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 in the culture medium or in the cells of the bacteria, and isolating the L-amino acid produced. The bacteria has an amplified gene encoding the Zwischenferment protein.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This is a continuation-in-part of U.S. application Ser. No. 09/531,269, filed 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 preparation of L-amino acids, in particular L-lysine, L-threonine and L-tryptophan, using coryneform bacteria in which at least the Zwischenferment protein encoded by the zwf gene is amplified.  
         DESCRIPTION OF BACKGROUND ART  
         [0003]    L-Amino acids are used in animal nutrition, in human medicine and in the pharmaceuticals industry. It is known that amino acids are prepared by fermentation of strains of coryneform bacteria, in particular  Corynebacterium glutamicum.  Because of its great importance, work is constantly being undertaken to improve the preparation process. Improvements to the process can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form by e.g. 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, such as e.g. the threonine analogue α-amino-β-hydroxyvaleric acid (AHV), the lysine analogue S-(2-aminoethyl)-L-cystein (AEC), or are auxotrophic for metabolites of regulatory importance and produce L-amino acids such as e.g. threonine or lysine are obtained in this manner.  
           [0005]    Methods of the recombinant DNA technique have also been employed for some years for improving the strain of  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 new improved processes for the preparation of amino acids.  
           [0007]    In general, the embodiments of the present invention provide new improved processes for the fermentative preparation of L-amino acids with coryneform bacteria. More specifically, the embodiments of the invention provide a process for the preparation of L-amino acids, in particular L-lysine, L-threonine, L-isoleucine and L-tryptophan, using coryneform bacteria in which the Zwischenferment protein (Zwf protein) encoded by the nucleotide sequence of the zwf gene is amplified, in particular over-expressed. The abbreviation “zwf” is a mnemonic for “Zwischenferment” (Jeffrey H. Miller: A Short Course In Bacterial Genetics, Cold Spring Harbor Laboratory Press, USA, 1992) and also referred to as glucose 6-phosphate dehydrogenase. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0008]    Embodiments of the invention will be described with reference to the following Figures, in which the base pair numbers stated are approximate values obtained in the context of reproducibility, and in which:  
         [0009]    [0009]FIG. 1 is a map of the plasmid pEC-T18mob2;  
         [0010]    [0010]FIG. 2 is a map of the plasmid pEC-T18mob2zwf;  
         [0011]    [0011]FIG. 3 is a map of the plasmid PAMC1;  
         [0012]    [0012]FIG. 4 is a map of the plasmid pMC1; and  
         [0013]    [0013]FIG. 5 is a map of the plasmid pCR2.1poxBint. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0014]    The strains employed preferably already produce L-amino acids before amplification of the zwf gene. The term “amplification” in this connection describes the increase in the intracellular activity of one or more enzymes or proteins 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 or protein having a high activity, and optionally combining these measures.  
         [0015]    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%, based on that of the wild-type enzyme or protein or the activity or concentration of the enzyme or protein in the starting microorganism.  
         [0016]    The microorganisms which the present invention provides 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, there may be mentioned in particular the species  Corynebacterium glutamicum,  which is known among specialists for its ability to produce L-amino acids.  
         [0017]    Suitable strains of the genus Corynebacterium, in particular of the species  Corynebacterium glutamicum,  are, for example, the known wild-type strains  
         [0018]    [0018] Corynebacterium glutamicum  ATCC13032  
         [0019]    [0019] Corynebacterium acetoglutamicum  ATCC15806  
         [0020]    [0020] Corynebacterium acetoacidophilum  ATCC13870  
         [0021]    [0021] Corynebacterium thermoaminogenes  FERM BP-1539  
         [0022]    [0022] Brevibacterium flavum  ATCC14067  
         [0023]    [0023] Brevibacterium lactofermentum  ATCC13869  
         [0024]    [0024] Brevibacterium divaricatum  ATCC14020  
         [0025]    and L-amino acid-producing mutants prepared therefrom, such as, for example, the L-threonine-producing strains  
         [0026]    [0026] Corynebacterium glutamicum  ATCC21649  
         [0027]    [0027] Brevibacterium flavum  BB69  
         [0028]    [0028] Brevibacterium flavum  DSM5399  
         [0029]    [0029] Brevibacterium lactofermentum  FERM-BP 269  
         [0030]    [0030] Brevibacterium lactofermentum  TBB-10  
         [0031]    and such as, for example, the L-isoleucine-producing strains  
         [0032]    [0032] Corynebacterium glutamicum  ATCC 14309  
         [0033]    [0033] Corynebacterium glutamicum  ATCC 14310  
         [0034]    [0034] Corynebacterium glutamicum  ATCC 14311  
         [0035]    [0035] Corynebacterium glutamicum  ATCC 15168  
         [0036]    [0036] Corynebacterium ammoniagenes  ATCC 6871  
         [0037]    and such as, for example, the L-tryptophan-producing strains  
         [0038]    [0038] Corynebacterium glutamicum  ATCC21850 and  
         [0039]    [0039] Corynebacterium glutamicum  KY9218(pKW9901)  
         [0040]    and such as, for example, the L-lysine-producing strains  
         [0041]    [0041] Corynebacterium glutamicum  FERM-P 1709  
         [0042]    [0042] Brevibacterium flavum  FERM-P 1708  
         [0043]    [0043] Brevibacterium lactofermentum  FERM-P 1712  
         [0044]    [0044] Corynebacterium glutamicum  FERM-P 6463  
         [0045]    [0045] Corynebacterium glutamicum  FERM-P 6464  
         [0046]    [0046] Corynebacterium glutamicum  ATCC13032  
         [0047]    [0047] Corynebacterium glutamicum  DM58-1  
         [0048]    [0048] Corynebacterium glutamicum  DSM12866.  
         [0049]    It has been found that coryneform bacteria produce L-amino acids, in particular L-lysine, L-threonine and L-tryptophan, in an improved manner after over-expression of the zwf gene which codes for the Zwf protein or Zwf polypeptide, respectively.  
         [0050]    JP-A-09224661 discloses the nucleotide sequence of the zwf gene of  Brevibacterium flavum  MJ-223 (FERM BP-1497) and refers to the protein encoded by the zwf-gene as glucose 6-phosphate dehydrogenase. The sequence information disclosed in JP-A-09224661 is shown in SEQ ID NO: 7 and 8. JP-A-09224661 describes the N-terminal amino acid sequence of the Zwf polypeptide as Met Val Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu (SEQ ID NO: 8).  
         [0051]    However, it has not been possible to confirm this. Instead, the following N-terminal amino acid sequence has been found: Met Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp (SEQ ID NO: 10). The nucleotide sequence of the corresponding zwf gene is shown in SEQ ID NO: 9. The methionine residue in the N-position can be split off in the context of post-translational modification, and Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp is then obtained as the N-terminal amino acid sequence.  
         [0052]    Accordingly, embodiments of this invention provide the nucleotide sequence of a novel zwf gene from a coryneform bacterium shown in SEQ ID NO: 9 nucleotides 538 to 2079. Genes encoding Zwf proteins from Gram-negative bacteria e.g.  Escherichia coli  or other Gram-positive bacteria e.g. Streptomyces or Bacillus may optionally be used. Alleles of the zwf gene which result from the degeneracy of the genetic code or due to sense mutations of neutral function can furthermore be used. The use of endogenous genes in particular endogenous genes from coryneform bacteria is preferred. “Endogenous genes” or “endogenous nucleotide sequences” refer to genes or nucleotide sequences which are available in the population of a species.  
         [0053]    To achieve an amplification (e.g., over-expression), the number of copies of the corresponding genes is increased, or the promoter and regulation region or the ribosome binding site upstream of the structural gene is 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 L-amino acid formation. The expression is likewise improved by measures to prolong the life of the mRNA. Furthermore, the enzyme activity is also increased by preventing the degradation of the enzyme protein. The genes or gene constructs are either present here in plasmids with a varying number of copies, or are 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.  
         [0054]    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 Pulhler (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.  
         [0055]    By way of example, the Zwf protein 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 zwf gene into the KpnI/SalI cleavage site of pEC-T18mob2, the plasmid pEC-T18mob2zwf shown in FIG. 2 was formed. Other plasmid vectors which are capable of replication in  C. glutamicum,  such as e.g. pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pZ8-1 (EP-B-0 375 889), can be used in the same way.  
         [0056]    In addition, it may be advantageous for the production of L-amino acids to amplify one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the pentose phosphate pathway or of amino acid export, in addition to amplification of the zwf gene.  
         [0057]    Thus, for example, in particular for the preparation of L-threonine, one or more genes chosen from the group consisting of:  
         [0058]    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),  
         [0059]    the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns et al., Journal of Bacteriology 174: 6076-6086 (1992)),  
         [0060]    the pyc gene which codes for pyruvate carboxylase (Peters-Wendisch et al., Microbiology 144: 915-927 (1998)),  
         [0061]    the mqo gene which codes for malate:quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),  
         [0062]    the tkt gene which codes for transketolase (accession number AB023377 of the European Molecular Biologies Laboratories databank (EMBL, Heidelberg, Germany)),  
         [0063]    the gnd gene which codes for 6-phosphogluconate dehydrogenase (JP-A-9-224662),  
         [0064]    the thrE gene which codes for the threonine export protein (DE 199 41 478.5; DSM 12840),  
         [0065]    the zwal gene (DE 199 59 328.0; DSM 13115),  
         [0066]    the eno gene which codes for enolase (DE: 199 41 478.5)  
         [0067]    can be amplified, in particular over-expressed, at the same time.  
         [0068]    Thus, for example, in particular for the preparation of L-lysine, one or more genes chosen from the group consisting of:  
         [0069]    the dapA gene which codes for dihydrodipicolinate synthase (EP-B 0 197 335),  
         [0070]    the lysC gene which codes for a feed back resistant aspartate kinase (Kalinowski et al. (1990), Molecular and General Genetics 224: 317-324),  
         [0071]    the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),  
         [0072]    the pyc gene which codes for pyruvate carboxylase (DE-A-198 31 609),  
         [0073]    the tkt gene which codes for transketolase (accession number AB023377 of the European Molecular Biologies Laboratories databank (EMBL, Heidelberg, Germany)),  
         [0074]    the gnd gene which codes for 6-phosphogluconate dehydrogenase (JP-A-9-224662),  
         [0075]    the lysE gene which codes for the lysine export protein (DE-A-195 48 222),  
         [0076]    the zwal gene (DE 199 59 328.0; DSM 13115),  
         [0077]    the eno gene which codes for enolase (DE 199 47 791.4)  
         [0078]    can be amplified, in particular over-expressed, at the same time. The use of endogenous genes is preferred.  
         [0079]    It may furthermore be advantageous for the production of L-amino acids at the same time to attenuate one of the genes chosen from the group consisting of  
         [0080]    the pck gene which codes for phosphoenol pyruvate carboxykinase (DE 199 50 409.1; DSM 13047),  
         [0081]    the pgi gene which codes for glucose 6-phosphate isomerase (U.S. patent application Ser. No. 09/396,478, DSM 12969),  
         [0082]    the poxB gene which codes for pyruvate oxidase (DE 199 51 975.7; DSM 13114),  
         [0083]    the zwa2 gene (DE: 199 59 327.2; DSM 13113)  
         [0084]    in addition to the amplification of the zwf gene.  
         [0085]    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, which enzymes or proteins are coded by the corresponding DNA, for example by using a weak promoter or a gene or allele which codes for a corresponding enzyme or protein which has a low activity or inactivates the corresponding enzyme or protein and optionally by combining these measures.  
         [0086]    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 protein or of the activity or concentration of the enzyme or protein in the starting microorganism.  
         [0087]    In addition to over-expression of the Zwf protein, it may furthermore be advantageous for the production of L-amino acids 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).  
         [0088]    The microorganisms prepared according to the invention 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 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)).  
         [0089]    The culture medium to be used should 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 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. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium should 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.  
         [0090]    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 e.g. 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.  
         [0091]    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).  
         [0092]    The following microorganism has been deposited at the Deutsche Sammlung fur 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.  
         [0093]    Referring now more particularly to the Figures, in FIGS. 1 and 2, the abbreviations used have the following meanings:  
                                       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       lacZalpha′:   5′-Terminus of the lacZα gene fragment       ′lacZalpha:   3′-Terminus of the lacZα gene fragment                  
 
