(en)Provided are a protein complex having the F 0 F 1 -ATPase activity; a DNA encoding the protein complex; a method for producing the protein complex, using the DNA; and a method for producing nucleoside 5′-triphosphate, using the protein. The present invention further provides a recombinant DNA with the DNA inserted therein; a transformant carrying the recombinant DNA; and a method for producing a protein complex, using the transformant.

1.ApplicationNumber: US-90188401-A
1.PublishNumber: US-2002037573-A1
2.Date Publish: 20020328
3.Inventor: TOMITA FUSAO
YOKOTA ATSUSHI

4.Inventor Harmonized: TOMITA FUSAO(JP)
YOKOTA ATSUSHI(JP)

5.Country: US
6.Claims:
(en)Provided are a protein complex having the F 0 F 1 -ATPase activity; a DNA encoding the protein complex; a method for producing the protein complex, using the DNA; and a method for producing nucleoside 5′-triphosphate, using the protein. The present invention further provides a recombinant DNA with the DNA inserted therein; a transformant carrying the recombinant DNA; and a method for producing a protein complex, using the transformant.

7.Description:
(en)BACKGROUND OF THE INVENTION
[0001] The present invention relates to a novel F 0 F 1 -ATPase, a DNA encoding the F 0 F 1 -ATPase, a method for producing the F 0 F 1 -ATPase, and a method for producing nucleoside 5′-triphosphate, using the F 0 F 1 -ATPase.
[0002] F 0 F 1 -ATPase plays principal roles in the biological energy metabolism, because the enzyme has an activity of generating adenosine 5′-triphosphate (ATP) as an energy source of organisms, by utilizing the gradient of proton concentration between the intramembrane and the extra membrane. Therefore, utilization of F 0 F 1 -ATPase enables us to develop a living thing with improved energy metabolism.
[0003] Herein, F 0 F 1 -ATPase is synonymous with H+-ATPase.
[0004] Because it has been known that the activity of F 0 F 1 -ATPase varies, depending on the change of outer environment, such as pH, the utilization of F 0 F 1 -ATPase can provide a living thing adaptable to the change of outer environment [Mol.Microbiol., 33,1152 (1999), J. Bacteriol., 176, 5167 (1994)].
[0005] F 0 F 1 -ATPase is a protein complex comprising a soluble catalytic sector F 1 and a transmembrane sector F 0 functioning as proton channel. In organisms such as Escherichia coli and Bacillus subtilis , F 1 is composed of five subunits of α, β, γ, δ and ε, while F 0 is composed of three subunits of a, b and c [Annu. Rev. Biochem., 66, 717 (1997)].
[0006] Concerning the F 0 F 1 -ATPase gene, the gene was isolated from Escherichia coli [Biochem. J., 224, 799 (1984)], Bacillus subtilis [J. Bacteriol., 176, 6802 (1994)], Bacillus megaterium [J. Biol. Chem., 264, 1528 (1989)], Bacillus firmus [Mol. Gen.Genet., 229,292(1991)], Bacillus sp. PS3 [Biochim. Biophys. Acta, 933, 141 (1988)], Methanosarcina barkeri [Biochem. Biophys. Res. Commun., 241, 427 (1997)], Lactobacillus acidophilus [Mol. Microbiol., 33, 1152 (1999)], Rhodobacter capsulatus [J. Bacteriol., 180, 416 (1998), Arch. Microbiol., 170, 385 (1998)] and the like, but no gene derived from microorganisms belonging to the genus Corynebacterium has been isolated yet.
[0007] Regarding the production of nucleoside 5′-triphosphate, methods using microorganisms (Japanese Published Unexamined Patent Application No.107593/1979; Japanese Published Unexamined Patent Application No. 51799/1984; J. Ferment. Bioeng., 68, 417 (1989)) and a method using enzymes (WO 98/22614) have been known. However, the productivity of nucleoside 5′-triphosphate is insufficient.
SUMMARY OF THE INVENTION
[0008] It is the purpose of the present invention to provide a protein complex having the F 0 F 1 -ATPase activity; a DNA encoding the protein complex; a method for producing the protein complex having the F 0 F 1 -ATPase activity, using the DNA; and a method for producing nucleoside 5′-triphosphate, using the protein complex.
[0009] For this purpose, the present inventors have made various investigations. Consequently, the inventors have successfully isolated the genes encoding component proteins of which a protein complex having the F 0 F 1 -ATPase activity is composed, from Corynebacterium ammoniagenes . Thus, the present invention has been accomplished.
[0010] The present invention relates to the following (1) to (32) subject matters.
[0011] (1) A protein selected from the group consisting of the following proteins (a) to (c):
[0012] (a) a protein having the amino acid sequence represented by SEQ ID NO: 1;
[0013] (b) a protein having a modified one of the amino acid sequence represented by SEQ ID NO: 1, where one or more amino acids are deleted, substituted or added, and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 2 to 8; and
[0014] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 1 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 2 to 8.
[0015] (2) A protein selected from the group consisting of the following proteins (a) to (c):
[0016] (a) a protein having the amino acid sequence represented by SEQ ID NO: 2;
[0017] (b) a protein having a modified one of the amino acid sequence represented by SEQ ID NO: 2, where one or more amino acids are deleted, substituted or added, and exreting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NO: 1 and SEQ ID NO: 3 to 8; and
[0018] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 2 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NO: 1 and SEQ ID NOS: 3 to 8.
[0019] (3) A protein selected from the group consisting of the following proteins (a) to (c):
[0020] (a) a protein having the amino acid sequence represented by SEQ ID NO: 3;
[0021] (b) a protein having a modified one of the amino acid sequence represented by SEQ ID NO: 3, where one or more amino acids are deleted, substituted or added, and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the proteins having the individual amino acid sequences represented by each of SEQ ID NOS: 1 and 2 and SEQ ID NOS: 4 to 8; and
[0022] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 3 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 and 2 and SEQ ID NOS: 4 to 8.
[0023] (4) A protein selected from the group consisting of the following proteins (a) to (c):
[0024] (a) a protein having the amino acid sequence represented by SEQ ID NO: 4;
[0025] (b) a protein having a modified one of the amino acid sequence represented by SEQ ID NO: 4, where one or more amino acids are deleted, substituted or added, and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 3 and SEQ ID NOS: 5 to 8; and
[0026] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 4 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NO: 1 to 3 and SEQ ID NOS: 5 to 8.
[0027] (5) A protein selected from the group consisting of the following proteins (a) to (c):
[0028] (a) a protein having the amino acid sequence represented by SEQ ID NO: 5;
[0029] (b) a protein comprising a modified one of the amino acid sequence represented by SEQ ID NO: 5, where one or more amino acids are deleted, substituted or added, and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 4 and SEQ ID NOS: 6 to 8; and
[0030] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 5 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 4 and SEQ ID NOS: 6 to 8.
[0031] (6) A protein selected from the group consisting of the following proteins (a) to (c):
[0032] (a) a protein having the amino acid sequence represented by SEQ ID NO: 6;
[0033] (b) a protein having a modified one of the amino acid sequence represented by SEQ ID NO: 6, where one or more amino acids are deleted, substituted or added, and which can exert the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 5 and SEQ ID NOS: 7 and 8; and
[0034] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 6 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 5 and SEQ ID NOS: 7 and 8.
[0035] (7) A protein selected from the group consisting of the following proteins(a) to (c):
[0036] (a) a protein having the amino acid sequence represented by SEQ ID NO: 7;
[0037] (b) a protein having a modified one of the amino acid sequence represented by SEQ ID NO: 7, where one or more amino acids are deleted, substituted or added, and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 6 and SEQ ID NO: 8; and
[0038] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 7 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 6 and SEQ ID NO: 8.
[0039] (8) A protein selected from the group consisting of the following proteins (a) to (c):
[0040] (a) a protein having the amino acid sequence represented by SEQ ID NO: 8;
[0041] (b) a protein having a modified one of the amino acid sequence represented by SEQ ID NO: 8, where one or more amino acids are deleted, substituted or added, and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 7; and
[0042] (c) a protein having an amino acid sequence having 70% or more identical to the amino acid sequence represented by SEQ ID NO: 8 and exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins comprising the amino acid sequences represented by each of SEQ ID NOS: 1 to 7.
[0043] (9) A protein complex comprising eight proteins respectively selected from the eight groups as defined by each of (1) to (8).
[0044] (10) A DNA encoding any one of the proteins of (1) to (8).
[0045] (11) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0046] (a) a DNA having the nucleotide sequence represented by SEQ ID NO:9; and
[0047] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 2 to 8.
[0048] (12) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0049] (a) a DNA having the nucleotide sequence represented by SEQ ID NO: 10; and
[0050] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NO: 1 and SEQ ID NOS: 3 to 8.
[0051] (13) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0052] (a) a DNA having the nucleotide sequence represented by SEQ ID NO: 11; and
[0053] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 and 2 and SEQ ID NOS: 4 to 8.
[0054] (14) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0055] (a) a DNA having the nucleotide sequence represented by SEQ ID NO:12; and
[0056] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the individual amino acid sequences represented by each of SEQ ID NOS: 1 to 3 and SEQ ID NOS: 5 to 8.
[0057] (15) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0058] (a) a DNA having the nucleotide sequence represented by SEQ ID NO:13; and
[0059] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 4 and SEQ ID NOS: 6 to 8.
[0060] (16) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0061] (a) a DNA having the nucleotide sequence represented by SEQ ID NO: 14; and
[0062] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 5 and SEQ ID NOS: 7 and 8.
[0063] (17) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0064] (a) a DNA having the nucleotide sequence represented by SEQ ID NO: 15; and
[0065] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 6 and SEQ ID NO: 8.
[0066] (18) A DNA selected from the group consisting of the following DNAs (a) and (b):
[0067] (a) a DNA having the nucleotide sequence represented by SEQ ID NO: 16; and
[0068] (b) a DNA hybridizing with the DNA under stringent conditions and encoding a protein exerting the F 0 F 1 -ATPase activity when the protein forms a complex with all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 7.
[0069] (19) A DNA comprising the eight DNAs respectively selected from the eight groups as defined by each of (11) to (18).
[0070] (20) A DNA having the nucleotide sequences represented by SEQ ID NOS: 9 to 16.
[0071] (21) A DNA having the nucleotide sequence represented by SEQ ID NO: 21.
[0072] (22) The DNA according to any one of (10) to (21), where the DNA is derived from a microorganism belonging to the genus Corynebacterium.
[0073] (23) The DNA according to any one of (10) to (21), where the DNA is derived from a microorganism of the species Corynebacterium ammoniagenes.
[0074] (24) A recombinant DNA constructed by inserting the DNA according to any one of (10) to (18) into a vector.
[0075] (25) A recombinant DNA constructed by inserting the DNA according to any one of (19) to (21) into a vector.
[0076] (26) A transformant obtained by transformation of a host cell with the recombinant DNA of (24) or (25).
[0077] (27) A transformant of (26), where the host cell is a microorganism of the species Escherichia coli, Corynebacterium glutamicum or Corynebacterium ammoniagenes.
[0078] (28) A method for producing a protein of any one of (1) to (8), which comprises culturing a transformant obtained by transformation of a host cell with the recombinant DNA of (24) in a culture medium, so as to allow the protein of any one of (1) to (8) to be expressed and accumulated in the culture and harvesting the protein from the culture.
[0079] (29) A method for producing a protein complex having the F 0 F 1 -ATPase activity, which comprises culturing a transformant obtained by transformation of a host cell with the recombinant DNA of (25) in a culture medium, so as to allow a protein complex having the F 0 F 1 -ATPase activity to be expressed and accumulated in the culture and recovering the protein complex from the culture.
[0080] (30) A method for producing nucleoside 5′-triphosphate, which comprises by use of a culture of a transformant obtained by transformation of a host cell with the recombinant DNA of (25) or a treated product of the culture as an enzyme source, allowing the enzyme source and a precursor of nucleoside 5′-triphosphate to co-exist with each other in an aqueous medium to generate and accumulate the nucleoside 5′-triphosphate and recovering the nucleoside 5′-triphosphate from the aqueous medium.
[0081] (31) The method of (30), where the precursor of nucleoside 5′-triphosphate is adenine, guanine, uracil, cytosine, hypoxanthine, adenosine, guanosine, uridine, cytidine, inosine, adenosine 5′-monophosphate, guanosine 5′-monophosphate, uridine 5′-monophosphate, cytidine 5′-monophosphate or inosine 5′-monophosphate.
[0082] (32) The method of (30), where the nucleoside 5′-triphosphate is adenosine 5′-triphosphate, guanosine 5′-triphosphate, uridine 5′-triphosphate or cytidine 5′-triphosphate.



BRIEF DESCRIPTION OF THE DRAWING
[0083]FIG. 1 shows restriction maps of fragments inserted in plasmids pUH71, pE61 and pDW31 and the open reading frames contained in the fragments.


