Modified strains for the production of recombinant silk

Patent metadata

Field	Value
Publication number	US-10647975-B2
Application number	US-201715724196-A
Country	US
Kind code	B2
Filing date	Oct 3, 2017
Priority date	Oct 3, 2017
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

Disclosed herein are modified strains for reducing degradation of recombinantly expressed products secreted from a host organism and methods of using the modified strains. In some embodiments, to attenuate a protease activity in Pichia pastoris , the genes encoding enzymes the degrade proteases are inactivated or mutated to reduce or eliminate activity. In preferred strains, the protease activity of proteases encoded by PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) (e.g., polypeptides comprising SEQ ID NO: 66 and 67) is attenuated.

First claim

The invention claimed is:

1. A Pichia pastoris microorganism, in which the activity of a YPS1-1 protease comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 67 and a YPS1-2 protease comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 68 has been attenuated or eliminated, wherein said polypeptide sequence at least 95% identical to SEQ ID NO: 67 and said polypeptide sequence at least 95% identical to SEQ ID NO: 68 each have a protease activity before said attenuation or elimination, and wherein said microorganism expresses a recombinant protein.

2. The microorganism of claim 1 , wherein said YPS1-1 protease comprises SEQ ID NO: 67.

3. The microorganism of claim 1 , wherein said YPS1-1 protease is encoded by a YPS1-1 gene comprising a polynucleotide sequence at least 95% identical to SEQ ID NO: 1 and encoding a polypeptide having protease activity.

4. The microorganism of claim 3 , wherein said YPS1-1 gene comprises SEQ ID NO: 1.

5. The microorganism of claim 1 , wherein said YPS1-2 protease comprises SEQ ID NO: 68.

6. The microorganism of claim 1 , wherein said YPS1-2 protease is encoded by a YPS1-2 gene comprising a polynucleotide sequence at least 95% identical to SEQ ID NO: 2 and encoding a polypeptide having protease activity.

7. The microorganism of claim 6 , wherein said YPS1-2 gene comprises SEQ ID NO: 2.

8. The microorganism of claim 1 , wherein said YPS1-1 protease is encoded by a YPS1-1 gene, wherein said YPS1-2 protease is encoded by a YPS1-2 gene, and wherein said YPS1-1 gene or said YPS1-2 gene, or both, has been mutated or knocked out.

9. The microorganism of claim 1 , wherein said recombinant protein comprises one or more repeat sequences {GGY-[GPG-X 1 ]n 1 -GPS-(A)n 2 }n 3 (SEQ ID NO: 514), wherein
X1=SGGQQ (SEQ ID NO: 515), GAGQQ (SEQ ID NO: 516), GQGPY (SEQ ID NO: 517), AGQQ (SEQ ID NO: 518) or SQ;
n1 is from 4 to 8;
n2 is from 6 to 20; and
n3 is from 2 to 20,
wherein said one or more repeat sequences are a silk-like polypeptide.

10. The microorganism of claim 9 , wherein said recombinant protein comprises SEQ ID NO: 463.

11. The microorganism of claim 1 , wherein the activity of one or more additional proteases has been attenuated or eliminated.

12. A Pichia pastoris engineered microorganism comprising YPS1-1 and YPS1-2 activity reduced by a mutation or deletion of the YPS1-1 gene comprising SEQ ID NO: 1 and the YPS1-2 gene comprising SEQ ID NO: 2, wherein said microorganism further comprises a recombinantly expressed protein comprising a polypeptide sequence comprising SEQ ID NO: 463.

13. A cell culture comprising the microorganism of claim 1 .

14. A cell culture comprising the microorganism of claim 1 , wherein said recombinantly expressed protein is less degraded than a cell culture comprising an otherwise identical Pichia pastoris microorganism whose YPS1-1 and YPS1-2 activity has not been attenuated or eliminated.

15. A method of producing a recombinant protein with a reduced degradation, comprising:
culturing the microorganism of claim 1 in a culture medium under conditions suitable for expression of the recombinantly expressed protein; and
isolating the recombinant protein from the microorganism or the culture medium.

16. The method of claim 15 , wherein said recombinant protein is secreted from said microorganism, and wherein isolating said recombinant protein comprises collecting a culture medium comprising said secreted recombinant protein.

17. The method of claim 15 , wherein said recombinant protein has a decreased level of degradation as compared to said recombinant protein produced by an otherwise identical microorganism wherein said YPS1-1 and said YPS1-2 protease activity has not been attenuated or eliminated.

18. A method of making the Pichia pastoris microorganism of claim 1 comprising knocking out or mutating a gene encoding the YPS 1-1 protein and a gene encoding the YPS 1-2 protein.

19. The method of claim 18 , wherein said recombinantly expressed protein comprises a polyA sequence comprising at least at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous alanine residues (SEQ ID NO: 519).

20. The method of claim 18 , wherein said recombinantly expressed protein comprises a silk-like polypeptide.

21. The method of claim 20 , wherein said silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X 1 ]n 1 -GPS-(A)n 2 }n 3 (SEQ ID NO: 514), wherein
X 1 =SGGQQ (SEQ ID NO: 515) or GAGQQ (SEQ ID NO: 516) or GQGPY (SEQ ID NO: 517) or AGQQ (SEQ ID NO: 518) or SQ;
n1 is from 4 to 8;
n2 is from 6 to 20; and
n3 is from 2 to 20.

22. The method of claim 18 , wherein said recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO: 462.

23. A Pichia pastoris microorganism, in which the activity of a YPS 1-1 protease comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 67 and a YPSI-2 protease comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 68 has been attenuated or eliminated, wherein said polypeptide sequence at least 95% identical to SEQ ID NO: 67 and said polypeptide sequence at least 95% identical to SEQ ID NO: 68 each have a protease activity before said attenuation or elimination.

Assignees

• Bolt Threads Inc

Inventors

• Gamboa Matthew Scott
• Kittleson Joshua Tyler

Classifications

• C12N9/60 Primary

  from yeast · CPC title

• C12Y304/23041

  Yapsin 1 (3.4.23.41) · CPC title

• C07K14/43518

  from spiders · CPC title