Pichia pastoris Mutant Strain for Phospholipase Expression
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Solution Overview
Problem
The Pichia pastoris expression system faces challenges in efficiently expressing exogenous proteins, particularly phospholipases and lipases, due to background gene expression and stability issues.
Innovation Solution
Engineered Pichia CICC32806 strains with specific mutations, including genes encoding BQ9382_C1-2260, EKK deletion, BQ9382_C1-3800, E129K, BQ9382_C1-5700, 1312M, BQ9382_C2-3950, Q145X, BQ9382_C3-2220, E188K, and BQ9382_C3-4370, W196X, are developed to enhance protein expression efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If the Pichia pastoris expression system is used to express exogenous proteins, then post-translational modification capabilities are achieved, but background gene expression and stability issues reduce expression efficiency
Solution Approach 1:
The patent extracts and eliminates harmful background gene expressions by knocking out specific genes (AOX1, AOX2, PRO4, PEP4, PRB1) that cause instability and unwanted protein production. This removes the disturbing elements from the expression system while preserving the beneficial post-translational modification capabilities.
Solution Approach 2:
The patent changes the genetic parameters of the Pichia pastoris strain by introducing multiple mutations and knockouts in specific genes. These parameter changes in the genome lead to improved expression stability and efficiency by eliminating background noise and enhancing the expression of exogenous proteins.
2Productivity
If methanol-utilizing yeast are used for protein expression, then high expression levels are achieved, but background AOX1 expression affects yield
Solution Approach 1:
The patent removes the harmful background AOX1 expression by knocking out the AOX1 gene in the Pichia pastoris strain. This eliminates the unwanted alcohol oxidase activity that would otherwise consume methanol and produce background protein expression, thereby improving the yield of the target exogenous protein.
3Reliability
If protease knockouts are performed to improve protein stability, then protein degradation is reduced, but growth rate decreases
Solution Approach 1:
The patent applies local quality by selectively knocking out specific proteases (PRO4, PEP4, PRB1) that are responsible for protein degradation, while maintaining other cellular functions. This localized modification improves protein stability without completely halting growth, as the knockouts are targeted at specific proteolytic pathways rather than all protein degradation.
Data Source
AI summary
Provided is a Pichia pastoris mutant strain for expressing an exogenous gene. Specifically, provided is a Pichia pastoris mutant strain comprising, with respect to Pichia pastoris mutant strain GS115 or CICC32806, one or more of the following six mutations: BQ9382_C1-2260, EKK deletions at positions 308-310, a hypothetical protein; BQ9382_C1-3800, E129K, 60S ribosomal subunit assembly/exported protein LOC1; BQ9382_C1-5700, 1312M, mitochondrial external NADH dehydrogenase, type II NAD(P)H: quinone oxidoreductase; BQ9382_C2-3950, Q145X, an essential protein having a binding partner Psr1p and used for completely activating a general stress response; BQ9382_C3-2220, E188K, a hypothetical protein; and BQ9382_C3-4370, W196X, orotidine 5\′-phosphate decarboxylase. The provided Pichia pastoris mutant strain is an effective commonly employed host for exogenous expression, and can efficiently express different proteins, especially phospholipase and lipase.