         [0094]    In FIGS. 3 and 4, the abbreviations used have the following meanings:  
                                                       Neo r:   Neomycin/kanamycin resistance           ColE1 ori:   Replication origin of the plasmid ColE1           CMV:   Cytomegalovirus promoter           lacP:   Lactose promoter           pgi:   Phosphoglucose isomerase gene           lacZ:   Part of the β-galactosidase gene           SV40 3′ splice   3′ splice site of Simian virus 40           SV40 polyA:   Polyadenylation site of Simian virus 40           f1(−)ori:   Replication origin of the filamentous phage f1           SV40 ori:   Replication origin of Simian virus 40           kan r:   Kanamycin resistance           pgi insert:   Internal fragment of the pgi gene           ori:   Replication origin of the plasmid pBGS8                      
 
         [0095]    In FIG. 5, the abbreviations used have the following meanings:  
                                                       ColE1 ori:   Replication origin of the plasmid ColE1           lacZ:   Cloning relict of the lacZα gene fragment           f1 ori:   Replication origin of phage f1           KmR:   Kanamycin resistance           ApR:   Ampicillin resistance           poxBint:   Internal fragment of the poxB gene.                      
 
         [0096]    The meaning of the abbreviations for the various restriction enzymes (e.g. BamHI, EcoRI etc.)are known from the prior art and are summarized, for example, by Kessler and Holtke (Gene 47, 1-153 (1986)) or Roberts et al. (Nucleic Acids Research 27, 312-313 (1999)).  
         [0097]    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), described by Sambrook et al., (Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratories, USA).  
       EXAMPLE 1  
       [0098]    Expression of the Zwf Protein  
         [0099]    1.1 Preparation of the Plasmid pEC-T18mob2  
         [0100]    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 lacZα 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). The vector constructed was transformed in the  E. coli  strain DH5α (Brown (ed.) Molecular Biology Labfax, BIOS Scientific Publishers, Oxford, UK, 1991). 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.), 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 and subsequent agarose gel electrophoresis (0.8%).  
         [0101]    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 DH5αpEC-T18mob2 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) as DSM 13244.  
         [0102]    1.2 Preparation of the Plasmid pEC-T18mob2zwf  
         [0103]    The gene from  Corynebacterium glutamicum  ATCC13032 was first amplified by a polymerase chain reaction (PCR) by means of the following oligonucleotide primer:  
                               zwf-forward:               5′-TCG ACG CGG TTC TGG AGC AG-3′   (SEQ ID NO:11)               zwf-reverse:       5′-CTA AAT TAT GGC CTG CGC CAG-3′.   (SEQ ID NO:12)          
 
