Cells expressing Pichia cytochrome C

• US 7,892,792 B2
• Filed: 06/25/2009
• Issued: 02/22/2011
• Est. Priority Date: 06/27/2008
• Status: Expired due to Fees

• Current Assignee: Indian Institute of Science
• Original Assignee: Indian Institute of Science
• Inventors: Mohanty, Abhishek, Rangarajan, Pundi N, Rajasekharan, Ram
• Primary Examiner(s): Nashed; Nashaat T
• Application Number:
  US12/491,909
• Publication Number:
  US 20100041099A1
• Time in Patent Office: 607 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/80 Cytochromes
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6431 Linoleic acids [18:2[n-6]]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

Described herein are recombinant yeast cells that express a cytochrome C gene the expression of which causes the recombinant yeast cells to (i) grow faster than wild type yeast cells of the same species when cultured on glucose medium, and (ii) accumulate high levels of polyunsaturated fatty acid precursor molecules or target gene expression products when grown on oleic acid.

Claims

1. A recombinant yeast cell comprising a recombinant expression cassette that expresses a gene encoding a cytochrome C polypeptide, wherein the cytochrome C polypeptide has at least 95% identity to SEQ ID NO. 2.

2. The recombinant yeast cell of claim 1, wherein the gene encoding the cytochrome C polypeptide is a Pichia pastoris cytochrome C gene.

3. The recombinant yeast cell of claim 1, wherein the gene encoding the cytochrome C polypeptide is operably linked to at least one gene regulatory element in the gene expression cassette.

4. The recombinant yeast cell of claim 1, wherein the expression cassette that expresses the gene encoding the cytochrome C polypeptide is an extrachromosomal self-replicating vector.

5. The recombinant yeast cell of claim 1, wherein the yeast cell is Saccharomyces cerevisiae.

6. The recombinant yeast cell of claim 1, wherein the gene encoding the cytochrome C polypeptide is encoded by SEQ ID NO:

1.

7. The recombinant yeast cell of claim 1, wherein the cytochrome C polypeptide is SEQ ID NO:

2.

8. The recombinant yeast cell of claim 1, wherein the recombinant cell further comprises a second recombinant gene cassette that expresses a target gene.

9. The recombinant yeast cell of claim 8, wherein the target gene is integrated into the cell genome or expressed extrachromosomally in a self-replicating plasmid.

10. The recombinant yeast cell of claim 8, wherein the target gene is a hepatitis B surface antigen gene, an insulin gene or an erythropoietin gene.

11. A method for producing a polyunsaturated fatty acid molecule in a recombinant yeast cell, comprising growing a recombinant yeast cell comprising a recombinant expression cassette that expresses a gene encoding a cytochrome C polypeptide, wherein the cytochrome C polypeptide has at least 95% identity to SEQ ID NO. 2, under conditions wherein a polyunsaturated fatty acid is produced.

12. The method of claim 11, wherein the polyunsaturated fatty acid is selected from the group consisting of:

a linoleic acid, a linolenic acid, an omega-3 fatty acid and an omega-6 fatty acid.

13. The method of claim 11, wherein the recombinant yeast cell does not express an endogenous gene encoding a biologically active cytochrome C polypeptide.

14. The method of claim 11, further comprising:

extracting the polyunsaturated fatty acid from the recombinant yeast cell, wherein the recombinant yeast cell is either grown on an oleic acid substrate or grown on a glucose substrate for at least about 12 hours before being grown on an oleic acid substrate.

15. A method of producing an isolated cytochrome C polypeptide comprising:

a) culturing a recombinant yeast cell comprising a recombinant expression cassette that expresses a gene encoding a cytochrome C polypeptide, wherein the cytochrome C polypeptide has at least 95% identity to SEQ ID NO. 2, under conditions wherein the yeast cell expresses the cytochrome C polypeptide, and

b) isolating the cytochrome C polypeptide, thereby producing an isolated cytochrome C polypeptide.

16. The method of claim 15, wherein the recombinant yeast cell is cultured on a glucose-containing substrate for more than about 12 hours.

17. The method of claim 15, wherein the recombinant expression cassette has a polynucleotide sequence that encodes a signal or secretion peptide, wherein the expressed cytochrome C polypeptide is transported out of or secreted from the recombinant yeast cell.

18. The method of claim 15, wherein step b) comprises centrifuging an aliquot of the yeast cell culture and then purifying the cytochrome C polypeptide from the culture supernatant.