose (autoclaved separately)   2%   (w/v)                                  
 
         [0145]    Tetracycline (5 mg/l) and kanamycin (25 mg/l) were added to this. The preculture was incubated for 16 hours at 33° C. at 240 rpm on a shaking machine. A main culture was seeded from this preculture such that the initial OD (660 nm) of the main culture was 0.1. Medium MM was used for the main culture.  
                                                 Medium MM                                    CSL (corn steep liquor)   5   g/l           MOPS (morpholinopropanesulfonic acid)   20   g/l           Glucose (autoclaved separately)   58   g/l           (NH 4 ) 2 SO 4     25   g/l           KH 2 PO 4     0.1   g/l           MgSO 4  * 7 H 2 O   1.0   g/l           CaCl 2  * 2 H 2 O   10   mg/l           FeSO 4  * 7 H 2 O   10   mg/l           MnSO 4  * H 2 O   5.0   mg/l           Biotin (sterile-filtered)   0.3   mg/l           Thiamine * HCl (sterile-filtered)   0.2   mg/l           L-Leucine (sterile-filtered)   0.1   g/l           CaCO 3     25   g/l                      
 
         [0146]    The CSL, MOPS and the salt solution were brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions were then added, as well as the CaCO 3  autoclaved in the dry state. Culturing was carried out in a 10 ml volume in a 100 ml conical flask with baffles. Tetracycline (5 mg/l) and kanamycin (25 mg/l) were added. Culturing was carried out at 33° C. and 80% atmospheric humidity.  
         [0147]    After 72 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, München). The amount of lysine formed was determined with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection. The result of the experiment is shown in Table 3.  
                                                 TABLE 3                                       OD   L-Lysine HCl           Strain   (660 nm)   g/l                                        DSM5715   10.8   16.0           DSM5715/pECgnd   7.6   16.5           DSM5715::pCR2.1poxBint   7.1   16.7           DSM5715::pCR2.1poxBint/   7.2   17.1           pECgnd                      
 
         [0148]    [0148] 
     
       
       
         1 
         
           
             16  
           
           
             1  
             252  
             DNA  
             Corynebacterium glutamicum  
           
            1 

atggtccaca acggcatcga gtacgccgac atgcaggtca tcggcgaggc ataccacctt     60 

ctgccctacg cagcaggcat gcagccagct gaaatcgctg aggttttcaa ggaatggaac    120 

gcaggcgacc tggattccta cctcatcgaa atcaccgcag aggttctctc ccaggtggat    180 

gctgaaaccg gcaagccact aatcgacgtc atcgttgacg ctgcaggtca gaagggcacc    240 

ggcaagtgga ct                                                        252 

 
           
             2  
             2335  
             DNA  
             Corynebacterium glutamicum  
             
               CDS  
               (474)..(1850)  
               gnd  
             
           
            2 

ttgttcggcc acgatgacac cggagctcac agcagaaatg aagtcggtgt tgttgttgat     60 

gccgacgacc atttttccag gggcggaaat catgctggcg actgatccag tggattcggc    120 

gatggcggcg tagacaccac cgttgaccaa gcccaccact tgcaggtgct tggatgccac    180 

gtgaagttcg ctgaccaccc ggccgggctc gatggtggtg tagcgcagcc ccagattgcg    240 

gtcgaggcca taattggcgt tgttgagtgc ttcaagttcg tctgtggtta aagctctggt    300 

ggcggcaagt tctgcaagcg aaagcagatc ttggggttga tcatcgcggg aagtcataat    360 

taattactct agtcggccta aaatggttgg attttcacct cctgtgacct ggtaaaatcg    420 

ccactacccc caaatggtca caccttttag gccgattttg ctgacaccgg gct atg       476 
                                                           Met 
                                                             1 

ccg tca agt acg atc aat aac atg act aat gga gat aat ctc gca cag      524 
Pro Ser Ser Thr Ile Asn Asn Met Thr Asn Gly Asp Asn Leu Ala Gln 
              5                  10                  15 

atc ggc gtt gta ggc cta gca gta atg ggc tca aac ctc gcc cgc aac      572 
Ile Gly Val Val Gly Leu Ala Val Met Gly Ser Asn Leu Ala Arg Asn 
         20                  25                  30 