         [0104]    The PCR reaction was carried out in 30 cycles in the presence of 200 μM deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), in each case 1 μM of the corresponding oligonucleotide, 100 ng chromosomal DNA from  Corynebacterium glutamicum  ATCC13032, 1/10 volume 10-fold reaction buffer and 2.6 units of a heat-stable Taq-/Pwo-DNA polymerase mixture (Expand High Fidelity PCR System from Roche Diagnostics, Mannheim, Germany) in a Thermocycler (PTC-100, MJ Research, Inc., Watertown, USA) under the following conditions: 94° C. for 30 seconds, 64° C. for 1 minute and 68° C. for 3 minutes.  
         [0105]    The amplified fragment about 1.8 kb in size was subsequently ligated with the aid of the SureClone Ligation Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) into the SmaI cleavage site of the vector pUC18 in accordance with the manufacturer&#39;s instructions. The  E. coli  strain DH5αmcr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America USA (1990) 87: 4645-4649) was transformed with the entire ligation batch. Transformants were identified with the aid of their carbenicillin resistance on LB-agar plates containing 50 μg/mL carbenicillin. The plasmids were prepared from 7 of the transformants and checked for the presence of the 1.8 kb PCR fragment as an insert by restriction analysis. The recombinant plasmid formed in this way is called pUC18zwf in the following.  
         [0106]    For construction of pEC-T18mob2zwf, pUC18zwf was digested with KpnI and SalI, and the product was isolated with the aid of the NucleoSpin Extraction Kit from Macherey-Nagel (Düren, Germany) in accordance with the manufacturer&#39;s instructions and then ligated with the vector pEC-T18mob2, which had also been cleaved with KpnI and SalI and dephosphorylated. The  E. coli  strain DH5αmcr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America USA (1990) 87: 4645-4649) was transformed with the entire ligation batch. Transformants were identified with the aid of their tetracycline resistance on LB-agar plates containing 5 μg/mL tetracycline. The plasmids were prepared from 12 of the transformants and checked for the presence of the 1.8 kb PCR fragment as an insert by restriction analysis. One of the recombinant plasmids isolated in this manner was called pEC-T18mob2zwf (FIG. 2).  
       EXAMPLE 2  
       [0107]    Preparation of Amino Acid Producers with an Amplified zwf Gene  
         [0108]    The L-lysine-producing strain  Corynebacterium glutamicum  DSM5715 is described in EP-B-0435132 and the L-threonine-producing strain  Brevibacterium flavum  DSM5399 is described in EP-B-0385940. Both strains are deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen [German Collection of Microorganisms and Cell Cultures] in Braunschweig (Germany) in accordance with the Budapest Treaty.  
         [0109]    2.1 Preparation of the Strains DSM5715/pEC-T18mob2zwf and DSM5399/pEC-T18mob2zwf  
         [0110]    The strains DSM5715 and DSM5399 were transformed with the plasmid pEC-T18mob2zwf 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 g/l 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. Incubation was carried out for 2 days at 33° C.  
         [0111]    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 endonucleases XbaI and KpnI, and the plasmid was checked by subsequent agarose gel electrophoresis. The strains obtained in this way were called DSM5715/pEC-T18mob2zwf and DSM5399/pEC-T18mob2zwf.  
         [0112]    2.2 Preparation of L-Threonine  
         [0113]    The  C. glutamicum  strain DSM5399/pEC-T18mob2zwf obtained in Example 2.1 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). 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)           The pH was brought to pH 7.4                      
 
         [0114]    Tetracycline (5 mg/l) was 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)   50   g/l           (NH 4 ) 2 SO 4     25   g/l           KH 2 PO 4     0.1   g/l           MgSO 4  * 7H 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                      
 
         [0115]    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 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.  
         [0116]    After 72 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount 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   12.3   0.74           DSM5399/pEC-T18mob2zwf   10.2   1.0                       
 
         [0117]    2.3 Preparation of L-Lysine  
         [0118]    The  C. glutamicum  strain DSM5715/pEC-T18mob2zwf obtained in Example 2.1 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)) 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)           The pH was brought to pH 7.4                      
 
         [0119]    Tetracycline (5 mg/l) was 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  * 2H 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                      
 
         [0120]    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 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 72 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). 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   L-Lysine HCl           Strain   (660 nm)   g/l                           DSM5715   10.8   16.0           DSM5715/pEC-T18mob2zwf    7.2   17.1                      
 
       EXAMPLE 3  
       [0121]    Construction of a Gene Library of  Corynebacterium glutamicum  Strain AS019  
         [0122]    A DNA library of  Corynebacterium glutamicum  strain ASO19 (Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985)) was constructed using λ Zap Expres™ 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 manufacturers instructions. AS019-DNA was digested with restriction enzyme Sau3A and ligated to BamHI treated and dephosphorylated λ Zap Express™ arms.  
       EXAMPLE 4  
       [0123]    Cloning and Sequencing of the pgi Gene  
         [0124]    1. Cloning  
         [0125]    [0125] Escherichia coli  strain DF1311, carrying mutations in the pgi and pgl genes as described by Kupor and Fraenkel, (Journal of Bacteriology 100: 1296-1301 (1969)), was transformed with approx. 500 ng of the AS019 λ Zap Express™ plasmid library described in Example 3. Selection for transformants was made on M9 minimal media, (Sambrook et al., (1989). Molecular Cloning. A Laboratory Manual Cold Spring Harbor Laboratories, USA), containing kanamycin at a concentration of 50 mg/l and incubation at 37° C. for 48 hours. Plasmid DNA was isolated from one transformant according to Birnboim and Doly (Nucleic Acids Research 7: 1513-1523 (1979)) and designated pAMC1 (FIG. 3).  
         [0126]    2. Sequencing  
         [0127]    For sequence analysis of the cloned insert of pAMC1 the method of Sanger et al. (Proceedings of the National Academy of Sciences USA 74,5463-5467 (1977)) was applied using primers differentially labeled with a colored fluorescent tag. It was carried out using the ABI prism 310 genetic analyzer from Perkin Elmer Applied Biosystems, (Perkin Elmer Corporation, Norwalk, Conn., U.S.A), and the ABI prism Big Dye™ Terminator Cycle Sequencing Ready Reaction kit also from Perkin Elmer.  
         [0128]    Initial sequence analysis was carried out using the universal forward and M13 reverse primers obtained from Pharmacia Biotech (St. Albans, Herts, AL1 3AW, UK):  
                                   Universal forward primer:   GTA ATA CGA CTC ACT ATA GGG C   (SEQ ID NO:13)                   M13 reverse primer:   GGA AAC AGC TAT GAC CAT G.   (SEQ ID NO:14)          
 
         [0129]    Internal primers were subsequently designed from the sequence obtained which allowed the entire pgi gene to be deduced. The sequence of the internal primers is as follows:  
                                           Internal primer 1:                   GGA AAC AGG GGA GCC GTC   (SEQ ID NO:15)                       Internal primer 2:           TGC TGA GAT ACC AGC GGT.   (SEQ ID NO:16)          
 
         [0130]    The sequence obtained was then 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 sequence obtained and those in EMBL and Genbank databases were achieved 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).  
         [0131]    The sequence thus obtained is shown in SEQ ID NO: 1. The analysis of the nucleotide sequence obtained revealed an open reading frame of 1650 base pairs which was designated as pgi gene. It codes for a protein of 550 amino acids shown in SEQ ID NO: 2.  
       EXAMPLE 5  
       [0132]    Preparation of an Integration Vector for Integration Mutagenesis of the pgi Gene  
         [0133]    An internal segment of the pgi gene was amplified by polymerase chain reaction (PCR) using genomic DNA isolated from  Corynebacterium glutamicum  AS019, (Heery and Dunican, (1993) Applied and Environmental Microbiology 59: 791-799), as template. The pgi primers used were:  
                                   fwd.   ATG GAR WCC AAY GGH AA   (SEQ ID NO:17)           Primer:               rev.   YTC CAC GCC CCA YTG RTC   (SEQ ID NO:18)       Primer:          
 
         [0134]    with R=A+G; Y=C+T; W=A+T; H=A+T+C.  
                                                                 PCR Parameters were as follows:    35 cycles                94° C. for 1 min.                47° C. for 1 min.                72° C. for 30 sec.               1.5 mM MgCl 2                  approx. 150-200 ng DNA template.                      
 
