Abstract:
The L-lysine-producing ability and the L-lysine-producing speed are improved in a coryneform bacterium harboring an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, by successively enhancing DNA coding for a dihydrodipicolinate reductase, DNA coding for a dihydrodipicolinate synthase, DNA coding for a diaminopimelate decarboxylase, and DNA coding for a diaminopimelate dehydrogenase.

Description:
TECHNICAL FIELD  
         [0001]    The present invention relates to a method for producing L-lysine by cultivating a microorganism obtained by modifying a coryneform bacterium used for fermentative production of amino acid or the like by means of a technique based on genetic engineering.  
         BACKGROUND ART  
         [0002]    L-Lysine, which is used as a fodder additive, is usually produced by a fermentative method by using an L-lysine-producing mutant strain belonging to the coryneform bacteria. Various L-lysine-producing bacteria known at present are those created by artificial mutation starting from wild type strains belonging to the coryneform bacteria.  
           [0003]    As for the coryneform bacteria, there are disclosed a vector plasmid which is autonomously replicable in bacterial cells and has a drug resistance marker gene (see U.S. Pat. No. 4,514,502), and a method for introducing a gene into bacterial cells (for example, Japanese Patent Laid-open No. 2-207791). There is also disclosed a possibility for breeding an L-threonine- or L-isoleucine-producing bacterium by using the techniques as described above (see U.S. Pat. Nos. 4,452,890 and 4,442,208). As for breeding of an L-lysine-producing bacterium, a technique is known, in which a gene participating in L-lysine biosynthesis is incorporated into a vector plasmid to amplify the gene in bacterial cells (for example, Japanese Patent Laid-open No. 56-160997).  
           [0004]    Known genes for L-lysine biosynthesis include, for example, a dihydrodipicolinate reductase gene (Japanese Patent Laid-open No. 7-75578) and a diaminopimelate dehydrogenase gene (Ishino, S. et al.,  Nucleic Acids Res.,  15, 3917 (1987)) in which a gene participating in L-lysine biosynthesis is cloned, as well as a phosphoenolpyruvate carboxylase gene (Japanese Patent Laid-open No. 60-87788), a dihydrodipicolinate synthase gene (Japanese Patent Publication No. 6-55149), and a diaminopimelate decarboxylase gene (Japanese Patent Laid-open No. 60-62994) in which amplification of a gene affects L-lysine productivity.  
           [0005]    As for enzymes participating in L-lysine biosynthesis, a case is known for an enzyme which undergoes feedback inhibition when used as a wild type. In this case, L-lysine productivity is improved by introducing an enzyme gene having such mutation that the feedback inhibition is desensitized. Those known as such a gene specifically include, for example, an aspartokinase gene (International Publication Pamphlet of WO 94/25605).  
           [0006]    As described above, certain successful results have been obtained by means of amplification of genes for the L-lysine biosynthesis system, or introduction of mutant genes. For example, a coryneform bacterium, which harbors a mutant aspartokinase gene with desensitized concerted inhibition by lysine and threonine, produces a considerable amount of L-lysine (about 25 g/L). However, this bacterium suffers decrease in growth speed as compared with a bacterium harboring no mutant aspartokinase gene. It is also reported that L-lysine productivity is improved by further introducing a dihydrodipicolinate synthase gene in addition to a mutant aspartokinase gene ( Applied and Environmental Microbiology,  57(6), 1746-1752 (1991)). However, such a bacterium suffers further decrease in growth speed.  
           [0007]    As for the dihydrodipicolinate reductase gene, it has been demonstrated that the activity of dihydrodipicolinate reductase is increased in a coryneform bacterium into which the gene has been introduced, however, no report is included for the influence on L-lysine productivity (Japanese Patent Laid-open No. 7-75578).  
           [0008]    In the present circumstances, no case is known for the coryneform bacteria, in which anyone has succeeded in remarkable improvement in L-lysine yield without restraining growth by combining a plurality of genes for L-lysine biosynthesis. No case has been reported in which growth is intended to be improved by enhancing a gene for L-lysine biosynthesis as well.  
         DISCLOSURE OF THE INVENTION  
         [0009]    An object of the present invention is to improve the L-lysine-producing ability and the growth speed of a coryneform bacterium by using genetic materials of DNA sequences each coding for aspartokinase (hereinafter referred to as “AK”, provided that a gene coding for an AK protein is hereinafter referred to as “lysC”, if necessary), dihydrodipicolinate reductase (hereinafter referred to as “DDPR”, provided that a gene coding for a DDPR protein is hereinafter referred to as “dapB”, if necessary), dihydrodipicolinate synthase (hereinafter abbreviate as “DDPS”, provided that a gene coding for a DDPS protein is hereinafter referred to as “dapA”, if necessary), diaminopimelate decarboxylase (hereinafter referred to as “DDC”, provided that a gene coding for a DDC protein is hereinafter referred to as “lysA”, if necessary), and diaminopimelate dehydrogenase (hereinafter referred to as “DDH”, provided that a gene coding for a DDH protein is hereinafter referred to as “ddh”, if necessary) which are important enzymes for L-lysine biosynthesis in cells of coryneform bacteria.  
           [0010]    When an objective substance is produced fermentatively by using a microorganism, the production speed, as well as the yield of the objective substance relative to an introduced material, is an extremely important factor. An objective substance may be produced remarkably inexpensively by increasing the production speed per a unit of fermentation equipment. Accordingly, it is industrially extremely important that the fermentative yield and the production speed are compatible with each other. The present invention proposes a solution for the problem as described above in order to fermentatively produce L-lysine by using a coryneform bacterium.  
           [0011]    The principle of the present invention is based on the fact that the growth of a coryneform bacterium can be improved, and the L-lysine-producing speed thereof can be improved by making enhancement while combining dapB with mutant lysC (hereinafter simply referred to as “mutant lysC”, if necessary) coding for mutant AK (hereinafter simply referred to as “mutant type AK”, if necessary) in which concerted inhibition by lysine and threonine is desensitized, as compared with a case in which lysC is enhanced singly, and that the L-lysine-producing speed can be further improved in a stepwise manner by successively enhancing dapA, lysA, and ddh.  
           [0012]    Namely, the present invention lies in a recombinant DNA autonomously replicable in cells of coryneform bacteria, comprising a DNA sequence coding for an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and a DNA sequence coding for a dihydrodipicolinate reductase. The present invention provides a recombinant DNA further comprising a DNA sequence coding for a dihydrodipicolinate synthase, in addition to each of the DNA sequences described above. The present invention provides a recombinant DNA further comprising a DNA sequence coding for a diaminopimelate decarboxylase, in addition to the three DNA sequences described above. The present invention provides a recombinant DNA further comprising a DNA sequence coding for a diaminopimelate dehydrogenase, in addition to the four DNA sequences described above.  
           [0013]    In another aspect, the present invention provides a coryneform bacterium harboring an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and comprising enhanced DNA coding for a dihydrodipicolinate reductase. The present invention provides a coryneform bacterium further comprising enhanced DNA coding for a dihydrodipicolinate synthase in the aforementioned coryneform bacterium. The present invention provides a coryneform bacterium further comprising enhanced DNA coding for a diaminopimelate decarboxylase in the aforementioned coryneform bacterium, in addition to the three DNA&#39;s described above. The present invention provides a coryneform bacterium further comprising enhanced DNA coding for a diaminopimelate dehydrogenase in the aforementioned coryneform bacterium, in addition to the four DNA&#39;s described above.  
           [0014]    In still another aspect, the present invention provides a method for producing L-lysine comprising the steps of cultivating any one of the coryneform bacteria described above in an appropriate medium, producing and accumulating L-lysine in a culture of the bacterium, and collecting L-lysine from the culture.  
           [0015]    The coryneform bacteria referred to in the present invention are a group of microorganisms as defined in  Bergey&#39;s Manual of Determinative Bacteriology,  8th ed., p. 599 (1974), which are aerobic Gram-positive rods having no acid resistance and no spore-forming ability. The coryneform bacteria include bacteria belonging to the genus  Corynebacterium,  bacteria belonging to the genus  Brevibacterium  having been hitherto classified into the genus  Brevibacterium  but united as bacteria belonging to the genus  Corynebacterium  at present, and bacteria belonging to the genus  Brevibacterium  closely relative to bacteria belonging to the genus  Corynebacterium.    
           [0016]    The present invention will be explained in detail below.  
           [0017]    &lt;1&gt; Preparation of Genes for L-lysine Biosynthesis Used for the Present Invention  
           [0018]    The genes for L-lysine biosynthesis used in the present invention are obtained respectively by preparing chromosomal DNA from a bacterium as a DNA donor, constructing a chromosomal DNA library by using a plasmid vector or the like, selecting a strain harboring a desired gene, and recovering, from the selected strain, recombinant DNA into which the gene has been inserted. The DNA donor for the gene for L-lysine biosynthesis used in the present invention is not specifically limited provided that the desired gene for L-lysine biosynthesis expresses an enzyme protein which functions in cells of coryneform bacteria. However, the DNA donor is preferably a coryneform bacterium.  
           [0019]    All of the genes of lysC, dapA, and dapB originating from coryneform bacteria have known sequences. Accordingly, they can be obtained by performing amplification in accordance with the polymerase chain reaction method (PCR; see White, T. J. et al.,  Trends Genet.,  5, 185 (1989)).  
           [0020]    Each of the genes for L-lysine biosynthesis used in the present invention is obtainable in accordance with certain methods as exemplified below.  
           [0021]    (1) Preparation of Mutant lysC  
           [0022]    A DNA fragment containing mutant lysC can be prepared from a mutant strain in which synergistic feedback inhibition on the AK activity by L-lysine and L-threonine is substantially desensitized (International Publication Pamphlet of WO 94/25605). Such a mutant strain can be obtained, for example, from a group of cells originating from a wild type strain of a coryneform bacterium subjected to a mutation treatment by applying an ordinary mutation treatment such as ultraviolet irradiation and treatment with a mutating agent such as N-methyl-N′-nitro-N-nitrosoguanidine. The AK activity can be measured by using a method described by Miyajima, R. et al. in  The Journal of Biochemistry  (1968), 63(2), 139-148. The most preferred as such a mutant strain is represented by an L-lysine-producing bacterium AJ3445 (FERM P-1944) derived by a mutation treatment from a wild type strain of  Brevibacterium lactofermentum  ATCC 13869 (having its changed present name of  Corynebacterium glutamicum ).  
           [0023]    Alternatively, mutant lysC is also obtainable by an in vitro mutation treatment of plasmid DNA containing wild type lysC. In another aspect, information is specifically known on mutation to desensitize synergistic feedback inhibition on AK by L-lysine and L-threonine (International Publication Pamphlet of WO 94/25605). Accordingly, mutant lysC can be also prepared from wild type lysC on the basis of the information in accordance with, for example, the site-directed mutagenesis method.  
           [0024]    A fragment comprising lysC can be isolated from a coryneform bacterium by preparing chromosomal DNA in accordance with, for example, a method of Saito and Miura (H. Saito and K. Miura,  Biochem. Biophys. Acta,  72, 619 (1963)), and amplifying lysC in accordance with the polymerase chain reaction method (PCR; see White, T. J. et al.,  Trends Genet.,  5, 185 (1989)).  
           [0025]    DNA primers are exemplified by single strand DNA&#39;s of 23-mer and 21-mer having nucleotide sequences shown in SEQ ID NOs: 1 and 2 in Sequence Listing in order to amplify, for example, a region of about 1,643 bp coding for lysC based on a sequence known for  Corynebacterium glutamicum  (see  Molecular Microbiology  (1991), 5(5), 1197-1204;  Mol. Gen. Genet.  (1990), 224, 317-324). DNA can be synthesized in accordance with an ordinary method by using DNA synthesizer model 380B produced by Applied Biosystems and using the phosphoamidite method (see  Tetrahedron Letters  (1981), 22, 1859). PCR can be performed by using DNA Thermal Cycler Model PJ2000 produced by Takara Shuzo, and using Taq DNA polymerase in accordance with a method designated by the supplier.  
           [0026]    It is preferred that lysC amplified by PCR is ligated with vector DNA autonomously replicable in cells of  E. coli  and/or coryneform bacteria to prepare recombinant DNA, and the recombinant DNA is introduced into cells of  E. coli  beforehand. Such provision makes following operations easy. The vector autonomously replicable in cells of  E. coli  is preferably a plasmid vector which is preferably autonomously replicable in cells of a host, including, for example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, and RSF1010.  
           [0027]    When a DNA fragment having an ability to allow a plasmid to be autonomously replicable in coryneform bacteria is inserted into these vectors, they can be used as a so-called shuttle vector autonomously replicable in both  E. coli  and coryneform bacteria.  
           [0028]    Such a shuttle vector includes the followings. Microorganisms harboring each of vectors and deposition numbers in international deposition facilities are shown in parentheses.  
           [0029]    pHC4:  Escherichia coli  AJ12617 (FERM BP-3532)  
           [0030]    pAJ655:  Escherichia coli  AJ11882 (FERM BP-136)  Corynebacterium glutamicum  SR8201 (ATCC 39135)  
           [0031]    pAJ1844:  Escherichia coli  AJ11883 (FERM BP-137)  Corynebacterium glutamicum SR 8202 (ATCC 39136)  
           [0032]    pAJ611:  Escherichia coli  AJ11884 (FERM BP-138)  
           [0033]    pAJ3148:  Corynebacterium glutamicum  SR8203 (ATCC 39137)  
           [0034]    pAJ440:  Bacillus subtilis  AJ11901 (FERM BP-140)  
           [0035]    These vectors are obtainable from the deposited microorganisms as follows. Cells collected at a logarithmic growth phase were lysed by using lysozyme and SDS, followed by separation from a lysate by centrifugation at 30,000×g to obtain a supernatant to which polyethylene glycol is added, followed by fractionation and purification by means of cesium chloride-ethidium bromide equilibrium density gradient centrifugation.  
           [0036]    [0036] E. coli  can be transformed by introducing a plasmid in accordance with, for example, a method of D. M. Morrison ( Methods in Enzymology,  68, 326 (1979)) or a method in which recipient cells are treated with calcium chloride to increase permeability for DNA (Mandel, M. and Higa, A.,  J. Mol. Biol.,  53, 159 (1970)).  
           [0037]    Wild type lysC is obtained when lysC is isolated from an AK wild type strain, while mutant lysC is obtained when lysC is isolated from an AK mutant strain in accordance with the method as described above.  
           [0038]    An example of a nucleotide sequence of a DNA fragment containing wild type lysC is shown in SEQ ID NO: 3 in Sequence Listing. An amino acid sequence of α-subunit of a wild type AK protein is deduced from the nucleotide sequence, which is shown in SEQ ID NO: 4 in Sequence Listing together with the DNA sequence. Only the amino acid sequence is shown in SEQ ID NO: 5. An amino acid sequence of β-subunit of the wild type AK protein is deduced from the nucleotide sequence of DNA, which is shown in SEQ ID NO: 6 in Sequence Listing together with the DNA. Only the amino acid sequence is shown in SEQ ID NO: 7. In each of the subunits, GTG is used as an initiation codon, and a corresponding amino acid is represented by methionine. However, this representation refers to methionine, valine, or formylmethionine.  
           [0039]    The mutant lysC used in the present invention is not specifically limited provided that it codes for AK in which synergistic feedback inhibition by L-lysine and L-threonine is desensitized. However, the mutant lysC is exemplified by one including mutation in which a 279th alanine residue as counted from the N-terminal is changed into an amino acid residue other than alanine and other than acidic amino acid in the α-subunit, and a 30th alanine residue is changed into an amino acid residue other than alanine and other than acidic amino acid in the β-subunit in the amino acid sequence of the wild type AK. The amino acid sequence of the wild type AK specifically includes the amino acid sequence shown in SEQ ID NO: 5 in Sequence Listing as the α-subunit, and the amino acid sequence shown in SEQ ID NO: 7 in Sequence Listing as the β-subunit.  
           [0040]    Those preferred as the amino acid residue other than alanine and other than acidic amino acid include threonine, arginine, cyteine, phenylanaline, proline, serine, tyrosine, and valine residues.  
           [0041]    The codon corresponding to an amino acid residue to be substituted is not specifically limited for its type provided that it codes for the amino acid residue. It is assumed that the amino acid sequence of possessed wild type AK may slightly differ depending on the difference in bacterial species and bacterial strains. AK&#39;s, which have mutation based on, for example, substitution, deletion, or insertion of one or more amino acid residues at one or more positions irrelevant to the enzyme activity as described above, can be also used for the present invention. Other AK&#39;s, which have mutation based on, for example, substitution, deletion, or insertion of other one or more amino acid residues, can be also used provided that no influence is substantially exerted on the AK activity, and on the desensitization of synergistic feedback inhibition by L-lysine and L-threonine.  
           [0042]    An AJ12691 strain obtained by introducing a mutant lysC plasmid p399AK9B into an AJ12036 strain (FERM BP-734)as a wild type strain of  Brevibacterium lactofermentum  has been deposited on Apr. 10, 1992 under a deposition number of FERM P-12918 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), transferred to international deposition based on the Budapest Treaty on Feb. 10, 1995, and deposited under a deposition number of FERM BP-4999.  
           [0043]    (2) Preparation of dapB  