ttc gcc cgc aac ggc aac act gtc gct gtc tac aac cgc agc act gac      620 
Phe Ala Arg Asn Gly Asn Thr Val Ala Val Tyr Asn Arg Ser Thr Asp 
     35                  40                  45 

aaa acc gac aag ctc atc gcc gat cac ggc tcc gaa ggc aac ttc atc      668 
Lys Thr Asp Lys Leu Ile Ala Asp His Gly Ser Glu Gly Asn Phe Ile 
 50                  55                  60                  65 

cct tct gca acc gtc gaa gag ttc gta gca tcc ctg gaa aag cca cgc      716 
Pro Ser Ala Thr Val Glu Glu Phe Val Ala Ser Leu Glu Lys Pro Arg 
                 70                  75                  80 

cgc gcc atc atc atg gtt cag gct ggt aac gcc acc gac gca gtc atc      764 
Arg Ala Ile Ile Met Val Gln Ala Gly Asn Ala Thr Asp Ala Val Ile 
             85                  90                  95 

aac cag ctg gca gat gcc atg gac gaa ggc gac atc atc atc gac ggc      812 
Asn Gln Leu Ala Asp Ala Met Asp Glu Gly Asp Ile Ile Ile Asp Gly 
        100                 105                 110 

ggc aac gcc ctc tac acc gac acc att cgt cgc gag aag gaa atc tcc      860 
Gly Asn Ala Leu Tyr Thr Asp Thr Ile Arg Arg Glu Lys Glu Ile Ser 
    115                 120                 125 

gca cgc ggt ctc cac ttc gtc ggt gct ggt atc tcc ggc ggc gaa gaa      908 
Ala Arg Gly Leu His Phe Val Gly Ala Gly Ile Ser Gly Gly Glu Glu 
130                 135                 140                 145 

ggc gca ctc aac ggc cca tcc atc atg cct ggt ggc cca gca aag tcc      956 
Gly Ala Leu Asn Gly Pro Ser Ile Met Pro Gly Gly Pro Ala Lys Ser 
                150                 155                 160 

tac gag tcc ctc gga cca ctg ctt gag tcc atc gct gcc aac gtt gac     1004 
Tyr Glu Ser Leu Gly Pro Leu Leu Glu Ser Ile Ala Ala Asn Val Asp 
            165                 170                 175 

ggc acc cca tgt gtc acc cac atc ggc cca gac ggc gcc ggc cac ttc     1052 
Gly Thr Pro Cys Val Thr His Ile Gly Pro Asp Gly Ala Gly His Phe 
        180                 185                 190 

gtc aag atg gtc cac aac ggc atc gag tac gcc gac atg cag gtc atc     1100 
Val Lys Met Val His Asn Gly Ile Glu Tyr Ala Asp Met Gln Val Ile 
    195                 200                 205 

ggc gag gca tac cac ctt ctg ccc tac gca gca ggc atg cag cca gct     1148 
Gly Glu Ala Tyr His Leu Leu Pro Tyr Ala Ala Gly Met Gln Pro Ala 
210                 215                 220                 225 

gaa atc gct gag gtt ttc aag gaa tgg aac gca ggc gac ctg gat tcc     1196 
Glu Ile Ala Glu Val Phe Lys Glu Trp Asn Ala Gly Asp Leu Asp Ser 
                230                 235                 240 

tac ctc atc gaa atc acc gca gag gtt ctc tcc cag gtg gat gct gaa     1244 
Tyr Leu Ile Glu Ile Thr Ala Glu Val Leu Ser Gln Val Asp Ala Glu 
            245                 250                 255 

acc ggc aag cca cta atc gac gtc atc gtt gac gct gca ggt cag aag     1292 
Thr Gly Lys Pro Leu Ile Asp Val Ile Val Asp Ala Ala Gly Gln Lys 
        260                 265                 270 

ggc acc ggc aag tgg act gtc aag gct gct ctt gat ctg ggt att gct     1340 
Gly Thr Gly Lys Trp Thr Val Lys Ala Ala Leu Asp Leu Gly Ile Ala 
    275                 280                 285 

acc acc ggc atc ggc gaa cgt gtt ttc gca cgt gca ctc tcc ggc gca     1388 
Thr Thr Gly Ile Gly Glu Arg Val Phe Ala Arg Ala Leu Ser Gly Ala 
290                 295                 300                 305 