         [0135]    The PCR product obtained was cloned into the commercially available pGEM-T vector received from Promega Corp., (Promega UK, Southampton.) using strain  E. coli  JM109, (Yanisch-Perron et al., 1985. Gene, 33: 103-119), as a host. The sequence of the PCR product is shown as SEQ ID NO: 3. The cloned insert was then excised as an EcoRI fragment and ligated to plasmid pBGS8 (Spratt et al., Gene 41: 337-342 (1986)) pretreated with EcoRI. The restriction enzymes used were obtained from Boehringer Mannheim UK Ltd., (Bell Lane, Lewes East Sussex BN7 1LG, UK.) and used according to manufacturers instructions.  E. coli  JM109 was then transformed with this ligation mixture and electrotransformants were selected on Luria agar supplemented with IPTG (isopropyl-β-D-thiogalactopyranoside), XGAL (5-bromo-4-chloro-3-indolyl-D-galactopyranoside) and kanamycin at a concentration of 1 mM, 0.02% and 50 mg/l respectively. Agar plates were incubated for twelve hours at 37° C. Plasmid DNA was isolated from one transformant, characterized by restriction enzyme analysis using EcoRI, BamHI and SalI designated pMC1 (FIG. 4).  
         [0136]    Plasmid pMC1 was deposited in the form of  Escherichia coli  strain DH5α/pMC1 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) as DSM 12969 according to the Budapest treaty.  
       EXAMPLE 6  
       [0137]    Integration Mutagenesis of the pgi Gene in the Lysine Producer DSM 5715  
         [0138]    The vector pMC1 mentioned in Example 5 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 pMC1 cannot replicate independently in DSM5715 and is retained in the cell only if it has integrated into the chromosome of DSM 5715. Selection of clones with pMC1 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 internal pgi fragment (Example 5) was labeled with the Dig hybridization kit from Boehringer Mannheim by the method of “The DIG System Users Guide for Filter Hybridization” of Boehringer Mannheim GmbH (Mannheim, Germany, 1993). Chromosomal DNA of a transformant 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. It was found in this way that the plasmid pMC1 was inserted within the chromosomal pgi gene of strain DSM5715. The strain was called DSM5715::pMC1.  
       EXAMPLE 7  
       [0139]    Effect of Over-Expression of the zwf Gene with Simultaneous Elimination of the pgi Gene on the Preparation of Lysine  
         [0140]    7.1 Preparation of the Strain DSM5715::pMC1/pEC-T18mob2zwf  
         [0141]    The vector pEC-T18mob2zwf mentioned in Example 1.2 was electroporated by the electroporation method of Tauch et al. (1994, FEMS Microbiological Letters, 123:343-347) in  Corynebacterium glutamicum  DSM 5715::pMC1. Selection for plasmid-carrying cells was made 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 15 mg/l kanamycin and with 5 mg/l tetracycline. Plasmid DNA was isolated from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology 144, 915-927) and checked by treatment with the restriction enzymes KpnI and SalI with subsequent agarose gel electrophoresis. The strain was called DSM5715::pMC1/pEC-T18mob2zwf.  
         [0142]    7.2 Preparation of Lysine  
         [0143]    The  C. glutamicum  strain DSM5715::pMC1/pEC-T18mob2zwf obtained in Example 7.1 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 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)           The pH was brought to pH 7.4                      
 
         [0144]    Tetracycline (5 mg/l) and kanamycin (5 mg/l) was 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)   50   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                      
 
         [0145]    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 is 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.  
         [0146]    After 72 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). 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 3.  
                                                 TABLE 3                                       OD   L-Lysine HCl           Strain   (660 nm)   g/l                                        DSM5715   7.3   14.3           DSM5715/pEC-T18mob2zwf   7.1   14.6           DSM5715::pMC1/   10.4   15.2           pECTmob2zwf                      
 
       EXAMPLE 8  
       [0147]    Preparation of a Genomic Cosmid Gene Library from  Corynebacterium glutamicum  ATCC 13032  
         [0148]    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. 27-0913-02). 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. 27-0948-02) and likewise dephosphorylated with shrimp alkaline phosphatase. 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 Res. 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  
       [0149]    Isolation and Sequencing of the poxB Gene  
         [0150]    The cosmid DNA of an individual colony (Example 7) was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer&#39; 3 s instructions and partly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche 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). 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).  
         [0151]    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).  
         [0152]    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  
       [0153]    Preparation of an Integration Vector for Integration Mutagenesis of the poxB Gene  
         [0154]    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 8, the following oligonucleotides were chosen for the polymerase chain reaction:  
                               poxBint1:               5′ TGC GAG ATG GTG AAT GGT GG 3′   (SEQ ID NO:19)               poxBint2:       5′ GCA TGA GGC AAC GCA TTA GC 3′   (SEQ ID NO:20)          
 
         [0155]    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.  
         [0156]    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  strain DH5α: was then electroporated with the ligation batch (Hanahan, In: DNA cloning. A Practical Approach. Vol. I, IRL-Press, Oxford, Washington DC, USA, 1985). Selection for plasmid-carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 nd  Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 25 mg/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. 5).  
         [0157]    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  
       [0158]    Integration Mutagenesis of the poxB Gene in the Lysine Producer DSM 5715  
         [0159]    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 in the cell only if it has integrated into the chromosome of DSM 5715. 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  
       [0160]    Effect of Over-Expression of the zwf Gene with Simultaneous Elimination of the poxB Gene on the Preparation of Lysine  
         [0161]    12.1 Preparation of the Strain DSM5715::pCR2.1poxBint/pEC-T18mob2zwf  
         [0162]    The strain DSM5715::pCR2.1poxBint was transformed with the plasmid pEC-T18mob2zwf 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 g/l 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.  
         [0163]    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 endonucleases XbaI and KpnI, and the plasmid was checked by subsequent agarose gel electrophoresis. The strain obtained in this way was called DSM5715:pCR2.1poxBint/pEC-T18mob2zwf.  
         [0164]    12.2 Preparation of L-Lysine  
         [0165]    The C. glutamicum strain DSM5715::pCR2.1poxBint/pEC-T18mob2zwf 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/i)) for 24 hours at 33° C. 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 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)           The pH was brought to pH 7.4                      
 
         [0166]    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                      
 
         [0167]    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 is 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.  
         [0168]    After 72 hours, the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). 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 4.  
                                                 TABLE 4                                       OD   L-Lysine HCl           Strain   (660 nm)   g/l                                        DSM5715   10.8   16.0           DSM5715/pEC-T18mob2zwf   8.3   17.1           DSM5715::pCR2.1poxBint   7.1   16.7           DSM5715::pCR2.1poxBint/   7.8   17.7           pEC-Tmob2zwf                      
 
         [0169]    [0169] 
     
       
       
         1 
         
           
             20  
           
           
             1  
             2811  
             DNA  
             Corynebacterium glutamicum  
             
               CDS  
               (373)..(2022)  
               pgi  
             
           
            1 

aaaacccgag gggcgaaaat tccaccctaa cttttttggg atcccctttt tccggggaat     60 

taattggttt gggtttcaat gggaaaacgg gaaacaatgg gccaaaggtt caaaaacccc    120 

aaaagggggc cgggttcaaa ttcccaaaaa aaatggcaaa aaaggggggg ccaaaaccaa    180 

gttggccccc aaaccaccgg ggcaacggcc cacccacaaa ggggttgggt taaaggaagg    240 

acgcccaaag taagcccgga atggcccacg ttcgaaaaag caggccccaa ttaaacgcac    300 

cttaaatttg tcgtgtttcc cactttgaac actcttcgat gcgcttggcc acaaaagcaa    360 

gctaacctga ag atg tta ttt aac gac aat aaa gga gtt ttc atg gcg gac    411 
              Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp 
                1               5                  10 

att tcg acc acc cag gtt tgg caa gac ctg acc gat cat tac tca aac      459 
Ile Ser Thr Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr Ser Asn 
     15                  20                  25 

ttc cag gca acc act ctg cgt gaa ctt ttc aag gaa gaa aac cgc gcc      507 
Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala 
 30                  35                  40                  45 

gag aag tac acc ttc tcc gcg gct ggc ctc cac gtc gac ctg tcg aag      555 
Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys 
                 50                  55                  60 

aat ctg ctt gac gac gcc acc ctc acc aag ctc ctt gca ctg acc gaa      603 
Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu 
             65                  70                  75 

gaa tct ggc ctt cgc gaa cgc att gac gcg atg ttt gcc ggt gaa cac      651 
Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His 
         80                  85                  90 

ctc aac aac acc gaa gac cgc gct gtc ctc cac acc gcg ctg cgc ctt      699 
Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu 
     95                 100                 105 

cct gcc gaa gct gat ctg tca gta gat ggc caa gat gtt gct gct gat      747 
Pro Ala Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp 
110                 115                 120                 125 

gtc cac gaa gtt ttg gga cgc atg cgt gac ttc gct act gcg ctg cgc      795 
Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg 
                130                 135                 140 

tca ggc aac tgg ttg gga cac acc ggc cac acg atc aag aag atc gtc      843 
Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val 
            145                 150                 155 

aac att ggt atc ggt ggc tct gac ctc gga cca gcc atg gct acg aag      891 
Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys 
        160                 165                 170 

gct ctg cgt gca tac gcg acc gct ggt atc tca gca gaa ttc gtc tcc      939 
Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser 
    175                 180                 185 

aac gtc gac cca gca gac ctc gtt tct gtg ttg gaa gac ctc gat gca      987 
Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala 
190                 195                 200                 205 

gaa tcc aca ttg ttc gtg atc gct tcg aaa act ttc acc acc cag gag     1035 
Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu 
                210                 215                 220 

acg ctg tcc aac gct cgt gca gct cgt gct tgg ctg gta gag aag ctc     1083 
Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu 
            225                 230                 235 

