Modified strains for the production of recombinant silk
AssigneeBolt Threads, Inc.
InventorsMatthew Scott Gamboa, Joshua Tyler Kittleson
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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.
Claims27
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.
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.
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.
Dependencies are inferred from the claim text.