acc agc cag cgc gct gca gca cag ggc aac cta cct gca ggt gtc ctc     1436 
Thr Ser Gln Arg Ala Ala Ala Gln Gly Asn Leu Pro Ala Gly Val Leu 
                310                 315                 320 

acc gat ctg gaa gca ctt ggc gtg gac aag gca cag ttc gtc gaa gga     1484 
Thr Asp Leu Glu Ala Leu Gly Val Asp Lys Ala Gln Phe Val Glu Gly 
            325                 330                 335 

ctt cgc cgt gca ctg tac gca tcc aag ctt gtt gct tac gca cag ggc     1532 
Leu Arg Arg Ala Leu Tyr Ala Ser Lys Leu Val Ala Tyr Ala Gln Gly 
        340                 345                 350 

ttc gac gag atc aag gct ggc tcc gac gag aac aac tgg gac gtt gac     1580 
Phe Asp Glu Ile Lys Ala Gly Ser Asp Glu Asn Asn Trp Asp Val Asp 
    355                 360                 365 

cct cgc gac ctc gct acc atc tgg cgc ggc ggc tgc atc att cgc gct     1628 
Pro Arg Asp Leu Ala Thr Ile Trp Arg Gly Gly Cys Ile Ile Arg Ala 
370                 375                 380                 385 

aag ttc ctc aac cgc atc gtc gaa gca tac gat gca aac gct gaa ctt     1676 
Lys Phe Leu Asn Arg Ile Val Glu Ala Tyr Asp Ala Asn Ala Glu Leu 
                390                 395                 400 

gag tcc ctg ctg ctc gat cct tac ttc aag agc gag ctc ggc gac ctc     1724 
Glu Ser Leu Leu Leu Asp Pro Tyr Phe Lys Ser Glu Leu Gly Asp Leu 
            405                 410                 415 

atc gat tca tgg cgt cgc gtg att gtc acc gcc acc cag ctt ggc ctg     1772 
Ile Asp Ser Trp Arg Arg Val Ile Val Thr Ala Thr Gln Leu Gly Leu 
        420                 425                 430 

cca atc cca gtg ttc gct tcc tcc ctg tcc tac tac gac agc ctg cgt     1820 
Pro Ile Pro Val Phe Ala Ser Ser Leu Ser Tyr Tyr Asp Ser Leu Arg 
    435                 440                 445 

gca gag cgt ctg cca gca gcc ctg atc cac tagtgtcgac ctgcaggcgc       1870 
Ala Glu Arg Leu Pro Ala Ala Leu Ile His 
450                 455 

gcgagctcca gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcaagg   1930 

tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa tatacgagcc   1990 

ggaagtataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac agtaattgcg   2050 

gctagcggat ctgacggttc actaaaccag ctctgcttat atagacctcc caccgtacac   2110 

gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt   2170 

gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc   2230 

gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa   2290 

tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtccc                   2335 

 
           
             3  
             459  
             PRT  
             Corynebacterium glutamicum  
           
            3 

Met Pro Ser Ser Thr Ile Asn Asn Met Thr Asn Gly Asp Asn Leu Ala 
  1               5                  10                  15 

Gln Ile Gly Val Val Gly Leu Ala Val Met Gly Ser Asn Leu Ala Arg 
             20                  25                  30 

Asn Phe Ala Arg Asn Gly Asn Thr Val Ala Val Tyr Asn Arg Ser Thr 
         35                  40                  45 

Asp Lys Thr Asp Lys Leu Ile Ala Asp His Gly Ser Glu Gly Asn Phe 
     50                  55                  60 

Ile Pro Ser Ala Thr Val Glu Glu Phe Val Ala Ser Leu Glu Lys Pro 
 65                  70                  75                  80 

Arg Arg Ala Ile Ile Met Val Gln Ala Gly Asn Ala Thr Asp Ala Val 
                 85                  90                  95 

Ile Asn Gln Leu Ala Asp Ala Met Asp Glu Gly Asp Ile Ile Ile Asp 
            100                 105                 110 

Gly Gly Asn Ala Leu Tyr Thr Asp Thr Ile Arg Arg Glu Lys Glu Ile 
        115                 120                 125 

Ser Ala Arg Gly Leu His Phe Val Gly Ala Gly Ile Ser Gly Gly Glu 
    130                 135                 140 

