SEC modified strains for improved secretion of recombinant proteins

Patent metadata

Field	Value
Publication number	US-11634729-B2
Application number	US-201916415605-A
Country	US
Kind code	B2
Filing date	May 17, 2019
Priority date	May 17, 2018
Publication date	Apr 25, 2023
Grant date	Apr 25, 2023

Abstract

Disclosed herein are modified strains for improving secretion of recombinantly expressed products secreted from a host organism with improved growth and productivity characteristics, as well as methods of using the modified strains.

First claim

The invention claimed is:

1. A Pichia pastoris microorganism, in which the activity of a SEC72 protein comprising a polypeptide sequence of SEQ ID NO: 2 has been eliminated, and wherein said microorganism expresses a recombinant silk-like protein comprising a secretion signal peptide derived from yeast and selected from a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and a MFα1 signal sequence.

2. The microorganism of claim 1 , wherein said SEC72 protein is encoded by an sec72 gene comprising SEQ ID NO: 1, and wherein said sec72 gene has been deleted.

3. The microorganism of claim 1 , further comprising a recombinantly expressed translocon complex.

4. The microorganism of claim 3 , wherein said recombinantly expressed translocon complex is a recombinantly expressed SSH1 translocon complex.

5. The microorganism of claim 4 , wherein said SSH1 translocon complex comprises a first polypeptide sequence at least 95% identical to SEQ ID NO: 4, a second polypeptide sequence at least 95% identical to SEQ ID NO: 6, and a third polypeptide sequence at least 95% identical to SEQ ID NO: 8.

6. A cell culture comprising a microorganism of claim 4 , wherein said cell culture has an improved strain growth rate and fermentation performance under standard cell culture conditions as compared to a cell culture that does not comprise a recombinantly expressed SSH1 translocon complex.

7. A cell culture comprising a microorganism of claim 4 , wherein said cell culture has an improved yield or specific productivity of said recombinant protein under standard cell culture conditions as compared to a cell culture of an otherwise identical microorgansims that comprises a functional sec72 gene and does not comprise a recombinantly expressed SSH1 translocon complex.

8. A method of producing a recombinant protein, comprising: culturing the microorganism of claim 4 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.

9. The method of claim 8 , 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.

10. The method of claim 8 , wherein said microorganism has an increased yield or specific productivity of said recombinant protein as compared to an otherwise identical microorganism not comprising said recombinantly expressed SSH1 translocon complex, and wherein said sec72 gene is not deleted.

11. The microorganism of claim 3 , wherein said translocon complex is expressed from a recombinant SSH1 gene, a recombinant SSS1 gene, and a recombinant SBH2 gene.

12. The microorganism of claim 11 , wherein said SSH1 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 3, wherein said SSS1 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 5, or wherein said SBH2 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 7.

13. The microorganism of claim 3 wherein said translocon complex comprises an SSH1 protein, an SSS1 protein, and an SBH2 protein.

14. The microorganism of claim 13 , wherein said SSH1 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 4, wherein said SSS1 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 6, or wherein said SBH2 protein comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 8.

15. The microorganism of claim 1 , wherein the activity of a YPS1-1 protease and a YPS1-2 protease has been attenuated or eliminated.

16. The microorganism of claim 15 , wherein said YPS1-1 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 10, or wherein said YPS1-2 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 12.

17. The microorganism of claim 1 , wherein said secretion signal peptide is selected from the group consisting of: SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, and SEQ ID NO: 90.

18. A cell culture comprising a microorganism of claim 1 .

19. The microorganism of claim 1 , wherein said secretion signal peptide is endogenous to P. pastoris.

20. The microorganism of claim 1 , wherein said secretion signal peptide is heterologous to P. pastoris.

21. The microorganism of claim 1 , wherein said secretion signal is derived from S. cerevisiae.

22. A method of modifying a Pichia pastoris microorganism to improve the secretion of a recombinantly expressed protein, sad method comprising knocking out a gene encoding an SEC72 protein, and wherein sad microorganism expresses a recombinant silk-like protein comprising a secretion signal peptide derived from yeast and selected from a PEP4 signal sequence, a CPY+4 signal sequence, a DAP2 signal sequence, and a MFa1 signal sequence.

23. The method of claim 22 , further comprising transforming said Pichia pastoris with a vector comprising genes encoding a recombinantly expressed SSH1 translocon complex.

Assignees

• Bolt Threads Inc

Inventors

• Stevens Thomas

Classifications

• C12R2001/84

  Pichia · CPC title

• C12P21/00

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

• C12N9/60

  from yeast · CPC title

• C07K14/39 Primary

  from yeasts · CPC title

• C12N1/16

  Yeasts; Culture media therefor · CPC title