Modified strains for the production of recombinant silk
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.
1 . A Pichia pastoris microorganism, in which the activities of a YPS1-1 protease comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 67, a YPS1-2 protease comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO; 68 and a MKC7 protease comprising a polypeptide sequence having at least 95% sequence identity to the full-length polypeptide sequence encoded by SEQ ID NO: 7 have been attenuated or eliminated as compared to an otherwise identical Pichia pastoris microorganism whose YPS1-1, YPS1-2, and MKC7 protease activities have not been attenuated or eliminated, wherein each of said polypeptide sequences has a protease activity before said attenuation or elimination, and wherein said microorganism expresses a recombinant protein.
2 . The microorganism of claim 1 , wherein the polypeptide sequence of 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 having at least 95% sequence identity to SEQ ID NO: 1.
4 . The microorganism of claim 3 , wherein said YPS1-1 gene comprises SEQ ID NO: 1.
5 . The microorganism of claim 1 , wherein the polypeptide sequence of 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 having at least 95% sequence identity to SEQ ID NO: 2.
7 . The microorganism of claim 6 , wherein said YPS1-2 gene comprises SEQ ID NO: 2.
8 . The microorganism of claim 1 , wherein said MKC7 protease is encoded by a MKC7 gene comprising a polynucleotide sequence having at least 95% sequence identity to SEQ ID NO: 7.
9 . The microorganism of claim 1 , wherein said MKC7 gene comprises SEQ ID NO: 7.
10 . 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 MKC7 protease is encoded by a MKC7 gene, and wherein said YPS1-1 gene, said YPS1-2 gene, and said MKC7 gene have been mutated or knocked out.
11 . The microorganism of claim 1 , wherein said recombinant protein is a silk-like polypeptide comprising one or more repeat sequences {GGY-[GPG-X 1 ] n1 -GPS-(A) n2 } n3 , wherein
(SEQ ID NO: 515)
X 1 = SGGQQ,
(SEQ ID NO: 516)
GAGQQ,
SEQ ID NO: 517)
GQGPY,
(SEQ ID NO: 518)
AGQQ,
or
SQ;
n1 is from 4 to 8;
n2 is from 6 to 20; and
n3 is from 2 to 20.
12 . The microorganism of claim 11 , wherein the polypeptide sequence of said silk-like polypeptide comprises SEQ ID NO: 463.
13 . The microorganism of claim 1 , wherein the activity of one or more additional proteases has been attenuated or eliminated as compared to an otherwise identical Pichia pastoris microorganism whose one or more additional protease activities has not been attenuated or eliminated.
14 . The microorganism of claim 13 , wherein the activity of one or more additional proteases comprises activity of a YPS1-5 protease.
15 . The microorganism of claim 13 , wherein the activity of one or more additional proteases comprises activity of a YPS1-3 protease.
16 . An engineered Pichia pastoris microorganism comprising a mutation or deletion of a YPS1-1 gene comprising SEQ ID NO: 1 and encoding a YPS-1 protease, a mutation or deletion of a YPS1-2 gene comprising SEQ ID NO: 2 and encoding a YPS1-2 protease, and a mutation or deletion of a MKC7 gene comprising SEQ ID NO: 7 and encoding a MKC7 protease, wherein the activities of the YPS1-1 protease, the YPS1-2 protease, and the MKC7 protease of the engineered microorganism are reduced as compared to an otherwise identical Pichia pastoris microorganism whose YPS1-1, YPS1-2, and MKC7 genes have not been mutated or deleted, and wherein said microorganism further comprises a recombinantly expressed protein comprising the polypeptide sequence of SEQ ID NO: 463.
17 . A cell culture comprising the microorganism of claim 1 .
18 . The cell culture of claim 17 , wherein said recombinant protein is less degraded than a cell culture comprising an otherwise identical Pichia pastoris microorganism whose YPS1-1, YPS1-2, and MKC7 protease activities have not been attenuated or eliminated.
19 . 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 recombinant protein; and
isolating the recombinant protein from the microorganism or the culture medium.
20 . The method of claim 19 , 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.
21 . The method of claim 19 , wherein said recombinant protein has a decreased level of degradation as compared to said recombinant protein produced by an otherwise identical microorganism whose YPS1-1, YPS1-2, and MKC7 protease activities have not been attenuated or eliminated.
22 . A method of making the Pichia pastoris of claim 1 , comprising knocking out or mutating a gene encoding the YPS1-1 protease, knocking out or mutating a gene encoding the YPS1-2 protease, knocking out or mutating a gene encoding the MKC7 protease, and transformation with a polynucleotide encoding the recombinant protein.
23 . The method of claim 22 , wherein said recombinant protein comprises a polyA sequence comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous alanine residues (SEQ ID NO: 519).
24 . The method of claim 22 , wherein said recombinant protein comprises a silk-like polypeptide.
25 . The method of claim 24 , wherein said silk-like polypeptide comprises one or more repeat sequences {GGY-[GPG-X 1 ] n1 -GPS-(A) n2 } n3 , wherein
(SEQ ID NO: 515)
X 1 = SGGQQ,
(SEQ ID NO: 516)
GAGQQ,
SEQ ID NO: 517)
GQGPY,
(SEQ ID NO: 518)
AGQQ,
or
SQ;
n1 is from 4 to 8;
n2 is from 6 to 20; and
n3 is from 2 to 20.
26 . The method of claim 22 , wherein the polypeptide sequence of said recombinant protein comprises SEQ ID NO: 463.
27 . A Pichia pastoris microorganism, in which the activities of a YPS1-1 protease comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO: 67, a YPS1-2 protease comprising a polypeptide sequence having at least 95% sequence identity to SEQ ID NO 68, and a MKC7 protease comprising a polypeptide sequence having at least 95% sequence identity to the full-length polypeptide sequence encoded by SEQ ID NO: 7 have been attenuated or eliminated as compared to an otherwise identical Pichia pastoris microorganism whose YPS1-1, YPS1-2, and MKC7 protease activities have not been attenuated or eliminated, wherein each of said polypeptide sequences has a protease activity before said attenuation or elimination.