Glu Gly Ala Leu Asn Gly Pro Ser Ile Met Pro Gly Gly Pro Ala Lys 
145                 150                 155                 160 

Ser Tyr Glu Ser Leu Gly Pro Leu Leu Glu Ser Ile Ala Ala Asn Val 
                165                 170                 175 

Asp Gly Thr Pro Cys Val Thr His Ile Gly Pro Asp Gly Ala Gly His 
            180                 185                 190 

Phe Val Lys Met Val His Asn Gly Ile Glu Tyr Ala Asp Met Gln Val 
        195                 200                 205 

Ile Gly Glu Ala Tyr His Leu Leu Pro Tyr Ala Ala Gly Met Gln Pro 
    210                 215                 220 

Ala Glu Ile Ala Glu Val Phe Lys Glu Trp Asn Ala Gly Asp Leu Asp 
225                 230                 235                 240 

Ser Tyr Leu Ile Glu Ile Thr Ala Glu Val Leu Ser Gln Val Asp Ala 
                245                 250                 255 

Glu Thr Gly Lys Pro Leu Ile Asp Val Ile Val Asp Ala Ala Gly Gln 
            260                 265                 270 

Lys Gly Thr Gly Lys Trp Thr Val Lys Ala Ala Leu Asp Leu Gly Ile 
        275                 280                 285 

Ala Thr Thr Gly Ile Gly Glu Arg Val Phe Ala Arg Ala Leu Ser Gly 
    290                 295                 300 

Ala Thr Ser Gln Arg Ala Ala Ala Gln Gly Asn Leu Pro Ala Gly Val 
305                 310                 315                 320 

Leu Thr Asp Leu Glu Ala Leu Gly Val Asp Lys Ala Gln Phe Val Glu 
                325                 330                 335 

Gly Leu Arg Arg Ala Leu Tyr Ala Ser Lys Leu Val Ala Tyr Ala Gln 
            340                 345                 350 

Gly Phe Asp Glu Ile Lys Ala Gly Ser Asp Glu Asn Asn Trp Asp Val 
        355                 360                 365 

Asp Pro Arg Asp Leu Ala Thr Ile Trp Arg Gly Gly Cys Ile Ile Arg 
    370                 375                 380 

Ala Lys Phe Leu Asn Arg Ile Val Glu Ala Tyr Asp Ala Asn Ala Glu 
385                 390                 395                 400 

Leu Glu Ser Leu Leu Leu Asp Pro Tyr Phe Lys Ser Glu Leu Gly Asp 
                405                 410                 415 

Leu Ile Asp Ser Trp Arg Arg Val Ile Val Thr Ala Thr Gln Leu Gly 
            420                 425                 430 

Leu Pro Ile Pro Val Phe Ala Ser Ser Leu Ser Tyr Tyr Asp Ser Leu 
        435                 440                 445 

Arg Ala Glu Arg Leu Pro Ala Ala Leu Ile His 
    450                 455 

 
           
             4  
             2160  
             DNA  
             Corynebacterium glutamicum  
             
               CDS  
               (327)..(2063)  
               poxB  
             
           
            4 

ttagaggcga ttctgtgagg tcactttttg tggggtcggg gtctaaattt ggccagtttt     60 

cgaggcgacc agacaggcgt gcccacgatg tttaaatagg cgatcggtgg gcatctgtgt    120 

ttggtttcga cgggctgaaa ccaaaccaga ctgcccagca acgacggaaa tcccaaaagt    180 

gggcatccct gtttggtacc gagtacccac ccgggcctga aactccctgg caggcgggcg    240 

aagcgtggca acaactggaa tttaagagca caattgaagt cgcaccaagt taggcaacac    300 

aatagccata acgttgagga gttcag atg gca cac agc tac gca gaa caa tta     353 
                             Met Ala His Ser Tyr Ala Glu Gln Leu 
                               1               5 

att gac act ttg gaa gct caa ggt gtg aag cga att tat ggt ttg gtg      401 
Ile Asp Thr Leu Glu Ala Gln Gly Val Lys Arg Ile Tyr Gly Leu Val 
 10                  15                  20                  25 

ggt gac agc ctt aat ccg atc gtg gat gct gtc cgc caa tca gat att      449 
Gly Asp Ser Leu Asn Pro Ile Val Asp Ala Val Arg Gln Ser Asp Ile 
                 30                  35                  40 

