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 activity of a YPS1-1 protease comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 67, a YPS1-2 protease comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 68, and a YPS 1-5 protease comprising a polypeptide sequence at least 95% identical to the polypeptide sequence encoded by the YPS1-5 gene set forth by SEQ ID NO: 5 have 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 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 YPS1-5 protease is encoded by a YPS1-5 gene comprising a polynucleotide sequence at least 95% identical to SEQ ID NO: 5 and encoding a polypeptide having protease activity.
9. The microorganism of claim 8 , wherein said YPS1-5 gene comprises SEQ ID NO: 5.
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 YPS1-5 protease is encoded by a YPS1-5 gene, and wherein said YPS1-1 gene, said YPS1-2 gene, said YPS1-5 gene have been mutated or knocked out.
11. The microorganism of claim 1 , wherein said recombinant protein comprises one or more repeat sequences {GGY-[GPG-X 1 ] n1 -GPS-(A) n2 } n3 , wherein
each X1 in each [GPG-X 1 ] n1 is separately one of 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.
12. The microorganism of claim 11 , wherein said recombinant protein comprises SEQ ID NO: 463.
13. A Pichia Pastoris engineered microorganism comprising YPS1-1, YPS1-2, and YPS1-5 activity reduced by a mutation or deletion of the YPS1-1 gene comprising SEQ ID NO: 1, the YPS1-2 gene comprising SEQ ID NO: 2, and the YPS1-5 gene comprising SEQ ID NO: 5, wherein said microorganism further comprises a recombinantly expressed protein comprising a polypeptide sequence comprising SEQ ID NO: 463.
14. A cell culture comprising the microorganism of claim 1 .
15. The cell culture of claim 14 , wherein said recombinantly expressed protein is less degraded than in a cell culture comprising an otherwise identical Pichia pastoris microorganism whose YPS1-1, YPS1-2, and YPS1-5 activities have not been attenuated or eliminated.
16. 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, a YPS1-2 protease comprising a polypeptide sequence at least 95% identical to SEQ ID NO: 68, and a YPS1-5 protease comprising a polypeptide sequence at least 95% identical to the polypeptide sequence encoded by the YPS1-5 gene set forth by SEQ ID NO: 5 have been attenuated or eliminated, wherein each of said polypeptide sequences has a protease activity before said attenuation or elimination.
17. The microorganism of claim 1 , wherein said YPS1-5 protease comprises an amino acid sequence encoded by SEQ ID NO: 5.