           [0044]    A DNA fragment containing dapB can be prepared from chromosome of a coryneform bacterium by means of PCR. The DNA donor is not specifically limited, however, it is exemplified by  Brevibacterium lactofermentum  ATCC 13869 strain.  
           [0045]    A DNA sequence coding for DDPR is known for  Brevibacterium lactofermentum  ( Journal of Bacteriology,  175(9), 2743-2749 (1993)), on the basis of which DNA primers for PCR can be prepared. Such DNA primers are specifically exemplified by DNA&#39;s of 23-mers respectively having nucleotide sequences depicted in SEQ ID NOs: 8 and 9 in Sequence Listing. Synthesis of DNA, PCR, and preparation of a plasmid containing obtained dapB can be performed in the same manner as those for lysC described above.  
           [0046]    A nucleotide sequence of a DNA fragment containing dapB and an amino acid sequence deduced from the nucleotide sequence are illustrated in SEQ ID NO: 10. Only the amino acid sequence is shown in SEQ ID NO: 11. In addition to DNA fragments coding for this amino acid sequence, the present invention can equivalently use DNA fragments coding for amino acid sequences substantially the same as the amino acid sequence shown in SEQ ID NO: 11, namely amino acid sequences having mutation based on, for example, substitution, deletion, or insertion of one or more amino acids provided that there is no substantial influence on the DDPR activity.  
           [0047]    A transformant strain AJ13107 obtained by introducing a plasmid pCRDAPB containing dapB obtained in Example described later on into  E. coli  JM109 strain has been internationally deposited since May 26, 1995 under a deposition number of FERM BP-5114 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) based on the Budapest Treaty.  
           [0048]    (3) Preparation of daPA  
           [0049]    A DNA fragment containing dapA can be prepared from chromosome of a coryneform bacterium by means of PCR. The DNA donor is not specifically limited, however, it is exemplified by  Brevibacterium lactofermentum  ATCC 13869 strain.  
           [0050]    A DNA sequence coding for DDPS is known for  Corynebacterium glutamicum  (see  Nucleic Acids Research,  18(21), 6421 (1990); EMBL accession No. X53993), on the basis of which DNA primers for PCR can be prepared. Such DNA primers are specifically exemplified by DNA&#39;s of 23-mers respectively having nucleotide sequences depicted in SEQ ID NOs: 12 and 13 in Sequence Listing. Synthesis of DNA, PCR, and preparation of a plasmid containing obtained dapA can be performed in the same manner as those for lysC described above.  
           [0051]    A nucleotide sequence of a DNA fragment containing dapA and an amino acid sequence deduced from the nucleotide sequence are exemplified in SEQ ID NO: 14. Only the amino acid sequence is shown in SEQ ID NO: 15. In addition to DNA fragments coding for this amino acid sequence, the present invention can equivalently use DNA fragments coding for amino acid sequences substantially the same as the amino acid sequence shown in SEQ ID NO: 15, namely amino acid sequences having mutation based on, for example, substitution, deletion, or insertion of one or more amino acids provided that there is no substantial influence on the DDPS activity.  
           [0052]    A transformant strain AJ13106 obtained by introducing a plasmid pCRDAPA containing dapA obtained in Example described later on into  E. coli  JM109 strain has been internationally deposited since May 26, 1995 under a deposition number of FERM BP-5113 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) based on the Budapest Treaty.  
           [0053]    (4) Preparation of lysA  
           [0054]    A DNA fragment containing lysA can be prepared from chromosome of a coryneform bacterium by means of PCR. The DNA donor is not specifically limited, however, it is exemplified by  Brevibacterium lactofermentum  ATCC 13869 strain.  
           [0055]    In the coryneform bacteria, lysA forms an operon together with argS (arginyl-tRNA synthase gene), and lysA exists downstream from argS. Expression of lysA is regulated by a promoter existing upstream from argS (see  Journal of Bacteriology, Nov.,  7356-7362 (1993)). DNA sequences of these genes are known for  Corynebacterium glutamicum  (see  Molecular Microbiology,  4(11), 1819-1830 (1990);  Molecular and General Genetics,  212, 112-119 (1988)), on the basis of which DNA primers for PCR can be prepared. Such DNA primers are specifically exemplified by DNA&#39;s of 23-mers respectively having nucleotide sequences shown in SEQ ID NO: 16 in Sequence Listing (corresponding to nucleotide numbers 11 to 33 in a nucleotide sequence described in  Molecular Microbiology,  4(11), 1819-1830 (1990)) and SEQ ID NO: 17 (corresponding to nucleotide numbers 1370 to 1392 in a nucleotide sequence described in  Molecular and General Genetics,  212, 112-119 (1988)). Synthesis of DNA, PCR, and preparation of a plasmid containing obtained lysA can be performed in the same manner as those for lysC described above.  
           [0056]    In Example described later on, a DNA fragment containing a promoter, argS, and lysA was used in order to enhance lysA. However, argS is not essential for the present invention. It is allowable to use a DNA fragment in which lysA is ligated just downstream from a promoter.  
           [0057]    A nucleotide sequence of a DNA fragment containing argS and lysA, and an amino acid sequence deduced to be encoded by the nucleotide sequence are exemplified in SEQ ID NO: 18. An example of an amino acid sequence encoded by argS is shown in SEQ ID NO: 19, and an example of an amino acid sequence encoded by lysA is shown in SEQ ID NO: 20. In addition to DNA fragments coding for these amino acid sequences, the present invention can equivalently use DNA fragments coding for amino acid sequences substantially the same as the amino acid sequence shown in SEQ ID NO: 20, namely amino acid sequences having mutation based on, for example, substitution, deletion, or insertion of one or more amino acids provided that there is no substantial influence on the DDC activity.  
           [0058]    (5) Preparation of ddh  
           [0059]    A DNA fragment containing ddh can be prepared from chromosome of a coryneform bacterium by means of PCR. The DNA donor is not specifically limited, however, it is exemplified by  Brevibacterium lactofermentum  ATCC 13869 strain.  
           [0060]    A DDH gene is known for  Corynebacterium glutamicum  (Ishino, S. et al.,  Nucleic Acids Res.,  15, 3917 (1987)), on the basis of which primers for PCR can be prepared. Such DNA primers are specifically exemplified by DNA&#39;s of 20-mers respectively having nucleotide sequences depicted in SEQ ID NOs: 21 and 22 in Sequence Listing. Synthesis of DNA, PCR, and preparation of a plasmid containing obtained ddh can be performed in the same manner as those for lysC described above.  
           [0061]    A nucleotide sequence of a DNA fragment containing ddh and an amino acid sequence deduced from the nucleotide sequence are illustrated in SEQ ID NO: 23. Only the amino acid sequence is shown in SEQ ID NO: 24. In addition to DNA fragments coding for this amino acid sequence, the present invention can equivalently use DNA fragments coding for amino acid sequences substantially the same as the amino acid sequence shown in SEQ ID NO: 24, namely amino acid sequences having mutation based on, for example, substitution, deletion, or insertion of one or more amino acids provided that there is no substantial influence on the DDH activity.  
           [0062]    &lt;2&gt; Recombinant DNA and Coryneform Bacterium of the Present Invention  
           [0063]    The coryneform bacterium of the present invention harbors an aspartokinase (mutant AK) in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, wherein DNA (dapB) coding for a dihydrodipicolinate reductase is enhanced. In a preferred embodiment, the coryneform bacterium of the present invention is a coryneform bacterium in which DNA (dapA) coding for dihydrodipicolinate synthase is further enhanced. In a more preferred embodiment, the coryneform bacterium of the present invention is a coryneform bacterium in which DNA (lysA) coding for diaminopimelate decarboxylase is further enhanced. In a more preferred embodiment, the coryneform bacterium of the present invention is a coryneform bacterium in which DNA (ddh) coding for diaminopimelate dehydrogenase is further enhanced.  
           [0064]    The term “enhance” DNA herein refers to the fact that the intracellular activity of an enzyme encoded by the DNA is raised by, for example, increasing the copy number of a gene, using a strong promoter, using a gene coding for an enzyme having a high specific activity, or combining these means.  
           [0065]    The coryneform bacterium harboring the mutant AK may be those which produce the mutant aspartokinase as a result of mutation, or those which are transformed by introducing mutant lysC.  
           [0066]    Examples of the coryneform bacterium used to introduce the DNA described above include, for example, the following lysine-producing wild type strains:  
           [0067]    [0067] Corynebacterium acetoacidophilum  ATCC 13870;  
           [0068]    [0068] Corynebacterium acetoglutamicum  ATCC 15806;  
           [0069]    [0069] Corynebacterium callunae  ATCC 15991;  
           [0070]    [0070] Corynebacterium glutamicum  ATCC 13032;  
           [0071]    ( Brevibacterium divaricatum ) ATCC 14020;  
           [0072]    ( Brevibacterium lactofermentum ) ATCC 13869;  
           [0073]    ( Corynebacterium lilium ) ATCC 15990;  
           [0074]    ( Brevibacterium flavum ) ATCC 14067;  
           [0075]    [0075] Corynebacterium melassecola  ATCC 17965;  
           [0076]    [0076] Brevibacterium saccharolyticum  ATCC 14066;  
           [0077]    [0077] Brevibacterium immariophilum  ATCC 14068;  
           [0078]    [0078] Brevibacterium roseum  ATCC 13825;  
           [0079]    [0079] Brevibacterium thiogenitalis  ATCC 19240;  
           [0080]    [0080] Microbacterium ammoniaphilum  ATCC 15354;  
           [0081]    [0081] Corynebacterium thermoaminogenes  AJ12340 (FERM BP-1539).  
           [0082]    Other than the bacterial strains described above, those usable as a host include, for example, mutant strains having an L-lysine-producing ability derived from the aforementioned strains. Such artificial mutant strains includes the followings: S-(2-aminoethyl)-cysteine (hereinafter abbreviated as “AEC”) resistant mutant strains ( Brevibacterium lactofermentum  AJ11082 (NRRL B-1147), Japanese Patent Publication Nos. 56-1914, 56-1915, 57-14157, 57-14158, 57-30474, 58-10075, 59-4993, 61-35840, 62-24074, 62-36673, 5-11958, 7-112437, and 7-112438); mutant strains which require amino acid such as L-homoserine for their growth (Japanese Patent Publication Nos. 48-28078 and 56-6499); mutant strains which exhibit resistance to AEC and require amino acids such as L-leucine, L-homoserine, L-proline, L-serine, L-arginine, L-alanine, and L-valine (U.S. Pat. Nos. 3,708,395 and 3,825,472); L-lysine-producing mutant strains which exhibit resistance to DL-α-amino-ε-caprolactam, α-amino-lauryllactam, aspartate-analog, sulfa drug, quinoid, and N-lauroylleucine; L-lysine-producing mutant strains which exhibit resistance to inhibitors of oxyaloacetate decarboxylase or respiratory system enzymes (Japanese Patent Laid-open Nos. 50-53588, 50-31093, 52-102498, 53-9394, 53-86089, 55-9783, 55-9759, 56-32995 and 56-39778, and Japanese Patent Publication Nos. 53-43591 and 53-1833); L-lysine-producing mutant strains which require inositol or acetic acid (Japanese Patent Laid-open Nos. 55-9784 and 56-8692); L-lysine-producing mutant strains which exhibit sensitivity to fluoropyruvic acid or temperature not less than 34° C. (Japanese Patent Laid-open Nos. 55-9783 and 53-86090); and producing mutant strains belonging to the genus  Brevibacterium  or  Corynebacterium  which exhibit resistance to ethylene glycol and produce L-lysine (U.S. Pat. No. 4,411,997).  
           [0083]    In a specified embodiment, in order to enhance the genes for L-lysine biosynthesis in the host as described above, the genes are introduced into the host by using a plasmid vector, transposon or phage vector or the like. Upon the introduction, it is expected to make enhancement to some extent even by using a low copy type vector. However, it is preferred to use a multiple copy type vector. Such a vector includes, for example, plasmid vectors, pAJ655, pAJ1844, pAJ611, pAJ3148, and pAJ440 described above. Besides, transposons derived from coryneform bacteria are described in International Publication Pamphlets of WO02/02627 and WO93/18151, European Patent Publication No. 445385, Japanese Patent Laid-open No. 6-46867, Vertes, A. A. et al., Mol. Microbiol., 11, 739-746 (1994), Bonamy, C., et al., Mol. Microbiol., 14, 571-581 (1994), Vertes, A. A. et al., Mol. Gen. Genet., 245, 397-405 (1994), Jagar, W. et al., FEMS Microbiology Letters, 126, 1-6 (1995), Japanese Patent Laid-open No. 7-107976, Japanese Patent Laid-open No. 7-327680 and the like.  
           [0084]    In the present invention, it is not indispensable that the mutant lysC is necessarily enhanced. It is allowable to use those which have mutation on lysC on chromosomal DNA, or in which the mutant lysC is incorporated into chromosomal DNA. Alternatively, the mutant lysC may be introduced by using a plasmid vector. On the other hand, dapA, dapB, lysA, and ddh are preferably enhanced in order to efficiently produce L-lysine.  
           [0085]    Each of the genes of lysC, dapA, dapB, lysA, and ddh may be successively introduced into the host by using different vectors respectively. Alternatively, two, three, four, or five species of the genes may be introduced together by using a single vector. When different vectors are used, the genes may be introduced in any order, however, it is preferred to use vectors which have a stable sharing and harboring mechanism in the host, and which are capable of co-existing with each other.  
           [0086]    A coryneform bacterium harboring the mutant AK and further comprising enhanced dapB is obtained, for example, by introducing, into a host coryneform bacterium, a recombinant DNA containing mutant lysC and dapB autonomously replicable in cells of coryneform bacteria.  
           [0087]    A coryneform bacterium further comprising enhanced dapA in addition to mutant lysC and dapB is obtained, for example, by introducing, into a host coryneform bacterium, a recombinant DNA containing mutant lysC, dapB, and dapA autonomously replicable in cells of coryneform bacteria.  
           [0088]    A coryneform bacterium further comprising enhanced lysA in addition to mutant lysC, dapB, and dapA is obtained, for example, by introducing, into a host coryneform bacterium, a recombinant DNA containing mutant lysC, dapB, dapA, and lysA autonomously replicable in cells of coryneform bacteria.  
           [0089]    A coryneform bacterium further comprising enhanced ddh in addition to mutant lysC, dapB, dapA, and lysA is obtained, for example, by introducing, into a host coryneform bacterium, a recombinant DNA containing mutant lysC, dapB, dapA, lysA, and ddh autonomously replicable in cells of coryneform bacteria.  
           [0090]    The above-mentioned recombinant DNAs can be obtained, for example, by inserting each of the genes participating in L-lysine biosynthesis into a vector such as plasmid vector, transposon or phage vector as described above.  
           [0091]    In the case in which a plasmid is used as a vector, the recombinant DNA can be introduced into the host in accordance with an electric pulse method (Sugimoto et al., Japanese Patent Laid-open No. 2-207791). Amplification of a gene using transposon can be performed by introducing a plasmid which carrying a transposon into the host cell and inducing transposition of the transposon.  
           [0092]    &lt;3&gt; Method for Producing L-lysine  
           [0093]    L-Lysine can be efficiently produced by cultivating, in an appropriate medium, the coryneform bacterium comprising the enhanced genes for L-lysine biosynthesis as described above, producing and accumulating L-lysine in a culture of the bacterium, and collecting L-lysine from the culture.  
           [0094]    The medium to be used is exemplified by an ordinary medium containing a carbon source, a nitrogen source, inorganic ions, and optionally other organic components.  
           [0095]    As the carbon source, it is possible to use sugars such as glucose, fructose, sucrose, molasses, and starch hydrolysate; and organic acids such as fumaric acid, citric acid, and succinic acid.  
           [0096]    As the nitrogen source, it is possible to use inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as soybean hydrolysate; ammonia gas; and aqueous ammonia.  
           [0097]    As organic trace nutrient sources, it is desirable to contain required substances such as vitamin B 1  and L-homoserine or yeast extract or the like in appropriate amounts. Other than the above, potassium phosphate, magnesium sulfate, iron ion, manganese ion and so on are added in small amounts, if necessary.  
           [0098]    Cultivation is preferably carried out under an aerobic condition for about 30 to 90 hours. The cultivation temperature is preferably controlled at 25° C. to 37° C., and pH is preferably controlled at 5 to 8 during cultivation. Inorganic or organic, acidic or alkaline substances, or ammonia gas or the like can be used for pH adjustment. L-lysine can be collected from a culture by combining an ordinary ion exchange resin method, a precipitation method, and other known methods.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0099]    [0099]FIG. 1 illustrates a process of construction of plasmids p399AKYB and p399AK9B comprising mutant lysC.  
         [0100]    [0100]FIG. 2 illustrates a process of construction of a plasmid pDPRB comprising dapB and Brevi.-or.  
         [0101]    [0101]FIG. 3 illustrates ia process of construction of a plasmid pDPSB comprising dapA and Brevi.-ori.  
         [0102]    [0102]FIG. 4 illustrates a process of construction of a plasmid p299LYSA comprising lysA.  
         [0103]    [0103]FIG. 5 illustrates a process of construction of a plasmid pLYSAB comprising lysA and Brevi.-ori.  
         [0104]    [0104]FIG. 6 illustrates a process of construction of a plasmid pPK4D comprising ddh and Brevi.-ori.  
         [0105]    [0105]FIG. 7 illustrates a process of construction of a plasmid pCRCAB comprising lysC, dapB and Brevi.-ori.  
         [0106]    [0106]FIG. 8 illustrates a process of construction of a plasmid pCB comprising mutant lysC, dapB, and Brevi.-ori.  
         [0107]    [0107]FIG. 9 illustrates a process of construction of a plasmid pAB comprising dapA, dapB and Brevi.-ori.  
         [0108]    [0108]FIG. 10 illustrates a process of construction of a plasmid p399DL comprising ddh and lysA.  
         [0109]    [0109]FIG. 11 illustrates a process of construction of a plasmid pDL comprising ddh, lysA and Brevi.-ori.  
         [0110]    [0110]FIG. 12 illustrates a process of construction of a plasmid pCAB comprising mutant lysC, dapA, dapB, and Brevi.-ori.  
         [0111]    [0111]FIG. 13 illustrates a process of construction of a plasmid pCABL comprising mutant lysC, dapA, dapB, lysA, and Brevi.-ori.  
         [0112]    [0112]FIG. 14 illustrates a process of construction of a plasmid pCABDL comprising mutant lysC, dapA, dapB, ddh, lysA, and Brevi.-ori. 
     