gag tgg gtg cac gtt cga aat gag gaa gcg gcg gcg ttt gca gcc ggt      497 
Glu Trp Val His Val Arg Asn Glu Glu Ala Ala Ala Phe Ala Ala Gly 
             45                  50                  55 

gcg gaa tcg ttg atc act ggg gag ctg gca gta tgt gct gct tct tgt      545 
Ala Glu Ser Leu Ile Thr Gly Glu Leu Ala Val Cys Ala Ala Ser Cys 
         60                  65                  70 

ggt cct gga aac aca cac ctg att cag ggt ctt tat gat tcg cat cga      593 
Gly Pro Gly Asn Thr His Leu Ile Gln Gly Leu Tyr Asp Ser His Arg 
     75                  80                  85 

aat ggt gcg aag gtg ttg gcc atc gct agc cat att ccg agt gcc cag      641 
Asn Gly Ala Lys Val Leu Ala Ile Ala Ser His Ile Pro Ser Ala Gln 
 90                  95                 100                 105 

att ggt tcg acg ttc ttc cag gaa acg cat ccg gag att ttg ttt aag      689 
Ile Gly Ser Thr Phe Phe Gln Glu Thr His Pro Glu Ile Leu Phe Lys 
                110                 115                 120 

gaa tgc tct ggt tac tgc gag atg gtg aat ggt ggt gag cag ggt gaa      737 
Glu Cys Ser Gly Tyr Cys Glu Met Val Asn Gly Gly Glu Gln Gly Glu 
            125                 130                 135 

cgc att ttg cat cac gcg att cag tcc acc atg gcg ggt aaa ggt gtg      785 
Arg Ile Leu His His Ala Ile Gln Ser Thr Met Ala Gly Lys Gly Val 
        140                 145                 150 

tcg gtg gta gtg att cct ggt gat atc gct aag gaa gac gca ggt gac      833 
Ser Val Val Val Ile Pro Gly Asp Ile Ala Lys Glu Asp Ala Gly Asp 
    155                 160                 165 

ggt act tat tcc aat tcc act att tct tct ggc act cct gtg gtg ttc      881 
Gly Thr Tyr Ser Asn Ser Thr Ile Ser Ser Gly Thr Pro Val Val Phe 
170                 175                 180                 185 

ccg gat cct act gag gct gca gcg ctg gtg gag gcg att aac aac gct      929 
Pro Asp Pro Thr Glu Ala Ala Ala Leu Val Glu Ala Ile Asn Asn Ala 
                190                 195                 200 

aag tct gtc act ttg ttc tgc ggt gcg ggc gtg aag aat gct cgc gcg      977 
Lys Ser Val Thr Leu Phe Cys Gly Ala Gly Val Lys Asn Ala Arg Ala 
            205                 210                 215 

cag gtg ttg gag ttg gcg gag aag att aaa tca ccg atc ggg cat gcg     1025 
Gln Val Leu Glu Leu Ala Glu Lys Ile Lys Ser Pro Ile Gly His Ala 
        220                 225                 230 

ctg ggt ggt aag cag tac atc cag cat gag aat ccg ttt gag gtc ggc     1073 
Leu Gly Gly Lys Gln Tyr Ile Gln His Glu Asn Pro Phe Glu Val Gly 
    235                 240                 245 

atg tct ggc ctg ctt ggt tac ggc gcc tgc gtg gat gcg tcc aat gag     1121 
Met Ser Gly Leu Leu Gly Tyr Gly Ala Cys Val Asp Ala Ser Asn Glu 
250                 255                 260                 265 

gcg gat ctg ctg att cta ttg ggt acg gat ttc cct tat tct gat ttc     1169 
Ala Asp Leu Leu Ile Leu Leu Gly Thr Asp Phe Pro Tyr Ser Asp Phe 
                270                 275                 280 

ctt cct aaa gac aac gtt gcc cag gtg gat atc aac ggt gcg cac att     1217 
Leu Pro Lys Asp Asn Val Ala Gln Val Asp Ile Asn Gly Ala His Ile 
            285                 290                 295 

ggt cga cgt acc acg gtg aag tat ccg gtg acc ggt gat gtt gct gca     1265 
Gly Arg Arg Thr Thr Val Lys Tyr Pro Val Thr Gly Asp Val Ala Ala 
        300                 305                 310 

