IP Library › Granted Patent US 8,030,042
Granted Patent B2
US 8,030,042 · App. 12/450,891 · Granted Oct 4, 2011

Polyamino acid synthetase and gene encoding the same

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Quick Facts
Patent No.
US 8,030,042
App. No.
12/450,891
Granted
Oct 4, 2011
Kind
B2
Abstract

The present invention provides an enzyme which catalyzes amino acid polymerization in the form of a non-ribosomal peptide synthetase (NRPS) and a gene encoding the same.

Claims (46)

1. An isolated polynucleotide comprising any one of the following nucleotide sequences:

(i) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2;

(ii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, wherein said polyamino acid synthetase polymerizes an amino acid in the presence of divalent cations including Mg 2+ and Mn 2+ utilizing the amino acid and ATP as its substrate;

(iii) the nucleotide sequence of SEQ ID NO: 1;

(iv) a nucleotide sequence encoding a polyamino acid synthetase, which can hybridize to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under highly stringent hybridization conditions (5 SSC; 5 Denhart solution; 0.5% (w/v) SDS; 50% (w/v) formamide; and 50° C.); and

(v) a nucleotide sequence encoding a polyamino acid synthetase, which has at least 95% identity to the nucleotide sequence of SEQ ID NO: 1.

2. An isolated polynucleotide comprising:

(i) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2; or

(ii) the nucleotide sequence of SEQ ID NO: 1.

3. The isolated polynucleotide according to claim 1 , wherein the polyamino acid synthetase is a polylysine synthetase.

4. The isolated polynucleotide according to any one of claims 1 to 3 , which is a DNA or RNA.

5. An isolated polynucleotide comprising any one of the following nucleotide sequences:

(i) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which consists of the amino acid sequence of SEQ ID NO: 13, 15, 17 or 19;

(ii) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof of a polyamino acid synthetase, wherein said condensation reaction catalytic domain consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13, 15, 17 or 19, and wherein said polyamino acid synthetase polymerizes an amino acid in the presence of divalent cations including Mg 2+ and Mn 2+ utilizing the amino acid and ATP as its substrate;

(iii) the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18;

(iv) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which can hybridize to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18 under highly stringent hybridization conditions (5 SSC; 5 Denhart solution; 0.5% (w/v) SDS; 50% (w/v) formamide; and 50° C.); and

(v) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which has at least 95% identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18.

6. An isolated polynucleotide comprising:

(i) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which consists of the amino acid sequence of SEQ ID NO: 13, 15, 17 or 19; or

(ii) the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18.

7. The isolated polynucleotide according to claim 5 or 6 , which is a DNA or RNA.

8. A recombinant vector comprising any one of the following nucleotide sequences:

(i) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2;

(ii) a nucleotide sequence encoding a polyamino acid synthetase consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, wherein said polyamino acid synthetase polymerizes an amino acid in the presence of divalent cations including Mg 2+ and Mn 2+ utilizing the amino acid and ATP as its substrate;

(iii) the nucleotide sequence of SEQ ID NO: 1;

(iv) a nucleotide sequence encoding a polyamino acid synthetase, which can hybridize to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under highly stringent hybridization conditions (5 SSC; 5 Denhart solution; 0.5% (w/v) SDS; 50% (w/v) formamide; and 50° C.); and

(v) a nucleotide sequence encoding a polyamino acid synthetase, which has at least 95% identity to the nucleotide sequence of SEQ ID NO: 1.

9. A recombinant vector comprising:

(i) a nucleotide sequence encoding a polyamino acid synthetase consisting of the amino acid sequence of SEQ ID NO: 2; or

(ii) the nucleotide sequence of SEQ ID NO: 1.

10. The recombinant vector according to claim 8 , wherein the polyamino acid synthetase is a polylysine synthetase.

11. A recombinant vector comprising any one of the following nucleotide sequences:

(i) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which consists of the amino acid sequence of SEQ ID NO: 13, 15, 17 or 19;

(ii) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof of a polyamino acid synthetase, which consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 13, 15, 17 or 19, wherein said polyamino acid synthetase polymerizes an amino acid in the presence of divalent cations including Mg 2+ and Mn 2+ utilizing the amino acid and ATP as its substrate;

(iii) the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18;

(iv) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which can hybridize to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18 under highly stringent hybridization conditions (5 SSC; 5 Denhart solution; 0.5% (w/v) SDS; 50% (w/v) formamide; and 50° C.); and

(v) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which has at least 95% identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18.

12. A recombinant vector comprising:

(i) a nucleotide sequence encoding a condensation reaction catalytic domain or a subdomain thereof, which consists of the amino acid sequence of SEQ ID NO: 13, 15, 17 or 19; or

(ii) the nucleotide sequence of SEQ ID NO: 12, 14, 16 or 18.

13. A transformant comprising the recombinant vector according to claim 8 , 10 or 11 .

14. A transformant comprising the recombinant vector according to claim 9 or 12 .

15. A method for producing a polyamino acid synthetase comprising transforming a host cell with the recombinant vector according to claim 8 , 10 or 11 and culturing the transformed cell.

16. A method for producing a polyamino acid synthetase, comprising performing in vitro transcription/translation of the recombinant vector according to claim 9 or 12 .

17. A method for producing a polyamino acid synthetase, comprising culturing the transformant according to claim 14 and isolating a desired protein.

18. A method for producing a polyamino acid synthetase comprising culturing the transformant according to claim 13 .