DETAILED DESCRIPTION OF THE INVENTION
[0084] (1) Preparation of the DNA of the Present Invention
[0085] (a) Preparation of a DNA Library
[0086] The protein complex of the present invention is a protein complex comprising the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 8 as components.
[0087] As long as the protein complex exerts the F 0 F 1 -ATPase activity, one or more amino acids can be deleted, substituted or added in the amino acid sequences of the individual proteins.
[0088] The protein having an amino acid sequence with one or more amino acids deleted, substitutied or added, which can be the component of the protein complex having the F 0 F 1 -ATPase activity, can be obtained by introducing mutation in the DNA encoding the protein having any one of SEQ ID NOS: 1 to 8, via the site-directed mutagenesis described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (abbreviated as Molecular Cloning, Second edition, hereinafter), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (abbreviated Current Protocols in Molecular Biology, hereinafter), Nucleic 14 Acids Research, 10, 6487 (1982),Proc. Natl. Acad. Sci. USA, 79, 6409(1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), and the like.
[0089] The number of the amino acids to be deleted, substituted, or added is not particularly limited, but should be such that deletion, substitution or addition according to well-known methods such as site-directed mutagenesis can be ocurred. The number is one to several tens, preferably one to 20, more preferably one to 10, still more preferably one to 5.
[0090] In order for the protein complex according to the present invention to have the F 0 F 1 -ATPase activity, each component protein of the protein complex has such identity to the corresponding amino acid sequences represented by each of SEQ ID NOS: 1 to 8, as at least 60%, preferably 80% or more, more preferably 95% or more. The identity of a nucleotide sequence or an amino acid sequence can be determined using the algorithm “BLAST” by Karlin and Altschl [Proc. Natl. Acad. Sci. USA, 90, 5873-5877 (1993)]. The programs called “BLASTN” and “BLASTX” have been developed based on the above algorithm [J. Mol. Biol., 215, 403-410 (1990)]. In the case of analyzing a nucleotide sequence based on BLAST, the parameter can be set to e.g. score=100, wordlength=12. In the case of analyzing an amino acid sequence based on BLASTX, the parameter can be set to e.g. score=50, wordlength=3. In the case of using BLAST or Gapped BLAST program, a default parameter of each program can be used. The specific analysis methods of using the above programs are known in the art (http://www.ncbi.nlm.nih.gov.).
[0091] However, the protein of the present invention does not include any proteins having the amino acid sequences in the public domain.
[0092] The DNAs of the present invention encode the proteins of the present invention or the protein complex comprising the proteins as the components and can be isolated from a microorganism of the genus Corynebacterium. The microorganism belonging to the genus Corynebacterium includes, for example, Corynebacterium ammoniagenes , specifically Corynebacterium ammoniagenes strain ATCC6872. Specific examples of the DNA of the present invention include DNAs having the nucleotide sequence represented by any one of SEQ ID NOS: 9 to 16, a DNA comprising all the individual nucleotide sequences, and a DNA having the nucleotide sequence represented by SEQ ID NO: 21.
[0093] Additionally, DNAs hybridizing under stringent conditions with the DNA having the nucleotide sequence represented by any one of SEQ ID NOS: 9 to 16, a DNA comprising all the individual nucleotide sequences, and a DNA represented by the nucleotide sequence represented by SEQ ID NO: 21 are also encompassed within the scope of the DNA of the present invention. The DNA hybridizing under stringent conditions can be isolated by colony hybridization, plaque hybridization or Southern hybridization or the like, using the DNAs of the nucleotide sequences represented by any one of SEQ ID NOS: 9 to 16, the DNA comprising all the individual nucleotide sequences or the DNA of the nucleotide sequence represented by SEQ ID NO: 21 as the probe. Specifically, the DNA includes a DNA isolated and identified by using a filter on which the colony- or plaque-derived DNA is immobilized, for hybridization in the presence of 0.7 to 1.0 mol/liter NaCl at 65° C. and subsequent washing of the filter with 0.1× to 2× SSC (saline-sodium citrate) solution [1× SSC solution (150 mmol/liter NaCl, 15 mmol/liter sodium citrate); n×means solution at n-fold concentration] under a condition of 65° C.
[0094] The hybridization can be promoted according to the method described in experimental text books, such as Molecular Cloning, Second edition; and Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995). Specifically, the hybridizable DNA includes DNA with at least 80%, preferably 95% or more identical to the DNA of the nucleotide sequence represented by any one of the nucleotide sequences of SEQ ID NOS: 9 to 16, the DNA comprising all the individual nucleotide sequences, or the DNA of the nucleotide sequence represented by SEQ ID NO: 21, when the identity is calculated by BLAST and the like as described above.
[0095] However, the DNA of the present invention does not include DNAs in the public domain.
[0096] The method for isolating the DNA of the present invention is described hereinbelow.
[0097] A microorganism belonging to the genus Corynebacterium is cultured according to a known method [for example, Appl. Microbiol. Biotechnol., 39, 318 (1993)]. After culturing, the chromosomal DNA of the microorganism is isolated and purified according to a known method [for example, Current Protocols in Molecular Biology, Agric. Biol. Chem., 49, 2925 (1985)].
[0098] A method for preparing a DNA library includes methods described in, for example, Molecular Cloning, Second edition; and Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995).
[0099] As the cloning vector for preparing the DNA library, any cloning vector autonomously replicable in Escherichia coli strain K12 can be used, including phage vector and plasmid vector. Specifically, the cloning vector includes ZAP Express [manufactured by Stratagene, Strategies, 5, 58 (1992)], λzap II (manufactured by Staratagene), λgt10 and λgt11 [DNA Cloning, A Practical Approach, 1, 49 (1985)], λTriplEx (manufactured by Clontech), λExCell (manufactured by Amersham Pharmacia Biotech), pBluescript II KS (−) and pBluescript IISK (+) [manufactured by Stratagene, Nucleic Acids Research, 17, 9494 (1989)], pUC18 [Gene, 33, 103 (1985)] and the like.
[0100] As the Escherichia coli for transformation with the vector in which the DNA is inserted, any microorganism belonging to the species Escherichia coli can be used. Specifically, the microorganism includes Escherichia coli XL1-Blue MRF′ [manufactured by Stratagene, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics,39, 440 (1954)], Escherichia coli Y1088 [Science, 222,778 (1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coli JM109 [Gene, 33, 103 (1985)] and the like.
[0101] (b) Acquisition of the DNA of the Present Invention
[0102] The objective clone can be selected from the DNA library by colony hybridization, plaque hybridization or Southern hybridization , as described in experimental textbooks such as Molecular Cloning, Second edition; and Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995).
[0103] The DNA probe for use in the hybridization includes, for example, DNA isolated by PCR [PCR Protocols, Academic Press (1990)] using DNA primers designed from known sequences, in addition to known genes or parts of the known genes and DNA synthesized on the basis of known sequences. The DNA probe includes, for example, a DNA fragment isolated from Escherichia coli chromosome by using the synthetic DNAs of SEQ ID NOS: 17 and 18 as primers, designed on the basis of the sequence of F 0 F 1 -ATPase β subunit gene of Escherichia coli.
[0104] The isolated DNA as it is or after cleavage with an appropriate restriction endonuclease is inserted into a vector. Then, the nucleotide sequence of the DNA is determined by methods for nucleotide sequencing for general use, for example the dideoxy method [Proc.Natl.Acad. Sci. USA, 74, 5463 (1977)] using 373A.DNA sequencer (manufactured by Perkin Elmer).
[0105] In case that the resulting DNA contains only a part of the DNA of the present invention, the full-length DNA can be isolated by hybridization with a DNA fragment as probe, isolated by PCR using primers designed from the isolated DNA sequence.
[0106] The vector in which the isolated DNA of the present invention is inserted includes pBluescript KS (−) (manufactured by Stratagene), pDIRECT [Nucleic Acids Research, 18, 6069 (1990)], pCR-Script Amp SK (+) (manufactured by Staratagene), pT7Blue (manufactured by Novagen), pCR II (manufactured by Invitrogen Corporation), pCR-TRAP (manufactured by Gene Hunter) and pNoTAT7 (manufactured by 5 Prime→3 Prime Co.).
[0107] The DNA having the novel nucleotide sequence isolated as described above includes, for example, the DNA having the nucleotide sequence represented by SEQ ID NO: 21.
[0108] The DNA having the nucleotide sequence represented by SEQ ID NO: 21 encodes all the individual proteins having the amino acid sequences represented by each of SEQ ID NOS: 1 to 8.
[0109] The bacterial strain carrying a plasmid comprising the DNA having the nucleotide sequence represented by SEQ ID NO: 21 includes, for example, Escherichia coli JM109/pE61, JM109/pDW31 and JM109/pUH71.
[0110] Further, the objective DNA can be isolated by preparing primers based on the nucleotide sequence thus determined and carrying out PCR using the chromosomal DNA as the template and the primers.
[0111] The DNA encoding any one of the component proteins of the protein complex of the present invention can be obtained by cleaving the DNA obtained above with a restriction endonuclease and the like.
[0112] For example, the DNA encoding any one of the proteins of SEQ ID NOS: 1 to 8 can be isolated as the DNA having any one of the nucleotide sequences repersented by SEQ ID NOS: 9 to 16, respectively, by cleaving the DNA of SEQ ID NO: 21 and individually isolating the resulting DNA.
[0113] Based on the thus determined nucleotide sequence of DNA, furthermore, the objective DNA can be prepared by chemical synthesis by means of DNA synthesizers such as the DNA synthesizer of Model 8905, manufactured by Perceptive Biosystems, Co.
[0114] [2] Production of the Proteins and the Protein Complex of the Present Invention
[0115] The proteins and the protein complex of the present invention can be prepared, for example, by expressing the DNA of the present invention in a host cell by the following procedures according to the method described in Molecular Cloning, Second edition, Current Protocols in Molecular Biology and the like.
[0116] More specifically, a recombinant DNA is prepared by inserting the DNA of the present invention downstream the promoter of an appropriate expression vector, which is then used for transformation of a host cell compatible with the expression vector, so that a transformant in which the protein or protein complex of the present invention is expressed can be obtained. As the host cell, any host cell capable of having the objective gene expressed, such as bacteria, yeast, an animal cell, an insect cell and a plant cell, can be used. As the expression vector, a vector having autonomous replication in the host cell or integration into the chromosome of the host cell and containing a promoter at a position where the DNA encoding the protein or protein complex of the present invention can be transcribed, is used.
[0117] In case that prokaryotic organisms such as bacteria are used as host cells, it is preferred that the recombinant DNA containing the DNA encoding the protein or protein complex of the present invention can autonomously replicate in the bacteria and simultaneously that, the recombinant DNA is a vector composed of promoter, ribosome binding sequence, the DNA encoding the protein or protein complex of the present invention and a transcription termination sequence. The recombinant DNA may contain a gene regulating the promoter.
[0118] The expression vector includes, for example, pHelix1 (manufactured by Roche Diagnostics), pKK233-2 (manufactured by Amersham Pharmacia Biotech), pSE280 (manufactured by Invitrogen Corporation), pGEMEX-1 (manufactured by Promega Corporation), pQE-8 (manufactured by Qiagen), pKYP10 (Japanese Published Unexamined Patent Application No.110600/1983, U.S. Pat. No. 4,686,191), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II KS(−) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERMBP-5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), Japanese Published Unexamined Patent Application No.221091/1985, U.S. Pat. No. 4,868,125], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Published Unexamined Patent Application No.221091/1985, U.S. Pat. No. 4,868,125], pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Amersham Pharmacia Biotech), pET system (manufactured by Novagen) and pSupex. Additionally, expression vectors with autonomous replication potencies in microorganisms of genus Corynebacterium include for example pCG1 (Japanese Published Unexamined Patent Application No.134500/1983); pCG2 (Japanese Published Unexamined Patent Application No. 35197/1983); pCG4 and pCG11 (both in Japanese Published Unexamined Patent Application No. 183799/1982, U.S. Pat. No. 4,500,640); pCE54 and pCB101 (both in Japanese Published Unexamined Patent Application No.105999/1983, U.S. Pat. No. 4,710,471); pCE51, pCE52 and pCE53 [all in Mol. Gen. Genet., 196, 175 (1984)]; pAJ1844 (Japanese Published Unexamined Patent Application No.21614/1983, U.S. Pat. No. 4,514,502); pHK4 (Japanese Published Unexamined Patent Application No.20399/1995, U.S. Pat. No. 5,616,480); pHM1519 [Agric. Biol. Chem., 48, 2901, (1985)]; pCV35 and pECM1 [both in J. Bacteriol., 172, 1663 (1990)]; pC2 [Plasmid, 36, 62 (1996)] and the like.
[0119] As the promoter, any promoter from which a gene can be expressed in the host cell can be used. For example, promoters derived from Escherichia coli and phages, such as trp promoter (P trp ), lac promoter, P L promoter, P R promoter and T7 promoter are mentioned. Additionally, artificially designed and modified promoters can also be used, such as two aligned P trp promoters in series (P trx ×2), tac promoter, lacT7 promoter, and let I promoter. Still additionally, the promoter capable of functioning in bacteria of the genus Corynebacterium [Microbiology, 142, 1297 (1996), Appl. Microbiol. Biotechnol., 53, 674 (2000) ]] and the like can also be used.
[0120] A plasmid with the distance between the ribosome binding sequence, namely the Shine-Dalgarno sequence and the initiation codon as adjusted to an appropriate length (for example, 6 to 18 bases) is preferably used.
[0121] By modifying the nucleotide sequence of the region encoding the protein or protein complex of the present invention to make codons optimum for the expression in hosts, the productivity of the objective protein or protein complex can be improved.
[0122] For the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA of the present invention. However, it is preferred that a transcription termination sequence is arranged immediately downstream the structural gene.