aca atc gaa aat att ttg cct cat gtg aag gaa aaa aca gat cgt tcc     1313 
Thr Ile Glu Asn Ile Leu Pro His Val Lys Glu Lys Thr Asp Arg Ser 
    315                 320                 325 

ttc ctt gat cgg atg ctc aag gca cac gag cgt aag ttg agc tcg gtg     1361 
Phe Leu Asp Arg Met Leu Lys Ala His Glu Arg Lys Leu Ser Ser Val 
330                 335                 340                 345 

gta gag acg tac aca cat aac gtc gag aag cat gtg cct att cac cct     1409 
Val Glu Thr Tyr Thr His Asn Val Glu Lys His Val Pro Ile His Pro 
                350                 355                 360 

gaa tac gtt gcc tct att ttg aac gag ctg gcg gat aag gat gcg gtg     1457 
Glu Tyr Val Ala Ser Ile Leu Asn Glu Leu Ala Asp Lys Asp Ala Val 
            365                 370                 375 

ttt act gtg gat acc ggc atg tgc aat gtg tgg cat gcg agg tac atc     1505 
Phe Thr Val Asp Thr Gly Met Cys Asn Val Trp His Ala Arg Tyr Ile 
        380                 385                 390 

gag aat ccg gag gga acg cgc gac ttt gtg ggt tca ttc cgc cac ggc     1553 
Glu Asn Pro Glu Gly Thr Arg Asp Phe Val Gly Ser Phe Arg His Gly 
    395                 400                 405 

acg atg gct aat gcg ttg cct cat gcg att ggt gcg caa agt gtt gat     1601 
Thr Met Ala Asn Ala Leu Pro His Ala Ile Gly Ala Gln Ser Val Asp 
410                 415                 420                 425 

cga aac cgc cag gtg atc gcg atg tgt ggc gat ggt ggt ttg ggc atg     1649 
Arg Asn Arg Gln Val Ile Ala Met Cys Gly Asp Gly Gly Leu Gly Met 
                430                 435                 440 

ctg ctg ggt gag ctt ctg acc gtt aag ctg cac caa ctt ccg ctg aag     1697 
Leu Leu Gly Glu Leu Leu Thr Val Lys Leu His Gln Leu Pro Leu Lys 
            445                 450                 455 

gct gtg gtg ttt aac aac agt tct ttg ggc atg gtg aag ttg gag atg     1745 
Ala Val Val Phe Asn Asn Ser Ser Leu Gly Met Val Lys Leu Glu Met 
        460                 465                 470 

ctc gtg gag gga cag cca gaa ttt ggt act gac cat gag gaa gtg aat     1793 
Leu Val Glu Gly Gln Pro Glu Phe Gly Thr Asp His Glu Glu Val Asn 
    475                 480                 485 

ttc gca gag att gcg gcg gct gcg ggt atc aaa tcg gta cgc atc acc     1841 
Phe Ala Glu Ile Ala Ala Ala Ala Gly Ile Lys Ser Val Arg Ile Thr 
490                 495                 500                 505 

gat ccg aag aaa gtt cgc gag cag cta gct gag gca ttg gca tat cct     1889 
Asp Pro Lys Lys Val Arg Glu Gln Leu Ala Glu Ala Leu Ala Tyr Pro 
                510                 515                 520 

gga cct gta ctg atc gat atc gtc acg gat cct aat gcg ctg tcg atc     1937 
Gly Pro Val Leu Ile Asp Ile Val Thr Asp Pro Asn Ala Leu Ser Ile 
            525                 530                 535 

cca cca acc atc acg tgg gaa cag gtc atg gga ttc agc aag gcg gcc     1985 
Pro Pro Thr Ile Thr Trp Glu Gln Val Met Gly Phe Ser Lys Ala Ala 
        540                 545                 550 

acc cga acc gtc ttt ggt gga gga gta gga gcg atg atc gat ctg gcc     2033 
Thr Arg Thr Val Phe Gly Gly Gly Val Gly Ala Met Ile Asp Leu Ala 
    555                 560                 565 

cgt tcg aac ata agg aat att cct act cca tgatgattga tacacctgct       2083 
Arg Ser Asn Ile Arg Asn Ile Pro Thr Pro 
570                 575 

gttctcattg accgcgagcg cttaactgcc aacatttcca ggatggcagc tcacgccggt   2143 

gcccatgaga ttgccct                                                  2160 

 
           