ggt gaa gag gct gtc gcg aag cac ttc gtc gca gtg tcc acc aat gct     1131 
Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala 
        240                 245                 250 

gaa aag gtc gca gag ttc ggt atc gac acg gac aac atg ttc ggc ttc     1179 
Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe 
    255                 260                 265 

tgg gac tgg gtc gga ggt cgt tac tcc gtg gac tcc gca gtt ggt ctt     1227 
Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu 
270                 275                 280                 285 

tcc ctc atg gca gtg atc ggc cct cgc gac ttc atg cgt ttc ctc ggt     1275 
Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly 
                290                 295                 300 

gga ttc cac gcg atg gat gaa cac ttc cgc acc acc aag ttc gaa gag     1323 
Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu 
            305                 310                 315 

aac gtt cca atc ttg atg gct ctg ctc ggt gtc tgg tac tcc gat ttc     1371 
Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe 
        320                 325                 330 

tat ggt gca gaa acc cac gct gtc cta cct tat tcc gag gat ctc agc     1419 
Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser 
    335                 340                 345 

cgt ttt gct gct tac ctc cag cag ctg acc atg gag acc aat ggc aag     1467 
Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Thr Asn Gly Lys 
350                 355                 360                 365 

tca gtc cac cgc gac ggc tcc cct gtt tcc act ggc act ggc gaa att     1515 
Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile 
                370                 375                 380 

tac tgg ggt gag cct ggc aca aat ggc cag cac gct ttc ttc cag ctg     1563 
Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu 
            385                 390                 395 

atc cac cag ggc act cgc ctt gtt cca gct gat ttc att ggt ttc gct     1611 
Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala 
        400                 405                 410 

cgt cca aag cag gat ctt cct gcc ggt gag cgc acc atg cat gac ctt     1659 
Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu 
    415                 420                 425 

ttg atg agc aac ttc ttc gca cag acc aag gtt ttg gct ttc ggt aag     1707 
Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys 
430                 435                 440                 445 

aac gct gaa gag atc gct gcg gaa ggt gtc gca cct gag ctg gtc aac     1755 
Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn 
                450                 455                 460 

cac aag gtc gtg cca ggt aat cgc cca acc acc acc att ttg gcg gag     1803 
His Lys Val Val Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu 
            465                 470                 475 

gaa ctt acc cct tct att ctc ggt gcg ttg atc gct ttg tac gaa cac     1851 
Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His 
        480                 485                 490 

acc gtg atg gtt cag ggc gtg att tgg gac atc aac tcc ttc gac caa     1899 
Thr Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln 
    495                 500                 505 

tgg ggt gtt gaa ctg ggc aaa cag cag gca aat gac ctc gct ccg gct     1947 
Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala 
510                 515                 520                 525 

gtc tct ggt gaa gag gat gtt gac tcg gga gat tct tcc act gat tca     1995 
Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser 
                530                 535                 540 

ctg att aag tgg tac cgc gca aat agg tagtcgcttg cttatagggt           2042 
Leu Ile Lys Trp Tyr Arg Ala Asn Arg 
            545                 550 

caggggcgtg aagaatcctc gcctcatagc actggccgct atcatcctga cctcgttcaa   2102 

tctgcgaaca gctattactg ctttagctcc gctggtttct gagattcggg atgatttagg   2162 

ggttagtgct tctcttattg gtgtgttggg catgatcccg actgctatgt tcgcggttgc   2222 

tgcgtttgcg cttccgtcgt tgaagaggaa gttcactact tcccaactgt tgatgtttgc   2282 

catgctgttg actgctgccg gtcagattat tcgtgtcgct ggacctgctt cgctgttgat   2342 

ggtcggtact gtgttcgcga tgtttgcgat cggagttacc aatgtgttgc ttccgattgc   2402 

tgttagggag tattttccgc gtcacgtcgg tggaatgtcg acaacttatc tggtgtcgtt   2462 

ccagattgtt caggcacttg ctccgacgct tgccgtgccg atttctcagt gggctacaca   2522 

tgtggggttg accggttgga gggtgtcgct cggttcgtgg gcgctgctgg ggttggttgc   2582 

ggcgatttcg tggattccgc tgttgagttt gcagggtgcc agggttgttg cggcgccgtc   2642 

gaaggtttct cttcctgtgt ggaagtcttc ggttggtgtg gggctcgggt tgatgtttgg   2702 

gtttacttcg tttgcgacgt atatcctcat gggttttatg ccgcagatgg taggtgatcc   2762 

aaagaattca aaaagcttct cgagagtact tctagagcgg ccgcgggcc               2811 

 
           
             2  
             550  
             PRT  
             Corynebacterium glutamicum  
           
            2 

Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp Ile Ser Thr 
  1               5                  10                  15 

Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr Ser Asn Phe Gln Ala 
             20                  25                  30 

Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala Glu Lys Tyr 
         35                  40                  45 

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

Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu Glu Ser Gly 
 65                  70                  75                  80 

Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His Leu Asn Asn 
                 85                  90                  95 

Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu Pro Ala Glu 
            100                 105                 110 

Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp Val His Glu 
        115                 120                 125 

Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg Ser Gly Asn 
    130                 135                 140 

Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val Asn Ile Gly 
145                 150                 155                 160 

Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys Ala Leu Arg 
                165                 170                 175 

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

Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala Glu Ser Thr 
        195                 200                 205 

Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu Thr Leu Ser 
    210                 215                 220 

Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu Gly Glu Glu 
225                 230                 235                 240 

Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala Glu Lys Val 
                245                 250                 255 

Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe Trp Asp Trp 
            260                 265                 270 

Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu Ser Leu Met 
        275                 280                 285 

Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly Gly Phe His 
    290                 295                 300 

Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu Asn Val Pro 
305                 310                 315                 320 

Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe Tyr Gly Ala 
                325                 330                 335 

Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser Arg Phe Ala 
            340                 345                 350 

Ala Tyr Leu Gln Gln Leu Thr Met Glu Thr Asn Gly Lys Ser Val His 
        355                 360                 365 

Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile Tyr Trp Gly 
    370                 375                 380 

Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu Ile His Gln 
385                 390                 395                 400 

Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala Arg Pro Lys 
                405                 410                 415 

Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu Leu Met Ser 
            420                 425                 430 

Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys Asn Ala Glu 
        435                 440                 445 

Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn His Lys Val 
    450                 455                 460 

Val Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu Glu Leu Thr 
465                 470                 475                 480 

Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His Thr Val Met 
                485                 490                 495 

Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln Trp Gly Val 
            500                 505                 510 

Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala Val Ser Gly 
        515                 520                 525 

Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser Leu Ile Lys 
    530                 535                 540 

Trp Tyr Arg Ala Asn Arg 
545                 550 

 
           
             3  
             462  
             DNA  
             Corynebacterium glutamicum  
           
            3 

atggagacca atggcaagtc agtccaccgc gacggctccc ctgtttccac tggcactggc     60 

gaaatttact ggggtgagcc tggcacaaat ggccagcacg ctttcttcca gctgatccac    120 

cagggcactc gccttgttcc agctgatttc attggtttcg ctcgtccaaa gcaggatctt    180 

cctgccggtg agcgcaccat gcatgacctt ttgatgagca acttcttcgc acagaccaag    240 

gttttggctt tcggtaagaa cgctgaagag atcgctgcgg aaggtgtcgc acctgagctg    300 

gtcaaccaca aggtcgtgcc aggtaatcgc ccaaccacca ccattttggc ggaggaactt    360 

accccttcta ttctcggtgc gttgatcgct ttgtacgaac acaccgtgat ggttcagggc    420 

gtgatttggg acatcaactc cttcgaccaa tggggcgtgg aa                       462 

 
           
             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  
             2260  
             DNA  
             Brevibacterium flavum MJ-233  
             
               CDS  
               (629)..(2080)  
               Glucose-6-Phosphate Dehydrogenase 
      (EC 1.1.1.49); JP-A-09-22461  
             