[0123] The host cell includes microorganisms of the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium and Pseudomonas, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000 , Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109 , Escherichia coli HB101 , Escherichia coli No.49 , Escherichia coli W3110 , Escherichia coli NY49 , Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium immariophilum ATCC14068 , Brevibacterium saccharolyticum ATCC14066 , Brevibacterium flavum ATCC14067 , Brevibacterium lactofermentum ATCC13869 , Corynebacterium ammoniagenes ATCC6872 , Corynebacterium ammoniagenes ATCC21170 , Corynebacterium glutamicum ATCC13032 , Corynebacterium acetoacidophilum ATCC13870 , Microbacterium ammoniaphilum ATCC15354 and Pseudomonas sp. D-0110.
[0124] As the method for transformation with the recombinant DNA, any method to introduce DNA into the host cell can be used, including, for example, the method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], protoplast method [Japanese Published Unexamined Patent Application No.248394/1988, Japanese Published Unexamined Patent Application No.186492/1982, U.S. Pat. No. 4,683,205, Japanese Published Unexamined Patent Application No.56678/1983, U.S. Pat. No. 4,681,847, J. Bacteriol., 159, 306 (1984)], electroporation method (Japanese Published Unexamined Patent Application No.207791/1990) or the methods described in Gene, 17, 107 (1982) and Mol. Gen. Genet., 168, 111 (1979) are mentioned. Otherwise, a DNA library of the chromosome of a microorganism of the genus Corynebacterium is prepared by using Escherichia coli , then DNA is transferred from Escherichia coli to a microorganism of the genus Corynebacterium via conjugation according to known methods [J. Bacteriol. 172, 1663 (1990), J. Bacteriol. 178, 5768 (1996)].
[0125] In case that yeast is used as the host cell, the expression vector includes, for example, YEp13 (ATCC37115), YEp24 (ATCC37051) and YCp50 (ATCC37419).
[0126] As the promoter, any promoter from which a gene can be expressed in yeast strains can be used, including, for example, the promoter of the gene of the glycolytic pathway such as hexokinase, PHO5 promoter, PGKpromoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MFα1 promoter and CUP 1 promoter.
[0127] The host cell includes, for example, microorganisms of the genus Saccharomyces, Kluyveromyces, Trichosporon or Schwanniomyces, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans and Schwanniomyces alluvius.
[0128] As the method for transformation with the recombinant DNA, any method to introduce DNA into yeast can be used, including, for example, electroporation method [Methods. Enzymol., 194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriology, 153, 163 (1983)], and the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
[0129] In case that an animal cell is used as the host cell, the expression vector includes, for example, pcDNAI, pcDM8 (commercially available from Funakoshi Co., Ltd.), pAGE107 [Japanese Published Unexamined Patent Application No.22979/1991, U.S. Pat. No. 516,735; Cytotechnology, 3, 133, (1990)], pAS3-3 (Japanese Published Unexamined Patent Application No.227075/1990, U.S. Pat. No. 5,218,092), pCDM8 [Nature, 329, 840, (1987)] pcDNAI/Amp (manufactured by Invitrogen Corporation), pREP4 (manufactured by Invitrogen Corporation), pAGE103 [J. Biochemistry, 101, 1307 (1987)] and pAGE210.
[0130] As the promoter, any promoter from which a gene can be expressed in animal cells can be used, including, for example, the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the SV40 early promoter, the promoter of retrovirus, metallothionein promoter, heat shock promoter and SRα promoter. Additionally, an enhancer of the IE gene of human CMV can be used together with such promoters.
[0131] The host cell includes Namalwa cell as a human cell, COS cell as a monkey cell, CHO cell as Chinese hamster cell, HBT5637 (Japanese Published Unexamined Patent Application No.299/1988, ATCC No. ATB-9) and the like.
[0132] As the method for transformation with the recombinant vector, any method to introduce DNA into animal cells can be used, including, for example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Published Unexamined Patent Application No.227075/1990, U.S. Pat. No. 5,218,092) and lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
[0133] In case that insect cells are used as the host cells, the protein or protein complex of the present invention can be expressed by methods described in, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992) and Bio/Technology, 6, 47 (1988).
[0134] Specifically, a recombinant virus is recovered in the culture supernatant of an insect cell via co-transfection of the insect cell with the recombinant gene transfer vector and baculovirus; further, an insect cell is infected with the recombinant virus, to express the protein or protein complex of the present invention.
[0135] The gene transfer vector for use in the method includes, for example, pVL1392, pVL1393 and pBlueBacIII (all manufactured by Invitrogen Corporation).
[0136] As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus can be used as a virus which infects insects of the family Noctuidae.
[0137] As the insect cells, Sf9 and Sf21 as the ovarian cells of Spodoptera frugiperda [Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)] and High 5 as the ovarian cell of Trichoplusia ni (manufactured by Invitrogen Corporation) can be used.
[0138] The method for the co-transfection of the insect cells with the recombinant gene transfer vector and the baculovirus for the preparation of the recombinant virus, includes, for example, the calcium phosphate method (Japanese Published Unexamined Patent Application No.227075/1990, U.S. Pat. No. 5,218,092) and the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
[0139] In case that plant cells are used as the host cells, the expression vector includes, for example, Ti plasmid and tobacco mosaic virus vector.
[0140] As the promoter, any promoter including, for example, the 35S promoter of cauliflower mosaic virus (CaMV) and rice actin 1 promoter can be used, as long as a gene can be expressed from the promoter in plant cells.
[0141] The host cell includes plant cells of, for example, tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, and barley.
[0142] As the method for transformation with the recombinant DNA, any method for introducing DNA into plant cells can be used, including, for example, the method using Agrobacterium (Japanese Published Unexamined Patent Application No.140885/1984, Japanese Published Unexamined Patent Application No. 70080/1985 and WO 94/00977), the electroporation method (Japanese Published Unexamined Patent Application No.251887/1985) and the method using particle gun (Japanese Patent No. 2606856 and Japanese Patent No. 2517813).
[0143] As the method for expressing the gene, secretory expression, or fusion protein expression or the like can be carried out according to the method described in Molecular Cloning, Second edition, in addition to direct expression.
[0144] In case of the expression in yeast, animal cells, insect cells or plant cells, a protein or protein complex with addition of sugar or sugar chain can be recovered.
[0145] The protein or protein complex of the present invention can be produced by culturing the transformant thus constructed in a culture medium to allow the transformant to express and accumulate the protein or protein complex of the present invention in the culture and recovering the protein or protein complex from the culture. Culturing the transformant of the present invention in a culture medium can be carried out according to a usual method to be applied for in culturing hosts.
[0146] As the culture medium for culturing the transformant obtained by using bacteria such as Escherichia coli or eukaryotic organisms such as yeast as the hosts, any of natural and synthetic culture media containing carbon sources, nitrogen sources, inorganic salts and the like, which can be assimilated by the biological organism and in which the transformant can be cultured efficiently can be used.
[0147] As the carbon source, any carbon source assimilated by the biological organism can be used, such as carbohydrates such as glucose, fructose, sucrose, molasses containing these substances, starch or starch hydrolyzates; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.
[0148] As the nitrogen source, ammonia, ammonium salts of inorganic acids or organic acids, such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; other nitrogen-containing compounds; and peptone, meat extract, yeast extract, corn steep liquor, case in hydrolyzates, soy bean bran, soy bean bran hydrolyzates, various fermenting bacteria and digested products thereof and the like can be used.
[0149] As the inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate and the like can be used.
[0150] The culturing is generally carried out under aerobic conditions, by shaking culture or submerged aeration agitation culture. The culturing temperature is preferably 15 to 40° C., and the culturing period is generally 16 hours to 7 days. The pH during the culturing is retained at 3.0 to 9.0. The pH is adjusted with inorganic or organic acids, alkali solutions, urea, calcium carbonate, ammonia and the like.
[0151] Additionally, antibiotics such as ampicillin and tetracycline can be added to the culture medium if required.
[0152] For culturing a microorganism transformed with a recombinant vector where an inducible promoter is used as promoter, an inducer can be added if required. For example, for culturing a microorganism transformed with a recombinant vector where lac promoter is used, isopropyl-β-D-thiogalactopyranoside and the like can be added to the culture medium, and for culturing a microorganism transformed with a recombinant vector where trp promoter is used, indole acrylic acid and the like can be added to the culture medium.
[0153] As the culture medium for culturing a transformant obtained by using an animal cell as the host, culture media for general use, such as RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM [Science, 122, 501 (1952)], Dulbecco's modified MEM [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] or culture media prepared by adding fetal calf serum to these culture media can be used.
[0154] The culturing is generally carried out under conditions, such as at pH 6 to 8 and 30 to 40° C. in the presence of 5% CO 2 for one to 7 days.
[0155] Additionally, antibiotics such as kanamycin and penicillin can be added to the culture medium during the culturing if required.
[0156] As the culture medium for culturing a transformant obtained by using an insect cell as the host, TNM-FH medium (manufactured by Pharmingen); Sf-900 II SFM (manufactured by Life Technologies); ExCell 400 and ExCell 405 (both manufactured by JRH Biosciences, Co.); Grace's Insect Medium [Nature, 195, 788 (1962)]; and the like can be used.
[0157] The culturing is generally conducted under conditions, for example at pH 6 to 7 and 25 to 30° C. for one to 5 days.
[0158] Additionally, antibiotics such as gentamycin can be added to the culture medium if reqired.
[0159] A transformant obtained by using a plant cell as the host can be cultured as a cell or after differentiation into a differentiated plant cell or a plant organ. As the culture medium for culturing the transformant, Murashige and Skoog (MS) medium, White's medium, or culture media prepared by adding plant hormones such as auxin and cytokinin or the like to these culture media can be used.
[0160] The culturing is generally conducted under conditions, for example at pH 5 to 9 and 20 to 40° C. for 3 to 60 days.
[0161] Additionally, antibiotics such as kanamycin and hygromycin can be added to the culture medium if required.
[0162] As described above, the protein or protein complex of the present invention can be produced, by culturing a microorganism-, animal cell- or plant cell-derived transformant carrying the recombinant DNA into which the DNA encoding the protein or protein complex of the present invention is inserted by general culturing methods, to allow the transformant to express and accumulate the protein or protein complex and recovering the protein or protein complex from the culture.
[0163] As the method for expressing the gene, secretory expression or expression as a fusion protein can be carried out according to the method described in Molecular Cloning, Second edition, in addition to direct expression.
[0164] The method for expressing the protein or protein complex of the present invention includes a method for expressing inside host cells, a method for secreting outside host cells, and a method for expressing on the outer membrane of host cells. By changing a host cell to be used or modifying the structure of the protein to be expressed, the method can be selected.
[0165] In case that the protein or protein complex of the present invention is expressed inside the host cell or on the outer membrane of the host cell, the protein or protein complex can be secreted outside the host cell, according to the method of Paulson, et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Law, et al. [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the methods described in Japanese Published Unexamined Patent Application No.336963/1993, WO 94/23021 and the like.
[0166] Specifically, the protein or protein complex of the present invention can be secreted outside the host cell, by expressing the protein or protein complex of the present invention in a form such that signal peptide is added to a protein containing the active site of the protein or protein complex of the present invention, by means of the genetic engineering.
[0167] According to the method described in Japanese Published Unexamined Patent Application No.227075/1990 (U.S. Pat. No. 5,218,092), the productivity can be improved by utilizing gene amplification systems using dihydrofolate reductase genes and the like. So as to isolate and purify the protein or protein complex of the present invention from the culture of the transformant, general methods for isolation and purification of enzymes can be used.
[0168] In case that the protein or protein complex of the present invention is expressed, for example, in the soluble state inside a cell, the cell is recovered by centrifugation after culturing and is then suspended in an aqueous buffer, which is subsequently disrupted with an ultrasonicator, French press, Manton-Gaulin homogenizer, Dinomill and the like, to recover a cell-free extract. From the supernatant recovered by centrifuging the cell-free extract, a purified product sample can be isolated using known methods to isolate and purify enzymes, including, for example, solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Industry, Co.), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Amersham Pharmacia Biotech, Co.), hydrophobic chromatography using resins such as phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing, singly or in combination.
[0169] In case that the protein or protein complex is expressed in the form of an inclusion body inside a cell, the cell is recovered and disrupted in the same manner as descrived above, followed by centrifugation to recover a precipitate fraction, from which the protein or protein complex is recovered by known methods. The inclusion body of the protein or protein complex is solubilized with a protein denaturant. The protein-solubilized solution is diluted with or dialyzed to against a solution without protein denaturant or a solution where the concentration of the protein denaturant is at lower level not to denature the protein or protein complex, to refold the protein or protein complex in a normal tertiary structure. Subsequently, the resulting protein or protein complex is subjected to the same method for isolation and purification as described above, to isolate a purified product sample.