    
     DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0113]    The present invention will be more specifically explained below with reference to Examples.  
       EXAMPLE 1  
       [0114]    Preparation of Wild Type lysC Gene and Mutant lysC Gene from  Brevibacterium lactofermentum    
         [0115]    &lt;1&gt; Preparation of Wild Type and Mutant lysC&#39;s and Preparation of Plasmids Containing Them  
         [0116]    A strain of  Brevibacterium lactofermentum  ATCC 13869, and an L-lysine-producing mutant strain AJ3445 (FERM P-1944) obtained from the ATCC 13869 strain by a mutation treatment were used as chromosomal DNA donors. The AJ3445 strain had been subjected to mutation so that lysC was changed to involve substantial desensitization from concerted inhibition by lysine and threonine ( Journal of Biochemistry,  68, 701-710 (1970)).  
         [0117]    A DNA fragment containing lysC was amplified from chromosomal DNA in accordance with the PCR method (polymerase chain reaction; see White, T. J. et al.,  Trends Genet.,  5, 185 (1989)). As for DNA primers used for amplification, single strand DNA&#39;s of 23-mer and 21-mer having nucleotide sequences shown in SEQ ID NOs: 1 and 2 were synthesized in order to amplify a region of about 1,643 bp coding for lysC on the basis of a sequence known for  Corynebacterium glutamicum  (see  Molecular Microbiology  (1991), 5(5), 1197-1204; and  Mol. Gen. Genet.  (1990), 224, 317-324). DNA was synthesized in accordance with an ordinary method by using DNA synthesizer model 380B produced by Applied Biosystems and using the phosphoamidite method (see  Tetrahedron Letters  (1981), 22, 1859).  
         [0118]    The gene was amplified by PCR by using DNA Thermal Cycler Model PJ2000 produced by Takara Shuzo, and using Taq DNA polymerase in accordance with a method designated by the supplier. An amplified gene fragment of 1,643 kb was confirmed by agarose gel electrophoresis. After that, the fragment excised from the gel was purified in accordance with an ordinary method, and it was digested with restriction enzymes NruI (produced by Takara Shuzo) and EcoRI (produced by Takara Shuzo).  
         [0119]    pHSG399 (see Takeshita, S. et al.,  Gene  (1987), 61, 63-74) was used as a cloning vector for the gene fragment. pHSG399 was digested with restriction enzymes SmaI (produced by Takara Shuzo) and EcoRI, and it was ligated with the amplified lysC fragment. DNA was ligated by using DNA ligation kit (produced by Takara Shuzo) in accordance with a designated method. Thus plasmids were prepared, in which the lysC fragments amplified from chromosomes of  Brevibacterium lactofermentum  were ligated with pHSG399 respectively. A plasmid comprising lysC from ATCC 13869 (wild type strain) was designated as p399AKY, and a plasmid comprising lysC from AJ3463 (L-lysine-producing bacterium) was designated as p399AK9.  
         [0120]    A DNA fragment (hereinafter referred to as “Brevi.-ori”) having an ability to make a plasmid autonomously replicable in bacteria belonging to the genus  Corynebacterium  was introduced into p399AKY and p399AK9 respectively to prepare plasmids carrying lysC autonomously replicable in bacteria belonging to the genus  Corynebacterium.  Brevi.-ori was prepared from a plasmid vector pHK4 containing Brevi.-ori and autonomously replicable in cells of both  Escherichia coli  and bacteria belonging to the genus  Corynebacterium.  pHK4 was constructed by digesting pHC4 with KpnI (produced by Takara Shuzo) and BamHI (produced by Takara Shuzo), extracting a Brevi.-ori fragment, and ligating it with pHSG298 having been also digested with KpnI and BamHI (see Japanese Patent Laid-open No. 5-7491). pHK4 gives kanamycin resistance to a host.  Escherichia coli  harboring pHK4 was designated as  Escherichia coli  AJ13136, and deposited on Aug. 1, 1995 under a deposition number of FERM BP-5186 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan).  
         [0121]    pHK4 was digested with restriction enzymes KpnI and BamHI, and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated BamHI linker (produced by Takara Shuzo) was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only BamHI. This plasmid was digested with BamHI, and the generated Brevi.-ori DNA fragment was ligated with p399AKY and p399AK9 having been also digested with BamHI respectively to prepare plasmids each containing the lysC gene autonomously replicable in bacteria belonging to the genus  Corynebacterium.    
         [0122]    A plasmid containing the wild type lysC gene originating from p399AKY was designated as p399AKYB, and a plasmid containing the mutant lysC gene originating from p399AK9 was designated as p399AK9B. The process of construction of p399AK9B and p399AKYB is shown in FIG. 1. A strain AJ12691 obtained by introducing the mutant lysC plasmid p399AK9B into a wild type strain of  Brevibacterium lactofermentum  (AJ12036 strain, FERM BP-734) was deposited on Apr. 10, 1992 under a deposition number of FERM P-12918 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), transferred to international deposition based on the Budapest Treaty on Feb. 10, 1995, and deposited under a deposition number of FERM BP-4999.  
         [0123]    &lt;2&gt; Determination of Nucleotide Sequences of Wild Type lysC and Mutant lysC from  Brevibacterium lactofermentum    
         [0124]    The plasmid p399AKY containing the wild type lysC and the plasmid p399AK9 containing the mutant lysC were prepared from the respective transformants to determine nucleotide sequences of the wild type and mutant lysC&#39;s. Nucleotide sequence determination was performed in accordance with a method of Sanger et al. (for example, F. Sanger et al.,  Proc. Natl. Acad. Sci.,  74, 5463 (1977)).  
         [0125]    The nucleotide sequence of wild type lysC encoded by p399AKY is shown in SEQ ID NO: 3 in Sequence Listing. On the other hand, the nucleotide sequence of mutant lysC encoded by p399AK9 had only mutation of one nucleotide such that 1051th G was changed into A in SEQ ID NO: 3 as compared with wild type lysC. It is known that lysC of  Corynebacterium glutamicum  has two subunits (α, β) encoded in an identical reading frame on an identical DNA strand (see Kalinowski, J. et al.,  Molecular Microbiology  (1991) 5(5), 1197-1204). Judging from homology, it is assumed that the gene sequenced herein also has two subunits (α, β) encoded in an identical reading frame on an identical DNA strand.  
         [0126]    An amino acid sequence of the α-subunit of the wild type AK protein deduced from the nucleotide sequence of DNA is shown in SEQ ID NO: 4 together with the DNA sequence. Only the amino acid sequence is shown in SEQ ID NO: 5. An amino acid sequence of the β-subunit of the wild type AK protein deduced from the nucleotide sequence of DNA is shown in SEQ ID NO: 6 together with DNA. Only the amino acid sequence is shown in SEQ ID NO: 7. In each of the subunits, GTG is used as an initiation codon, and a corresponding amino acid is represented by methionine. However, this representation refers to methionine, valine, or formylmethionine.  
         [0127]    On the other hand, mutation on the sequence of mutant lysC means occurrence of amino acid residue substitution such that a 279th alanine residue of the α-subunit is changed into a threonine residue, and a 30th alanine residue of the β-subunit is changed into a threonine residue in the amino acid sequence of the wild type AK protein (SEQ ID NOs: 5, 7).  
       EXAMPLE 2  
       [0128]    Preparation of dapB from  Brevibacterium lactofermentum    
         [0129]    &lt;1&gt; Preparation of dapB and Construction of Plasmid Containing dapB  
         [0130]    A wild type strain of  Brevibacterium lactofermentum  ATCC 13869 was used as a chromosomal DNA donor. Chromosomal DNA was prepared from the ATCC 13869 strain in accordance with an ordinary method. A DNA fragment containing dapB was amplified from the chromosomal DNA in accordance with PCR. As for DNA primers used for amplification, DNA&#39;s of 23-mers having nucleotide sequences depicted in SEQ ID NOs: 8 and 9 in Sequence Listing respectively were synthesized in order to amplify a region of about 2.0 kb coding for DDPR on the basis of a sequence known for  Brevibacterium lactofermentum  (see  Journal of Bacteriology,  157(9), 2743-2749 (1993)). Synthesis of DNA and PCR were performed in the same manner as described in Example 1. pCR-Script (produced by Invitrogen) was used as a cloning vector for the amplified gene fragment of 2,001 bp, which was ligated with the amplified dapB fragment. Thus a plasmid was constructed, in which the dapB fragment of 2,001 bp amplified from chromosome of  Brevibacterium lactofermentum  was ligated with pCR-Script. The plasmid obtained as described above, which had dapB originating from ATCC 13869, was designated as pCRDAPB. A transformant strain AJ13107 obtained by introducing pCRDAPB into  E. coli  JM109 strain has been internationally deposited since May 26, 1995 under a deposition number of FERM BP-5114 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) based on the Budapest Treaty.  
         [0131]    A fragment of 1,101 bp containing a structural gene of DDPR was extracted by digesting pCRDAPB with EcoRV and SphI. This fragment was ligated with pHSG399 having been digested with HincII and SphI to prepare a plasmid. The prepared plasmid was designated as p399DPR.  
         [0132]    Brevi.-ori was introduced into the prepared p399DPR to construct a plasmid carrying dapB autonomously replicable in coryneform bacteria. pHK4 was digested with a restriction enzyme KpnI (produced by Takara Shuzo), and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated BamHI linker (produced by Takara Shuzo) was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only BamHI. This plasmid was digested with BamHI, and the generated Brevi.-ori DNA fragment was ligated with p399DPR having been also digested with BamHI to prepare a plasmid containing dapB autonomously replicable in coryneform bacteria. The prepared plasmid was designated as pDPRB. The process of construction of pDPRB is shown in FIG. 2.  
         [0133]    &lt;2&gt; Determination of Nucleotide Sequence of dapB from  Brevibacterium lactofermentum    
         [0134]    Plasmid DNA was prepared from the AJ13107 strain harboring p399DPR, and its nucleotide sequence was determined in the same manner as described in Example 1. A determined nucleotide sequence and an amino acid sequence deduced from the nucleotide sequence are shown in SEQ ID NO: 10. Only the amino acid sequence is shown in SEQ ID NO: 11.  
       EXAMPLE 3  
       [0135]    Preparation of dapA from  Brevibacterium lactofermentum    
         [0136]    &lt;1&gt; Preparation of dapA and Construction of Plasmid Containing dapA  
         [0137]    A wild type strain of  Brevibacterium lactofermentum  ATCC 13869 was used as a chromosomal DNA donor. Chromosomal DNA was prepared from the ATCC 13869 strain in accordance with an ordinary method. A DNA fragment containing dapA was amplified from the chromosomal DNA in accordance with PCR. As for DNA primers used for amplification, DNA&#39;s of 20-mers having nucleotide sequences shown in SEQ ID NOs: 12 and 13 in Sequence Listing respectively were synthesized in order to amplify a region of about 1.5 kb coding for DDPS on the basis of a sequence known for  Corynebacterium glutamicum  (see  Nucleic Acids Research,  18(21), 6421 (1990); EMBL accession No. X53993). Synthesis of DNA and PCR were performed in the same manner as described in Example 1. pCR1000 (produced by Invitrogen, see  Bio/Technology,  9, 657-663 (1991)) was used as a cloning vector for the amplified gene fragment of 1,411 bp, which was ligated with the amplified dapA fragment. Ligation of DNA was performed by using DNA ligation kit (produced by Takara Shuzo) in accordance with a designated method. Thus a plasmid was constructed, in which the dapA fragment of 1,411 bp amplified from chromosome of  Brevibacterium lactofermentum  was ligated with pCR1000. The plasmid obtained as described above, which had dapA originating from ATCC 13869, was designated as pCRDAPA.  
         [0138]    A transformant strain AJ13106 obtained by introducing pCRDAPA into  E. coli  JM109 strain has been internationally deposited since May 26, 1995 under a deposition number of FERM BP-5113 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) based on the Budapest Treaty.  
         [0139]    Brevi.-ori was introduced into the prepared pCRDAPA to construct a plasmid carrying dapA autonomously replicable in coryneform bacteria. pHK4 was digested with restriction enzymes KpnI and BamHI (produced by Takara Shuzo), and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated SmaI linker (produced by Takara Shuzo) was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only SmaI. This plasmid was digested with SmaI, and the generated Brevi.-ori DNA fragment was ligated with pCRDAPA having been also digested with SmaI to prepare a plasmid containing dapA autonomously replicable in coryneform bacteria. This plasmid was designated as pDPSB. The process of construction of pDPSB(Km r ) is shown in FIG. 3.  
         [0140]    &lt;2&gt; Determination of Nucleotide Sequence of dapA from  Brevibacterium lactofermentum    
         [0141]    Plasmid DNA was prepared from the AJ13106 strain harboring pCRDAPA, and its nucleotide sequence was determined in the same manner as described in Example 1. A determined nucleotide sequence and an amino acid sequence deduced from the nucleotide sequence are shown in SEQ ID NO: 14. Only the amino acid sequence is shown in SEQ ID NO: 15.  
       EXAMPLE 4  
       [0142]    Preparation of lysA from  Brevibacterium lactofermentum    
         [0143]    &lt;1&gt; Preparation of lysA and Construction of Plasmid Containing lysA  
         [0144]    A wild type strain of  Brevibacterium lactofermentum  ATCC 13869 was used as a chromosomal DNA donor. Chromosomal DNA was prepared from the ATCC 13869 strain in accordance with an ordinary method. A DNA fragment containing argS, lysA, and a promoter of an operon containing them was amplified from the chromosomal DNA in accordance with PCR. As for DNA primers used for amplification, synthetic DNA&#39;s of 23-mers having nucleotide sequences depicted in SEQ ID NOs: 16 and 17 in Sequence Listing respectively were used in order to amplify a region of about 3.6 kb coding for arginyl-tRNA synthase and DDC on the basis of a sequence known for  Corynebacterium glutamicum  (see  Molecular Microbiology,  4(11), 1819-1830 (1990);  Molecular and General Genetics,  212, 112-119 (1988)). Synthesis of DNA and PCR were performed in the same manner as described in Example 1. pHSG399 was used as a cloning vector for the amplified gene fragment of 3,579 bp. pHSG399 was digested with a restriction enzyme SmaI (produced by Takara Shuzo), which was ligated with the DNA fragment containing amplified lysA. A plasmid obtained as described above, which had lysA originating from ATCC 13869, was designated as p399LYSA.  
         [0145]    A DNA fragment containing lysA was extracted by digesting p399LYSA with KpnI (produced by Takara Shuzo) and BamHI (produced by Takara Shuzo). This DNA fragment was ligated with pHSG299 having been digested with KpnI and BamHI. An obtained plasmid was designated as p299LYSA. The process of construction of p299LYSA is shown in FIG. 4.  
         [0146]    Brevi.-ori was introduced into the obtained p299LYSA to construct a plasmid carrying lysA autonomously replicable in coryneform bacteria. pHK4 was digested with restriction enzymes KpnI and BamHI, and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated KpnI linker (produced by Takara Shuzo) was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only KpnI. This plasmid was digested with KpnI, and the generated Brevi.-ori DNA fragment was ligated with p299LYSA having been also digested with KpnI to prepare a plasmid containing lysA autonomously replicable in coryneform bacteria. The prepared plasmid was designated as pLYSAB. The process of construction of pLYSAB is shown in FIG. 5.  
         [0147]    &lt;2&gt; Determination of Nucleotide Sequence of lysA from  Brevibacterium lactofermentum    
         [0148]    Plasmid DNA of p299LYSA was prepared, and its nucleotide sequence was determined in the same manner as described in Example 1. A determined nucleotide sequence and an amino acid sequence deduced to be encoded by the nucleotide sequence are shown in SEQ ID NO: 18. Concerning the nucleotide sequence, an amino acid sequence encoded by argS and an amino acid sequence encoded by lysA are shown in SEQ ID NOs: 19 and 20 respectively.  
       EXAMPLE 5  
       [0149]    Preparation of ddh from  Brevibacterium lactofermentum    
         [0150]    A ddh gene was obtained by amplifying the ddh gene from chromosomal DNA of  Brevibacterium lactofermentum ATCC  13869 in accordance with the PCR method by using two oligonucleotide primers (SEQ ID NOs: 21, 22) prepared on the basis of a known nucleotide sequence of a ddh gene of  Corynebacterium glutamicum  (Ishino, S. et al.,  Nucleic Acids Res.,  15, 3917 (1987)). An obtained amplified DNA fragment was digested with EcoT22I and AvaI, and cleaved edges were blunt-ended. After that, the fragment was inserted into a SmaI site of pMW119 to obtain a plasmid pDDH.  
         [0151]    Next, pDDH was digested with SalI and EcoRI, followed by blunt end formation. After that, an obtained fragment was ligated with pUC18 having been digested with SmaI. A plasmid thus obtained was designated as pUC18DDH.  
         [0152]    Brevi.-ori was introduced into pUC18DDH to construct a plasmid carrying ddh autonomously replicable in coryneform bacteria. pHK4 was digested with restriction enzymes KpnI and BamHI, and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated PstI linker (produced by Takara Shuzo) was ligated so that it was inserted into a PstI site of pHSG299. A plasmid constructed as described above was designated as pPK4. Next, pUC18DDH was digested with XbaI and KpnI, and a generated fragment was ligated with pPK4 having been digested with KpnI and XbaI. Thus a plasmid containing ddh autonomously replicable in coryneform bacteria was constructed. This plasmid was designated as pPK4D. The process of construction of pPK4D is shown in FIG. 6.  
       EXAMPLE 6  
       [0153]    Construction of Plasmid Comprising Combination of Mutant lysC and dapA  
         [0154]    A plasmid comprising mutant lysC, dapA, and replication origin of coryneform bacteria was constructed from the plasmid pCRDAPA comprising dapA and the plasmid p399AK9B comprising mutant lysC and Brevi.-ori. p399AK9B was completely degraded with SalI, and then it was blunt-ended, with which an EcoRI linker was ligated to construct a plasmid in which the SalI site was modified into an EcoRI site. The obtained plasmid was designated as p399AK9BSE. The mutant lysC and Brevi.-ori were excised as one fragment by partially degrading p399AK9BSE with EcoRI. This fragment was ligated with pCRDAPA having been digested with EcoRI. An obtained plasmid was designated as pCRCAB. This plasmid is autonomously replicable in  E. coli  and coryneform bacteria, and it gives kanamycin resistance to a host, the plasmid comprising a combination of mutant lysC and dapA. The process of construction of pCRCAB is shown in FIG. 7.  
       EXAMPLE 7  
       [0155]    Construction of Plasmid Comprising Combination of Mutant lysC and dapB  
         [0156]    A plasmid comprising mutant lysC and dapB was constructed from the plasmid p399AK9 having mutant lysC and the plasmid p399DPR having dapB. A fragment of 1,101 bp containing a structural gene of DDPR was extracted by digesting p399DPR with EcoRV and SphI. This fragment was ligated with p399AK9 having been digested with SalI and then blunt-ended and having been further digested with SphI to construct a plasmid comprising a combination of mutant lysC and dapB. This plasmid was designated as p399AKDDPR.  
         [0157]    Next, Brevi.-ori was introduced into the obtained p399AKDDPR. The plasmid pHK4 containing Brevi.-ori was digested with a restriction enzyme KpnI (produced by Takara Shuzo), and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated BamHI linker (produced by Takara Shuzo) was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only BamHI. This plasmid was digested with BamHI, and the generated Brevi.-ori DNA fragment was ligated with p399AKDDPR having been also digested with BamHI to construct a plasmid containing mutant lysC and dapb autonomously replicable in coryneform bacteria. The constructed plasmid was designated as pCB. The process of construction of pCB is shown in FIG. 8.  
       EXAMPLE 8  
       [0158]    Construction of Plasmid Comprising Combination of dapA and dapB  
         [0159]    The plasmid pCRDAPA comprising dapA was digested with KpnI and EcoRI to extract a DNA fragment containing daPA which was ligated with the vector plasmid pHSG399 having been digested with KpnI and EcoRI. An obtained plasmid was designated as p399DPS.  
         [0160]    On the other hand, the plasmid pCRDAPB comprising dapB was digested with SacII and EcoRI to extract a DNA fragment of 2.0 kb containing a region coding for DDPR which was ligated with p399DPS having been digested with SacII and EcoRI to construct a plasmid comprising a combination of dapA and dapB. The obtained plasmid was designated as p399AB.  
         [0161]    Next, Brevi.-ori was introduced into p399AB. pHK4 containing Brevi.-ori was digested with a restriction enzyme BamHI (produced by Takara Shuzo), and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated KpnI linker (produced by Takara Shuzo) was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only KpnI. This plasmid was digested with KpnI, and the generated Brevi.-ori DNA fragment was ligated with p399AB having been also digested with KpnI to construct a plasmid containing dapA and dapB autonomously replicable in coryneform bacteria. The constructed plasmid was designated as pAB. The process of construction of pAB is shown in FIG. 9.  
       EXAMPLE 9  
       [0162]    Construction of Plasmid Comprising Combination of ddh and lysA  
         [0163]    The plasmid pUC18DDH comprising ddh was digested with EcoRI and XbaI to extract a DNA fragment containing ddh. This ddh fragment was ligated with the plasmid p399LYSA comprising lysA having been digested with BamHI and XbaI with cleaved edges having been blunt-ended after the digestion. An obtained plasmid was designated as p399DL. The process of construction of p399DL is shown in FIG. 10.  
         [0164]    Next, Brevi.-ori was introduced into p399DL. pHK4 was digested with XbaI and BamHI, and cleaved edges were blunt-ended. After the blunt end formation, a phosphorylated XbaI linker was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only XbaI. This plasmid was digested with XbaI, and the generated Brevi.-ori DNA fragment was ligated with p399DL having been also digested with XbaI to construct a plasmid containing ddh and lysA autonomously replicable in coryneform bacteria. The constructed plasmid was designated as pDL. The process of construction of pDL is shown in FIG. 11.  
       EXAMPLE 10  
       [0165]    Construction of Plasmid Comprising Combination of Mutant lysC, dapA, and dapB  
         [0166]    p399DPS was degraded with EcoRI and SphI to form blunt ends followed by extraction of a dapA gene fragment. This fragment was ligated with the p399AK9 having been digested with SalI and blunt-ended to construct a plasmid p399CA in which mutant lysC and dapA co-existed.  
         [0167]    The plasmid pCRDAPB comprising dapB was digested with EcoRI and blunt-ended, followed by digestion with SacI to extract a DNA fragment of 2.0 kb comprising dapB. The plasmid p399CA comprising dapA and mutant lysC was digested with SpeI and blunt-ended, which was thereafter digested with SacI and ligated with the extracted dapB fragment to obtain a plasmid comprising mutant lysC, dapA, and dapB. This plasmid was designated as p399CAB.  
         [0168]    Next, Brevi.-ori was introduced into p399CAB. The plasmid pHK4 comprising Brevi.-ori was digested with a restriction enzyme BamHI (produced by Takara Shuzo), and cleaved edges were blunt-ended. Blunt end formation was performed by using DNA Blunting kit (produced by Takara Shuzo) in accordance with a designated method. After the blunt end formation, a phosphorylated KpnI linker (produced by Takara Shuzo) was ligated to make modification so that the DNA fragment corresponding to the Brevi.-ori portion might be excised from pHK4 by digestion with only KpnI. This plasmid was digested with KpnI, and the generated Brevi.-ori DNA fragment was ligated with p399CAB having been also digested with KpnI to construct a plasmid comprising a combination of mutant lysC, dapA, and dapB autonomously replicable in coryneform bacteria. The constructed plasmid was designated as pCAB. The process of construction of pCAB is shown in FIG. 12.  
       EXAMPLE 11  
       [0169]    Construction of Plasmid Comprising Combination of Mutant lysC, dapA, dapB, and lysA  
         [0170]    The plasmid p299LYSA comprising lysA was digested with KpnI and BamHI and blunt-ended, and then a lysA gene fragment was extracted. This fragment was ligated with pCAB having been digested with HpaI (produced by Takara Shuzo) and blunt-ended to construct a plasmid comprising a combination of mutant lysC, dapA, dapB, and lysA autonomously replicable in coryneform bacteria. The constructed plasmid was designated as pCABL. The process of construction of pCABL is shown in FIG. 13. It is noted that the lysA gene fragment is inserted into a HpaI site in a DNA fragment containing the dapB gene in pCABL, however, the HpaI site is located upstream from a promoter for the dapB gene (nucleotide numbers 611 to 616 in SEQ ID NO: 10), and the dapB gene is not decoupled.  
       EXAMPLE 12  
       [0171]    Construction of Plasmid Comprising Combination of Mutant lysC, dapA, dapB, ddh, and lysA  
         [0172]    pHSG299 was digested with XbaI and KpnI, which was ligated with p399DL comprising ddh and lysA having been digested with XbaI and KpnI. A constructed plasmid was designated as p299DL. p299DL was digested with XbaI and KpnI and blunt-ended. After the blunt end formation, a DNA fragment comprising ddh and lysA was extracted. This DNA fragment was ligated with the plasmid pCAB comprising the combination of mutant lysC, dapA, and dapB having been digested with HpaI and blunt-ended to construct a plasmid comprising a combination of mutant lysC, dapA, dapB, lysA and ddh autonomously replicable in coryneform bacteria. The constructed plasmid was designated as pCABDL. The process of construction of pCABDL is shown in FIG. 14.  
       EXAMPLE 13  
       [0173]    Introduction of Plasmids Comprising Genes for L-Lysine Biosynthesis into L-Lysine-Producing Bacterium of  Brevibacterium lactofermentum    
         [0174]    The plasmids comprising the genes for L-lysine biosynthesis constructed as described above, namely p399AK9B(Cm r ), pDPSB(Km r ), pDPRB(Cm r ), pLYSAB(Cm r ), pPK4D(Cm r ), pCRCAB(Km r ), pAB(Cm r ), pCB(Cm r ), pDL(Cm r ), pCAB(Cm r ), pCABL(Cm r ), and pCABDL(Cm r ) were introduced into an L-lysine-producing bacterium AJ11082 (NRRL B-11470) of  Brevibacterium lactofermentum  respectively. AJ11082 strain has a property of AEC resistance. The plasmids were introduced in accordance with an electric pulse method (Sugimoto et al., Japanese Patent Laid-open No. 2-207791). Transformants were selected based on drug resistance markers possessed by the respective plasmids. Transformants were selected on a complete medium containing 5 μg/ml of chloramphenicol when a plasmid comprising a chloramphenicol resistance gene was introduced, or transformants were selected on a complete medium containing 25 μg/ml of kanamycin when a plasmid comprising a kanamycin resistance gene was introduced.  
       EXAMPLE 14  
     Production of L-Lysine  
       [0175]    Each of the transformants obtained in Example 13 was cultivated in an L-lysine-producing medium to evaluate its L-lysine productivity. The L-lysine-producing medium had the following composition.  
         [0176]    [L-Lysine-producing Medium] 
         [0177]    The following components other than calcium carbonate (per 1 L) were dissolved to make adjustment at pH 8.0 with KOH. The medium was sterilized at 115° C. for 15 minutes, to which calcium carbonate (50 g) having been separately sterilized in hot air in a dry state was thereafter added.  
                                                           Glucose   100   g           (NH 4 ) 2 SO 4     55   g           KH 2 PO 4     1   g           MgSO 4 .7H 2 O   1   g           Biotin   500   μg           Thiamin   2000   μg           FeSO 4 .7H 2 O   0.01   g           MnSO 4 .7H 2 O   0.01   g           Nicotinamide   5   mg           Protein hydrolysate (Mamenou)   30   ml           Calcium carbonate   50   g                      
 