           
            7 

gatccgatga ggctttggct ctgcgtggca aggcaggcgt tgccaacgct cagcgcgctt     60 

acgctgtgta caaggagctt ttcgacgccg ccgagctgcc tgtaaggcgc caacactcag    120 

cgcccactgt gggcatccac cggcgtgaag aaccctgcgt acgctgcaac tctttacgtt    180 

tccgagctgg ctggtccaaa caccgtcaac accatgccag aaggcaccat cgacgctgtt    240 

ctggaactgg gcaacctgca cggtgacaac ctgtccaact ccgcggcaga agctgacgct    300 

gtgttctccc agcttgaggc tctgggcgtt gacttggcag atgtcttcca ggtcctggag    360 

accgaggccg tggacaagtt cgttgcttct tggagcgaac tgcttgagtc catggaagct    420 

cgcctgaagt agaatcagca cgctgcatca gtaacggcga catgaaatcg aattagttcg    480 

atcttatgtg gccgttacac atctttcatt aaagaaagga tcgtgacgct taccatcgtg    540 

agcacaaaac acgaccccct ccagctggac aaacccactg cgcgacccgc aggataaacg    600 

actcccccgc atcgctggcc cttccggc atg gtg atc ttc ggt gtc act ggc       652 
                               Met Val Ile Phe Gly Val Thr Gly 
                                 1               5 

gac ttg gct cga aag aag ctg ctc ccc gcc att tat gat cta gca aac      700 
Asp Leu Ala Arg Lys Lys Leu Leu Pro Ala Ile Tyr Asp Leu Ala Asn 
     10                  15                  20 

cgc gga ttg ctg ccc cca gga ttc tcg ttg gta ggt tac ggc cgc cgc      748 
Arg Gly Leu Leu Pro Pro Gly Phe Ser Leu Val Gly Tyr Gly Arg Arg 
 25                  30                  35                  40 

gaa tgg tcc aaa gaa gac ttt gaa aaa tac gta cgc gat gcc gca agt      796 
Glu Trp Ser Lys Glu Asp Phe Glu Lys Tyr Val Arg Asp Ala Ala Ser 
                 45                  50                  55 

gct ggt gct cgt acg gaa ttc cgt gaa aat gtt tgg gag cgc ctc gcc      844 
Ala Gly Ala Arg Thr Glu Phe Arg Glu Asn Val Trp Glu Arg Leu Ala 
             60                  65                  70 

gag ggt atg gaa ttt gtt cgc ggc aac ttt gat gat gat gca gct ttc      892 
Glu Gly Met Glu Phe Val Arg Gly Asn Phe Asp Asp Asp Ala Ala Phe 
         75                  80                  85 

gac aac ctc gct gca aca ctc aag cgc atc gac aaa acc cgc ggc acc      940 
Asp Asn Leu Ala Ala Thr Leu Lys Arg Ile Asp Lys Thr Arg Gly Thr 
     90                  95                 100 

gcc ggc aac tgg gct tac tac ctg tcc att cca cca gat tcc ttc gca      988 
Ala Gly Asn Trp Ala Tyr Tyr Leu Ser Ile Pro Pro Asp Ser Phe Ala 
105                 110                 115                 120 

gcg gtc tgc cac cag ctg gag cgt tcc ggc atg gct gaa tcc acc gaa     1036 
Ala Val Cys His Gln Leu Glu Arg Ser Gly Met Ala Glu Ser Thr Glu 
                125                 130                 135 

gaa gca tgg cgc cgc gtg atc atc gag aag cct ttc ggc cac aac ctc     1084 
Glu Ala Trp Arg Arg Val Ile Ile Glu Lys Pro Phe Gly His Asn Leu 
            140                 145                 150 

gaa tcc gca cac gag ctc aac cag ctg gtc aac gca gtc ttc cca gaa     1132 
Glu Ser Ala His Glu Leu Asn Gln Leu Val Asn Ala Val Phe Pro Glu 
        155                 160                 165 

tct tct gtg ttc cgc atc gac cac tat ttg ggc aag gaa aca gtt caa     1180 
Ser Ser Val Phe Arg Ile Asp His Tyr Leu Gly Lys Glu Thr Val Gln 
    170                 175                 180 

aac atc ctg gct ctg cgt ttt gct aac cag ctg ttt gag cca ctg tgg     1228 
Asn Ile Leu Ala Leu Arg Phe Ala Asn Gln Leu Phe Glu Pro Leu Trp 
185                 190                 195                 200 

aac tcc aac tac gtt gac cac gtc cag atc acc atg gct gaa gat att     1276 
Asn Ser Asn Tyr Val Asp His Val Gln Ile Thr Met Ala Glu Asp Ile 
                205                 210                 215 

ggc ttg ggt gga cgt gct ggt tac tac gac ggc atc ggc gca gcc cgc     1324 
Gly Leu Gly Gly Arg Ala Gly Tyr Tyr Asp Gly Ile Gly Ala Ala Arg 
            220                 225                 230 

gac gtc atc cag aac cac ctg atc cag ctc ttg gct ctg gtt gcc atg     1372 
Asp Val Ile Gln Asn His Leu Ile Gln Leu Leu Ala Leu Val Ala Met 
        235                 240                 245 

gaa gaa cca att tct ttc gtg cca gcg cag ctg cag gca gaa aag atc     1420 
Glu Glu Pro Ile Ser Phe Val Pro Ala Gln Leu Gln Ala Glu Lys Ile 
    250                 255                 260 

aag gtg ctc tct gcg aca aag ccg tgc tac cca ttg gat aaa acc tcc     1468 
Lys Val Leu Ser Ala Thr Lys Pro Cys Tyr Pro Leu Asp Lys Thr Ser 
265                 270                 275                 280 

gct cgt ggt cag tac gct gcc ggt tgg cag ggc tct gag tta gtc aag     1516 
Ala Arg Gly Gln Tyr Ala Ala Gly Trp Gln Gly Ser Glu Leu Val Lys 
                285                 290                 295 

gga ctt cgc gaa gaa gat ggc ttc aac cct gag tcc acc act gag act     1564 
Gly Leu Arg Glu Glu Asp Gly Phe Asn Pro Glu Ser Thr Thr Glu Thr 
            300                 305                 310 

ttt gcg gct tgt acc tta gag atc acg tct cgt cgc tgg gct ggt gtg     1612 
Phe Ala Ala Cys Thr Leu Glu Ile Thr Ser Arg Arg Trp Ala Gly Val 
        315                 320                 325 

ccg ttc tac ctg cgc acc ggt aag cgt ctt ggt cgc cgt gtt act gag     1660 
Pro Phe Tyr Leu Arg Thr Gly Lys Arg Leu Gly Arg Arg Val Thr Glu 
    330                 335                 340 

att gcc gtg gtg ttt aaa gac gca cca cac cag cct ttc gac ggc gac     1708 
Ile Ala Val Val Phe Lys Asp Ala Pro His Gln Pro Phe Asp Gly Asp 
345                 350                 355                 360 

atg act gta tcc ctt ggc caa aac gcc atc gtg att cgc gtg cag cct     1756 
Met Thr Val Ser Leu Gly Gln Asn Ala Ile Val Ile Arg Val Gln Pro 
                365                 370                 375 

gat gaa ggt gtg ctc atc cgc ttc ggt tcc aag gtt cca ggt tct gcc     1804 
Asp Glu Gly Val Leu Ile Arg Phe Gly Ser Lys Val Pro Gly Ser Ala 
            380                 385                 390 

atg gaa gtc cgt gac gtc aac atg gac ttc tcc tac tca gaa tcc ttc     1852 
Met Glu Val Arg Asp Val Asn Met Asp Phe Ser Tyr Ser Glu Ser Phe 
        395                 400                 405 

act gaa gaa tca cct gaa gca tac gag cgc ctt atc ttg gat gcg ctg     1900 
Thr Glu Glu Ser Pro Glu Ala Tyr Glu Arg Leu Ile Leu Asp Ala Leu 
    410                 415                 420 

ttg gat gaa tcc agc ctt ttc cct acc aac gag gaa gtg gaa ctg agc     1948 
Leu Asp Glu Ser Ser Leu Phe Pro Thr Asn Glu Glu Val Glu Leu Ser 
425                 430                 435                 440 

tgg aag att ctg gat cca att ctt gaa gca tgg gat gcc gat gga gaa     1996 
Trp Lys Ile Leu Asp Pro Ile Leu Glu Ala Trp Asp Ala Asp Gly Glu 
                445                 450                 455 

cca gag gat tac cca gca ggt acg tgg ggt cca aag agc gct gat gaa     2044 
Pro Glu Asp Tyr Pro Ala Gly Thr Trp Gly Pro Lys Ser Ala Asp Glu 
            460                 465                 470 

atg ctt tcc cgc aac ggt cac acc tgg cgc agg cca taatttaggg          2090 
Met Leu Ser Arg Asn Gly His Thr Trp Arg Arg Pro 
        475                 480 

gcaaaaaatg atctttgaac ttccggatac caccacccag caaatttcca agaccctaac   2150 

tcgactgcgt gaatcgggca cccaggtcac caccggccga gtgctcaccc tcatcgtggt   2210 

cactgactcc gaaagcgatg tcgctgcagt taccgagtcc accaatgaag              2260 

 
           