[0170] In case that the protein or protein complex of the present invention or derivatives thereof such as a glycosylated form are secreted extracellularly, the protein or protein complex or the derivatives thereof, such as a glycosylated form, can be recovered in the culture supernatant. More specifically, the culture is treated by the same means as described above, such as centrifugation, to recover the soluble fraction, from which a purified product sample can be isolated by the method for isolation and purification as described above.
[0171] [3] Production of Nucleoside 5′-triphosphate
[0172] Using as an enzyme source the culture of the transformant prepared described above in [2] or a treated product of the culture, nucleoside 5′-triphosphate can be produced by reacting a precursor of nucleoside 5′-triphosphate with the enzyme source in an aqueous medium.
[0173] The treated product of the culture includes, for example, a concentrate of the culture, a dried product of the culture, transformant cells recovered by centrifuging the culture, a dried product of the transformant cells, a freeze-dried product of the transformant cells, a detergent-treated product of the transformant cells, a sonicated product of the transformant cells, a mechanically disrupted product of the transformant cells, a solvent-treated product of the transformant cells, a enzyme-treated product of the transformant cells, a protein fraction of the transformant cells, an immobilized product of the transformant cells or an enzyme sample extracted from the transformant cells.
[0174] As the enzyme source for generating nucleoside 5′-triphosphate, the transformant cells are used at a concentration of 1 g/liter to 500 g/liter, preferably 10 g/liter to 300 g/liter based on wet weight.
[0175] The precursor of nucleoside 5′-triphosphate includes, for example, adenine, guanine, uracil, cytosine, hypoxanthine, adenosine, guanosine, uridine, cytidine, inosine, adenosine 5′-monophosphate, guanosine 5′-monophosphate, uridine 5′-monophosphate, cytidine 5′-monophosphate, and inosine 5′-monophosphate.
[0176] The nucleoside 5′-triphosphate includes for example adenosine 5′-triphosphate, guanosine 5′-triphosphate, uridine 5′-triphosphate, and cytidine 5′-triphosphate.
[0177] The aqueous medium for use in generating nucleoside 5′-triphosphate includes, for example, water; buffers such as phosphate buffer, carbonate buffer, acetate buffer, borate buffer, citrate buffer and Tris buffer; alcohols such as methanol and ethanol; esters such as ethyl acetate; ketones such as acetone; and amides such as acetamide. Additionally, the liquid culture of the microorganism used as the enzyme source can also be used as the aqueous medium.
[0178] For the generation of nucleoside 5′-triphosphate, a detergent or an organic solvent can be added if required. As the detergent, any of detergents promoting the generation of nucleoside 5′-triphosphate can be used, including, for example, nonionic detergents such as polyoxyethylene octadecylamine (for example, Nymeen S-215, manufactured by Nippon Oil & Fats Co., Ltd.), cationic detergents such as cetyltrimethylammonium bromide and alkyldimethyl benzylammonium chloride (for example, Cation F2-40E, manufactured by Nippon Oil & Fats Co., Ltd.) and anionic detergents such as lauroyl sarcosinate and tertiary amines such as alkyldimethylamine (for example, tertiary amine FB, manufactured by Nippon Oil & Fats Co., Ltd.), singly or in combination of several types thereof. The detergent is generally used at a concentration of 0.1 to 50 g/liter. The organic solvent includes, for example, xylene, toluene, aliphatic alcohol, acetone and ethyl acetate and is generally used at a concentration of 0.1 to 50 ml/liter.
[0179] The nucleoside 5′-triphosphate generating reaction is carried out in an aqueous medium under conditions of pH 5 to 10, preferably pH 6 to 8 and 20 to 60° C., for one to 96 hours. For the generating reaction, inorganic salts such as magnesium chloride can be added if required.
[0180] The nucleoside 5 ′-triphosphate generated in an aqueous medium is determined by known methods (for example, WO 98/12343) using HPLC.
[0181] The nucleoside 5′-triphosphate generated in the reaction solution can be isolated by known methods using activated charcoal, ion exchange resins and the like.
[0182] The present invention is illustrated in the following examples, but the present invention is not limited to these examples.
EXAMPLE 1
Preparation of the Chromosomal DNA of Corynebacterium ammoniagenes strain ATCC6872
[0183] Corynebacterium ammoniagenes strain ATCC6872 was inoculated in 8 ml of a culture medium prepared by adding glycine (10 mg/ml) to CM medium (10 mg/ml polypeptone, 10 mg/ml meat extract, 5 mg/ml yeast extract, 3 mg/ml sodium chloride, 30 μg/ml biotin, pH7.2), for culturing at 30° C. overnight.
[0184] After culturing, the cells were collected from the resulting culture via centrifugation.
[0185] The cells were washed with TE buffer [10 mmol/liter Tris-HCl, 1 mmol/liter ethylenediaminetetraacetic acid (EDTA), pH8.0] and subsequently suspended in 800 μl of the same buffer. To the suspension were added 40 μl of 50 mg/ml lysozyme solution and 20 μl of 10 mg/ml RNase A solution, and the resulting solution was incubated at 37° C. for one hour. To the resulting solution was added 20 μl of 20% sodium dodecylsulfate (SDS) solution, and the resulting solution was incubated at 70° C. for one hour. 24 μl of 20mg/ml proteinase K solution was added to the resulting reaction solution, and the resulting solution was incubated at 50° C. for one hour. To the resulting reaction solution was added an equal volume of phenol, followed by agitation. The resulting solution was left to stand overnight at 4° C., to extract DNA into the aqueous layer. The aqueous layer was then recovered. To the aqueous layer was added an equal volume of phenol/chloroform, followed by agitation and extraction for 2 hours, and the resulting aqueous layer was recovered. To the resulting aqueous layer was added an equal volume of chloroform/isoamyl alcohol, followed by agitation and extraction for 30 minutes, and the resulting aqueous layer was recovered. To the aqueous layer was added a 2-fold volume of ethanol, to precipitate DNA. The resulting precipitate was dissolved in 300 μl of TE buffer, and the resulting solution was used as chromosomal DNA.
EXAMPLE 2
Probe-labeling
[0186] According to the method described in Current Protocols in Molecular Biology, chromosomal DNA was prepared from Escherichia coli strain W3110. The DNA primer of SEQ ID NO: 17 and the DNA primer of SEQ ID NO: 18, corresponding to parts of the gene coding for the β subunit of Escherichia coli F 0 F 1 -ATPase, were synthesized using the DNA synthesizer of Model 8905, manufactured by Perceptive Biosystems, Co. Using the synthesized DNAs as primers and the chromosomal DNA of Escherichia coli strain W3110 as a template, the probe was labeled by PCR DIG Probe Synthesis Kit (manufactured by Roche Diagnostics KK). Labeling reaction was carried out according to the manual of the kit, using 0.1 μg of the chromosomal DNA and 0.5 pmol of each of the primers in 50 μl of the reaction solution, and reaction step of 94° C. for 30 seconds, of 55° C. for one minute and of 72° C. for one minute were repeated 30 times.
EXAMPLE 3
Southern Hybridization
[0187] 10 μg of the chromosomal DNA as isolated in Example 1 was completely digested with restriction endonuclease EcoRI. Similarly, the chromosome was thoroughly cleaved with BamHI. 1 μg each of the samples cleaved with the respective restriction endonucleases was subjected to agarose gel electrophoresis. After electrophoresis, the DNA was transferred onto nylon membrane according to the method described in Molecular Cloning, Second edition.
[0188] Hybridization was carried out by using DIG Luminescent Detection Kit (manufactured by Roche Diagnostics KK.). The nylon membrane (Hybond N+) with the DNA transferred thereon was subjected to prehybridization in 1 ml of prehybridization solution [0.5 mol/liter Na 2 HPO 4 -12H 2 O (pH7.2), 7% SDS, 1 mmol/liter EDTA] per 10 cm 2 of membrane at 65° C. for 30 minutes. Then, 1 ml of a prehybridization solution containing 1 μl of the probe prepared in Example 2 per 3 ml of the solution was used per 5 cm 2 of membrane, for hybridization at 65° C. for 16 hours. After the hybridization, the membrane was washed with a wash buffer [40 mmol/liter Na 2 HPO 4 -12H 2 O (pH 7.2), 1%SDS] at 65° C. for 20 minutes. The wash procedure was repeated three times. Then, the treatment using 1 ml of DIG Buffer 1 [100 mmol/liter Tris-HCl (pH7.5), 150 mmol/liter NaCl] per 2 cm 2 of membrane at room temperature for 10 minutes was repeated twice. Subsequently, blocking against antibodies was carried out, using 0.5 w/v % blocking solution at room temperature for one hour. Labeling with an antibody was carried out at room temperature, using 62.5 μl of DIG Buffer 1 containing 75 mU/ml anti-DIG AP Fab fragment and 0.2% Tween 20 per 1 cm 2 of membrane for 30 minutes. Additionally, wash procedure using DIG Buffer 1 containing 0.2% Tween 20 at a ratio of 0.125 ml per 1 cm 2 of membrane at room temperature for 15 minutes was repeated twice. Subsequently, the membrane was treated with DIG Buffer 3 [100 mmol/liter Tris-HCl (pH9.5), 100 mmol/liter NaCl, 50 mmol/liter MgCl 2 ] for 3minutes. After dropwise addition of CSPD solution, the resulting mixture was incubated at 37° C. for 15 minutes, luminescent reaction was promoted.
[0189] After termination of the luminescent reaction, the membrane was dried in air. Subsequently, the membrane was exposed to an X-ray film for 30 minutes.
[0190] As a result of hybridization, the probe strongly hybridized with the EcoRI-cleaved 6.5-Kb fragment and the BamHI-cleaved 6-Kb fragment of Corynebacterium ammoniagenes chromosomal DNA.
EXAMPLE 4
Colony Hybridization
[0191] 1 μg of Corynebacterium ammoniagenes ATCC6872 chromosomal DNA was completely digested with restriction endonuclease EcoRI or BamHI, and the individual digested fragments were separated by agarose gel electrophoresis to recover fragments around the EcoRI-cleaved 6.5-kb fragment and the BamHI-cleaved 6-Kb fragment, by RECOCHIP (manufactured by Takara Shuzo Co., Ltd.). 0.1 μg of a plasmid vector pBluescript II KS (−) (manufactured by Stratagene) was thoroughly digested with EcoRI or BamHI, which was then subjected to dephosphorylation reaction with temperature-sensitive alkaliphosphatase (manufactured by GIBCO BRL).
[0192] The EcoRI cleavage fragment around 6.5 kb and the EcoRI-cleaved and phosphatase-treated pBluescript II KS (−) were subjected to a ligation reaction at 16° C. for 16 hours, using a ligation kit. Using the ligation reaction solution, Escherichia coli strain JM109 was transformed by the method using calcium ion described in Molecular Cloning, Second edition. The resulting transformant was spread on an LB agar medium containing 100 μg/ml ampicillin, for overnight culturing at 37° C. Similarly, the BamHI cleavage fragment around 6 kb and the BamHI-cleaved and phosphatase-treated pBluescript II KS (−) were subjected to a ligation reaction at 16° C. for 16 hours, using a ligation kit. Using the ligation reaction solution, the Escherichia coli strain JM109 was transformed by the method described above. The resulting transformant was spread on an LB agar culture medium containing 100 μg/ml ampicillin, for overnight culturing at 37° C. The growing colony was transferred on the membrane (Hybond N+) and lysed to fix the DNA on the membrane according to the method described in Molecular Cloning, Second edition. Colony hybridization was carried out by the same method as for Southern hybridization in Example 3.
[0193] Consequently, a bacterial strain harboring the plasmid pE61 carrying the 6.5-kb EcoRI cleavage fragment of Corynebacterium ammoniagenes ATCC6872 chromosomal DNA and a bacterial strain harboring the plasmid pDW31 carrying the 6-kb BamHI cleavage fragment thereof were selected as positive clones.
[0194] From the colonies of the two clones were isolated the plasmids, which were the plasmids pE61 and pDW31, so that the structures of the plasmids were confirmed by digestion with restriction endonucleases (FIG. 1).
EXAMPLE 5
Recovery of Upstream Gene
[0195] The plasmids pE61 and pDW31 as obtained in Example 4 were found not to carry the genes predicted to be present upstream among the genes coding for the proteins composing the F 0 F 1 -ATPase protein complex. Therefore, the genes present upstream were isolated by the following method.
[0196] The DNA primer having the nucleotide sequence represented by SEQ ID NO: 19 and the DNA primer having the nucleotide sequence represented by SEQ ID NO: 20, corresponding to parts of the F 0 F 1 -ATPase b subunit gene which exists in the plasmid pE61, were synthesized using a DNA synthesizer of Model 8905, which was manufactured by Perceptive Biosystems, Co. Using the synthesized DNAs as primers and the plasmid pE61 DNA as a template, the probe was labeled by PCR DIG Probe Synthesis Kit (manufactured by Roche Diagnostics). Using the resulting probe, Southern hybridization was carried out by the same method as in Example 3. Strong hybridization with a 5.0-Kb HindIII-digested fragment of the chromosomal DNA of Corynebacterium ammoniagenes strain ATCC6872 was observed.
[0197] Using a bacterial strain harboring a plasmid constructed by inserting a fragment of the HindIII-digested chromosome DNA around 5.0 kb into pbluescript II KS (−),colony hybridization was carried out. Then, a bacterial strain harboring a plasmid pUH71 carrying a Hind III fragment of 5-kb was hybridized strongly and thus, was selected as a positive clone.
[0198] From the colony of the clone was isolated the plasmid, which was named pUH71. Then, the structure of the plasmid was confirmed by digestion with restriction endonucleases.(FIG. 1)
EXAMPLE 6
Determination of a Nucleotide Sequence
[0199] The nucleotide sequences of the inserted fragments in the plasmids pE61 and the pDW31 and in the plasmid pUH71 were determined with ABI 377 Sequencer. Open reading frames consisting of individual nucleotide sequences represented by each of SEQ ID NOS: 9 to 16 encoding the amino acid sequences represented by each of SEQ ID NOS: 1 to 8, respectively, existed in the nucleotide sequences of the fragments.
[0200] As a result of comparative analysis the nucleotide sequences of the fragments with other bacterial F 0 F 1 -ATPase genes, it is shown that the nucleotide sequences correspond to an operon of genes of the subunits a, c, b, δ, α, γ, β and ε located in this order, as in many other bacteria. The nucleotide sequence of the operon is shown as SEQ ID NO: 21.
[0201] Further, Table 1 shows the amino acid sequence identity (%) of each subunit of Bacillus subtilis F 0 F 1 -ATPase [J. Bacteriol., 176, 6802 (1994)] and each subunit of Escherichia coli F 0 F 1 -ATPase[Biochem. J., 224, 799 (1984)] with each subunit of the F 0 F 1 -ATPase of the present invention, respectively.
TABLE 1 subunits a b c α β γ δ ε Bucillus subtilis 26 29 47 54 65 35 24 31 Escherichia coli 23 31 35 48 61 38 24 32