             5  
             579  
             PRT  
             Corynebacterium glutamicum  
           
            5 

Met Ala His Ser Tyr Ala Glu Gln Leu Ile Asp Thr Leu Glu Ala Gln 
  1               5                  10                  15 

Gly Val Lys Arg Ile Tyr Gly Leu Val Gly Asp Ser Leu Asn Pro Ile 
             20                  25                  30 

Val Asp Ala Val Arg Gln Ser Asp Ile Glu Trp Val His Val Arg Asn 
         35                  40                  45 

Glu Glu Ala Ala Ala Phe Ala Ala Gly Ala Glu Ser Leu Ile Thr Gly 
     50                  55                  60 

Glu Leu Ala Val Cys Ala Ala Ser Cys Gly Pro Gly Asn Thr His Leu 
 65                  70                  75                  80 

Ile Gln Gly Leu Tyr Asp Ser His Arg Asn Gly Ala Lys Val Leu Ala 
                 85                  90                  95 

Ile Ala Ser His Ile Pro Ser Ala Gln Ile Gly Ser Thr Phe Phe Gln 
            100                 105                 110 

Glu Thr His Pro Glu Ile Leu Phe Lys Glu Cys Ser Gly Tyr Cys Glu 
        115                 120                 125 

Met Val Asn Gly Gly Glu Gln Gly Glu Arg Ile Leu His His Ala Ile 
    130                 135                 140 

Gln Ser Thr Met Ala Gly Lys Gly Val Ser Val Val Val Ile Pro Gly 
145                 150                 155                 160 

Asp Ile Ala Lys Glu Asp Ala Gly Asp Gly Thr Tyr Ser Asn Ser Thr 
                165                 170                 175 

Ile Ser Ser Gly Thr Pro Val Val Phe Pro Asp Pro Thr Glu Ala Ala 
            180                 185                 190 

Ala Leu Val Glu Ala Ile Asn Asn Ala Lys Ser Val Thr Leu Phe Cys 
        195                 200                 205 

Gly Ala Gly Val Lys Asn Ala Arg Ala Gln Val Leu Glu Leu Ala Glu 
    210                 215                 220 

Lys Ile Lys Ser Pro Ile Gly His Ala Leu Gly Gly Lys Gln Tyr Ile 
225                 230                 235                 240 

Gln His Glu Asn Pro Phe Glu Val Gly Met Ser Gly Leu Leu Gly Tyr 
                245                 250                 255 

Gly Ala Cys Val Asp Ala Ser Asn Glu Ala Asp Leu Leu Ile Leu Leu 
            260                 265                 270 

Gly Thr Asp Phe Pro Tyr Ser Asp Phe Leu Pro Lys Asp Asn Val Ala 
        275                 280                 285 

Gln Val Asp Ile Asn Gly Ala His Ile Gly Arg Arg Thr Thr Val Lys 
    290                 295                 300 

Tyr Pro Val Thr Gly Asp Val Ala Ala Thr Ile Glu Asn Ile Leu Pro 
305                 310                 315                 320 

His Val Lys Glu Lys Thr Asp Arg Ser Phe Leu Asp Arg Met Leu Lys 
                325                 330                 335 

Ala His Glu Arg Lys Leu Ser Ser Val Val Glu Thr Tyr Thr His Asn 
            340                 345                 350 

Val Glu Lys His Val Pro Ile His Pro Glu Tyr Val Ala Ser Ile Leu 
        355                 360                 365 

Asn Glu Leu Ala Asp Lys Asp Ala Val Phe Thr Val Asp Thr Gly Met 
    370                 375                 380 

Cys Asn Val Trp His Ala Arg Tyr Ile Glu Asn Pro Glu Gly Thr Arg 
385                 390                 395                 400 

Asp Phe Val Gly Ser Phe Arg His Gly Thr Met Ala Asn Ala Leu Pro 
                405                 410                 415 

His Ala Ile Gly Ala Gln Ser Val Asp Arg Asn Arg Gln Val Ile Ala 
            420                 425                 430 

Met Cys Gly Asp Gly Gly Leu Gly Met Leu Leu Gly Glu Leu Leu Thr 
        435                 440                 445 