             8  
             484  
             PRT  
             Brevibacterium flavum MJ-233  
           
            8 

Met Val Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu 
  1               5                  10                  15 

Pro Ala Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe 
             20                  25                  30 

Ser Leu Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu 
         35                  40                  45 

Lys Tyr Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg 
     50                  55                  60 

Glu Asn Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly 
 65                  70                  75                  80 

Asn Phe Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys 
                 85                  90                  95 

Arg Ile Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu 
            100                 105                 110 

Ser Ile Pro Pro Asp Ser Phe Ala Ala Val Cys His Gln Leu Glu Arg 
        115                 120                 125 

Ser Gly Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val Ile Ile 
    130                 135                 140 

Glu Lys Pro Phe Gly His Asn Leu Glu Ser Ala His Glu Leu Asn Gln 
145                 150                 155                 160 

Leu Val Asn Ala Val Phe Pro Glu Ser Ser Val Phe Arg Ile Asp His 
                165                 170                 175 

Tyr Leu Gly Lys Glu Thr Val Gln Asn Ile Leu Ala Leu Arg Phe Ala 
            180                 185                 190 

Asn Gln Leu Phe Glu Pro Leu Trp Asn Ser Asn Tyr Val Asp His Val 
        195                 200                 205 

Gln Ile Thr Met Ala Glu Asp Ile Gly Leu Gly Gly Arg Ala Gly Tyr 
    210                 215                 220 

Tyr Asp Gly Ile Gly Ala Ala Arg Asp Val Ile Gln Asn His Leu Ile 
225                 230                 235                 240 

Gln Leu Leu Ala Leu Val Ala Met Glu Glu Pro Ile Ser Phe Val Pro 
                245                 250                 255 

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

Cys Tyr Pro Leu Asp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly 
        275                 280                 285 

Trp Gln Gly Ser Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe 
    290                 295                 300 

Asn Pro Glu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile 
305                 310                 315                 320 

Thr Ser Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys 
                325                 330                 335 

Arg Leu Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala 
            340                 345                 350 

Pro His Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn 
        355                 360                 365 

Ala Ile Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile Arg Phe 
    370                 375                 380 

Gly Ser Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp Val Asn Met 
385                 390                 395                 400 

Asp Phe Ser Tyr Ser Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr 
                405                 410                 415 

Glu Arg Leu Ile Leu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro 
            420                 425                 430 

Thr Asn Glu Glu Val Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu 
        435                 440                 445 

Glu Ala Trp Asp Ala Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr 
    450                 455                 460 

Trp Gly Pro Lys Ser Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr 
465                 470                 475                 480 

Trp Arg Arg Pro 

 
           
             9  
             2259  
             DNA  
             Corynebacterium glutamicum  
             
               CDS  
               (538)..(2079)  
               Zwf-Protein  
             
           
            9 

gatccgatga ggctttggct ctgcgtggca aggcaggcgt tgccaacgct cagcgcgctt     60 

acgctgtgta caaggagctt ttcgacgccg ccgagctgcc tgtaaggcgc caacactcag    120 

cgcccactgt gggcatccac cggcgtgaag aaccctgcgt acgctgcaac tctttacgtt    180 

tccgagctgg ctggtccaaa caccgtcaac accatgccag aaggcaccat cgacgctgtt    240 

ctggaactgg gcaacctgca cggtgacaac ctgtccaact ccgcggcaga agctgacgct    300 

gtgttctccc agcttgaggc tctgggcgtt gacttggcag atgtcttcca ggtcctggag    360 

accgaggccg tggacaagtt cgttgcttct tggagcgaac tgcttgagtc catggaagct    420 

cgcctgaagt agaatcagca cgctgcatca gtaacggcga catgaaatcg aattagttcg    480 

atcttatgtg gccgttacac atctttcatt aaagaaagga tcgtgacgct taccatc       537 

gtg agc aca aac acg acc ccc tcc agc tgg aca aac cca ctg cgc gac      585 
Met Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp 
  1               5                  10                  15 

ccg cag gat aaa cga ctc ccc cgc atc gct ggc cct tcc ggc atg gtg      633 
Pro Gln Asp Lys Arg Leu Pro Arg Ile Ala Gly Pro Ser Gly Met Val 
             20                  25                  30 

atc ttc ggt gtc act ggc gac ttg gct cga aag aag ctg ctc ccc gcc      681 
Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu Pro Ala 
         35                  40                  45 

att tat gat cta gca aac cgc gga ttg ctg ccc cca gga ttc tcg ttg      729 
Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe Ser Leu 
     50                  55                  60 

gta ggt tac ggc cgc cgc gaa tgg tcc aaa gaa gac ttt gaa aaa tac      777 
Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu Lys Tyr 
 65                  70                  75                  80 

gta cgc gat gcc gca agt gct ggt gct cgt acg gaa ttc cgt gaa aat      825 
Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg Glu Asn 
                 85                  90                  95 

gtt tgg gag cgc ctc gcc gag ggt atg gaa ttt gtt cgc ggc aac ttt      873 
Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly Asn Phe 
            100                 105                 110 

gat gat gat gca gct ttc gac aac ctc gct gca aca ctc aag cgc atc      921 
Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys Arg Ile 
        115                 120                 125 

gac aaa acc cgc ggc acc gcc ggc aac tgg gct tac tac ctg tcc att      969 
Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu Ser Ile 
    130                 135                 140 

cca cca gat tcc ttc gca gcg gtc tgc cac cag ctg gag cgt tcc ggc     1017 
Pro Pro Asp Ser Phe Ala Ala Val Cys His Gln Leu Glu Arg Ser Gly 
145                 150                 155                 160 

atg gct gaa tcc acc gaa gaa gca tgg cgc cgc gtg atc atc gag aag     1065 
Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val Ile Ile Glu Lys 
                165                 170                 175 

cct ttc ggc cac aac ctc gaa tcc gca cac gag ctc aac cag ctg gtc     1113 
Pro Phe Gly His Asn Leu Glu Ser Ala His Glu Leu Asn Gln Leu Val 
            180                 185                 190 

aac gca gtc ttc cca gaa tct tct gtg ttc cgc atc gac cac tat ttg     1161 
Asn Ala Val Phe Pro Glu Ser Ser Val Phe Arg Ile Asp His Tyr Leu 
        195                 200                 205 

ggc aag gaa aca gtt caa aac atc ctg gct ctg cgt ttt gct aac cag     1209 
Gly Lys Glu Thr Val Gln Asn Ile Leu Ala Leu Arg Phe Ala Asn Gln 
    210                 215                 220 

ctg ttt gag cca ctg tgg aac tcc aac tac gtt gac cac gtc cag atc     1257 
Leu Phe Glu Pro Leu Trp Asn Ser Asn Tyr Val Asp His Val Gln Ile 
225                 230                 235                 240 

acc atg gct gaa gat att ggc ttg ggt gga cgt gct ggt tac tac gac     1305 
Thr Met Ala Glu Asp Ile Gly Leu Gly Gly Arg Ala Gly Tyr Tyr Asp 
                245                 250                 255 

ggc atc ggc gca gcc cgc gac gtc atc cag aac cac ctg atc cag ctc     1353 
Gly Ile Gly Ala Ala Arg Asp Val Ile Gln Asn His Leu Ile Gln Leu 
            260                 265                 270 

ttg gct ctg gtt gcc atg gaa gaa cca att tct ttc gtg cca gcg cag     1401 
Leu Ala Leu Val Ala Met Glu Glu Pro Ile Ser Phe Val Pro Ala Gln 
        275                 280                 285 

ctg cag gca gaa aag atc aag gtg ctc tct gcg aca aag ccg tgc tac     1449 
Leu Gln Ala Glu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro Cys Tyr 
    290                 295                 300 