         [0178]    Each of the various types of the transformants and the parent strain was inoculated to the medium having the composition described above to perform cultivation at 31.5° C. with reciprocating shaking. The amount of produced L-lysine after 40 or 72 hours of cultivation, and the growth after 72 hours (OD 562 ) are shown in Table 1. In the table, lysC* represents mutant lysC. The growth was quantitatively determined by measuring OD at 560 nm after 101-fold dilution.  
                                                   TABLE 1                           Accumulation of L-Lysine after Cultivation for 40 or 72 Hours                Amount of               produced           L-lysine(g/L)            Bacterial   Introduced   after   after   Growth       strain/plasmid   gene   40 hrs   72 hrs   (OD 562 /101)               AJ11082       22.0   29.8   0.450       AJ11082/p399AK9B     lysC*     16.8   34.5   0.398       AJ11082/pDPSB     dapA     18.7   33.8   0.410       AJ11082/pDRB     dapB     19.9   29.9   0.445       AJ11082/pLYSAB     lysA     19.8   32.5   0.356       AJ11082/pPK4D     ddh     19.0   33.4   0.330       AJ11082/pCRCAB     lysC* ,  dapA     19.7   36.5   0.360       AJ11082/pAB     dapA ,  dapB     19.0   34.8   0.390       AJ11082/pCB     lysC* ,  dapB     23.3   35.0   0.440       AJ11082/pDL     ddh ,  lysA     23.3   31.6   0.440       AJ11082/pCAB     lysC* ,  dapA ,   23.0   45.0   0.425             dapB         AJ11082/pCABL     lysC* ,  dapA ,   26.2   46.5   0.379             dapB ,  lysA         AJ11082/pCABDL     lysC* ,  dapA ,   26.5   47.0   0.409             dapB ,  lysA ,             ddh                    
 
         [0179]    As shown in Table 1, when mutant lysC, dapA, or dapB was enhanced singly, the amount of produced L-lysine was larger than or equivalent to that produced by the parent strain after 72 hours of cultivation, however, the amount of produced L-lysine was smaller than that produced by the parent strain after 40 hours of cultivation. Namely, the L-lysine-producing speed was lowered in cultivation for a short period. Similarly, when mutant lysC and dapA, or dapA and dapB were enhanced in combination, the amount of produced L-lysine was larger than that produced by the parent strain after 72 hours of cultivation, however, the amount of produced L-lysine was smaller than that produced by the parent strain after 40 hours of cultivation. Thus the L-lysine-producing speed was lowered.  
         [0180]    On the other hand, when lysA or ddh was enhanced singly, or when lysA and ddh were enhanced in combination, the amount of produced L-lysine was larger than that produced by the parent strain after 40 hours of cultivation, however, the amount of produced L-lysine was consequently smaller than that produced by the parent strain after the long period of cultivation because of decrease in growth.  
         [0181]    On the contrary, in the case of the strain in which dapB was enhanced together with mutant lysC, the growth was improved, the L-lysine-producing speed was successfully restored in the short period of cultivation, and the accumulated amount of L-lysine was also improved in the long period of cultivation. In the case of the strain in which three of mutant lysC, dapA, and dapB were simultaneously enhanced, the L-lysine productivity was further improved. Both of the L-lysine-producing speed and the amount of accumulated L-lysine were improved in a stepwise manner by successively enhancing lysA and ddh.  
       Industrial Applicability  
       [0182]    According to the present invention, the L-lysine-producing ability of coryneform bacteria can be improved, and the growth speed can be also improved.  
         [0183]    The L-lysine-producing speed can be improved, and the productivity can be also improved in coryneform L-lysine-producing bacteria by enhancing dapB together with mutant lysC. The L-lysine-producing speed and the productivity can be further improved by successively enhancing dapA, lysA, and ddh in addition to the aforementioned genes.   
     
       
       
         1 
         
           
             
24 
 
           
           
             
               23 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             NO  
             1 

TCGCGAAGTA GCACCTGTCA CTT                                             23 

 
           
           
             
               21 bases  
               nucleic acid  
               single  
               linear  
             
             
               other..synthetic DNA  
               /desc = “synthetic DNA” 
             
             YES  
             2 

ACGGAATTCA ATCTTACGGC C                                               21 

 
           
           
             
               1643 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               DNA (genomic)  
             
             NO  
             
               Brevibacterium lactofermentum  
               ATCC 13869  
             
             3 

TCGCGAAGTA GCACCTGTCA CTTTTGTCTC AAATATTAAA TCGAATATCA ATATACGGTC     60 

TGTTTATTGG AACGCATCCC AGTGGCTGAG ACGCATCCGC TAAAGCCCCA GGAACCCTGT    120 

GCAGAAAGAA AACACTCCTC TGGCTAGGTA GACACAGTTT ATAAAGGTAG AGTTGAGCGG    180 

GTAACTGTCA GCACGTAGAT CGAAAGGTGC ACAAAGGTGG CCCTGGTCGT ACAGAAATAT    240 

GGCGGTTCCT CGCTTGAGAG TGCGGAACGC ATTAGAAACG TCGCTGAACG GATCGTTGCC    300 

ACCAAGAAGG CTGGAAATGA TGTCGTGGTT GTCTGCTCCG CAATGGGAGA CACCACGGAT    360 

GAACTTCTAG AACTTGCAGC GGCAGTGAAT CCCGTTCCGC CAGCTCGTGA AATGGATATG    420 

CTCCTGACTG CTGGTGAGCG TATTTCTAAC GCTCTCGTCG CCATGGCTAT TGAGTCCCTT    480 

GGCGCAGAAG CTCAATCTTT CACTGGCTCT CAGGCTGGTG TGCTCACCAC CGAGCGCCAC    540 

GGAAACGCAC GCATTGTTGA CGTCACACCG GGTCGTGTGC GTGAAGCACT CGATGAGGGC    600 

AAGATCTGCA TTGTTGCTGG TTTTCAGGGT GTTAATAAAG AAACCCGCGA TGTCACCACG    660 

TTGGGTCGTG GTGGTTCTGA CACCACTGCA GTTGCGTTGG CAGCTGCTTT GAACGCTGAT    720 

GTGTGTGAGA TTTACTCGGA CGTTGACGGT GTGTATACCG CTGACCCGCG CATCGTTCCT    780 

AATGCACAGA AGCTGGAAAA GCTCAGCTTC GAAGAAATGC TGGAACTTGC TGCTGTTGGC    840 

TCCAAGATTT TGGTGCTGCG CAGTGTTGAA TACGCTCGTG CATTCAATGT GCCACTTCGC    900 

GTACGCTCGT CTTATAGTAA TGATCCCGGC ACTTTGATTG CCGGCTCTAT GGAGGATATT    960 

CCTGTGGAAG AAGCAGTCCT TACCGGTGTC GCAACCGACA AGTCCGAAGC CAAAGTAACC   1020 

GTTCTGGGTA TTTCCGATAA GCCAGGCGAG GCTGCCAAGG TTTTCCGTGC GTTGGCTGAT   1080 

GCAGAAATCA ACATTGACAT GGTTCTGCAG AACGTCTCCT CTGTGGAAGA CGGCACCACC   1140 

GACATCACGT TCACCTGCCC TCGCGCTGAC GGACGCCGTG CGATGGAGAT CTTGAAGAAG   1200 

CTTCAGGTTC AGGGCAACTG GACCAATGTG CTTTACGACG ACCAGGTCGG CAAAGTCTCC   1260 

CTCGTGGGTG CTGGCATGAA GTCTCACCCA GGTGTTACCG CAGAGTTCAT GGAAGCTCTG   1320 

CGCGATGTCA ACGTGAACAT CGAATTGATT TCCACCTCTG AGATCCGCAT TTCCGTGCTG   1380 

ATCCGTGAAG ATGATCTGGA TGCTGCTGCA CGTGCATTGC ATGAGCAGTT CCAGCTGGGC   1440 

GGCGAAGACG AAGCCGTCGT TTATGCAGGC ACCGGACGCT AAAGTTTTAA AGGAGTAGTT   1500 

TTACAATGAC CACCATCGCA GTTGTTGGTG CAACCGGCCA GGTCGGCCAG GTTATGCGCA   1560 

CCCTTTTGGA AGAGCGCAAT TTCCCAGCTG ACACTGTTCG TTTCTTTGCT TCCCCGCGTT   1620 

CCGCAGGCCG TAAGATTGAA TTC                                           1643 

 
           
           
             
               1643 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               DNA (genomic)  
             
