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.
Claims17
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.
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.
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.
Dependencies are inferred from the claim text.