CELLS EXPRESSING PICHIA CYTOCHROME C

• US 20100041099A1
• Filed: 06/25/2009
• Published: 02/18/2010
• Est. Priority Date: 06/27/2008
• Status: Active Grant

• Current Assignee: Indian Institute of Science
• Original Assignee: Indian Institute of Science
• Inventors: Mohanty, Abhishek, Rangarajan, Pundi Narasimhan, Rajasekharan, Ram
• Granted Patent: US 7,892,792 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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 gene encoding a cytochrome C polypeptide,

2. The recombinant yeast cell of claim 1 wherein the gene encoding the cytochrome C polypeptide is selected from the group consisting of a Pichia pastoris cytochrome C gene;

a Debaryomyces hansenii cytochrome C gene;

a Debryomyces occidentalis cytochrome C gene;

a Pichia guilliermondii cytochrome C gene;

a Pichia stipitis cytochrome C gene;

a Candida albicans cytochrome C gene;

a Kluyveromyces lactis cytochrome C gene;

a Pachysolen tannophilus cytochrome C gene; and

a Lodderomyces elongisporus cytochrome C gene.

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

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

8. The recombinant yeast cell of claim 1, wherein the cytochrome C polypeptide encoded by the gene comprises the amino acid sequence of SEQ ID NO. 2 or is a homolog with at least about 85% homology to the sequence of SEQ ID NO. 2.

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

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

11. The recombinant yeast cell of claim 9, wherein the target gene is a hepatitis B surface antigen gene, an insulin gene or an erythropoietin gene.

12. A method for producing a polyunsaturated fatty acid molecule in a recombinant yeast cell, comprising growing a recombinant yeast cell comprising a gene encoding a cytochrome C polypeptide, wherein the yeast cell further comprises at least one gene encoding a polypeptide selected from the group consisting of:

a Δ

6-desaturase, an elongase, a Δ

12-desaturase, an ω

3-desaturase and a Δ

5-desaturase, under conditions wherein a polyunsaturated fatty acid is produced.

13. The method of claim 12, 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,

14. The method of claim 12, wherein the recombinant yeast cell does not express an endogenous gene encoding a biologically active cytochrome C polypeptide

15. The method of claim 12, further comprising:

a) extracting intracellular oleic 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; and

either

b) chemically or enzymatically converting the extracted oleic acid in vitro into at least one polyunsaturated acid, or

c) feeding the extracted oleic acid to a cell that converts the internalized oleic acid to at least one polyunsaturated acid.

16. A method of producing an isolated target polypeptide comprising:

a) culturing the recombinant yeast cell comprising a gene encoding a cytochrome C polypeptide and a recombinant gene cassette that expresses a target gene under conditions wherein the yeast cell expresses the target gene, and

b) isolating the polypeptide product of the expressed target gene, thereby producing an isolated target polypeptide.

4. The recombinant yeast cell of claim 17 wherein the gene encoding the cytochrome C polypeptide is operably linked to at least one gene regulatory element in a gene expression cassette.

5. The recombinant yeast cell of claim 17 wherein the gene encoding the cytochrome C polypeptide is encoded by an extraceromosomal self-replicating vector.

7. The recombinant yeast cell of claim 17 wherein the gene encoding the cytochrome C polypeptide is encoded by SEQ ID NO. 1 or a fragment thereof.

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

18. The method of claim 16, wherein the target gene has a polynucleotide sequence that encodes a signal or secretion peptide, wherein the expressed polypeptide product is transported out of or secreted from the recombinant yeast cell.

19. The method of claim 16, wherein step b) comprises centrifuging an aliquot of the yeast cell culture and then purifying the polypeptide product from the culture supernatant.