             NO  
             
               Brevibacterium lactofermentum  
               ATCC 13869  
             
             
               CDS  
                217..1482 

 
             
             4 

TCGCGAAGTA GCACCTGTCA CTTTTGTCTC AAATATTAAA TCGAATATCA ATATACGGTC     60 

TGTTTATTGG AACGCATCCC AGTGGCTGAG ACGCATCCGC TAAAGCCCCA GGAACCCTGT    120 

GCAGAAAGAA AACACTCCTC TGGCTAGGTA GACACAGTTT ATAAAGGTAG AGTTGAGCGG    180 

GTAACTGTCA GCACGTAGAT CGAAAGGTGC ACAAAG GTG GCC CTG GTC GTA CAG      234 
                                        Met Ala Leu Val Val Gln 
                                          1               5 

AAA TAT GGC GGT TCC TCG CTT GAG AGT GCG GAA CGC ATT AGA AAC GTC      282 
Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala Glu Arg Ile Arg Asn Val 
             10                  15                  20 

GCT GAA CGG ATC GTT GCC ACC AAG AAG GCT GGA AAT GAT GTC GTG GTT      330 
Ala Glu Arg Ile Val Ala Thr Lys Lys Ala Gly Asn Asp Val Val Val 
         25                  30                  35 

GTC TGC TCC GCA ATG GGA GAC ACC ACG GAT GAA CTT CTA GAA CTT GCA      378 
Val Cys Ser Ala Met Gly Asp Thr Thr Asp Glu Leu Leu Glu Leu Ala 
     40                  45                  50 

GCG GCA GTG AAT CCC GTT CCG CCA GCT CGT GAA ATG GAT ATG CTC CTG      426 
Ala Ala Val Asn Pro Val Pro Pro Ala Arg Glu Met Asp Met Leu Leu 
 55                  60                  65                  70 

ACT GCT GGT GAG CGT ATT TCT AAC GCT CTC GTC GCC ATG GCT ATT GAG      474 
Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu Val Ala Met Ala Ile Glu 
                 75                  80                  85 

TCC CTT GGC GCA GAA GCT CAA TCT TTC ACT GGC TCT CAG GCT GGT GTG      522 
Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr Gly Ser Gln Ala Gly Val 
             90                  95                 100 

CTC ACC ACC GAG CGC CAC GGA AAC GCA CGC ATT GTT GAC GTC ACA CCG      570 
Leu Thr Thr Glu Arg His Gly Asn Ala Arg Ile Val Asp Val Thr Pro 
        105                 110                 115 

GGT CGT GTG CGT GAA GCA CTC GAT GAG GGC AAG ATC TGC ATT GTT GCT      618 
Gly Arg Val Arg Glu Ala Leu Asp Glu Gly Lys Ile Cys Ile Val Ala 
    120                 125                 130 

GGT TTT CAG GGT GTT AAT AAA GAA ACC CGC GAT GTC ACC ACG TTG GGT      666 
Gly Phe Gln Gly Val Asn Lys Glu Thr Arg Asp Val Thr Thr Leu Gly 
135                 140                 145                 150 

CGT GGT GGT TCT GAC ACC ACT GCA GTT GCG TTG GCA GCT GCT TTG AAC      714 
Arg Gly Gly Ser Asp Thr Thr Ala Val Ala Leu Ala Ala Ala Leu Asn 
                155                 160                 165 

GCT GAT GTG TGT GAG ATT TAC TCG GAC GTT GAC GGT GTG TAT ACC GCT      762 
Ala Asp Val Cys Glu Ile Tyr Ser Asp Val Asp Gly Val Tyr Thr Ala 
            170                 175                 180 

GAC CCG CGC ATC GTT CCT AAT GCA CAG AAG CTG GAA AAG CTC AGC TTC      810 
Asp Pro Arg Ile Val Pro Asn Ala Gln Lys Leu Glu Lys Leu Ser Phe 
        185                 190                 195 

GAA GAA ATG CTG GAA CTT GCT GCT GTT GGC TCC AAG ATT TTG GTG CTG      858 
Glu Glu Met Leu Glu Leu Ala Ala Val Gly Ser Lys Ile Leu Val Leu 
    200                 205                 210 

CGC AGT GTT GAA TAC GCT CGT GCA TTC AAT GTG CCA CTT CGC GTA CGC      906 
Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn Val Pro Leu Arg Val Arg 
215                 220                 225                 230 

TCG TCT TAT AGT AAT GAT CCC GGC ACT TTG ATT GCC GGC TCT ATG GAG      954 
Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu Ile Ala Gly Ser Met Glu 
                235                 240                 245 

GAT ATT CCT GTG GAA GAA GCA GTC CTT ACC GGT GTC GCA ACC GAC AAG     1002 
Asp Ile Pro Val Glu Glu Ala Val Leu Thr Gly Val Ala Thr Asp Lys 
            250                 255                 260 

TCC GAA GCC AAA GTA ACC GTT CTG GGT ATT TCC GAT AAG CCA GGC GAG     1050 
Ser Glu Ala Lys Val Thr Val Leu Gly Ile Ser Asp Lys Pro Gly Glu 
        265                 270                 275 

GCT GCC AAG GTT TTC CGT GCG TTG GCT GAT GCA GAA ATC AAC ATT GAC     1098 
Ala Ala Lys Val Phe Arg Ala Leu Ala Asp Ala Glu Ile Asn Ile Asp 
    280                 285                 290 

ATG GTT CTG CAG AAC GTC TCC TCT GTG GAA GAC GGC ACC ACC GAC ATC     1146 
Met Val Leu Gln Asn Val Ser Ser Val Glu Asp Gly Thr Thr Asp Ile 
295                 300                 305                 310 

ACG TTC ACC TGC CCT CGC GCT GAC GGA CGC CGT GCG ATG GAG ATC TTG     1194 
Thr Phe Thr Cys Pro Arg Ala Asp Gly Arg Arg Ala Met Glu Ile Leu 
                315                 320                 325 

AAG AAG CTT CAG GTT CAG GGC AAC TGG ACC AAT GTG CTT TAC GAC GAC     1242 
Lys Lys Leu Gln Val Gln Gly Asn Trp Thr Asn Val Leu Tyr Asp Asp 
            330                 335                 340 

CAG GTC GGC AAA GTC TCC CTC GTG GGT GCT GGC ATG AAG TCT CAC CCA     1290 
Gln Val Gly Lys Val Ser Leu Val Gly Ala Gly Met Lys Ser His Pro 
        345                 350                 355 

GGT GTT ACC GCA GAG TTC ATG GAA GCT CTG CGC GAT GTC AAC GTG AAC     1338 
Gly Val Thr Ala Glu Phe Met Glu Ala Leu Arg Asp Val Asn Val Asn 
    360                 365                 370 

ATC GAA TTG ATT TCC ACC TCT GAG ATC CGC ATT TCC GTG CTG ATC CGT     1386 
Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg Ile Ser Val Leu Ile Arg 
375                 380                 385                 390 

GAA GAT GAT CTG GAT GCT GCT GCA CGT GCA TTG CAT GAG CAG TTC CAG     1434 
Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala Leu His Glu Gln Phe Gln 
                395                 400                 405 

CTG GGC GGC GAA GAC GAA GCC GTC GTT TAT GCA GGC ACC GGA CGC TAA     1482 
Leu Gly Gly Glu Asp Glu Ala Val Val Tyr Ala Gly Thr Gly Arg 
            410                 415                 420 

AGTTTTAAAG GAGTAGTTTT ACAATGACCA CCATCGCAGT TGTTGGTGCA ACCGGCCAGG   1542 

TCGGCCAGGT TATGCGCACC CTTTTGGAAG AGCGCAATTT CCCAGCTGAC ACTGTTCGTT   1602 

TCTTTGCTTC CCCGCGTTCC GCAGGCCGTA AGATTGAATT C                       1643 

 
           
           
             
               421 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             5 

Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala 
  1              5                  10                  15 

Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala 
             20                  25                  30 

Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp 
         35                  40                  45 

Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg 
     50                  55                  60 

Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu 
 65                  70                  75                  80 

Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr 
                 85                  90                  95 

Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg 
            100                 105                 110 

Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly 
        115                 120                 125 

Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg 
    130                 135                 140 

Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala 
145                 150                 155                160 

Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val 
                165                 170                 175 

Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys 
            180                 185                 190 

Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly 
        195                 200                 205 

Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn 
    210                 215                 220 

Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu 
225                 230                 235                 240 

Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr 
                245                 250                 255 

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

Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp 
        275                 280                 285 

Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu 
    290                 295                 300 

Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ala Asp Gly Arg 
305                 310                 315                 320 

Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr 
                325                 330                 335 

Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala 
            340                 345                 350 

Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu 
        355                 360                 365 

Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg 
    370                 375                 380 

Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala 
385                 390                 395                 400 

Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr 
                405                 410                 415 

Ala Gly Thr Gly Arg 
            420 

 
           
           
             
               1643 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               DNA (genomic)  
             
             NO  
             
               Brevibacterium lactofermentum  
               ATCC 13869  
             
             
               CDS  
                964..1482 

 
             
             6 

TCGCGAAGTA GCACCTGTCA CTTTTGTCTC AAATATTAAA TCGAATATCA ATATACGGTC     60 

TGTTTATTGG AACGCATCCC AGTGGCTGAG ACGCATCCGC TAAAGCCCCA GGAACCCTGT    120 

GCAGAAAGAA AACACTCCTC TGGCTAGGTA GACACAGTTT ATAAAGGTAG AGTTGAGCGG    180 

GTAACTGTCA GCACGTAGAT CGAAAGGTGC ACAAAGGTGG CCCTGGTCGT ACAGAAATAT    240 

GGCGGTTCCT CGCTTGAGAG TGCGGAACGC ATTAGAAACG TCGCTGAACG GATCGTTGCC    300 

ACCAAGAAGG CTGGAAATGA TGTCGTGGTT GTCTGCTCCG CAATGGGAGA CACCACGGAT    360 

GAACTTCTAG AACTTGCAGC GGCAGTGAAT CCCGTTCCGC CAGCTCGTGA AATGGATATG    420 

CTCCTGACTG CTGGTGAGCG TATTTCTAAC GCTCTCGTCG CCATGGCTAT TGAGTCCCTT    480 

GGCGCAGAAG CTCAATCTTT CACTGGCTCT CAGGCTGGTG TGCTCACCAC CGAGCGCCAC    540 

GGAAACGCAC GCATTGTTGA CGTCACACCG GGTCGTGTGC GTGAAGCACT CGATGAGGGC    600 

AAGATCTGCA TTGTTGCTGG TTTTCAGGGT GTTAATAAAG AAACCCGCGA TGTCACCACG    660 

TTGGGTCGTG GTGGTTCTGA CACCACTGCA GTTGCGTTGG CAGCTGCTTT GAACGCTGAT    720 

GTGTGTGAGA TTTACTCGGA CGTTGACGGT GTGTATACCG CTGACCCGCG CATCGTTCCT    780 

AATGCACAGA AGCTGGAAAA GCTCAGCTTC GAAGAAATGC TGGAACTTGC TGCTGTTGGC    840 

TCCAAGATTT TGGTGCTGCG CAGTGTTGAA TACGCTCGTG CATTCAATGT GCCACTTCGC    900 

GTACGCTCGT CTTATAGTAA TGATCCCGGC ACTTTGATTG CCGGCTCTAT GGAGGATATT    960 

CCT  GTG GAA GAA GCA GTC CTT ACC GGT GTC GCA ACC GAC AAG TCC GAA    1008 
     Met Glu Glu Ala Val Leu Thr Gly Val Ala Thr Asp Lys Ser Glu 
       1               5                  10                  15 

GCC AAA GTA ACC GTT CTG GGT ATT TCC GAT AAG CCA GGC GAG GCT GCC     1056 
Ala Lys Val Thr Val Leu Gly Ile Ser Asp Lys Pro Gly Glu Ala Ala 
                 20                  25                  30 

AAG GTT TTC CGT GCG TTG GCT GAT GCA GAA ATC AAC ATT GAC ATG GTT     1104 
Lys Val Phe Arg Ala Leu Ala Asp Ala Glu Ile Asn Ile Asp Met Val 
             35                  40                  45 

CTG CAG AAC GTC TCC TCT GTG GAA GAC GGC ACC ACC GAC ATC ACG TTC     1152 
Leu Gln Asn Val Ser Ser Val Glu Asp Gly Thr Thr Asp Ile Thr Phe 
         50                  55                  60 

ACC TGC CCT CGC GCT GAC GGA CGC CGT GCG ATG GAG ATC TTG AAG AAG     1200 
Thr Cys Pro Arg Ala Asp Gly Arg Arg Ala Met Glu Ile Leu Lys Lys 
    65                   70                  75 

CTT CAG GTT CAG GGC AAC TGG ACC AAT GTG CTT TAC GAC GAC CAG GTC     1248 
Leu Gln Val Gln Gly Asn Trp Thr Asn Val Leu Tyr Asp Asp Gln Val 
80                   85                  90                  95 

GGC AAA GTC TCC CTC GTG GGT GCT GGC ATG AAG TCT CAC CCA GGT GTT     1296 
Gly Lys Val Ser Leu Val Gly Ala Gly Met Lys Ser His Pro Gly Val 
                100                 105                 110 

ACC GCA GAG TTC ATG GAA GCT CTG CGC GAT GTC AAC GTG AAC ATC GAA     1344 
Thr Ala Glu Phe Met Glu Ala Leu Arg Asp Val Asn Val Asn Ile Glu 
           115                  120                 125 

TTG ATT TCC ACC TCT GAG ATC CGC ATT TCC GTG CTG ATC CGT GAA GAT     1392 
Leu Ile Ser Thr Ser Glu Ile Arg Ile Ser Val Leu Ile Arg Glu Asp 
        130                 135                 140 

GAT CTG GAT GCT GCT GCA CGT GCA TTG CAT GAG CAG TTC CAG CTG GGC     1440 
Asp Leu Asp Ala Ala Ala Arg Ala Leu His Glu Gln Phe Gln Leu Gly 
    145                 150                 155 

GGC GAA GAC GAA GCC GTC GTT TAT GCA GGC ACC GGA CGC TAAAGTTTTAA     1490 
Gly Glu Asp Glu Ala Val Val Tyr Ala Gly Thr Gly Arg 
160                 165                 170 

AGGAGTAGTT TTACAATGAC CACCATCGCA GTTGTTGGTG CAACCGGCCA GGTCGGCCAG   1550 

GTTATGCGCA CCCTTTTGGA AGAGCGCAAT TTCCCAGCTG ACACTGTTCG TTTCTTTGCT   1610 

TCCCCGCGTT CCGCAGGCCG TAAGATTGAA TTC                                1643 

 
           
           
             
               172 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             7 

Met Glu Glu Ala Val Leu Thr Gly Val Ala Thr Asp Lys Ser Glu Ala 
  1               5                  10                  15 

Lys Val Thr Val Leu Gly Ile Ser Asp Lys Pro Gly Glu Ala Ala Lys 
             20                  25                  30 

Val Phe Arg Ala Leu Ala Asp Ala Glu Ile Asn Ile Asp Met Val Leu 
         35                  40                  45 

Gln Asn Val Ser Ser Val Glu Asp Gly Thr Thr Asp Ile Thr Phe Thr 
     50                  55                  60 

Cys Pro Arg Ala Asp Gly Arg Arg Ala Met Glu Ile Leu Lys Lys Leu 
 65                  70                  75                  80 

Gln Val Gln Gly Asn Trp Thr Asn Val Leu Tyr Asp Asp Gln Val Gly 
                 85                  90                  95 

Lys Val Ser Leu Val Gly Ala Gly Met Lys Ser His Pro Gly Val Thr 
            100                 105                 110 

Ala Glu Phe Met Glu Ala Leu Arg Asp Val Asn Val Asn Ile Glu Leu 
        115                 120                 125 

Ile Ser Thr Ser Glu Ile Arg Ile Ser Val Leu Ile Arg Glu Asp Asp 
    130                 135                 140 

Leu Asp Ala Ala Ala Arg Ala Leu His Glu Gln Phe Gln Leu Gly Gly 
145                 150                 155                160 

Glu Asp Glu Ala Val Val Tyr Ala Gly Thr Gly Arg 
                165                 170 

 
           
           
             
               23 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             NO  
             8 

GGATCCCCAA TCGATACCTG GAA                                             23 

 
           
           
             
               23 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             YES  
             9 

CGGTTCATCG CCAAGTTTTT CTT                                             23 

 
           
           
             
               2001 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               DNA (genomic)  
             
             NO  
             
               Brevibacterium lactofermentum  
               ATCC 13869  
             
             
               CDS  
                730..1473 

 
             
             10 

GGATCCCCAA TCGATACCTG GAACGACAAC CTGATCAGGA TATCCAATGC CTTGAATATT     60 

GACGTTGAGG AAGGAATCAC CAGCCATCTC AACTGGAAGA CCTGACGCCT GCTGAATTGG    120 

ATCAGTGGCC CAATCGACCC ACCAACCAGG TTGGCTATTA CCGGCGATAT CAAAAACAAC    180 

TCGCGTGAAC GTTTCGTGCT CGGCAACGCG GATGCCAGCG ATCGACATAT CGGAGTCACC    240 

AACTTGAGCC TGCTGCTTCT GATCCATCGA CGGGGAACCC AACGGCGGCA AAGCAGTGGG    300 

GGAAGGGGAG TTGGTGGACT CTGAATCAGT GGGCTCTGAA GTGGTAGGCG ACGGGGCAGC    360 

ATCTGAAGGC GTGCGAGTTG TGGTGACCGG GTTAGCGGTT TCAGTTTCTG TCACAACTGG    420 

AGCAGGACTA GCAGAGGTTG TAGGCGTTGA GCCGCTTCCA TCACAAGCAC TTAAAAGTAA    480 

AGAGGCGGAA ACCACAAGCG CCAAGGAACT ACCTGCGGAA CGGGCGGTGA AGGGCAACTT    540 

AAGTCTCATA TTTCAAACAT AGTTCCACCT GTGTGATTAA TCTCCAGAAC GGAACAAACT    600 

GATGAACAAT CGTTAACAAC ACAGACCAAA ACGGTCAGTT AGGTATGGAT ATCAGCACCT    660 

TCTGAATGGG TACGTCTAGA CTGGTGGGCG TTTGAAAAAC TCTTCGCCCC ACGAAAATGA    720 

AGGAGCATA ATG GGA ATC AAG GTT GGC GTT CTC GGA GCC AAA GGC CGT        768 
          Met Gly Ile Lys Val Gly Val Leu Gly Ala Lys Gly Arg 
            1               5                  10 

