Pichia pastoris formate dehydrogenase and uses therefor

• US 20040038237A1
• Filed: 12/16/2002
• Published: 02/26/2004
• 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.
• Granted Patent: US 7,087,418 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 nucleic acid comprising a nucleotide sequence encoding amino acid sequence SEQ ID NO:

5.

2. An isolated nucleic acid comprising a fragment of the nucleic acid of claim 1, wherein said nucleotide sequence encodes at least 32 contiguous residues of amino acid sequence SEQ ID NO:

5.

3. The isolated nucleic acid of claim 2, wherein said nucleic acid encodes a functional equivalent of a polypeptide comprising amino acid sequence SEQ ID NO:

5.

4. The isolated nucleic acid of claim 2, wherein said nucleic acid or its complement is useful as a primer or a probe.

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

5.

6. An isolated nucleic acid of claim 2 comprising a nucleotide sequence comprising the catalytic domain of amino acid sequence SEQ ID NO:

5.

7. An isolated nucleic acid comprising a fragment of the nucleic acid of claim 1, wherein said isolated nucleic acid comprises at least 21 contiguous amino acids of the catalytic domain of SEQ ID NO:

5.

8. 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.

9. An isolated nucleic acid comprising at least 21 contiguous nucleotides of the nucleic acid of claim 8.

10. An isolated nucleic acid comprising a nucleotide sequence which is complementary to a nucleotide sequence of claim 1.

12. A vector comprising the isolated nucleic acid claim 1.

13. A host cell comprising a vector of claim 12, wherein the host cell is selected from the group consisting of bacterial, fungal, insect, mammalian, and plant cells.

21. A method for producing a recombinant polypeptide comprising:

a) culturing a host cell of claim 13 under conditions suitable for the production of a recombinant polypeptide; and

b) recovering the recombinant polypeptide from the host cell or host cell culture, thereby producing the recombinant polypeptide.

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

incubating a recombinant polypeptide of claim 15 or a host cell of claim 13 with formate and NAD+ under conditions to allow oxidation of the formate and reduction of the NAD+, thereby producing NADH.

11. An isolated nucleic acid which hybridizes under stringent conditions to any one of:

(a) the nucleotide sequence SEQ ID NO;

1;

(b) the nucleotide squence 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.

14. An isolated nucleic acid comprising the nucleotide sequence encoding a formate deydrogenase contained in the plasmid in the ATCC deposit designated PTA-3691.

15. A recombinant polypeptide comprising amino acid sequence SEQ ID NO:

5.

16. A recombinant polypeptide of claim 15 comprising the NAD-binding domain of amino acid sequence SEQ ID NO:

5.

17. A recombinant polypeptide of claim 15 comprising the catalytic domain of amino acid sequence SEQ ID NO:

5.

18. A recombinant polypeptide comprising an amino acid sequence which is at least 86% identical to the recombinant polypeptide of claim 15, wherein said isolated polypeptide is a functional equivalent of a polypeptide comprising amino acid sequence SEQ ID NO:

5.

20. An antibody which binds to an isolated polypeptide of claim 15.

23. A method of reducing a substrate comprising:

incubating a recombinant polypeptide of claim 15 with formate, nicotinamide adenine dinucleotide (NAD+), substrate, and a reducing enzyme under conditions to allow oxidation of the formate, reduction of the NAD+, and thereby allow reduction of the substrate.

24. A method of reducing a ketone to produce an alcohol comprising:

incubating a recombinant polypeptide of claim 15 with formate, nicotinamide adenine dinucleotide (NAD+), the ketone and ketone reductase under conditions to allow oxidation of the formate, reduction of the NAD+, and reduction of the ketone, thereby producing the alcohol.

25. The method of claim 24 wherein said ketone is a 2-pentanone ketone and said ketone reductase is an isolated Gluconobacter oxydans 2-keto reductase reducing enzyme having the amino acid sequence as set forth in SEQ ID NO:

24.

19. A recombinant polypeptide that is a formate dehydrogenase encoded by the nucleotide sequence contained in the plasmid in the ATCC deposit designated PTA-3691.