[0202]



0









SEQUENCE LISTING





<160> NUMBER OF SEQ ID NOS: 21



<210> SEQ ID NO 1

<211> LENGTH: 304

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 1



Met Cys Asp Gly Val Arg Ser Cys Asp Arg Glu Phe Glu Thr Ser Ile

1 5 10 15



Ala Pro Tyr Asp Val Asp Asn Arg Thr Ala Arg Thr Arg Glu Arg Thr

20 25 30



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

35 40 45



Leu Asp Pro Glu Phe Phe Pro Gly Gln Tyr Tyr Gly Asp Ile Leu Phe

50 55 60



Asp Asp Val Leu Gly Gly Trp Phe Ala Leu Asp Arg Ile Met Leu Val

65 70 75 80



Arg Leu Leu Met Thr Ala Val Leu Val Leu Leu Phe Ile Ala Ala Phe

85 90 95



Arg Asn Pro Lys Leu Val Pro Lys Gly Leu Gln Asn Val Ala Glu Tyr

100 105 110



Ala Leu Asp Phe Val Arg Ile His Ile Ala Glu Asp Ile Leu Gly Lys

115 120 125



Lys Glu Gly Arg Arg Phe Leu Pro Leu Leu Ala Ala Ile Phe Phe Gly

130 135 140



Thr Leu Phe Trp Asn Val Ser Thr Ile Ile Pro Ala Leu Asn Ile Ser

145 150 155 160



Ala Asn Ala Arg Ile Gly Met Pro Ile Val Leu Ala Gly Ala Ala Tyr

165 170 175



Ile Ala Met Ile Tyr Ala Gly Thr Lys Arg Tyr Gly Phe Gly Lys Tyr

180 185 190



Val Lys Ser Ser Leu Val Ile Pro Asn Leu Pro Pro Ala Leu His Leu

195 200 205



Leu Val Val Pro Ile Glu Phe Phe Ser Thr Phe Ile Leu Arg Pro Val

210 215 220



Thr Leu Ala Ile Arg Leu Met Ala Asn Phe Leu Ala Gly His Ile Ile

225 230 235 240



Leu Val Leu Leu Tyr Ser Ala Thr Asn Phe Phe Phe Trp Gln Leu Asn

245 250 255



Gly Trp Thr Ala Met Ser Gly Val Thr Leu Leu Ala Ala Val Leu Phe

260 265 270



Thr Val Tyr Glu Ile Ile Ile Ile Phe Leu Gln Ala Tyr Ile Phe Ala

275 280 285



Leu Leu Thr Ala Val Tyr Ile Glu Leu Ser Leu His Ala Asp Ser His

290 295 300





<210> SEQ ID NO 2

<211> LENGTH: 79

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 2



Met Asn Asp Ile Ile Leu Ala Gln Ala Thr Glu Thr Ser Phe Asp Gly

1 5 10 15



Leu Gln Ser Ile Gly Tyr Gly Leu Ala Thr Ile Gly Pro Gly Leu Gly

20 25 30



Ile Gly Ile Leu Val Gly Lys Thr Val Glu Gly Met Ala Arg Gln Pro

35 40 45



Glu Met Ala Gly Gln Leu Arg Thr Thr Met Phe Leu Gly Ile Ala Phe

50 55 60



Val Glu Ala Leu Ala Leu Ile Gly Leu Val Ala Gly Phe Leu Phe

65 70 75





<210> SEQ ID NO 3

<211> LENGTH: 189

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 3



Met Asn Asn Val Phe Tyr Tyr Leu Ala Ala Glu Gly Glu Ser Leu Pro

1 5 10 15



Leu Glu Gly Gly Asn Ser Leu Leu Phe Pro Lys Ser Tyr Asp Ile Val

20 25 30



Trp Ser Leu Ile Pro Phe Leu Ile Ile Leu Ile Val Phe Ala Met Phe

35 40 45



Val Ile Pro Lys Phe Gln Glu Leu Leu Gln Glu Arg Glu Asp Arg Ile

50 55 60



Glu Gly Gly Ile Lys Arg Ala Glu Ala Gln Gln Ala Glu Ala Lys Ala

65 70 75 80



Ala Leu Glu Lys Tyr Asn Ala Gln Leu Ala Asp Ala Arg Ala Glu Ala

85 90 95



Ala Glu Ile Arg Glu Gln Ala Arg Glu Arg Gly Lys Gln Ile Glu Ala

100 105 110



Glu Ala Lys Thr Gln Ala Glu Glu Glu Ala Arg Arg Ile Val Ala Gly

115 120 125



Gly Glu Lys Gln Leu Glu Ala Ser Arg Ala Gln Val Val Ala Glu Leu

130 135 140



Arg Ser Asp Ile Gly Gln Asn Ser Ile Asn Leu Ala Glu Lys Leu Leu

145 150 155 160



Gly Gly Glu Leu Ser Glu Ser Thr Lys Gln Ser Ser Thr Ile Asp Asn

165 170 175



Phe Leu Ser Glu Leu Asp Ser Val Ala Ser Ala Gly Lys

180 185





<210> SEQ ID NO 4

<211> LENGTH: 271

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 4



Met Lys Ala Ala Ser Arg Glu Ser Leu Ala Ser Ala Thr Glu Ser Leu

1 5 10 15



Asp Ser Asn Leu Ala Ala Gln Ala Gly Val Ala Val Ser Thr Met Thr

20 25 30



Gly Met Glu Leu Phe Glu Val Ser Gln Val Leu Gly Asp Asp Arg Glu

35 40 45



Leu Arg Gly Ala Val Ile Asp Glu Ser Ala Ser Thr Glu Ser Arg Lys

50 55 60



Lys Leu Val Asn Asp Leu Phe Gly Ala Lys Val Ser Pro Ala Thr Leu

65 70 75 80



Gln Val Leu Glu Gln Ile Ala Ser Ser Lys Trp Ser Ser Ala Arg Glu

85 90 95



Met Val Ser Gly Leu Ile Ala Leu Ser Arg Arg Ala Leu Met Arg Gly

100 105 110



Ala Glu Ser Glu Gly Gln Leu Gly Gln Val Glu Asp Glu Leu Phe Arg

115 120 125



Leu Ser Arg Ile Leu Asp Arg Glu Gly Glu Leu Thr Gln Leu Leu Ser

130 135 140



Asp Arg Ala Ala Glu Pro Ala Arg Lys Arg Lys Leu Leu Ala Asp Val

145 150 155 160



Leu Tyr Gly Lys Val Thr Lys Phe Thr Glu Ala Leu Ala Leu Gln Val

165 170 175



Ile Asp Arg Pro Glu His Asn Pro Ile Asp Asp Ile Ala Asn Leu Ala

180 185 190



Ala Glu Ala Ala Gln Leu Gln Gly Arg Thr Val Ala His Val Val Ser

195 200 205



Ala Gly Glu Leu Asn Glu Gly Gln Gln Ala Val Leu Ala Glu Lys Leu

210 215 220



Gly Lys Ile Tyr Gly Arg Ala Met Ser Ile His Ser Glu Val Asp Thr

225 230 235 240



Ser Leu Leu Gly Gly Met Thr Ile Arg Val Gly Asp Glu Val Ile Asp

245 250 255



Gly Ser Thr Ala Gly Lys Ile Glu Arg Leu Arg Thr Ala Leu Lys

260 265 270





<210> SEQ ID NO 5

<211> LENGTH: 546

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 5



Met Ala Glu Leu Thr Ile Ser Ser Asp Glu Ile Arg Ser Ala Ile Ala

1 5 10 15



Asn Tyr Thr Ser Ser Tyr Ser Ala Glu Ala Ser Arg Glu Glu Val Gly

20 25 30



Val Val Ile Ser Ala Ala Asp Gly Ile Ala Gln Val Ser Gly Leu Pro

35 40 45



Ser Val Met Ala Asn Glu Leu Leu Glu Phe Pro Gly Gly Val Ile Gly

50 55 60



Val Ala Gln Asn Leu Glu Thr Asn Ser Ile Gly Val Val Ile Leu Gly

65 70 75 80



Asn Tyr Glu Ser Leu Lys Glu Gly Asp Gln Val Lys Arg Thr Gly Glu

85 90 95



Val Leu Ser Ile Pro Val Gly Glu Glu Phe Leu Gly Arg Val Ile Asn

100 105 110



Pro Leu Gly Gln Ala Ile Asp Gly Leu Gly Pro Ile Ala Gly Glu Glu

115 120 125



Asp Arg Val Leu Glu Leu Gln Ala Pro Ser Val Leu Gln Arg Gln Pro

130 135 140



Val Glu Glu Pro Met Gln Thr Gly Ile Lys Ala Ile Asp Ala Met Thr

145 150 155 160



Pro Ile Gly Arg Gly Gln Arg Gln Leu Ile Ile Gly Asp Arg Lys Thr

165 170 175



Gly Lys Thr Ala Val Cys Ile Asp Thr Ile Leu Asn Gln Lys Ala Asn

180 185 190



Trp Glu Ser Gly Asp Lys Asn Lys Gln Val Arg Cys Ile Tyr Val Ala

195 200 205



Ile Gly Gln Lys Gly Ser Thr Ile Ala Gly Val Arg Lys Thr Leu Glu

210 215 220



Glu Gln Gly Ala Leu Glu Tyr Thr Thr Ile Val Ala Ala Pro Ala Ser

225 230 235 240



Asp Ser Ala Gly Phe Lys Trp Leu Ala Pro Phe Ser Gly Ala Ala Leu

245 250 255



Gly Gln His Trp Met Tyr Gln Gly Asn His Val Leu Val Ile Tyr Asp

260 265 270



Asp Leu Thr Lys Gln Ala Glu Ala Tyr Arg Ala Ile Ser Leu Leu Leu

275 280 285



Arg Arg Pro Pro Gly Arg Glu Ala Tyr Pro Gly Asp Val Phe Tyr Leu

290 295 300



His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu Ser Asp Asp Leu Gly

305 310 315 320



Ala Gly Ser Leu Thr Ala Leu Pro Ile Ile Glu Thr Lys Ala Asn Asp

325 330 335



Val Ser Ala Phe Ile Pro Thr Asn Val Ile Ser Ile Thr Asp Gly Gln

340 345 350



Val Phe Leu Glu Ser Asp Leu Phe Asn Gln Gly Val Arg Pro Ala Ile

355 360 365



Asn Val Gly Val Ser Val Ser Arg Val Gly Gly Ala Ala Gln Thr Lys

370 375 380



Gly Met Lys Lys Val Ala Gly Asn Leu Arg Leu Asp Leu Ala Ser Tyr

385 390 395 400



Arg Asp Leu Gln Gly Phe Ala Ala Phe Ala Ser Asp Leu Asp Pro Val

405 410 415



Ser Lys Ala Gln Leu Glu Arg Gly Glu Arg Leu Val Glu Ile Leu Lys

420 425 430



Gln Ser Glu Ser Ser Pro Gln Ala Val Glu Tyr Gln Met Val Ser Ile

435 440 445



Phe Leu Ala Glu Glu Gly Val Phe Asp Val Val Pro Val Glu Asp Val

450 455 460



Arg Arg Phe Glu Ala Asp Val Gln Glu Tyr Leu Gln Gln Asn Thr Pro

465 470 475 480



Glu Val Tyr Glu Gln Ile Ala Gly Gly Lys Ala Phe Thr Asp Glu Ser

485 490 495



Lys Glu Ala Leu Leu Ala Ala Ala Lys Asp Phe Thr Pro Ser Phe Arg

500 505 510



Thr Thr Glu Gly His Asn Leu Gly Thr Glu Ala Pro Val Asp Pro Leu

515 520 525



Ala Glu Glu Glu Val Lys Lys Thr Glu Val Thr Val Ser Arg Lys Ser

530 535 540



Ala Lys

545





<210> SEQ ID NO 6

<211> LENGTH: 327

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 6



Met Ala Asn Leu Arg Glu Leu Arg Asp Arg Ile Arg Ser Val Asn Ser