Val Lys Leu His Gln Leu Pro Leu Lys Ala Val Val Phe Asn Asn Ser 
    450                 455                 460 

Ser Leu Gly Met Val Lys Leu Glu Met Leu Val Glu Gly Gln Pro Glu 
465                 470                 475                 480 

Phe Gly Thr Asp His Glu Glu Val Asn Phe Ala Glu Ile Ala Ala Ala 
                485                 490                 495 

Ala Gly Ile Lys Ser Val Arg Ile Thr Asp Pro Lys Lys Val Arg Glu 
            500                 505                 510 

Gln Leu Ala Glu Ala Leu Ala Tyr Pro Gly Pro Val Leu Ile Asp Ile 
        515                 520                 525 

Val Thr Asp Pro Asn Ala Leu Ser Ile Pro Pro Thr Ile Thr Trp Glu 
    530                 535                 540 

Gln Val Met Gly Phe Ser Lys Ala Ala Thr Arg Thr Val Phe Gly Gly 
545                 550                 555                 560 

Gly Val Gly Ala Met Ile Asp Leu Ala Arg Ser Asn Ile Arg Asn Ile 
                565                 570                 575 

Pro Thr Pro 

 
           
             6  
             875  
             DNA  
             Corynebacterium glutamicum  
           
            6 

tgcgagatgg tgaatggtgg tgagcagggt gaacgcattt tgcatcacgc gattcagtcc     60 

accatggcgg gtaaaggtgt gtcggtggta gtgattcctg gtgatatcgc taaggaagac    120 

gcaggtgacg gtacttattc caattccact atttcttctg gcactcctgt ggtgttcccg    180 

gatcctactg aggctgcagc gctggtggag gcgattaaca acgctaagtc tgtcactttg    240 

ttctgcggtg cgggcgtgaa gaatgctcgc gcgcaggtgt tggagttggc ggagaagatt    300 

aaatcaccga tcgggcatgc gctgggtggt aagcagtaca tccagcatga gaatccgttt    360 

gaggtcggca tgtctggcct gcttggttac ggcgcctgcg tggatgcgtc caatgaggcg    420 

gatctgctga ttctattggg tacggatttc ccttattctg atttccttcc taaagacaac    480 

gttgcccagg tggatatcaa cggtgcgcac attggtcgac gtaccacggt gaagtatccg    540 

gtgaccggtg atgttgctgc aacaatcgaa aatattttgc ctcatgtgaa ggaaaaaaca    600 

gatcgttcct tccttgatcg gatgctcaag gcacacgagc gtaagttgag ctcggtggta    660 

gagacgtaca cacataacgt cgagaagcat gtgcctattc accctgaata cgttgcctct    720 

attttgaacg agctggcgga taaggatgcg gtgtttactg tggataccgg catgtgcaat    780 

gtgtggcatg cgaggtacat cgagaatccg gagggaacgc gcgactttgt gggttcattc    840 

cgccacggca cgatggctaa tgcgttgcct catgc                               875 

 
           
             7  
             23  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence Primer gnd1  
             
           
            7 

atggtkcaca cyggyatyga rta                                             23 

 
           
             8  
             21  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence Primer gnd2  
             
           
            8 

rgtccayttr ccrgtrccyt t                                               21 

 
           
             9  
             17  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence Internal 
      primer 1  
             
           
            9 

ggtggatgct gaaaccg                                                    17 

 
           
             10  
             17  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence Internal 
      primer 2  
             
           
            10 

gctgcatgcc tgctgcg                                                    17 

 
           
             11  
             17  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence  Internal 
      primer 3  
             
           
            11 

ttgttgctta cgcacag                                                    17 

 
           
             12  
             17  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence Internal 
      primer 4  
             
           
            12 

tcgtaggact ttgctgg                                                    17 

 
           
             13  
             21  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence gnd fwd. 
      primer  
             
           
            13 

actctagtcg gcctaaaatg g                                               21 

 
           
             14  
             21  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence gnd rev. 
      primer  
             
           
            14 

cacacaggaa acagatatga c                                               21 

 
           
             15  
             20  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence Primer 
      poxBint1  
             
           
            15 

tgcgagatgg tgaatggtgg                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial sequence  
             
               Description of artificial sequence Primer 
      poxBint2  
             
           
            16 

gcatgaggca acgcattagc                                                 20