cca ttg gat aaa acc tcc gct cgt ggt cag tac gct gcc ggt tgg cag     1497 
Pro Leu Asp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly Trp Gln 
305                 310                 315                 320 

ggc tct gag tta gtc aag gga ctt cgc gaa gaa gat ggc ttc aac cct     1545 
Gly Ser Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe Asn Pro 
                325                 330                 335 

gag tcc acc act gag act ttt gcg gct tgt acc tta gag atc acg tct     1593 
Glu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile Thr Ser 
            340                 345                 350 

cgt cgc tgg gct ggt gtg ccg ttc tac ctg cgc acc ggt aag cgt ctt     1641 
Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys Arg Leu 
        355                 360                 365 

ggt cgc cgt gtt act gag att gcc gtg gtg ttt aaa gac gca cca cac     1689 
Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala Pro His 
    370                 375                 380 

cag cct ttc gac ggc gac atg act gta tcc ctt ggc caa aac gcc atc     1737 
Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn Ala Ile 
385                 390                 395                 400 

gtg att cgc gtg cag cct gat gaa ggt gtg ctc atc cgc ttc ggt tcc     1785 
Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile Arg Phe Gly Ser 
                405                 410                 415 

aag gtt cca ggt tct gcc atg gaa gtc cgt gac gtc aac atg gac ttc     1833 
Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp Val Asn Met Asp Phe 
            420                 425                 430 

tcc tac tca gaa tcc ttc act gaa gaa tca cct gaa gca tac gag cgc     1881 
Ser Tyr Ser Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr Glu Arg 
        435                 440                 445 

ctt atc ttg gat gcg ctg ttg gat gaa tcc agc ctt ttc cct acc aac     1929 
Leu Ile Leu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro Thr Asn 
    450                 455                 460 

gag gaa gtg gaa ctg agc tgg aag att ctg gat cca att ctt gaa gca     1977 
Glu Glu Val Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu Glu Ala 
465                 470                 475                 480 

tgg gat gcc gat gga gaa cca gag gat tac cca gca ggt acg tgg ggt     2025 
Trp Asp Ala Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr Trp Gly 
                485                 490                 495 

cca aag agc gct gat gaa atg ctt tcc cgc aac ggt cac acc tgg cgc     2073 
Pro Lys Ser Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr Trp Arg 
            500                 505                 510 

agg cca taatttaggg gcaaaaaatg atctttgaac ttccggatac caccacccag      2129 
Arg Pro 

caaatttcca agaccctaac tcgactgcgt gaatcgggca cccaggtcac caccggccga   2189 

gtgctcaccc tcatcgtggt cactgactcc gaaagcgatg tcgctgcagt taccgagtcc   2249 

accaatgaag                                                          2259 

 
           
             10  
             514  
             PRT  
             Corynebacterium glutamicum  
           
            10 

Met Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp 
  1               5                  10                  15 

Pro Gln Asp Lys Arg Leu Pro Arg Ile Ala Gly Pro Ser Gly Met Val 
             20                  25                  30 

Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu Leu Pro Ala 
         35                  40                  45 

Ile Tyr Asp Leu Ala Asn Arg Gly Leu Leu Pro Pro Gly Phe Ser Leu 
     50                  55                  60 

Val Gly Tyr Gly Arg Arg Glu Trp Ser Lys Glu Asp Phe Glu Lys Tyr 
 65                  70                  75                  80 

Val Arg Asp Ala Ala Ser Ala Gly Ala Arg Thr Glu Phe Arg Glu Asn 
                 85                  90                  95 

Val Trp Glu Arg Leu Ala Glu Gly Met Glu Phe Val Arg Gly Asn Phe 
            100                 105                 110 

Asp Asp Asp Ala Ala Phe Asp Asn Leu Ala Ala Thr Leu Lys Arg Ile 
        115                 120                 125 

Asp Lys Thr Arg Gly Thr Ala Gly Asn Trp Ala Tyr Tyr Leu Ser Ile 
    130                 135                 140 

Pro Pro Asp Ser Phe Ala Ala Val Cys His Gln Leu Glu Arg Ser Gly 
145                 150                 155                 160 

Met Ala Glu Ser Thr Glu Glu Ala Trp Arg Arg Val Ile Ile Glu Lys 
                165                 170                 175 

Pro Phe Gly His Asn Leu Glu Ser Ala His Glu Leu Asn Gln Leu Val 
            180                 185                 190 

Asn Ala Val Phe Pro Glu Ser Ser Val Phe Arg Ile Asp His Tyr Leu 
        195                 200                 205 

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

Leu Phe Glu Pro Leu Trp Asn Ser Asn Tyr Val Asp His Val Gln Ile 
225                 230                 235                 240 

Thr Met Ala Glu Asp Ile Gly Leu Gly Gly Arg Ala Gly Tyr Tyr Asp 
                245                 250                 255 

Gly Ile Gly Ala Ala Arg Asp Val Ile Gln Asn His Leu Ile Gln Leu 
            260                 265                 270 

Leu Ala Leu Val Ala Met Glu Glu Pro Ile Ser Phe Val Pro Ala Gln 
        275                 280                 285 

Leu Gln Ala Glu Lys Ile Lys Val Leu Ser Ala Thr Lys Pro Cys Tyr 
    290                 295                 300 

Pro Leu Asp Lys Thr Ser Ala Arg Gly Gln Tyr Ala Ala Gly Trp Gln 
305                 310                 315                 320 

Gly Ser Glu Leu Val Lys Gly Leu Arg Glu Glu Asp Gly Phe Asn Pro 
                325                 330                 335 

Glu Ser Thr Thr Glu Thr Phe Ala Ala Cys Thr Leu Glu Ile Thr Ser 
            340                 345                 350 

Arg Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly Lys Arg Leu 
        355                 360                 365 

Gly Arg Arg Val Thr Glu Ile Ala Val Val Phe Lys Asp Ala Pro His 
    370                 375                 380 

Gln Pro Phe Asp Gly Asp Met Thr Val Ser Leu Gly Gln Asn Ala Ile 
385                 390                 395                 400 

Val Ile Arg Val Gln Pro Asp Glu Gly Val Leu Ile Arg Phe Gly Ser 
                405                 410                 415 

Lys Val Pro Gly Ser Ala Met Glu Val Arg Asp Val Asn Met Asp Phe 
            420                 425                 430 

Ser Tyr Ser Glu Ser Phe Thr Glu Glu Ser Pro Glu Ala Tyr Glu Arg 
        435                 440                 445 

Leu Ile Leu Asp Ala Leu Leu Asp Glu Ser Ser Leu Phe Pro Thr Asn 
    450                 455                 460 

Glu Glu Val Glu Leu Ser Trp Lys Ile Leu Asp Pro Ile Leu Glu Ala 
465                 470                 475                 480 

Trp Asp Ala Asp Gly Glu Pro Glu Asp Tyr Pro Ala Gly Thr Trp Gly 
                485                 490                 495 

Pro Lys Ser Ala Asp Glu Met Leu Ser Arg Asn Gly His Thr Trp Arg 
            500                 505                 510 

Arg Pro 

 
           
             11  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer 
      zwf-forward  
             
           
            11 

tcgacgcggt tctggagcag                                                 20 

 
           
             12  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer 
      zwf-reverse  
             
           
            12 

ctaaattatg gcctgcgcca g                                               21 

 
           
             13  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Universal 
      forward Primer  
             
           
            13 

gtaatacgac tcactatagg gc                                              22 

 
           
             14  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence M13 reverse 
      primer  
             
           
            14 

ytccacgccc caytgrtc                                                   18 

 
           
             15  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Internal 
      Primer 1  
             
           
            15 

ggaaacaggg gagccgtc                                                   18 

 
           
             16  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Internal 
      Primer 2  
             
           
            16 

tgctgagata ccagcggt                                                   18 

 
           
             17  
             17  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence fwd primer  
             
           
            17 

atggarwcca aygghaa                                                    17 

 
           
             18  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence rev. primer  
             
           
            18 

ytccacgccc caytgrtc                                                   18 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer 
      poxBint1  
             
           
            19 

tgcgagatgg tgaatggtgg                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer 
      poxBint2  
             
           
            20 

gcatgaggca acgcattagc                                                 20