1 5 10 15



Thr Lys Lys Ile Thr Lys Ala Gln Glu Leu Ile Ala Thr Ser Arg Ile

20 25 30



Thr Lys Ala Gln Ala Lys Val Asp Ala Ala Ala Pro Tyr Ala His Glu

35 40 45



Met Ser Asn Met Met Asp Arg Leu Ala Ser Ala Ser Ser Leu Glu His

50 55 60



Pro Met Leu Arg His Arg Glu Asn Gly Lys Val Ala Ala Val Leu Val

65 70 75 80



Val Ser Ser Asp Arg Gly Met Cys Gly Gly Tyr Asn Asn Asn Val Phe

85 90 95



Lys Lys Ala Ala Glu Leu Glu Gly Leu Leu Arg Gly Gln Gly Phe Asp

100 105 110



Val Val Arg Tyr Val Thr Gly Ser Lys Gly Val Gly Tyr Tyr Asn Phe

115 120 125



Arg Glu Lys Glu Val Val Gly Ala Trp Thr Gly Phe Ser Gln Asp Pro

130 135 140



Ser Trp Glu Gly Thr His Asp Val Arg His His Leu Val Asp Gly Phe

145 150 155 160



Ile Ala Gly Ser Glu Gly Thr Thr Pro Ala Arg Gln Gly Val Asn Thr

165 170 175



Glu Asp Gln Thr Val Arg Gly Phe Asp Gln Val His Val Val Tyr Thr

180 185 190



Glu Phe Glu Ser Met Leu Val Gln Thr Pro Arg Ala His Gln Leu Leu

195 200 205



Pro Ile Glu Pro Val Ile Lys Glu Glu Glu Leu His Leu Gly Asp Ser

210 215 220



Ala Leu Glu Ala Asn Pro Asp Ala Gln Gly Leu Ser Ala Asp Tyr Glu

225 230 235 240



Phe Glu Pro Asp Ala Asp Thr Leu Leu Ser Ala Leu Leu Pro Gln Tyr

245 250 255



Val Ser Arg Ile Leu Phe Ser Met Phe Leu Glu Ala Ser Ala Ser Glu

260 265 270



Ser Ala Ala Arg Arg Thr Ala Met Lys Ala Ala Thr Asp Asn Ala Asn

275 280 285



Asp Leu Val Thr Asp Leu Ser Arg Val Ala Asn Gln Ala Arg Gln Ala

290 295 300



Gln Ile Thr Gln Glu Ile Thr Glu Ile Val Gly Gly Ala Gly Ala Leu

305 310 315 320



Ala Glu Ser Ala Glu Ser Asp

325





<210> SEQ ID NO 7

<211> LENGTH: 481

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 7



Met Thr Thr Ala Leu His Glu Gln Asn Thr Gln Glu Ser Ala Ile Ala

1 5 10 15



Gly Arg Val Val Arg Val Ile Gly Pro Val Val Asp Val Glu Phe Pro

20 25 30



Arg Gly Gly Leu Pro Ala Leu Tyr Asn Ala Leu Thr Val Glu Ile Asn

35 40 45



Leu Glu Ser Val Ala Arg Thr Ile Thr Leu Glu Val Ala Gln His Leu

50 55 60



Gly Asp Asn Leu Val Arg Thr Val Ser Met Ala Pro Thr Asp Gly Leu

65 70 75 80



Val Arg Arg Ala Ala Val Thr Asp Thr Glu Ala Pro Ile Ser Val Pro

85 90 95



Val Gly Asp Val Val Lys Gly His Val Phe Asn Ala Leu Gly Asp Cys

100 105 110



Leu Asp Glu Pro Gly Leu Gly Arg Asp Gly Glu Gln Trp Gly Ile His

115 120 125



Arg Glu Pro Pro Ala Phe Asp Gln Leu Glu Gly Lys Thr Glu Ile Leu

130 135 140



Glu Thr Gly Ile Lys Val Ile Asp Leu Leu Thr Pro Tyr Val Lys Gly

145 150 155 160



Gly Lys Ile Gly Leu Phe Gly Gly Ala Gly Val Gly Lys Thr Val Leu

165 170 175



Ile Gln Glu Met Ile Thr Arg Ile Ala Arg Glu Phe Ser Gly Thr Ser

180 185 190



Val Phe Ala Gly Val Gly Glu Arg Thr Arg Glu Gly Thr Asp Leu Phe

195 200 205



Leu Glu Met Glu Glu Met Gly Val Leu Gln Asp Thr Ala Leu Val Phe

210 215 220



Gly Gln Met Asp Glu Pro Pro Gly Val Arg Met Arg Val Ala Leu Ser

225 230 235 240



Gly Leu Thr Met Ala Glu Tyr Phe Arg Asp Val Gln Asn Gln Asp Val

245 250 255



Leu Leu Phe Ile Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu

260 265 270



Val Ser Thr Leu Leu Gly Arg Met Pro Ser Ala Val Gly Tyr Gln Pro

275 280 285



Thr Leu Ala Asp Glu Met Gly Val Leu Gln Glu Arg Ile Thr Ser Thr

290 295 300



Lys Gly Lys Ser Ile Thr Ser Leu Gln Ala Val Tyr Val Pro Ala Asp

305 310 315 320



Asp Tyr Thr Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala

325 330 335



Thr Thr Glu Leu Asp Arg Ala Ile Ala Ser Lys Gly Ile Tyr Pro Ala

340 345 350



Val Asn Pro Leu Ser Ser Thr Ser Arg Ile Leu Glu Pro Ser Ile Val

355 360 365



Gly Glu Arg His Tyr Ala Val Ala Gln Arg Val Ile Asn Ile Leu Gln

370 375 380



Lys Asn Lys Glu Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu

385 390 395 400



Leu Ser Glu Glu Asp Lys Ile Thr Val Gln Arg Ala Arg Arg Ile Glu

405 410 415



Arg Phe Leu Gly Gln Asn Phe Phe Val Ala Glu Lys Phe Thr Gly Leu

420 425 430



Pro Gly Ser Tyr Val Pro Leu Ala Asp Thr Ile Asp Ala Phe Glu Arg

435 440 445



Ile Cys Asn Gly Glu Phe Asp His Tyr Pro Glu Gln Ala Phe Asn Gly

450 455 460



Leu Gly Gly Leu Asp Asp Val Glu Ala Ala Tyr Lys Lys Leu Thr Glu

465 470 475 480



Lys





<210> SEQ ID NO 8

<211> LENGTH: 123

<212> TYPE: PRT

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 8



Met Ala Asp Ile Thr Val Glu Leu Val Ser Val Glu Arg Met Leu Trp

1 5 10 15



Ser Gly Lys Ala Thr Ile Ile Ser Ala Glu Thr Thr Glu Gly Glu Ile

20 25 30



Gly Val Leu Pro Gly His Glu Pro Leu Leu Gly Gln Leu Ala Glu Asn

35 40 45



Gly Val Val Thr Phe Arg Pro Val Asp Gly Asp Arg Lys Val Ala Ala

50 55 60



Val Gln Gly Gly Phe Leu Ser Val Ser Thr Glu Lys Ile Thr Val Leu

65 70 75 80



Ala Asp Trp Ala Val Trp Ala Asp Glu Val Asn Glu Ser Gln Ala Gln

85 90 95



Glu Asp Ala Leu Ser Ser Asp Glu Leu Val Ser Ser Arg Gly Gln Ala

100 105 110



Ala Leu Arg Ala Leu Ala Arg Ser Arg Glu Ser

115 120





<210> SEQ ID NO 9

<211> LENGTH: 912

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 9



atgtgcgacg gagtccgtag ctgtgacaga gagtttgaga cgtccatcgc accgtacgac 60



gtcgacaatc gtacggcccg aacacgggag agaacgctga gcgttacaac attggccatg 120



aagggtagct tccacgcgcc cgaactggac ccagaatttt tcccggggca atattacggc 180



gacatcctgt tcgacgatgt gttgggcgga tggttcgcac ttgatcgcat catgctggtt 240



cgtctgttga tgaccgccgt cttggtgctt ttatttattg cagcatttag gaacccaaag 300



ctggttccta agggactaca gaacgtcgca gaatacgcgt tagatttcgt ccgaattcac 360



attgctgagg acatcctggg caagaaggag ggtcgtcgct tcctaccgtt gctggcggct 420



atcttcttcg gcaccctttt ctggaacgtc tccacgatta ttccggcact gaacatctcc 480



gcaaacgctc gtattggcat gcctattgtc ttggctggcg cagcgtatat cgcaatgatt 540



tacgcaggca ccaagcgcta tggcttcggt aagtacgtca agtcgtcgtt ggttattcct 600



aaccttccac cggctttgca cttgctggtt gttccaattg agtttttctc gaccttcatc 660



ttgcgtcccg tcactctggc aattcgtctt atggcgaact tccttgccgg ccacatcatt 720



ttggttctgc tgtactctgc cacgaacttc ttcttctggc agctcaacgg ctggacagcg 780



atgtccggtg tgaccctgct cgcagcggtt ctgtttacgg tctacgagat catcatcatc 840



ttcctgcagg catacatctt tgctctgctg acggcggtgt acatcgagtt gtcacttcac 900



gcagactcgc ac 912





<210> SEQ ID NO 10

<211> LENGTH: 237

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 10



atgaacgaca tcatcttggc tcaggcaacc gagacctcct tcgatggcct tcagtccatc 60



ggctacggcc ttgcaaccat cggccctggc ttgggtattg gtatcctcgt cggcaagacc 120



gttgagggca tggcacgtca gcctgagatg gctggccagc tgcgtaccac catgttcctg 180



ggtatcgcct tcgttgaggc tcttgcactt atcggcctgg ttgcaggctt cctgttc 237





<210> SEQ ID NO 11

<211> LENGTH: 567

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 11



atgaacaacg tcttttacta tcttgcagcg gaaggagagt cccttccact ggaaggtggc 60



aactcccttc tgtttcccaa gagctatgac atcgtctggt ctctgatccc gttcttaatc 120



atccttattg tcttcgcaat gtttgtcatt ccgaagttcc aggaactgtt gcaagagcgt 180



gaagaccgga ttgagggcgg catcaagcgc gctgaagccc aacaggcaga agcaaaggcc 240



gcacttgaga agtacaacgc acagctagct gacgctcgcg cagaggcagc tgaaatccgt 300



gagcaggcgc gtgagcgcgg caagcagatt gaagcagagg caaagaccca ggcagaggaa 360



gaagcacgcc gtatcgtcgc aggtggcgaa aaacagcttg aagcttcccg cgcacaggta 420



gttgctgaac tgcgttccga tatcggacag aactccatca acttggctga gaagctgctc 480



ggcggtgaac tctctgagtc caccaagcag tcttcaacca ttgataactt cctgtccgag 540



ctcgactctg tggcatcggc cggaaag 567





<210> SEQ ID NO 12

<211> LENGTH: 813

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 12



atgaaggcag ctagccgcga atcgctcgca tccgctaccg agtcgctgga ttccaatctg 60



gcagctcaag caggtgtagc agtgtccacc atgaccggca tggaactgtt cgaggtttcc 120



caagtattgg gtgatgaccg cgaactccgt ggagcagtca ttgatgaatc tgcttccact 180



gaatcccgca agaagctcgt taatgatctc ttcggtgcca aagtttctcc tgctaccttg 240