GTT GGT CAA ACT ATT GTG GCA GCA GTC AAT GAG TCC GAC GAT CTG GAG      816 
Val Gly Gln Thr Ile Val Ala Ala Val Asn Glu Ser Asp Asp Leu Glu 
     15                  20                  25 

CTT GTT GCA GAG ATC GGC GTC GAC GAT GAT TTG AGC CTT CTG GTA GAC      864 
Leu Val Ala Glu Ile Gly Val Asp Asp Asp Leu Ser Leu Leu Val Asp 
 30                  35                  40                  45 

AAC GGC GCT GAA GTT GTC GTT GAC TTC ACC ACT CCT AAC GCT GTG ATG      912 
Asn Gly Ala Glu Val Val Val Asp Phe Thr Thr Pro Asn Ala Val Met 
                 50                  55                  60 

GGC AAC CTG GAG TTC TGC ATC AAC AAC GGC ATT TCT GCG GTT GTT GGA      960 
Gly Asn Leu Glu Phe Cys Ile Asn Asn Gly Ile Ser Ala Val Val Gly 
             65                  70                  75 

ACC ACG GGC TTC GAT GAT GCT CGT TTG GAG CAG GTT CGC GCC TGG CTT     1008 
Thr Thr Gly Phe Asp Asp Ala Arg Leu Glu Gln Val Arg Ala Trp Leu 
         80                  85                  90 

GAA GGA AAA GAC AAT GTC GGT GTT CTG ATC GCA CCT AAC TTT GCT ATC     1056 
Glu Gly Lys Asp Asn Val Gly Val Leu Ile Ala Pro Asn Phe Ala Ile 
     95                 100                 105 

TCT GCG GTG TTG ACC ATG GTC TTT TCC AAG CAG GCT GCC CGC TTC TTC     1104 
Ser Ala Val Leu Thr Met Val Phe Ser Lys Gln Ala Ala Arg Phe Phe 
110                 115                 120                 125 

GAA TCA GCT GAA GTT ATT GAG CTG CAC CAC CCC AAC AAG CTG GAT GCA     1152 
Glu Ser Ala Glu Val Ile Glu Leu His His Pro Asn Lys Leu Asp Ala 
                130                 135                 140 

CCT TCA GGC ACC GCG ATC CAC ACT GCT CAG GGC ATT GCT GCG GCA CGC     1200 
Pro Ser Gly Thr Ala Ile His Thr Ala Gln Gly Ile Ala Ala Ala Arg 
            145                 150                 155 

AAA GAA GCA GGC ATG GAC GCA CAG CCA GAT GCG ACC GAG CAG GCA CTT     1248 
Lys Glu Ala Gly Met Asp Ala Gln Pro Asp Ala Thr Glu Gln Ala Leu 
        160                 165                 170 

GAG GGT TCC CGT GGC GCA AGC GTA GAT GGA ATC CCA GTT CAC GCA GTC     1296 
Glu Gly Ser Arg Gly Ala Ser Val Asp Gly Ile Pro Val His Ala Val 
    175                 180                 185 

CGC ATG TCC GGC ATG GTT GCT CAC GAG CAA GTT ATC TTT GGC ACC CAG     1344 
Arg Met Ser Gly Met Val Ala His Glu Gln Val Ile Phe Gly Thr Gln 
190                 195                 200                 205 

GGT CAG ACC TTG ACC ATC AAG CAG GAC TCC TAT GAT CGC AAC TCA TTT     1392 
Gly Gln Thr Leu Thr Ile Lys Gln Asp Ser Tyr Asp Arg Asn Ser Phe 
                210                 215                 220 

GCA CCA GGT GTC TTG GTG GGT GTG CGC AAC ATT GCA CAG CAC CCA GGC     1440 
Ala Pro Gly Val Leu Val Gly Val Arg Asn Ile Ala Gln His Pro Gly 
            225                 230                 235 

CTA GTC GTA GGA CTT GAG CAT TAC CTA GGC CTG TAAAGGCTCA TTTCAGCAGC   1493 
Leu Val Val Gly Leu Glu His Tyr Leu Gly Leu 
        240                 245 

GGGTGGAATT TTTTAAAAGG AGCGTTTAAA GGCTGTGGCC GAACAAGTTA AATTGAGCGT   1553 

GGAGTTGATA GCGTGCAGTT CTTTTACTCC ACCCGCTGAT GTTGAGTGGT CAACTGATGT   1613 

TGAGGGCGCG GAAGCACTCG TCGAGTTTGC GGGTCGTGCC TGCTACGAAA CTTTTGATAA   1673 

GCCGAACCCT CGAACTGCTT CCAATGCTGC GTATCTGCGC CACATCATGG AAGTGGGGCA   1733 

CACTGCTTTG CTTGAGCATG CCAATGCCAC GATGTATATC CGAGGCATTT CTCGGTCCGC   1793 

GACCCATGAA TTGGTCCGAC ACCGCCATTT TTCCTTCTCT CAACTGTCTC AGCGTTTCGT   1853 

GCACAGCGGA GAATCGGAAG TAGTGGTGCC CACTCTCATC GATGAAGATC CGCAGTTGCG   1913 

TGAACTTTTC ATGCACGCCA TGGATGAGTC TCGGTTCGCT TTCAATGAGC TGCTTAATGC   1973 

GCTGGAAGAA AAACTTGGCG ATGAACCG                                      2001 

 
           
           
             
               248 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             11 

Met Gly Ile Lys Val Gly Val Leu Gly Ala Lys Gly Arg Val Gly Gln 
  1               5                  10                  15 

Thr Ile Val Ala Ala Val Asn Glu Ser Asp Asp Leu Glu Leu Val Ala 
             20                  25                  30 

Glu Ile Gly Val Asp Asp Asp Leu Ser Leu Leu Val Asp Asn Gly Ala 
         35                  40                  45 

Glu Val Val Val Asp Phe Thr Thr Pro Asn Ala Val Met Gly Asn Leu 
     50                  55                  60 

Glu Phe Cys Ile Asn Asn Gly Ile Ser Ala Val Val Gly Thr Thr Gly 
 65                  70                  75                  80 

Phe Asp Asp Ala Arg Leu Glu Gln Val Arg Ala Trp Leu Glu Gly Lys 
                 85                  90                  95 

Asp Asn Val Gly Val Leu Ile Ala Pro Asn Phe Ala Ile Ser Ala Val 
            100                 105                 110 

Leu Thr Met Val Phe Ser Lys Gln Ala Ala Arg Phe Phe Glu Ser Ala 
        115                 120                 125 

Glu Val Ile Glu Leu His His Pro Asn Lys Leu Asp Ala Pro Ser Gly 
    130                 135                 140 

Thr Ala Ile His Thr Ala Gln Gly Ile Ala Ala Ala Arg Lys Glu Ala 
145                 150                 155                 160 

Gly Met Asp Ala Gln Pro Asp Ala Thr Glu Gln Ala Leu Glu Gly Ser 
                165                 170                 175 

Arg Gly Ala Ser Val Asp Gly Ile Pro Val His Ala Val Arg Met Ser 
            180                 185                 190 

Gly Met Val Ala His Glu Gln Val Ile Phe Gly Thr Gln Gly Gln Thr 
        195                 200                 205 

Leu Thr Ile Lys Gln Asp Ser Tyr Asp Arg Asn Ser Phe Ala Pro Gly 
    210                 215                 220 

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

Gly Leu Glu His Tyr Leu Gly Leu 
                245 

 
           
           
             
               23 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             NO  
             12 

GTCGACGGAT CGCAAATGGC AAC                                             23 

 
           
           
             
               23 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             YES  
             13 

GGATCCTTGA GCACCTTGCG CAG                                             23 

 
           
           
             
               1411 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               DNA (genomic)  
             
             NO  
             
               Brevibacterium lactofermentum  
               ATCC 13869  
             
             
               CDS  
                311..1213 

 
             
             14 

CTCTCGATAT CGAGAGAGAA GCAGCGCCAC GGTTTTTCGG TGATTTTGAG ATTGAAACTT     60 

TGGCAGACGG ATCGCAAATG GCAACAAGCC CGTATGTCAT GGACTTTTAA CGCAAAGCTC    120 

ACACCCACGA GCTAAAAATT CATATAGTTA AGACAACATT TTTGGCTGTA AAAGACAGCC    180 

GTAAAAACCT CTTGCTCATG TCAATTGTTC TTATCGGAAT GTGGCTTGGG CGATTGTTAT    240 

GCAAAAGTTG TTAGGTTTTT TGCGGGGTTG TTTAACCCCC AAATGAGGGA AGAAGGTAAC    300 

CTTGAACTCT ATG AGC ACA GGT TTA ACA GCT AAG ACC GGA GTA GAG CAC       349 
           Met Ser Thr Gly Leu Thr Ala Lys Thr Gly Val Glu His 
             1               5                  10 

TTC GGC ACC GTT GGA GTA GCA ATG GTT ACT CCA TTC ACG GAA TCC GGA      397 
Phe Gly Thr Val Gly Val Ala Met Val Thr Pro Phe Thr Glu Ser Gly 
     15                  20                  25 

GAC ATC GAT ATC GCT GCT GGC CGC GAA GTC GCG GCT TAT TTG GTT GAT      445 
Asp Ile Asp Ile Ala Ala Gly Arg Glu Val Ala Ala Tyr Leu Val Asp 
 30                  35                  40                  45 

AAG GGC TTG GAT TCT TTG GTT CTC GCG GGC ACC ACT GGT GAA TCC CCA      493 
Lys Gly Leu Asp Ser Leu Val Leu Ala Gly Thr Thr Gly Glu Ser Pro 
                 50                  55                  60 

ACG ACA ACC GCC GCT GAA AAA CTA GAA CTG CTC AAG GCC GTT CGT GAG      541 
Thr Thr Thr Ala Ala Glu Lys Leu Glu Leu Leu Lys Ala Val Arg Glu 
             65                  70                  75 

GAA GTT GGG GAT CGG GCG AAC GTC ATC GCC GGT GTC GGA ACC AAC AAC      589 
Glu Val Gly Asp Arg Ala Asn Val Ile Ala Gly Val Gly Thr Asn Asn 
         80                  85                  90 

ACG CGG ACA TCT GTG GAA CTT GCG GAA GCT GCT GCT TCT GCT GGC GCA      637 
Thr Arg Thr Ser Val Glu Leu Ala Glu Ala Ala Ala Ser Ala Gly Ala 
     95                 100                 105 

GAC GGC CTT TTA GTT GTA ACT CCT TAT TAC TCC AAG CCG AGC CAA GAG      685 
Asp Gly Leu Leu Val Val Thr Pro Tyr Tyr Ser Lys Pro Ser Gln Glu 
110                 115                 120                 125 

GGA TTG CTG GCG CAC TTC GGT GCA ATT GCT GCA GCA ACA GAG GTT CCA      733 
Gly Leu Leu Ala His Phe Gly Ala Ile Ala Ala Ala Thr Glu Val Pro 
                130                 135                 140 

ATT TGT CTC TAT GAC ATT CCT GGT CGG TCA GGT ATT CCA ATT GAG TCT      781 
Ile Cys Leu Tyr Asp Ile Pro Gly Arg Ser Gly Ile Pro Ile Glu Ser 
            145                 150                 155 

GAT ACC ATG AGA CGC CTG AGT GAA TTA CCT ACG ATT TTG GCG GTC AAG      829 
Asp Thr Met Arg Arg Leu Ser Glu Leu Pro Thr Ile Leu Ala Val Lys 
        160                 165                 170 

GAC GCC AAG GGT GAC CTC GTT GCA GCC ACG TCA TTG ATC AAA GAA ACG      877 
Asp Ala Lys Gly Asp Leu Val Ala Ala Thr Ser Leu Ile Lys Glu Thr 
    175                 180                 185 

GGA CTT GCC TGG TAT TCA GGC GAT GAC CCA CTA AAC CTT GTT TGG CTT      925 
Gly Leu Ala Trp Tyr Ser Gly Asp Asp Pro Leu Asn Leu Val Trp Leu 
190                 195                 200                 205 

GCT TTG GGC GGA TCA GGT TTC ATT TCC GTA ATT GGA CAT GCA GCC CCC      973 
Ala Leu Gly Gly Ser Gly Phe Ile Ser Val Ile Gly His Ala Ala Pro 
                210                 215                 220 

ACA GCA TTA CGT GAG TTG TAC ACA AGC TTC GAG GAA GGC GAC CTC GTC     1021 
Thr Ala Leu Arg Glu Leu Tyr Thr Ser Phe Glu Glu Gly Asp Leu Val 
            225                 230                 235 

CGT GCG CGG GAA ATC AAC GCC AAA CTA TCA CCG CTG GTA GCT GCC CAA     1069 
Arg Ala Arg Glu Ile Asn Ala Lys Leu Ser Pro Leu Val Ala Ala Gln 
        240                 245                 250 

GGT CGC TTG GGT GGA GTC AGC TTG GCA AAA GCT GCT CTG CGT CTG CAG     1117 
Gly Arg Leu Gly Gly Val Ser Leu Ala Lys Ala Ala Leu Arg Leu Gln 
    255                 260                 265 

GGC ATC AAC GTA GGA GAT CCT CGA CTT CCA ATT ATG GCT CCA AAT GAG     1165 
Gly Ile Asn Val Gly Asp Pro Arg Leu Pro Ile Met Ala Pro Asn Glu 
270                 275                 280                 285 

CAG GAA CTT GAG GCT CTC CGA GAA GAC ATG AAA AAA GCT GGA GTT CTA     1213 
Gln Glu Leu Glu Ala Leu Arg Glu Asp Met Lys Lys Ala Gly Val Leu 
                290                 295                 300 

TAAATATGAA TGATTCCCGA AATCGCGGCC GGAAGGTTAC CCGCAAGGCG GCCCACCAGA   1273 

AGCTGGTCAG GAAAACCATC TGGATACCCC TGTCTTTCAG GCACCAGATG CTTCCTCTAA   1333 

CCAGAGCGCT GTAAAAGCTG AGACCGCCGG AAACGACAAT CGGGATGCTG CGCAAGGTGC   1393 

TCAAGGATCC CAACATTC                                                 1411 

 
           
           
             
               301 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             15 

Met Ser Thr Gly Leu Thr Ala Lys Thr Gly Val Glu His Phe Gly Thr 
  1               5                  10                  15 

Val Gly Val Ala Met Val Thr Pro Phe Thr Glu Ser Gly Asp Ile Asp 
             20                  25                  30 

Ile Ala Ala Gly Arg Glu Val Ala Ala Tyr Leu Val Asp Lys Gly Leu 
         35                  40                  45 

Asp Ser Leu Val Leu Ala Gly Thr Thr Gly Glu Ser Pro Thr Thr Thr 
     50                  55                  60 

Ala Ala Glu Lys Leu Glu Leu Leu Lys Ala Val Arg Glu Glu Val Gly 
 65                  70                  75                  80 

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

Ser Val Glu Leu Ala Glu Ala Ala Ala Ser Ala Gly Ala Asp Gly Leu 
            100                 105                 110 

Leu Val Val Thr Pro Tyr Tyr Ser Lys Pro Ser Gln Glu Gly Leu Leu 
        115                 120                 125 

Ala His Phe Gly Ala Ile Ala Ala Ala Thr Glu Val Pro Ile Cys Leu 
    130                 135                 140 

Tyr Asp Ile Pro Gly Arg Ser Gly Ile Pro Ile Glu Ser Asp Thr Met 
145                 150                 155                 160 

Arg Arg Leu Ser Glu Leu Pro Thr Ile Leu Ala Val Lys Asp Ala Lys 
                165                 170                 175 

Gly Asp Leu Val Ala Ala Thr Ser Leu Ile Lys Glu Thr Gly Leu Ala 
            180                 185                 190 

Trp Tyr Ser Gly Asp Asp Pro Leu Asn Leu Val Trp Leu Ala Leu Gly 
        195                 200                 205 

Gly Ser Gly Phe Ile Ser Val Ile Gly His Ala Ala Pro Thr Ala Leu 
    210                 215                 220 

Arg Glu Leu Tyr Thr Ser Phe Glu Glu Gly Asp Leu Val Arg Ala Arg 
225                 230                 235                 240 

Glu Ile Asn Ala Lys Leu Ser Pro Leu Val Ala Ala Gln Gly Arg Leu 
                245                 250                 255 

Gly Gly Val Ser Leu Ala Lys Ala Ala Leu Arg Leu Gln Gly Ile Asn 
            260                 265                 270 

Val Gly Asp Pro Arg Leu Pro Ile Met Ala Pro Asn Glu Gln Glu Leu 
        275                 280                 285 

Glu Ala Leu Arg Glu Asp Met Lys Lys Ala Gly Val Leu 
    290                 295                 300 

 
           
           
             
               23 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             NO  
             16 

GTGGAGCCGA CCATTCCGCG AGG                                             23 

 
           
           
             
               23 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             YES  
             17 

CCAAAACCGC CCTCCACGGC GAA                                             23 

 
           
           
             
               3579 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               DNA (genomic)  
             
             NO  
             
               Brevibacterium lactofermentum  
               ATCC 13869  
             
             
               CDS  
                533..2182 

 
             
             
               CDS  
                2188..3522 

 
             
