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.
Claims50
A Pichia pastoris microorganism, in which the activity of a YPS1-1 protease and a YPS1-2 protease has been attenuated or eliminated, wherein said microorganism expresses a recombinant polypeptide.
The microorganism of any of the above claims, wherein said YPS1-2 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO:
- 68.
The microorganism of any of the above claims, wherein said YPS1-2 protease is encoded by a YPS1-2 gene.
The microorganism of any of the above claims, wherein, said YPS1-1 gene or said YPS1-2 gene, or both, has been mutated or knocked out.
The microorganism of any of the above claims, wherein said microorganism expresses a recombinant protein.
The microorganism of any of the above claims, wherein the activity of one or more additional proteases has been attenuated or eliminated.
A Pichia pastoris engineered microorganism comprising YPS1-1 and YPS1-2 activity reduced by a mutation or deletion of the YPS1-1 gene comprising SEQ ID NO:
- 1 and the YPS1-2 gene comprising SEQ ID NO:
- 2, wherein said microorganism further comprises a recombinantly expressed protein comprising a polypeptide sequence encoded by SEQ ID NO:
- 462.
A method of modifying Pichia pastoris to reduce the degradation of a recombinantly expressed protein, comprising knocking out or mutating a gene encoding a YPS1-1 protein and a YPS1-2 protein.
Dependencies are inferred from the claim text.