caggttctgg aacagattgc atcgtcgaag tggtcgagcg cccgcgagat ggtttccgga 300



ctgatcgctc tttcacgtcg tgctttgatg cgcggcgcag aaagcgaagg acaactagga 360



caggtcgaag atgaactctt ccgcttgtcc cggatcctgg accgcgaagg cgaactcacc 420



cagctgcttt ctgaccgagc tgcagaacct gcgcgtaagc gcaagttgct ggcagatgtg 480



ctttacggaa aggtcaccaa attcactgag gcgcttgcgc tgcaggtgat tgaccgccct 540



gagcacaatc ccattgatga cattgcgaat ctggcggctg aagcagcaca gcttcagggt 600



cgcactgttg cgcacgttgt tagtgcgggt gaactcaatg aaggccagca ggcagtactc 660



gccgagaagc tgggcaagat ttatggtcgt gcgatgtcca tccactctga ggttgacacc 720



agcctcctcg gtggtatgac aatccgcgta ggcgatgaag ttattgacgg ttctaccgca 780



ggcaaaattg agcgcctgcg taccgctttg aag 813





<210> SEQ ID NO 13

<211> LENGTH: 1638

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 13



atggcggagc tgacgatctc ctccgatgag atccgtagcg cgatagcgaa ctacacctcg 60



agctactccg cggaggcctc ccgtgaggag gtcggcgtgg tcatttcggc agctgacggt 120



attgcacagg tttctgggct accttcagtt atggcgaatg agctgctcga gttccctggc 180



ggcgtaatcg gcgtcgcaca aaaccttgaa accaactcca ttggcgttgt tattcttggt 240



aactacgagt ccctcaaaga aggcgaccaa gttaagcgaa ctggcgaagt tctctccatc 300



ccagtgggtg aagagttcct cggccgcgtt attaacccat tgggtcaggc aattgacggc 360



ctgggcccaa tcgctggcga agaggaccgc gtcctcgagc tgcaggcacc ttccgtgttg 420



cagcgtcagc cagttgaaga gccaatgcag accggcatca aggctattga tgctatgacc 480



ccaatcggtc gcggtcagcg tcagctcatc attggtgacc gtaagactgg taaaaccgca 540



gtctgcatcg acaccatcct taaccagaag gctaactggg aatccggcga caagaacaag 600



caagttcgtt gtatctacgt cgctattggt cagaagggct ccaccatcgc tggtgtccgc 660



aagaccctcg aagagcaggg cgctctggag tacaccacca tcgtggctgc tcctgcttct 720



gactccgcgg gcttcaagtg gttggcacca ttctccggtg ctgctcttgg tcagcactgg 780



atgtaccagg gcaaccacgt cttggtcatc tatgatgact tgaccaagca ggctgaggct 840



taccgtgcga tttccctgtt gctgcgtcgc ccgccgggcc gcgaagctta cccaggtgac 900



gtcttctact tgcactcccg tctgctggag cgtgctgcga agctctccga tgatttgggt 960



gcaggttctt tgaccgcact gccaattatt gaaaccaagg cgaatgacgt gtctgcgttc 1020



attccaacca acgttatttc cattaccgac ggccaggtct tcctggagtc cgacctgttc 1080



aaccaaggcg tccgtccggc aattaacgtc ggtgtgtcgg tttcccgtgt tggtggcgct 1140



gctcagacca agggtatgaa gaaggttgca ggtaacctgc gtcttgacct cgcttcctac 1200



cgtgatctgc agggctttgc tgccttcgct tctgacttgg acccagtgtc caaggcccag 1260



cttgagcgcg gtgagcgtct ggttgagatc ctgaagcagt ctgagtcttc tcctcaggca 1320



gtcgagtacc agatggtttc catcttcttg gctgaagaag gcgtcttcga cgtcgttcct 1380



gtcgaagatg ttcgtcgctt tgaggctgac gttcaggaat acctgcagca gaacacccca 1440



gaggtttacg agcagattgc cggcggtaag gcatttaccg acgagtccaa ggaagccctg 1500



ttggctgcag ctaaggactt cactccttcc ttccgcacca ccgagggcca caacttgggc 1560



actgaagctc cagttgatcc tttggctgaa gaagaagtca agaagactga agtcaccgtc 1620



tcccgtaagt cggctaag 1638





<210> SEQ ID NO 14

<211> LENGTH: 981

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 14



atggcaaatc ttcgcgaatt gcgcgaccgt atccggtccg tgaactcgac caagaagatc 60



accaaggcgc aggagctgat tgcaacttct cgcattacca aggcgcaagc caaggttgat 120



gcagcagcac cgtacgcaca cgagatgtcg aacatgatgg accgtcttgc atcggctagc 180



tcgttggagc acccaatgct gcgccaccgt gaaaacggca aagttgcagc cgtactcgtg 240



gtctcttctg accgcggtat gtgtggtggc tacaacaaca acgtctttaa gaaggctgct 300



gagctcgaag gactccttcg cggtcaaggc ttcgacgttg tccgctacgt aaccggtagc 360



aagggcgtcg gctactacaa cttccgtgag aaggaagttg tgggcgcgtg gactggcttt 420



tctcaggatc cgtcctggga aggcactcac gacgttcgtc accacttggt tgacggcttc 480



attgctggct ccgaaggtac aactccggcc cgtcagggcg tgaacaccga agaccaaacg 540



gtacgtggtt tcgaccaggt acacgttgtt tacaccgagt tcgaatccat gctggttcag 600



actccacgtg ctcaccagtt gttgccgatt gaaccggtaa ttaaagaaga ggaacttcac 660



ctgggcgact cggcgctaga agccaaccct gatgctcagg gcctgtctgc tgactacgag 720



tttgagccgg atgcagatac tttgctctcg gcacttctgc cgcagtatgt atcacgtatc 780



cttttctcga tgttcttgga ggcttcggct tctgagtccg cagctcgtcg aactgcaatg 840



aaggctgcga ctgacaacgc taatgacttg gtaaccgact tgtctcgtgt tgctaaccag 900



gctcgtcagg cgcagattac ccaggaaatc acagaaatcg tcggtggcgc tggcgcgctc 960



gccgaaagcg cagaaagtga c 981





<210> SEQ ID NO 15

<211> LENGTH: 1443

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 15



atgactacag ctctgcatga gcagaacaca caggagtcgg caattgccgg ccgtgtggtg 60



cgtgtcatcg gtccggtcgt cgacgtggag ttcccgcgtg gcggactacc ggcactgtat 120



aatgcactga ccgtcgagat taacctcgag tctgttgcac gcaccattac ccttgaggtt 180



gcacagcacc tcggcgacaa cctggttcgt accgtttcga tggcacctac cgatggtctt 240



gttcggcgtg cggcagtaac cgataccgag gcaccaatct ccgtgccagt tggcgatgtt 300



gttaagggcc acgtctttaa cgcattgggc gactgcttgg atgagccagg tctgggccgc 360



gatggcgagc agtggggcat ccaccgcgag ccaccagcat tcgaccagct cgaaggcaag 420



accgagatcc tcgaaaccgg cattaaggtc atcgaccttt tgaccccata cgtcaagggc 480



ggcaagattg gcctcttcgg cggtgcgggt gttggtaaga ccgttctgat tcaggaaatg 540



attactcgta tcgcacgcga gttctctggt acctccgtgt tcgctggtgt tggcgagcgt 600



acccgtgagg gcaccgacct gttcttggaa atggaagaaa tgggcgtact gcaggacacc 660



gccctcgtgt tcggccagat ggacgaaccg ccaggagttc gtatgcgcgt agctctgtcc 720



ggtctgacca tggcggagta cttccgcgat gttcagaacc aggacgtgct gctgttcatc 780



gataacatct tccgtttcac tcaggctggt tctgaagttt cgacccttct gggccgtatg 840



ccttccgctg tgggctacca gccaaccttg gctgatgaga tgggtgtact ccaggagcgc 900



attacctcta ctaagggtaa gtcgattacc tctctgcagg ccgtttacgt tcctgccgat 960



gactacactg acccagctcc agcgaccacc ttcgctcact tggatgcaac caccgagctt 1020



gaccgtgcga ttgcttccaa gggtatctac ccagcagtga acccactgtc gtcgacttct 1080



cgtattctcg agccaagcat cgtcggtgag cgtcactacg ctgttgctca gcgcgtgatc 1140



aacattttgc agaagaacaa ggaactgcag gatattatcg cgattctggg tatggacgag 1200



ctgtctgaag aggacaagat caccgttcag cgcgcacgtc gcattgagcg cttcttgggc 1260



cagaacttct tcgtcgcaga gaagttcacc ggcctgccag gctcttacgt acctttggca 1320



gataccatcg acgctttcga gcgcatttgc aacggcgaat tcgaccacta cccagagcag 1380



gccttcaacg gcttgggtgg cttggacgac gtcgaagcag cgtacaagaa gctgactgag 1440



aag 1443





<210> SEQ ID NO 16

<211> LENGTH: 369

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes



<400> SEQUENCE: 16



atggctgaca tcaccgtgga actggtttct gttgagcgca tgctgtggtc tggaaaggcc 60



accatcattt ccgcagagac caccgagggt gagatcggcg tgcttccggg tcacgaacca 120



ttgcttggcc agctggctga gaatggcgta gttaccttcc gtcctgtcga cggtgaccgc 180



aaggtcgccg ctgttcaggg tggcttcctc tccgtatcca ccgagaagat caccgtcttg 240



gcggactggg cagtttgggc agatgaggtt aatgaatctc aggctcaaga agatgccttg 300



tcttccgatg aattggtttc ttctcgtgga caggcagcgc ttcgcgcctt ggctcgttcc 360



cgcgaaagc 369





<210> SEQ ID NO 17

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

DNA



<400> SEQUENCE: 17



acgttctgct gttcgttgac 20





<210> SEQ ID NO 18

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

DNA



<400> SEQUENCE: 18



ccggtgaata cttctgc 17





<210> SEQ ID NO 19

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

DNA



<400> SEQUENCE: 19



tggttgcagg cttcctgttc 20





<210> SEQ ID NO 20

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic

DNA



<400> SEQUENCE: 20



ctgcttcaat ctgcttg 17





<210> SEQ ID NO 21

<211> LENGTH: 9500

<212> TYPE: DNA

<213> ORGANISM: Corynebacterium ammoniagenes

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1387)..(2298)

<221> NAME/KEY: CDS

<222> LOCATION: (2404)..(2640)

<221> NAME/KEY: CDS

<222> LOCATION: (2691)..(3257)

<221> NAME/KEY: CDS

<222> LOCATION: (3267)..(4079)

<221> NAME/KEY: CDS

<222> LOCATION: (4147)..(5784)

<221> NAME/KEY: CDS

<222> LOCATION: (5850)..(6830)

<221> NAME/KEY: CDS

<222> LOCATION: (6837)..(8279)

<221> NAME/KEY: CDS

<222> LOCATION: (8293)..(8661)