             18 

GTGGAGCCGA CCATTCCGCG AGGCTGCACT GCAACGAGGT CGTAGTTTTG GTACATGGCT     60 

TCTGGCCAGT TCATGGATTG GCTGCCGAAG AAGCTATAGG CATCGCACCA GGGCCACCGA    120 

GTTACCGAAG ATGGTGCCGT GCTTTTCGCC TTGGGCAGGG ACCTTGACAA AGCCCACGCT    180 

GATATCGCCA AGTGAGGGAT CAGAATAGTG CATGGGCACG TCGATGCTGC CACATTGAGC    240 

GGAGGCAATA TCTACCTGAG GTGGGCATTC TTCCCAGCGG ATGTTTTCTT GCGCTGCTGC    300 

AGTGGGCATT GATACCAAAA AGGGGCTAAG CGCAGTCGAG GCGGCAAGAA CTGCTACTAC    360 

CCTTTTTATT GTCGAACGGG GCATTACGGC TCCAAGGACG TTTGTTTTCT GGGTCAGTTA    420 

CCCCAAAAAG CATATACAGA GACCAATGAT TTTTCATTAA AAAGGCAGGG ATTTGTTATA    480 

AGTATGGGTC GTATTCTGTG CGACGGGTGT ACCTCGGCTA GAATTTCTCC CC ATG        535 
                                                          Met 

                                                            1 

ACA CCA GCT GAT CTC GCA ACA TTG ATT AAA GAG ACC GCG GTA GAG GTT      583 
Thr Pro Ala Asp Leu Ala Thr Leu Ile Lys Glu Thr Ala Val Glu Val 
              5                  10                  15 

TTG ACC TCC CGC GAG CTC GAT ACT TCT GTT CTT CCG GAG CAG GTA GTT      631 
Leu Thr Ser Arg Glu Leu Asp Thr Ser Val Leu Pro Glu Gln Val Val 
         20                  25                  30 

GTG GAG CGT CCG CGT AAC CCA GAG CAC GGC GAT TAC GCC ACC AAC ATT      679 
Val Glu Arg Pro Arg Asn Pro Glu His Gly Asp Tyr Ala Thr Asn Ile 
     35                  40                  45 

GCA TTG CAG GTG GCT AAA AAG GTC GGT CAG AAC CCT CGG GAT TTG GCT      727 
Ala Leu Gln Val Ala Lys Lys Val Gly Gln Asn Pro Arg Asp Leu Ala 
 50                  55                  60                  65 

ACC TGG CTG GCA GAG GCA TTG GCT GCA GAT GAC GCC ATT GAT TCT GCT      775 
Thr Trp Leu Ala Glu Ala Leu Ala Ala Asp Asp Ala Ile Asp Ser Ala 
                 70                  75                  80 

GAA ATT GCT GGC CCA GGC TTT TTG AAC ATT CGC CTT GCT GCA GCA GCA      823 
Glu Ile Ala Gly Pro Gly Phe Leu Asn Ile Arg Leu Ala Ala Ala Ala 
             85                  90                  95 

CAG GGT GAA ATT GTG GCC AAG ATT CTG GCA CAG GGC GAG ACT TTC GGA      871 
Gln Gly Glu Ile Val Ala Lys Ile Leu Ala Gln Gly Glu Thr Phe Gly 
        100                 105                 110 

AAC TCC GAT CAC CTT TCC CAC TTG GAC GTG AAC CTC GAG TTC GTT TCT      919 
Asn Ser Asp His Leu Ser His Leu Asp Val Asn Leu Glu Phe Val Ser 
    115                 120                 125 

GCA AAC CCA ACC GGA CCT ATT CAC CTT GGC GGA ACC CGC TGG GCT GCC      967 
Ala Asn Pro Thr Gly Pro Ile His Leu Gly Gly Thr Arg Trp Ala Ala 
130                 135                 140                 145 

GTG GGT GAC TCT TTG GGT CGT GTG CTG GAG GCT TCC GGC GCG AAA GTG     1015 
Val Gly Asp Ser Leu Gly Arg Val Leu Glu Ala Ser Gly Ala Lys Val 
                150                 155                 160 

ACC CGC GAA TAC TAC TTC AAC GAT CAC GGT CGC CAG ATC GAT CGT TTC     1063 
Thr Arg Glu Tyr Tyr Phe Asn Asp His Gly Arg Gln Ile Asp Arg Phe 
            165                 170                 175 

GCT TTG TCC CTT CTT GCA GCG GCG AAG GGC GAG CCA ACG CCA GAA GAC     1111 
Ala Leu Ser Leu Leu Ala Ala Ala Lys Gly Glu Pro Thr Pro Glu Asp 
        180                 185                 190 

GGT TAT GGC GGC GAA TAC ATT AAG GAA ATT GCG GAG GCA ATC GTC GAA     1159 
Gly Tyr Gly Gly Glu Tyr Ile Lys Glu Ile Ala Glu Ala Ile Val Glu 
    195                 200                 205 

AAG CAT CCT GAA GCG TTG GCT TTG GAG CCT GCC GCA ACC CAG GAG CTT     1207 
Lys His Pro Glu Ala Leu Ala Leu Glu Pro Ala Ala Thr Gln Glu Leu 
210                 215                 220                 225 

TTC CGC GCT GAA GGC GTG GAG ATG ATG TTC GAG CAC ATC AAA TCT TCC     1255 
Phe Arg Ala Glu Gly Val Glu Met Met Phe Glu His Ile Lys Ser Ser 
                230                 235                 240 

CTG CAT GAG TTC GGC ACC GAT TTC GAT GTC TAC TAC CAC GAG AAC TCC     1303 
Leu His Glu Phe Gly Thr Asp Phe Asp Val Tyr Tyr His Glu Asn Ser 
            245                 250                 255 

CTG TTC GAG TCC GGT GCG GTG GAC AAG GCC GTG CAG GTG CTG AAG GAC     1351 
Leu Phe Glu Ser Gly Ala Val Asp Lys Ala Val Gln Val Leu Lys Asp 
        260                 265                 270 

AAC GGC AAC CTG TAC GAA AAC GAG GGC GCT TGG TGG CTG CGT TCC ACC     1399 
Asn Gly Asn Leu Tyr Glu Asn Glu Gly Ala Trp Trp Leu Arg Ser Thr 
    275                 280                 285 

GAA TTC GGC GAT GAC AAA GAC CGC GTG GTG ATC AAG TCT GAC GGC GAC     1447 
Glu Phe Gly Asp Asp Lys Asp Arg Val Val Ile Lys Ser Asp Gly Asp 
290                 295                 300                 305 

GCA GCC TAC ATC GCT GGC GAT ATC GCG TAC GTG GCT GAT AAG TTC TCC     1495 
Ala Ala Tyr Ile Ala Gly Asp Ile Ala Tyr Val Ala Asp Lys Phe Ser 
                310                 315                 320 

CGC GGA CAC AAC CTA AAC ATC TAC ATG TTG GGT GCT GAC CAC CAT GGT     1543 
Arg Gly His Asn Leu Asn Ile Tyr Met Leu Gly Ala Asp His His Gly 
            325                 330                 335 

TAC ATC GCG CGC CTG AAG GCA GCG GCG GCG GCA CTT GGC TAC AAG CCA     1591 
Tyr Ile Ala Arg Leu Lys Ala Ala Ala Ala Ala Leu Gly Tyr Lys Pro 
        340                 345                 350 

GAA GGC GTT GAA GTC CTG ATT GGC CAG ATG GTG AAC CTG CTT CGC GAC     1639 
Glu Gly Val Glu Val Leu Ile Gly Gln Met Val Asn Leu Leu Arg Asp 
    355                 360                 365 

GGC AAG GCA GTG CGT ATG TCC AAG CGT GCA GGC ACC GTG GTC ACC CTA     1687 
Gly Lys Ala Val Arg Met Ser Lys Arg Ala Gly Thr Val Val Thr Leu 
370                 375                 380                 385 

GAT GAC CTC GTT GAA GCA ATC GGC ATC GAT GCG GCG CGT TAC TCC CTG     1735 
Asp Asp Leu Val Glu Ala Ile Gly Ile Asp Ala Ala Arg Tyr Ser Leu 
                390                 395                 400 

ATC CGT TCC TCC GTG GAT TCT TCC CTG GAT ATC GAT CTC GGC CTG TGG     1783 
Ile Arg Ser Ser Val Asp Ser Ser Leu Asp Ile Asp Leu Gly Leu Trp 
            405                 410                 415 

GAA TCC CAG TCC TCC GAC AAC CCT GTG TAC TAC GTG CAG TAC GGA CAC     1831 
Glu Ser Gln Ser Ser Asp Asn Pro Val Tyr Tyr Val Gln Tyr Gly His 
        420                 425                 430 

GCT CGT CTG TGC TCC ATC GCG CGC AAG GCA GAG ACC TTG GGT GTC ACC     1879 
Ala Arg Leu Cys Ser Ile Ala Arg Lys Ala Glu Thr Leu Gly Val Thr 
    435                 440                 445 

GAG GAA GGC GCA GAC CTA TCT CTA CTG ACC CAC GAC CGC GAA GGC GAT     1927 
Glu Glu Gly Ala Asp Leu Ser Leu Leu Thr His Asp Arg Glu Gly Asp 
450                 455                 460                 465 

CTC ATC CGC ACA CTC GGA GAG TTC CCA GCA GTG GTG AAG GCT GCC GCT     1975 
Leu Ile Arg Thr Leu Gly Glu Phe Pro Ala Val Val Lys Ala Ala Ala 
                470                 475                 480 

GAC CTA CGT GAA CCA CAC CGC ATT GCC CGC TAT GCT GAG GAA TTA GCT     2023 
Asp Leu Arg Glu Pro His Arg Ile Ala Arg Tyr Ala Glu Glu Leu Ala 
            485                 490                 495 

GGA ACT TTC CAC CGC TTC TAC GAT TCC TGC CAC ATC CTT CCA AAG GTT     2071 
Gly Thr Phe His Arg Phe Tyr Asp Ser Cys His Ile Leu Pro Lys Val 
        500                 505                 510 

GAT GAG GAT ACG GCA CCA ATC CAC ACA GCA CGT CTG GCA CTT GCA GCA     2119 
Asp Glu Asp Thr Ala Pro Ile His Thr Ala Arg Leu Ala Leu Ala Ala 
    515                 520                 525 

GCA ACC CGC CAG ACC CTC GCT AAC GCC CTG CAC CTG GTT GGC GTT TCC     2167 
Ala Thr Arg Gln Thr Leu Ala Asn Ala Leu His Leu Val Gly Val Ser 
530                 535                 540                 545 

GCA CCG GAG AAG ATG TAACA ATG GCT ACA GTT GAA AAT TTC AAT GAA       2214 
Ala Pro Glu Lys Met       Met Ala Thr Val Glu Asn Phe Asn Glu 
                550         1               5 

CTT CCC GCA CAC GTA TGG CCA CGC AAT GCC GTG CGC CAA GAA GAC GGC     2262 
Leu Pro Ala His Val Trp Pro Arg Asn Ala Val Arg Gln Glu Asp Gly 
 10                  15                  20                  25 

GTT GTC ACC GTC GCT GGT GTG CCT CTG CCT GAC CTC GCT GAA GAA TAC     2310 
Val Val Thr Val Ala Gly Val Pro Leu Pro Asp Leu Ala Glu Glu Tyr 
                 30                  35                  40 

GGA ACC CCA CTG TTC GTA GTC GAC GAG GAC GAT TTC CGT TCC CGC TGT     2358 
Gly Thr Pro Leu Phe Val Val Asp Glu Asp Asp Phe Arg Ser Arg Cys 
             45                  50                  55 

CGC GAC ATG GCT ACC GCA TTC GGT GGA CCA GGC AAT GTG CAC TAC GCA     2406 
Arg Asp Met Ala Thr Ala Phe Gly Gly Pro Gly Asn Val His Tyr Ala 
         60                  65                  70 

TCT AAA GCG TTC CTG ACC AAG ACC ATT GCA CGT TGG GTT GAT GAA GAG     2454 
Ser Lys Ala Phe Leu Thr Lys Thr Ile Ala Arg Trp Val Asp Glu Glu 
     75                  80                  85 

GGG CTG GCA CTG GAC ATT GCA TCC ATC AAC GAA CTG GGC ATT GCC CTG     2502 
Gly Leu Ala Leu Asp Ile Ala Ser Ile Asn Glu Leu Gly Ile Ala Leu 
 90                  95                 100                 105 

GCC GCT GGT TTC CCC GCC AGC CGT ATC ACC GCG CAC GGC AAC AAC AAA     2550 
Ala Ala Gly Phe Pro Ala Ser Arg Ile Thr Ala His Gly Asn Asn Lys 
                110                 115                 120 

GGC GTA GAG TTC CTG CGC GCG TTG GTT CAA AAC GGT GTG GGA CAC GTG     2598 
Gly Val Glu Phe Leu Arg Ala Leu Val Gln Asn Gly Val Gly His Val 
            125                 130                 135 

GTG CTG GAC TCC GCA CAG GAA CTA GAA CTG TTG GAT TAC GTT GCC GCT     2646 
Val Leu Asp Ser Ala Gln Glu Leu Glu Leu Leu Asp Tyr Val Ala Ala 
        140                 145                 150 

GGT GAA GGC AAG ATT CAG GAC GTG TTG ATC CGC GTA AAG CCA GGC ATC     2694 
Gly Glu Gly Lys Ile Gln Asp Val Leu Ile Arg Val Lys Pro Gly Ile 
    155                 160                 165 

GAA GCA CAC ACC CAC GAG TTC ATC GCC ACT AGC CAC GAA GAC CAG AAG     2742 
Glu Ala His Thr His Glu Phe Ile Ala Thr Ser His Glu Asp Gln Lys 
170                 175                 180                 185 

TTC GGA TTC TCC CTG GCA TCC GGT TCC GCA TTC GAA GCA GCA AAA GCC     2790 
Phe Gly Phe Ser Leu Ala Ser Gly Ser Ala Phe Glu Ala Ala Lys Ala 
                190                 195                 200 

GCC AAC AAC GCA GAA AAC CTG AAC CTG GTT GGC CTG CAC TGC CAC GTT     2838 
Ala Asn Asn Ala Glu Asn Leu Asn Leu Val Gly Leu His Cys His Val 
            205                 210                 215 

GGT TCC CAG GTG TTC GAC GCC GAA GGC TTC AAG CTG GCA GCA GAA CGC     2886 
Gly Ser Gln Val Phe Asp Ala Glu Gly Phe Lys Leu Ala Ala Glu Arg 
        220                 225                 230 

GTG TTG GGC CTG TAC TCA CAG ATC CAC AGC GAA CTG GGC GTT GCC CTT     2934 
Val Leu Gly Leu Tyr Ser Gln Ile His Ser Glu Leu Gly Val Ala Leu 
    235                 240                 245 

CCT GAA CTG GAT CTC GGT GGC GGA TAC GGC ATT GCC TAT ACC GCA GCT     2982 
Pro Glu Leu Asp Leu Gly Gly Gly Tyr Gly Ile Ala Tyr Thr Ala Ala 
250                 255                 260                 265 

GAA GAA CCA CTC AAC GTC GCA GAA GTT GCC TCC GAC CTG CTC ACC GCA     3030 
Glu Glu Pro Leu Asn Val Ala Glu Val Ala Ser Asp Leu Leu Thr Ala 
                270                 275                 280 

GTC GGA AAA ATG GCA GCG GAA CTA GGC ATC GAC GCA CCA ACC GTG CTT     3078 
Val Gly Lys Met Ala Ala Glu Leu Gly Ile Asp Ala Pro Thr Val Leu 
            285                 290                 295 

GTT GAG CCC GGC CGC GCT ATC GCA GGC CCC TCC ACC GTG ACC ATC TAC     3126 
Val Glu Pro Gly Arg Ala Ile Ala Gly Pro Ser Thr Val Thr Ile Tyr 
        300                 305                 310 

GAA GTC GGC ACC ACC AAA GAC GTC CAC GTA GAC GAC GAC AAA ACC CGC     3174 
Glu Val Gly Thr Thr Lys Asp Val His Val Asp Asp Asp Lys Thr Arg 
    315                 320                 325 

CGT TAC ATC GCC GTG GAC GGA GGC ATG TCC GAC AAC ATC CGC CCA GCA     3222 
Arg Tyr Ile Ala Val Asp Gly Gly Met Ser Asp Asn Ile Arg Pro Ala 
330                 335                 340                 345 

CTC TAC GGC TCC GAA TAC GAC GCC CGC GTA GTA TCC CGC TTC GCC GAA     3270 
Leu Tyr Gly Ser Glu Tyr Asp Ala Arg Val Val Ser Arg Phe Ala Glu 
                350                 355                 360 

GGA GAC CCA GTA AGC ACC CGC ATC GTG GGC TCC CAC TGC GAA TCC GGC     3318 
Gly Asp Pro Val Ser Thr Arg Ile Val Gly Ser His Cys Glu Ser Gly 
            365                 370                 375 

GAT ATC CTG ATC AAC GAT GAA ATC TAC CCA TCT GAC ATC ACC AGC GGC     3366 
Asp Ile Leu Ile Asn Asp Glu Ile Tyr Pro Ser Asp Ile Thr Ser Gly 
        380                 385                 390 

GAC TTC CTT GCA CTC GCA GCC ACC GGC GCA TAC TGC TAC GCC ATG AGC     3414 
Asp Phe Leu Ala Leu Ala Ala Thr Gly Ala Tyr Cys Tyr Ala Met Ser 
    395                 400                 405 

TCC CGC TAC AAC GCC TTC ACA CGG CCC GCC GTC GTG TCC GTC CGC GCT     3462 
Ser Arg Tyr Asn Ala Phe Thr Arg Pro Ala Val Val Ser Val Arg Ala 
410                 415                 420                 425 

GGC AGC TCC CGC CTC ATG CTG CGC CGC GAA ACG CTC GAC GAC ATC CTC     3510 
Gly Ser Ser Arg Leu Met Leu Arg Arg Glu Thr Leu Asp Asp Ile Leu 
                430                 435                 440 

