Pichia pastoris formate dehydrogenase and uses therefor

• US 7,087,418 B2
• Filed: 12/16/2002
• Issued: 08/08/2006
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Goldberg, Steven L., Patel, Ramesh N., Johnston, Robert M., Nanduri, Venkata B., Cino, Paul M.
• Primary Examiner(s): Rao, Manjunath
• Assistant Examiner(s): PAK, YONG D
• Application Number:
  US10/320,300
• Publication Number:
  US 20040038237A1
• Time in Patent Office: 1,331 Days
• Field of Search: 435/190, 435/4, 435/6, 435/252.3, 435/320.1, 435/69.1, 435/71.1, 435/440, 435/325, 435/410, 536/23.2, 536/23.74, 536/23.1
• US Class Current: 435/190
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 2333/04 Varicella-zoster virus

Abstract

This invention relates to a recombinant Pichia pastoris formate dehydrogenase (FDH) enzyme that catalyzes the oxidation of formate to carbon dioxide and the simultaneous reduction of nicotinamide adenine dinucleotide (NAD+) to its reduced form (NADH). Also related are isolated nucleic acids encoding P. pastoris FDH polypeptides, and fragments and variants thereof, as well as vectors and host cells comprising these nucleic acids. Further related are isolated, recombinant P. pastoris FDH polypeptides, and fragments and variants thereof, and antibodies that specifically bind to P. pastoris FDH polypeptides, fragments, or variants. The invention also relates to methods of obtaining isolated P. pastoris FDH nucleic acids, polypeptides, and antibodies, and methods of using P. pastoris FDH in various reactions for industrial or pharmaceutical applications.

Claims

1. An isolated polynucleotide comprising a nucleotide sequence encoding amino acid sequence SEQ ID NO:

5.

4. An isolated nucleic acid of claim 1 comprising:

(a) the nucleotide sequence SEQ ID NO;

1;

(b) the nucleotide sequence SEQ ID NO;

3;

or (c) a nucleotide sequence that differs from (a) or (b) due to degeneracy of the genetic code.

5. An isolated polynucleotide comprising a nucleotide sequence which is complementary to a nucleotide sequence of claim 1.

7. A vector comprising the isolated polynucleotide of claim 1.

8. A host cell transformed or transfected with the vector of claim 7, wherein the host cell is selected from the group consisting of bacterial, fungal, insect, mammalian, and plant cells.

10. A method for producing a recombinant polypeptide comprising the amino acid sequence SEQ ID NO:

5 comprising;

a) culturing a host cell of claim 8 under conditions suitable for the production of a recombinant polypeptide; and

b) recovering the recombinant polypeptide comprising the amino acid sequence SEQ ID NO;

5 from the host cell or host cell culture, thereby producing the recombinant polypeptide.

11. A method of producing nicotinamide adenine dinucleotide (NAD+) in a reduced form (NADH) comprising:

incubating the host cell of claim 8 with formate and NAD+ under conditions to allow oxidation of the formate and reduction of the NAD+, thereby producing NADH.

2. An isolated polynucleotide comprising a nucleotide sequence encoding the NAD-binding domain of amino acid sequence SEQ ID NO:

5, wherein said polynucleotide encodes a polypeotide having formate dehydrogenase activity, and wherein the NAD-binding domain of amino acid sequence SEQ ID NO;

5 is amino acids 117-309 of SEQ ID NO;

5.

12. An isolated nucleic acid of claim 2, wherein said isolated polynucleotide further comprises a nucleotide sequence encoding the catalytic domain of amino acid sequence SEQ ID NO:

5, and wherein the catalytic domain of amino acid sequence SEQ ID NO;

5 is amino acids 16-115 of SEQ ID NO;

5.

13. The isolated polynucleotide of claim 12, wherein said polypeptide shares at least 95% sequence identity with the amino acid sequence SEQ ID NO:

5.

15. A vector comprising the isolated nucleic acid of claim 6, 13, or 14.

3. An isolated polynucleotide comprising a nucleotide sequence encoding the catalytic domain of amino acid sequence SEQ ID NO:

5, wherein said polynucleotide encodes a polypeptide having formate dehydrogenase activity, and wherein the catalytic domain of amino acid sequence SEQ ID NO;

5 is amino acids 16-115 of SEQ ID NO;

5.

6. An isolated polynucleotide which hybridizes under high stringency conditions to any one of:

(a) the nucleotide sequence SEQ ID NO;

1;

(b) the nucleotide sequence SEQ ID NO;

3;

(c) the complement of (a);

(d) the complement of (b);

or (e) a nucleotide sequence that differs from (a), (b), (c) or (d) due to degeneracy of the genetic code;

wherein said polynucleotide encodes a polypeptide having formate dehydrogenase activity; and

wherein said high stringency conditions are hybridization in 50% formamide, 5×

Denhardt'"'"'s solution. 5×

SSPE, and 0.2% SDS at 42°

C., followed by washing in 0.1×

SSPE and 0.1% SDS at 65°

C.

9. An isolated polynucleotide comprising the nucleotide sequence encoding a formate dehydrogenase contained in the plasmid in the ATCC deposit designated PTA-3691.

14. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that shares at least 95% sequence identity with amino acid sequence SEQ ID NO:

5, wherein said polynucleotide encodes a polypeptide having formate dehydrogenase activity.