<400> SEQUENCE: 21



acctcgtatg ggcagtaatt cgccgtgtct cccaaggtaa gtctcccttt gctgcagata 60



aggcgcacat ccaccaccgg ctgttgtctt taggccatac gcaccgccgc accgtattgg 120



tgctctacct gtgggtctcg gctgttgcct ttggcgcagt tagctactcg attgttccgc 180



cactgatcgc aaccatcgca acaattttgg cgctggtggt tgcaagtggc gtgacactaa 240



ttccgttgcg tcgtgggaaa atcgacttcc cagccaagcg ttgacgtaga caggcttttc 300



gatgatatcc tgcgccacaa gcgctactta gagggttcaa gttttcctta cgtctgtagt 360



agagtgtccc agcgtgagtg atactttaaa tggcgatggc gccagcacca gtagttttga 420



tgacccgcgc gtgccactgc agcgcgccct gcggctgggc tcgattgcgc tagccataat 480



caccgtttta tctctagcta tctggggtgg agttcgtggt ctacccggaa tctggggagt 540



agtcatcggc gctgctattg gcggtggctt cgttctgatt acggcagcgc ttgtcctgtt 600



tactgcaaag tctgcaccgt ccaccaccat ggctgttgtc ctcggtggat ggctactcaa 660



ggttgtactc ttgattgtcg tcctgatgat cattcgggac ctggaatttt atgacactat 720



ggctatgttc atcacggttg tcttggcgat gattgcggta ctcggtactg aggtctgggg 780



aatcatcacc tcccgcgtga cttatatttc ttaatagcta ctagtgacat gacgcccgct 840



ggggcgtcat aggttgcgac aaaggggagt gcatctttag ggatgtgctc ccgtttgtga 900



tcgctaaacg ggtggagtga tggggtgtag ggaaataggc aaaacagtat tttgatttat 960



ttgaattccc tggtcgagac atggcatgtg acataaagca tagaaaggca aaggggtgac 1020



gtatggcgaa aaaatgcctt taacgtgcac ctaaggggaa ttgcaagggg tgaaagcagg 1080



cagtgttatc gggggtaatt caagggggta aaagactgtg aaacggctat tcaaaaaata 1140



actgtgatca accttactaa tgcctgtaaa ccggggttgg gctggggata tgggctaatc 1200



cagggggagc aatcgcaggg ttggctcaaa cctcggaagt tcggatgact gcagatcgaa 1260



ataagcctca atgagctgat attatttcct ccgggttacc gcggaagcgg cattagtaag 1320



attcgtgtcc tgcgaaaaat ttatgccgta tcccatttac ggtgattccc tgttacagct 1380



tcgctgatgt gcgacggagt ccgtagctgt gacagagagt ttgagacgtc catcgcaccg 1440



tacgacgtcg acaatcgtac ggcccgaaca cgggagagaa cgctgagcgt tacaacattg 1500



gccatgaagg gtagcttcca cgcgcccgaa ctggacccag aatttttccc ggggcaatat 1560



tacggcgaca tcctgttcga cgatgtgttg ggcggatggt tcgcacttga tcgcatcatg 1620



ctggttcgtc tgttgatgac cgccgtcttg gtgcttttat ttattgcagc atttaggaac 1680



ccaaagctgg ttcctaaggg actacagaac gtcgcagaat acgcgttaga tttcgtccga 1740



attcacattg ctgaggacat cctgggcaag aaggagggtc gtcgcttcct accgttgctg 1800



gcggctatct tcttcggcac ccttttctgg aacgtctcca cgattattcc ggcactgaac 1860



atctccgcaa acgctcgtat tggcatgcct attgtcttgg ctggcgcagc gtatatcgca 1920



atgatttacg caggcaccaa gcgctatggc ttcggtaagt acgtcaagtc gtcgttggtt 1980



attcctaacc ttccaccggc tttgcacttg ctggttgttc caattgagtt tttctcgacc 2040



ttcatcttgc gtcccgtcac tctggcaatt cgtcttatgg cgaacttcct tgccggccac 2100



atcattttgg ttctgctgta ctctgccacg aacttcttct tctggcagct caacggctgg 2160



acagcgatgt ccggtgtgac cctgctcgca gcggttctgt ttacggtcta cgagatcatc 2220



atcatcttcc tgcaggcata catctttgct ctgctgacgg cggtgtacat cgagttgtca 2280



cttcacgcag actcgcacta aggcgcgaac taaccggcct tgtaataacc cccactttaa 2340



gaccaactcc aacttcgctg gaataaacga aaagcgaaga tttttcgaaa gggaacgact 2400



ttcatgaacg acatcatctt ggctcaggca accgagacct ccttcgatgg ccttcagtcc 2460



atcggctacg gccttgcaac catcggccct ggcttgggta ttggtatcct cgtcggcaag 2520



accgttgagg gcatggcacg tcagcctgag atggctggcc agctgcgtac caccatgttc 2580



ctgggtatcg ccttcgttga ggctcttgca cttatcggcc tggttgcagg cttcctgttc 2640



taaaaagcgc gcgtttaaaa ccaaaccacc gtttttaaga ctggagactt atgaacaacg 2700



tcttttacta tcttgcagcg gaaggagagt cccttccact ggaaggtggc aactcccttc 2760



tgtttcccaa gagctatgac atcgtctggt ctctgatccc gttcttaatc atccttattg 2820



tcttcgcaat gtttgtcatt ccgaagttcc aggaactgtt gcaagagcgt gaagaccgga 2880



ttgagggcgg catcaagcgc gctgaagccc aacaggcaga agcaaaggcc gcacttgaga 2940



agtacaacgc acagctagct gacgctcgcg cagaggcagc tgaaatccgt gagcaggcgc 3000



gtgagcgcgg caagcagatt gaagcagagg caaagaccca ggcagaggaa gaagcacgcc 3060



gtatcgtcgc aggtggcgaa aaacagcttg aagcttcccg cgcacaggta gttgctgaac 3120



tgcgttccga tatcggacag aactccatca acttggctga gaagctgctc ggcggtgaac 3180



tctctgagtc caccaagcag tcttcaacca ttgataactt cctgtccgag ctcgactctg 3240



tggcatcggc cggaaagtag gcaactatga aggcagctag ccgcgaatcg ctcgcatccg 3300



ctaccgagtc gctggattcc aatctggcag ctcaagcagg tgtagcagtg tccaccatga 3360



ccggcatgga actgttcgag gtttcccaag tattgggtga tgaccgcgaa ctccgtggag 3420



cagtcattga tgaatctgct tccactgaat cccgcaagaa gctcgttaat gatctcttcg 3480



gtgccaaagt ttctcctgct accttgcagg ttctggaaca gattgcatcg tcgaagtggt 3540



cgagcgcccg cgagatggtt tccggactga tcgctctttc acgtcgtgct ttgatgcgcg 3600



gcgcagaaag cgaaggacaa ctaggacagg tcgaagatga actcttccgc ttgtcccgga 3660



tcctggaccg cgaaggcgaa ctcacccagc tgctttctga ccgagctgca gaacctgcgc 3720



gtaagcgcaa gttgctggca gatgtgcttt acggaaaggt caccaaattc actgaggcgc 3780



ttgcgctgca ggtgattgac cgccctgagc acaatcccat tgatgacatt gcgaatctgg 3840



cggctgaagc agcacagctt cagggtcgca ctgttgcgca cgttgttagt gcgggtgaac 3900



tcaatgaagg ccagcaggca gtactcgccg agaagctggg caagatttat ggtcgtgcga 3960



tgtccatcca ctctgaggtt gacaccagcc tcctcggtgg tatgacaatc cgcgtaggcg 4020



atgaagttat tgacggttct accgcaggca aaattgagcg cctgcgtacc gctttgaagt 4080



agtcaactac aacgacagaa ttgatttaag taagtgctgg acgaatctac cgagagtagg 4140



aagaacatgg cggagctgac gatctcctcc gatgagatcc gtagcgcgat agcgaactac 4200



acctcgagct actccgcgga ggcctcccgt gaggaggtcg gcgtggtcat ttcggcagct 4260



gacggtattg cacaggtttc tgggctacct tcagttatgg cgaatgagct gctcgagttc 4320



cctggcggcg taatcggcgt cgcacaaaac cttgaaacca actccattgg cgttgttatt 4380



cttggtaact acgagtccct caaagaaggc gaccaagtta agcgaactgg cgaagttctc 4440



tccatcccag tgggtgaaga gttcctcggc cgcgttatta acccattggg tcaggcaatt 4500



gacggcctgg gcccaatcgc tggcgaagag gaccgcgtcc tcgagctgca ggcaccttcc 4560



gtgttgcagc gtcagccagt tgaagagcca atgcagaccg gcatcaaggc tattgatgct 4620



atgaccccaa tcggtcgcgg tcagcgtcag ctcatcattg gtgaccgtaa gactggtaaa 4680



accgcagtct gcatcgacac catccttaac cagaaggcta actgggaatc cggcgacaag 4740



aacaagcaag ttcgttgtat ctacgtcgct attggtcaga agggctccac catcgctggt 4800



gtccgcaaga ccctcgaaga gcagggcgct ctggagtaca ccaccatcgt ggctgctcct 4860



gcttctgact ccgcgggctt caagtggttg gcaccattct ccggtgctgc tcttggtcag 4920



cactggatgt accagggcaa ccacgtcttg gtcatctatg atgacttgac caagcaggct 4980



gaggcttacc gtgcgatttc cctgttgctg cgtcgcccgc cgggccgcga agcttaccca 5040



ggtgacgtct tctacttgca ctcccgtctg ctggagcgtg ctgcgaagct ctccgatgat 5100



ttgggtgcag gttctttgac cgcactgcca attattgaaa ccaaggcgaa tgacgtgtct 5160



gcgttcattc caaccaacgt tatttccatt accgacggcc aggtcttcct ggagtccgac 5220



ctgttcaacc aaggcgtccg tccggcaatt aacgtcggtg tgtcggtttc ccgtgttggt 5280



ggcgctgctc agaccaaggg tatgaagaag gttgcaggta acctgcgtct tgacctcgct 5340



tcctaccgtg atctgcaggg ctttgctgcc ttcgcttctg acttggaccc agtgtccaag 5400



gcccagcttg agcgcggtga gcgtctggtt gagatcctga agcagtctga gtcttctcct 5460



caggcagtcg agtaccagat ggtttccatc ttcttggctg aagaaggcgt cttcgacgtc 5520



gttcctgtcg aagatgttcg tcgctttgag gctgacgttc aggaatacct gcagcagaac 5580



accccagagg tttacgagca gattgccggc ggtaaggcat ttaccgacga gtccaaggaa 5640



gccctgttgg ctgcagctaa ggacttcact ccttccttcc gcaccaccga gggccacaac 5700



ttgggcactg aagctccagt tgatcctttg gctgaagaag aagtcaagaa gactgaagtc 5760



accgtctccc gtaagtcggc taagtaaaga cctccgggta cttactcaca ctgactgaat 5820



agaaatttag aagggaggag cgaaacaaca tggcaaatct tcgcgaattg cgcgaccgta 5880



tccggtccgt gaactcgacc aagaagatca ccaaggcgca ggagctgatt gcaacttctc 5940



gcattaccaa ggcgcaagcc aaggttgatg cagcagcacc gtacgcacac gagatgtcga 6000



acatgatgga ccgtcttgca tcggctagct cgttggagca cccaatgctg cgccaccgtg 6060



aaaacggcaa agttgcagcc gtactcgtgg tctcttctga ccgcggtatg tgtggtggct 6120



acaacaacaa cgtctttaag aaggctgctg agctcgaagg actccttcgc ggtcaaggct 6180



tcgacgttgt ccgctacgta accggtagca agggcgtcgg ctactacaac ttccgtgaga 6240



aggaagttgt gggcgcgtgg actggctttt ctcaggatcc gtcctgggaa ggcactcacg 6300



acgttcgtca ccacttggtt gacggcttca ttgctggctc cgaaggtaca actccggccc 6360



gtcagggcgt gaacaccgaa gaccaaacgg tacgtggttt cgaccaggta cacgttgttt 6420



acaccgagtt cgaatccatg ctggttcaga ctccacgtgc tcaccagttg ttgccgattg 6480



aaccggtaat taaagaagag gaacttcacc tgggcgactc ggcgctagaa gccaaccctg 6540



atgctcaggg cctgtctgct gactacgagt ttgagccgga tgcagatact ttgctctcgg 6600



cacttctgcc gcagtatgta tcacgtatcc ttttctcgat gttcttggag gcttcggctt 6660



ctgagtccgc agctcgtcga actgcaatga aggctgcgac tgacaacgct aatgacttgg 6720



taaccgactt gtctcgtgtt gctaaccagg ctcgtcaggc gcagattacc caggaaatca 6780



cagaaatcgt cggtggcgct ggcgcgctcg ccgaaagcgc agaaagtgac tagattatga 6840



ctacagctct gcatgagcag aacacacagg agtcggcaat tgccggccgt gtggtgcgtg 6900



tcatcggtcc ggtcgtcgac gtggagttcc cgcgtggcgg actaccggca ctgtataatg 6960



cactgaccgt cgagattaac ctcgagtctg ttgcacgcac cattaccctt gaggttgcac 7020



agcacctcgg cgacaacctg gttcgtaccg tttcgatggc acctaccgat ggtcttgttc 7080



ggcgtgcggc agtaaccgat accgaggcac caatctccgt gccagttggc gatgttgtta 7140



agggccacgt ctttaacgca ttgggcgact gcttggatga gccaggtctg ggccgcgatg 7200



gcgagcagtg gggcatccac cgcgagccac cagcattcga ccagctcgaa ggcaagaccg 7260



agatcctcga aaccggcatt aaggtcatcg accttttgac cccatacgtc aagggcggca 7320



agattggcct cttcggcggt gcgggtgttg gtaagaccgt tctgattcag gaaatgatta 7380



ctcgtatcgc acgcgagttc tctggtacct ccgtgttcgc tggtgttggc gagcgtaccc 7440



gtgagggcac cgacctgttc ttggaaatgg aagaaatggg cgtactgcag gacaccgccc 7500



tcgtgttcgg ccagatggac gaaccgccag gagttcgtat gcgcgtagct ctgtccggtc 7560



tgaccatggc ggagtacttc cgcgatgttc agaaccagga cgtgctgctg ttcatcgata 7620



acatcttccg tttcactcag gctggttctg aagtttcgac ccttctgggc cgtatgcctt 7680



ccgctgtggg ctaccagcca accttggctg atgagatggg tgtactccag gagcgcatta 7740



cctctactaa gggtaagtcg attacctctc tgcaggccgt ttacgttcct gccgatgact 7800



acactgaccc agctccagcg accaccttcg ctcacttgga tgcaaccacc gagcttgacc 7860



gtgcgattgc ttccaagggt atctacccag cagtgaaccc actgtcgtcg acttctcgta 7920



ttctcgagcc aagcatcgtc ggtgagcgtc actacgctgt tgctcagcgc gtgatcaaca 7980



ttttgcagaa gaacaaggaa ctgcaggata ttatcgcgat tctgggtatg gacgagctgt 8040



ctgaagagga caagatcacc gttcagcgcg cacgtcgcat tgagcgcttc ttgggccaga 8100



acttcttcgt cgcagagaag ttcaccggcc tgccaggctc ttacgtacct ttggcagata 8160



ccatcgacgc tttcgagcgc atttgcaacg gcgaattcga ccactaccca gagcaggcct 8220



tcaacggctt gggtggcttg gacgacgtcg aagcagcgta caagaagctg actgagaagt 8280



agggagaggc acatggctga catcaccgtg gaactggttt ctgttgagcg catgctgtgg 8340



tctggaaagg ccaccatcat ttccgcagag accaccgagg gtgagatcgg cgtgcttccg 8400



ggtcacgaac cattgcttgg ccagctggct gagaatggcg tagttacctt ccgtcctgtc 8460



gacggtgacc gcaaggtcgc cgctgttcag ggtggcttcc tctccgtatc caccgagaag 8520



atcaccgtct tggcggactg ggcagtttgg gcagatgagg ttaatgaatc tcaggctcaa 8580



gaagatgcct tgtcttccga tgaattggtt tcttctcgtg gacaggcagc gcttcgcgcc 8640



ttggctcgtt cccgcgaaag ctaatccttt caaagactcg ttctttctaa aggttgctta 8700



agcaccgagg attcggagat ttagggcagc aatgaccgcc cttcctctct cataaagaac 8760



ccgcagtaat aattgctgcg ggttcttttt tgccgtttta gccagcttgt gtgcgaagac 8820



tatcgctgta gcccgagcgg ggcctacagc agaggtctgg gcggaggtag tcaaccaaac 8880



gatttattgt gactctatgc aggcgctctg tagactattc aacaacttga gattgattga 8940



accgcttcta ggcagtttgg ttgaggcagt tcagataagg tgttctgcct cgcccgtgcc 9000



atgagcggtt gtgacggaga agggaagagc gggactgatg agcgtggttt cggtaatcct 9060



ttggttgctc gccattattg ccatactatg tattttcttc gcagcgatgc gctttttcac 9120



cttgcggtca cgcggtgctt cagtgttgat gcgcaagctc ccggccaagg gctaccacgg 9180



ctggcgccat ggtgtgctgc gctacaaggg agacactgta gatttctaca agcttcgctc 9240



cgtgtggcct atggccgatc actcatttag tcgcctcgac atcgagttgc tggattctcg 9300



tcccgcaact gatggcgagg ctgctttcat ttccaaggac tatttgatct tctgcttcag 9360



cgcagctggc aagggctacg agctcgcgtg tacgcagcac gccatgatgg catttggcgc 9420



gtgggtggaa gcctcgccgt cgcagcgtaa ggaacagatt gattttcgtc gtttgcgcga 9480



acgggcaacg cgtccgcggg 9500