Promoters for expressing genes in a fungal cell

Patent metadata

Field	Value
Publication number	US-9359630-B2
Application number	US-201113883931-A
Country	US
Kind code	B2
Filing date	Nov 30, 2011
Priority date	Nov 30, 2010
Publication date	Jun 7, 2016
Grant date	Jun 7, 2016

Abstract

The present invention relates to isolated promoters and constructs, vectors, and fungal host cells comprising such promoters operably linked to polynucleotides encoding polypeptides. The present invention also relates to methods for producing such polypeptides.

First claim

What is claimed is:

1. A method for producing a polypeptide, comprising:
(a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a polynucleotide encoding the polypeptide operably linked to a promoter selected from the group consisting of (i) a promoter comprising SEQ ID NO: 1 or a subsequence thereof that retains promoter activity, (ii) a promoter comprising a nucleotide sequence that hybridizes under at least high stringency conditions with SEQ ID NO: 1 or the full-length complement thereof, wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; and (iii) a hybrid or tandem promoter of (i) or (ii); wherein the polynucleotide encoding the polypeptide is foreign to the promoter; and
(b) isolating the polypeptide from the cultivation medium.

2. The method of claim 1 , wherein the promoter comprises a nucleotide sequence that hybridizes under high stringency conditions with SEQ ID NO: 1 or the full-length complement thereof, wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

3. The method of claim 1 , wherein the promoter comprises a nucleotide sequence that hybridizes under very high stringency conditions with SEQ ID NO: 1 or the full-length complement thereof, wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

4. The method of claim 1 , wherein the promoter comprises the polynucleotide sequence of SEQ ID NO: 1; or a subsequence thereof that retains promoter activity.

5. The method of claim 1 , wherein the promoter is a hybrid promoter comprising a portion of SEQ ID NO: 1 and one or more portions of the polynucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 31, and SEQ ID NO: 32.

6. The method of claim 1 , wherein the promoter is a tandem promoter comprising SEQ ID NO: 1 and one or more polynucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 31, and SEQ ID NO: 32; or a subsequence(s) thereof that retains promoter activity.

7. The method of claim 1 , wherein the polypeptide is native or foreign to the fungal host cell.

8. The method of claim 1 , wherein the fungal host cell is a filamentous fungal cell.

9. The method of claim 1 , wherein the fungal host cell is a yeast cell.

10. A nucleic acid construct comprising a polynucleotide encoding a polypeptide operably linked to a promoter, wherein the polynucleotide encoding the polypeptide is foreign to the promoter and wherein the promoter is selected from the group consisting of (i) a promoter comprising SEQ ID NO: 1 or a subsequence thereof that retains promoter activity, (ii) a promoter comprising a nucleotide sequence that hybridizes under at least high stringency conditions with SEQ ID NO: 1 or the full-length complement thereof, wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; and (iii) a hybrid or tandem promoter of (i) or (ii).

11. The nucleic acid construct of claim 10 , which comprises a nucleotide sequence that hybridizes under high stringency conditions with SEQ ID NO: 1; or the full-length complement thereof, wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

12. The nucleic acid construct of claim 10 , which comprises the polynucleotide sequence of SEQ ID NO: 1 or a subsequence thereof that retains promoter activity.

13. The nucleic acid construct of claim 10 , which is a hybrid promoter comprising a portion of SEQ ID NO: 1 and one or more portions of the polynucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 31, and SEQ ID NO: 32.

14. The nucleic acid construct of claim 10 , which is a tandem promoter comprising SEQ ID NO: 1 and one or more polynucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 31, and SEQ ID NO: 32; or a subsequence(s) thereof that retains promoter activity.

15. A recombinant host cell comprising the nucleic acid construct of claim 10 .

16. The recombinant host cell of claim 15 , which is a filamentous fungal cell.

17. The recombinant host cell of claim 15 , which is a yeast cell.

18. The nucleic acid construct of claim 10 , which comprises a nucleotide sequence that hybridizes under very high stringency conditions with SEQ ID NO: 1 or the full-length complement thereof, wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

19. The method of claim 1 , wherein the promoter consists of the polynucleotide sequence of SEQ ID NO: 1 or a subsequence thereof that retains promoter activity.

20. The nucleic acid construct of claim 10 , which consists of the polynucleotide sequence of SEQ ID NO: 1 or a subsequence thereof that retains promoter activity.

Assignees

• Catlett Michael
• Yaver Debbie
• Novozymes Inc

Inventors

• Catlett Michael
• Yaver Debbie

Classifications

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12N15/815 Primary

  for yeasts other than Saccharomyces · CPC title

• C12N15/80 Primary

  for fungi · CPC title

• C12P21/06 Primary

  produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00) · CPC title