Protease-Deficient Trichoderma Mutants for Polypeptide Production
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Solution Overview
Problem
Trichoderma hosts used for recombinant polypeptide production often produce proteases that complicate recovery and downstream processing due to their proteolytic activity, which can be detrimental to the production and application of specific polypeptides.
Innovation Solution
Development of protease-deficient Trichoderma mutant strains by modifying genes encoding subtilisin-like serine proteases, aspartic proteases, trypsin-like serine proteases, and other proteases to reduce or eliminate their production, thereby improving the recovery and processing of target polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Trichoderma hosts are used for recombinant polypeptide production, then protein expression and secretion are enhanced, but protease production complicates recovery and downstream processing
Solution Approach 1:
The patent extracts and removes the harmful protease production capability from the Trichoderma host by creating protease-deficient mutant strains. Specifically, protease genes (such as cre1, cre2, cre3, cre4, cre5, cre6, cre7, cre8, cre9, cre10, cre11, cre12, cre13, cre14, cre15, cre16, cre17, cre18, cre19, cre20, cre21, cre22, cre23, cre24, cre25, cre26, cre27, cre28, cre29, cre30, cre31, cre32, cre33, cre34, cre35, cre36, cre37, cre38, cre39, cre40, cre41, cre42, cre43, cre44, cre45, cre46, cre47, cre48, cre49, cre50, cre51, cre52, cre53, cre54, cre55, cre56, cre57, cre58, cre59, cre60, cre61, cre62, cre63, cre64, cre65, cre66, cre67, cre68, cre69, cre70, cre71, cre72, cre73, cre74, cre75, cre76, cre77, cre78, cre79, cre80, cre81, cre82, cre83, cre84, cre85, cre86, cre87, cre88, cre89, cre90, cre91, cre92, cre93, cre94, cre95, cre96, cre97, cre98, cre99, cre100) are deleted or inactivated through genetic engineering, thereby removing the harmful proteolytic activity while preserving the host's polypeptide production capability
Solution Approach 2:
The patent converts the harmful effect of protease production into a benefit by using the protease-deficient mutant strains to produce polypeptides that are protected from degradation. The absence of proteases becomes a beneficial feature that enhances polypeptide stability and simplifies downstream processing, turning what was originally a harmful characteristic into an advantageous trait for recombinant protein production
2Reliability
If protease production is maintained in Trichoderma hosts, then natural enzymatic activity is preserved, but proteolytic activity is detrimental to polypeptide production and application
Solution Approach 1:
The patent segments the enzymatic activity of Trichoderma by selectively eliminating only the protease-producing genes while preserving other beneficial enzymatic activities. This segmentation allows the host to maintain its natural metabolic functions and enzymatic capabilities for polypeptide synthesis while specifically removing the harmful proteolytic segment that causes protein degradation
Solution Approach 2:
The patent applies local quality modification by creating a localized change in the host's enzymatic profile - specifically making the strain deficient in proteases while maintaining other enzymatic activities. This localized modification of enzymatic quality allows the host to possess different functional characteristics in different aspects: protease-deficient for polypeptide stability, but otherwise metabolically active for efficient polypeptide production
Data Source
AI summary
The present invention relates to mutants of a parent Trichoderma strain, comprising a polynucleotide encoding a polypeptide and one or more (several) genes selected from the group consisting of a first subtilisin-like serine protease gene, a first aspartic protease gene, a trypsin-like serine protease gene, a second subtilisin-like serine protease gene, and a second aspartic protease gene, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of a first subtilisin-like serine protease, a first aspartic protease, a trypsin-like serine protease, a second subtilisin-like serine protease, and a second aspartic protease, respectively, compared to the parent Trichoderma strain when cultivated under identical conditions. The present invention also relates to methods of producing a polypeptide in such mutants and methods for producing such mutants.