Pichia Strains With SEC72 Deletion for Protein Secretion
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Solution Overview
Problem
Existing methods for producing recombinant proteins in Pichia pastoris face challenges with intracellular retention and degradation, leading to low yields and negative impacts on cell growth and longevity.
Innovation Solution
Engineering Pichia pastoris strains by deleting the SEC72 gene and introducing a recombinantly expressed SSH1 translocon complex, along with attenuating YPS1-1 and YPS1-2 proteases, to enhance protein secretion and maintain cell growth and productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If recombinant proteins are expressed in Pichia pastoris, then protein production is achieved, but intracellular retention and degradation occur leading to low yields
Solution Approach 1:
The patent extracts and removes the SEC72 gene from the Pichia pastoris genome through deletion. This gene removal eliminates the harmful function of SEC72 in retaining and degrading recombinant proteins intracellularly, thereby improving protein secretion efficiency and yield without compromising cell growth
Solution Approach 2:
The patent introduces the SSH1 translocon complex as an intermediary system to facilitate protein secretion. By overexpressing SSH1, the patent creates an enhanced translocon pathway that mediates the transport of recombinant proteins from the endoplasmic reticulum to the extracellular environment, improving secretion efficiency
2Reliability
If SEC72 gene is deleted to improve secretion, then protein secretion is enhanced, but cell growth and productivity may be negatively impacted
Solution Approach 1:
The patent compensates for the growth impact of SEC72 deletion by changing the expression level parameter of the SSH1 translocon complex through overexpression. This parameter change restores cellular functionality and maintains growth rate while preserving the secretion enhancement benefit
Solution Approach 2:
The SSH1 translocon complex serves multiple functions: it compensates for the loss of SEC72-mediated secretion, maintains cellular protein transport efficiency, and supports cell growth. This multi-functionality allows the system to achieve both improved secretion and maintained productivity
3Quantity of substance
If recombinant proteins are expressed, then protein production is achieved, but proteolytic degradation occurs reducing full-length protein yield
Solution Approach 1:
The patent removes the SEC72 gene which is responsible for proteolytic degradation of recombinant proteins. By extracting this harmful function from the cell, the patent prevents degradation of full-length proteins while maintaining production capacity
Data Source
AI summary
Disclosed herein are modified strains for improving secretion of recombinantly expressed products secreted from a host organism with improved growth and productivity characteristics, as well as methods of using the modified strains.