Chaperone Coexpression Enhances Protein Secretion in Trichoderma
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Solution Overview
Problem
Protein secretion is often the bottleneck in over-expressing proteins in cellular systems due to intermolecular aggregation reactions competing with intramolecular folding, and there is a need to reduce enzymatic side activities in protein production for use in food products containing hydrocolloids and polysaccharides.
Innovation Solution
Engineered Trichoderma filamentous fungus host cells with endogenous and exogenously introduced secretion enhancing proteins, such as bip1, ppi1, or sil1, are used to increase protein secretion and produce chymosin as a fusion protein with reduced catalytic activity, and mutant strains are created to be deficient in certain enzyme genes to minimize side activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If protein over-expression is attempted in cellular systems, then protein production quantity increases, but intermolecular aggregation reactions compete with intramolecular folding causing secretion bottlenecks
Solution Approach 1:
The patent introduces chaperone proteins (BiP, PDI, calnexin, calreticulin) as intermediary molecules that mediate between the over-expressed target protein and the folding machinery. These chaperones bind to hydrophobic regions of nascent proteins, preventing aggregation and facilitating proper folding, thus resolving the contradiction between high production and folding efficiency
Solution Approach 2:
The patent modifies the expression system by co-expressing multiple chaperone genes alongside the target protein gene. This changes the cellular environment parameters (chaperone concentration, folding capacity) to accommodate high-level protein production without aggregation, thereby maintaining both productivity and folding reliability
2Productivity
If secreted enzymatic activities are present in the expression host, then protein secretion function is provided, but unwanted side activities interfere with food products containing hydrocolloids and polysaccharides
Solution Approach 1:
The patent removes or inactivates endogenous secreted enzymes in the Trichoderma reesei host that cause unwanted side activities on hydrocolloids and polysaccharides. By eliminating these harmful enzymatic functions while retaining the protein secretion capability, the system produces clean protein products suitable for food applications
Solution Approach 2:
The patent utilizes the host's natural protein secretion machinery while simultaneously removing harmful enzymatic activities. The secretion pathway itself is preserved and enhanced for producing the desired protein, while the harmful side activities are eliminated, converting a potentially problematic system into a beneficial one for food-grade protein production
Data Source
AI summary
The present teachings provide methods for increasing protein secretion, e.g., chymosin in filamentous fungi by coexpressing certain chaperone(s) and/or foldase(s). The present teachings also provide filamentous fungi containing certain chaperone(s) and/or foldase(s) and a protein of interest for increased secretion.