TCA CTA GAG GCA TAACGCTTTT CGACGCCTGA CCCCGCCCTT CACCTTCGCC         3562 
Ser Leu Glu Ala 
            445 

GTGGAGGGCG GTTTTGG                                                  3579 

 
           
           
             
               550 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             19 

Met Thr Pro Ala Asp Leu Ala Thr Leu Ile Lys Glu Thr Ala Val Glu 
  1               5                  10                  15 

Val Leu Thr Ser Arg Glu Leu Asp Thr Ser Val Leu Pro Glu Gln Val 
             20                  25                  30 

Val Val Glu Arg Pro Arg Asn Pro Glu His Gly Asp Tyr Ala Thr Asn 
         35                  40                  45 

Ile Ala Leu Gln Val Ala Lys Lys Val Gly Gln Asn Pro Arg Asp Leu 
     50                  55                  60 

Ala Thr Trp Leu Ala Glu Ala Leu Ala Ala Asp Asp Ala Ile Asp Ser 
 65                  70                  75                  80 

Ala Glu Ile Ala Gly Pro Gly Phe Leu Asn Ile Arg Leu Ala Ala Ala 
                 85                  90                  95 

Ala Gln Gly Glu Ile Val Ala Lys Ile Leu Ala Gln Gly Glu Thr Phe 
            100                 105                 110 

Gly Asn Ser Asp His Leu Ser His Leu Asp Val Asn Leu Glu Phe Val 
        115                 120                 125 

Ser Ala Asn Pro Thr Gly Pro Ile His Leu Gly Gly Thr Arg Trp Ala 
    130                 135                 140 

Ala Val Gly Asp Ser Leu Gly Arg Val Leu Glu Ala Ser Gly Ala Lys 
145                 150                 155                 160 

Val Thr Arg Glu Tyr Tyr Phe Asn Asp His Gly Arg Gln Ile Asp Arg 
                165                 170                 175 

Phe Ala Leu Ser Leu Leu Ala Ala Ala Lys Gly Glu Pro Thr Pro Glu 
            180                 185                 190 

Asp Gly Tyr Gly Gly Glu Tyr Ile Lys Glu Ile Ala Glu Ala Ile Val 
        195                 200                 205 

Glu Lys His Pro Glu Ala Leu Ala Leu Glu Pro Ala Ala Thr Gln Glu 
    210                 215                 220 

Leu Phe Arg Ala Glu Gly Val Glu Met Met Phe Glu His Ile Lys Ser 
225                 230                 235                 240 

Ser Leu His Glu Phe Gly Thr Asp Phe Asp Val Tyr Tyr His Glu Asn 
                245                 250                 255 

Ser Leu Phe Glu Ser Gly Ala Val Asp Lys Ala Val Gln Val Leu Lys 
            260                 265                 270 

Asp Asn Gly Asn Leu Tyr Glu Asn Glu Gly Ala Trp Trp Leu Arg Ser 
        275                 280                 285 

Thr Glu Phe Gly Asp Asp Lys Asp Arg Val Val Ile Lys Ser Asp Gly 
    290                 295                 300 

Asp Ala Ala Tyr Ile Ala Gly Asp Ile Ala Tyr Val Ala Asp Lys Phe 
305                 310                 315                 320 

Ser Arg Gly His Asn Leu Asn Ile Tyr Met Leu Gly Ala Asp His His 
                325                 330                 335 

Gly Tyr Ile Ala Arg Leu Lys Ala Ala Ala Ala Ala Leu Gly Tyr Lys 
            340                 345                 350 

Pro Glu Gly Val Glu Val Leu Ile Gly Gln Met Val Asn Leu Leu Arg 
        355                 360                 365 

Asp Gly Lys Ala Val Arg Met Ser Lys Arg Ala Gly Thr Val Val Thr 
    370                 375                 380 

Leu Asp Asp Leu Val Glu Ala Ile Gly Ile Asp Ala Ala Arg Tyr Ser 
385                 390                 395                 400 

Leu Ile Arg Ser Ser Val Asp Ser Ser Leu Asp Ile Asp Leu Gly Leu 
                405                 410                 415 

Trp Glu Ser Gln Ser Ser Asp Asn Pro Val Tyr Tyr Val Gln Tyr Gly 
            420                 425                 430 

His Ala Arg Leu Cys Ser Ile Ala Arg Lys Ala Glu Thr Leu Gly Val 
        435                 440                 445 

Thr Glu Glu Gly Ala Asp Leu Ser Leu Leu Thr His Asp Arg Glu Gly 
    450                 455                 460 

Asp Leu Ile Arg Thr Leu Gly Glu Phe Pro Ala Val Val Lys Ala Ala 
465                 470                 475                 480 

Ala Asp Leu Arg Glu Pro His Arg Ile Ala Arg Tyr Ala Glu Glu Leu 
                485                 490                 495 

Ala Gly Thr Phe His Arg Phe Tyr Asp Ser Cys His Ile Leu Pro Lys 
            500                 505                 510 

Val Asp Glu Asp Thr Ala Pro Ile His Thr Ala Arg Leu Ala Leu Ala 
        515                 520                 525 

Ala Ala Thr Arg Gln Thr Leu Ala Asn Ala Leu His Leu Val Gly Val 
    530                 535                 540 

Ser Ala Pro Glu Lys Met 
545                 550 

 
           
           
             
               445 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             20 

Met Ala Thr Val Glu Asn Phe Asn Glu Leu Pro Ala His Val Trp Pro 
  1               5                  10                  15 

Arg Asn Ala Val Arg Gln Glu Asp Gly Val Val Thr Val Ala Gly Val 
             20                  25                  30 

Pro Leu Pro Asp Leu Ala Glu Glu Tyr Gly Thr Pro Leu Phe Val Val 
         35                  40                  45 

Asp Glu Asp Asp Phe Arg Ser Arg Cys Arg Asp Met Ala Thr Ala Phe 
     50                  55                  60 

Gly Gly Pro Gly Asn Val His Tyr Ala Ser Lys Ala Phe Leu Thr Lys 
 65                  70                  75                  80 

Thr Ile Ala Arg Trp Val Asp Glu Glu Gly Leu Ala Leu Asp Ile Ala 
                 85                  90                  95 

Ser Ile Asn Glu Leu Gly Ile Ala Leu Ala Ala Gly Phe Pro Ala Ser 
            100                 105                 110 

Arg Ile Thr Ala His Gly Asn Asn Lys Gly Val Glu Phe Leu Arg Ala 
        115                 120                 125 

Leu Val Gln Asn Gly Val Gly His Val Val Leu Asp Ser Ala Gln Glu 
    130                 135                 140 

Leu Glu Leu Leu Asp Tyr Val Ala Ala Gly Glu Gly Lys Ile Gln Asp 
145                 150                 155                 160 

Val Leu Ile Arg Val Lys Pro Gly Ile Glu Ala His Thr His Glu Phe 
                165                 170                 175 

Ile Ala Thr Ser His Glu Asp Gln Lys Phe Gly Phe Ser Leu Ala Ser 
            180                 185                 190 

Gly Ser Ala Phe Glu Ala Ala Lys Ala Ala Asn Asn Ala Glu Asn Leu 
        195                 200                 205 

Asn Leu Val Gly Leu His Cys His Val Gly Ser Gln Val Phe Asp Ala 
    210                 215                 220 

Glu Gly Phe Lys Leu Ala Ala Glu Arg Val Leu Gly Leu Tyr Ser Gln 
225                 230                 235                 240 

Ile His Ser Glu Leu Gly Val Ala Leu Pro Glu Leu Asp Leu Gly Gly 
                245                 250                 255 

Gly Tyr Gly Ile Ala Tyr Thr Ala Ala Glu Glu Pro Leu Asn Val Ala 
            260                 265                 270 

Glu Val Ala Ser Asp Leu Leu Thr Ala Val Gly Lys Met Ala Ala Glu 
        275                 280                 285 

Leu Gly Ile Asp Ala Pro Thr Val Leu Val Glu Pro Gly Arg Ala Ile 
    290                 295                 300 

Ala Gly Pro Ser Thr Val Thr Ile Tyr Glu Val Gly Thr Thr Lys Asp 
305                 310                 315                 320 

Val His Val Asp Asp Asp Lys Thr Arg Arg Tyr Ile Ala Val Asp Gly 
                325                 330                 335 

Gly Met Ser Asp Asn Ile Arg Pro Ala Leu Tyr Gly Ser Glu Tyr Asp 
            340                 345                 350 

Ala Arg Val Val Ser Arg Phe Ala Glu Gly Asp Pro Val Ser Thr Arg 
        355                 360                 365 

Ile Val Gly Ser His Cys Glu Ser Gly Asp Ile Leu Ile Asn Asp Glu 
    370                 375                 380 

Ile Tyr Pro Ser Asp Ile Thr Ser Gly Asp Phe Leu Ala Leu Ala Ala 
385                 390                 395                 400 

Thr Gly Ala Tyr Cys Tyr Ala Met Ser Ser Arg Tyr Asn Ala Phe Thr 
                405                 410                 415 

Arg Pro Ala Val Val Ser Val Arg Ala Gly Ser Ser Arg Leu Met Leu 
            420                 425                 430 

Arg Arg Glu Thr Leu Asp Asp Ile Leu Ser Leu Glu Ala 
        435                 440                 445 

 
           
           
             
               20 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             NO  
             21 

CATCTAAGTA TGCATCTCGG                                                 20 

 
           
           
             
               20 bases  
               nucleic acid  
               single  
               linear  
             
             
               other nucleic acid  
               /desc = “synthetic DNA” 
             
             YES  
             22 

TGCCCCTCGA GCTAAATTAG                                                20 

 
           
           
             
               1034 base pairs  
               nucleic acid  
               double  
               linear  
             
             
               DNA (genomic)  
             
             NO  
             
               Brevibacterium lactofermentum  
               ATCC 13869  
             
             
               CDS  
                61..1020 

 
             
             23 

ATGCATCTCG GTAAGCTCGA CCAGGACAGT GCCACCACAA TTTTGGAGGA TTACAAGAAC     60 

ATG ACC AAC ATC CGC GTA GCT ATC GTG GGC TAC GGA AAC CTG GGA CGC      108 
Met Thr Asn Ile Arg Val Ala Ile Val Gly Tyr Gly Asn Leu Gly Arg 
  1               5                  10                  15 

AGC GTC GAA AAG CTT ATT GCC AAG CAG CCC GAC ATG GAC CTT GTA GGA      156 
Ser Val Glu Lys Leu Ile Ala Lys Gln Pro Asp Met Asp Leu Val Gly 
             20                  25                  30 

ATC TTC TCG CGC CGG GCC ACC CTC GAC ACA AAG ACG CCA GTC TTT GAT      204 
Ile Phe Ser Arg Arg Ala Thr Leu Asp Thr Lys Thr Pro Val Phe Asp 
         35                  40                  45 

GTC GCC GAC GTG GAC AAG CAC GCC GAC GAC GTG GAC GTG CTG TTC CTG      252 
Val Ala Asp Val Asp Lys His Ala Asp Asp Val Asp Val Leu Phe Leu 
     50                  55                  60 

TGC ATG GGC TCC GCC ACC GAC ATC CCT GAG CAG GCA CCA AAG TTC GCG      300 
Cys Met Gly Ser Ala Thr Asp Ile Pro Glu Gln Ala Pro Lys Phe Ala 
 65                  70                  75                  80 

CAG TTC GCC TGC ACC GTA GAC ACC TAC GAC AAC CAC CGC GAC ATC CCA      348 
Gln Phe Ala Cys Thr Val Asp Thr Tyr Asp Asn His Arg Asp Ile Pro 
                 85                  90                  95 

CGC CAC CGC CAG GTC ATG AAC GAA GCC GCC ACC GCA GCC GGC AAC GTT      396 
Arg His Arg Gln Val Met Asn Glu Ala Ala Thr Ala Ala Gly Asn Val 
            100                 105                 110 

GCA CTG GTC TCT ACC GGC TGG GAT CCA GGA ATG TTC TCC ATC AAC CGC      444 
Ala Leu Val Ser Thr Gly Trp Asp Pro Gly Met Phe Ser Ile Asn Arg 
        115                 120                 125 

GTC TAC GCA GCG GCA GTC TTA GCC GAG CAC CAG CAG CAC ACC TTC TGG      492 
Val Tyr Ala Ala Ala Val Leu Ala Glu His Gln Gln His Thr Phe Trp 
    130                 135                 140 

GGC CCA GGT TTG TCA CAG GGC CAC TCC GAT GCT TTG CGA CGC ATC CCT      540 
Gly Pro Gly Leu Ser Gln Gly His Ser Asp Ala Leu Arg Arg Ile Pro 
145                 150                 155                 160 

GGC GTT CAA AAG GCA GTC CAG TAC ACC CTC CCA TCC GAA GAC GCC CTG      588 
Gly Val Gln Lys Ala Val Gln Tyr Thr Leu Pro Ser Glu Asp Ala Leu 
                165                 170                 175 

GAA AAG GCC CGC CGC GGC GAA GCC GGC GAC CTT ACC GGA AAG CAA ACC      636 
Glu Lys Ala Arg Arg Gly Glu Ala Gly Asp Leu Thr Gly Lys Gln Thr 
            180                 185                 190 

CAC AAG CGC CAA TGC TTC GTG GTT GCC GAC GCG GCC GAT CAC GAG CGC      684 
His Lys Arg Gln Cys Phe Val Val Ala Asp Ala Ala Asp His Glu Arg 
        195                 200                 205 

ATC GAA AAC GAC ATC CGC ACC ATG CCT GAT TAC TTC GTT GGC TAC GAA      732 
Ile Glu Asn Asp Ile Arg Thr Met Pro Asp Tyr Phe Val Gly Tyr Glu 
    210                 215                 220 

GTC GAA GTC AAC TTC ATC GAC GAA GCA ACC TTC GAC TCC GAG CAC ACC      780 
Val Glu Val Asn Phe Ile Asp Glu Ala Thr Phe Asp Ser Glu His Thr 
225                 230                 235                 240 

GGC ATG CCA CAC GGT GGC CAC GTG ATT ACC ACC GGC GAC ACC GGT GGC      828 
Gly Met Pro His Gly Gly His Val Ile Thr Thr Gly Asp Thr Gly Gly 
                245                 250                 255 

TTC AAC CAC ACC GTG GAA TAC ATC CTC AAG CTG GAC CGA AAC CCA GAT      876 
Phe Asn His Thr Val Glu Tyr Ile Leu Lys Leu Asp Arg Asn Pro Asp 
            260                 265                 270 

TTC ACC GCT TCC TCA CAG ATC GCT TTC GGT CGC GCA GCT CAC CGC ATG      924 
Phe Thr Ala Ser Ser Gln Ile Ala Phe Gly Arg Ala Ala His Arg Met 
        275                 280                 285 

AAG CAG CAG GGC CAA AGC GGA GCT TTC ACC GTC CTC GAA GTT GCT CCA      972 
Lys Gln Gln Gly Gln Ser Gly Ala Phe Thr Val Leu Glu Val Ala Pro 
    290                 295                 300 

TAC CTG CTC TCC CCA GAG AAC TTG GAC GAT CTG ATC GCA CGC GAC GTC     1020 
Tyr Leu Leu Ser Pro Glu Asn Leu Asp Asp Leu Ile Ala Arg Asp Val 
305                 310                 315                 320 

TAATTTAGCT CGAG                                                     1034 

 
           
           
             
               320 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
             24 

Met Thr Asn Ile Arg Val Ala Ile Val Gly Tyr Gly Asn Leu Gly Arg 
  1               5                  10                  15 

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

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

Val Ala Asp Val Asp Lys His Ala Asp Asp Val Asp Val Leu Phe Leu 
     50                  55                  60 

Cys Met Gly Ser Ala Thr Asp Ile Pro Glu Gln Ala Pro Lys Phe Ala 
 65                  70                  75                  80 

Gln Phe Ala Cys Thr Val Asp Thr Tyr Asp Asn His Arg Asp Ile Pro 
                 85                  90                  95 

Arg His Arg Gln Val Met Asn Glu Ala Ala Thr Ala Ala Gly Asn Val 
            100                 105                 110 

Ala Leu Val Ser Thr Gly Trp Asp Pro Gly Met Phe Ser Ile Asn Arg 
        115                 120                 125 

Val Tyr Ala Ala Ala Val Leu Ala Glu His Gln Gln His Thr Phe Trp 
    130                 135                 140 

Gly Pro Gly Leu Ser Gln Gly His Ser Asp Ala Leu Arg Arg Ile Pro 
145                 150                 155                 160 

Gly Val Gln Lys Ala Val Gln Tyr Thr Leu Pro Ser Glu Asp Ala Leu 
                165                 170                 175 

Glu Lys Ala Arg Arg Gly Glu Ala Gly Asp Leu Thr Gly Lys Gln Thr 
            180                 185                 190 

His Lys Arg Gln Cys Phe Val Val Ala Asp Ala Ala Asp His Glu Arg 
        195                 200                 205 

Ile Glu Asn Asp Ile Arg Thr Met Pro Asp Tyr Phe Val Gly Tyr Glu 
    210                 215                 220 

Val Glu Val Asn Phe Ile Asp Glu Ala Thr Phe Asp Ser Glu His Thr 
225                 230                 235                 240 

Gly Met Pro His Gly Gly His Val Ile Thr Thr Gly Asp Thr Gly Gly 
                245                 250                 255 

Phe Asn His Thr Val Glu Tyr Ile Leu Lys Leu Asp Arg Asn Pro Asp 
            260                 265                 270 

Phe Thr Ala Ser Ser Gln Ile Ala Phe Gly Arg Ala Ala His Arg Met 
        275                 280                 285 

Lys Gln Gln Gly Gln Ser Gly Ala Phe Thr Val Leu Glu Val Ala Pro 
    290                 295                 300 

Tyr Leu Leu Ser Pro Glu Asn Leu Asp Asp Leu Ile Ala Arg Asp Val 
305                 310                 315                 320