Chrysosporium lucknowense mutant strains for high-purity enzyme production
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Solution Overview
Problem
Current fungal hosts like Chrysosporium lucknowense secrete a mix of enzymes, making it difficult to produce specific enzymes at high purity levels, as they also secrete non-relevant or counterproductive proteins, complicating screening and purification processes.
Innovation Solution
Isolation of mutant strains with reduced secretion of non-desired proteins, such as proteases and cellulases, to produce specific enzymes at high levels without co-production of background proteins, using methods like mutagenesis and genetic disruption, and employing specific promoters for transcriptional control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If fungal hosts like Chrysosporium lucknowense are used for enzyme production, then high protein secretion capacity is achieved, but the secreted protein mixture becomes undefined with many non-desired enzymes
Solution Approach 1:
The patent extracts and removes non-desired protease activities from the fungal host system through isolation of mutants with reduced protease secretion. This allows the host to maintain high overall protein secretion capacity while selectively eliminating unwanted proteolytic activities that contaminate the target enzyme preparation.
Solution Approach 2:
The patent applies local quality by creating fungal strains with differentiated secretion profiles - specifically reducing protease secretion in certain strains while maintaining or enhancing secretion of target enzymes. This localized modification of secretion characteristics enables high purity production without sacrificing overall productivity.
2Manufacturing precision
If mutant strains with reduced protease secretion are isolated, then enzyme purity increases, but screening and isolation processes become more complex
Solution Approach 1:
The patent employs self-service by utilizing the mutant strains' inherent reduced protease secretion capability as a built-in purification feature. The strains naturally produce cleaner protein preparations without requiring extensive external purification steps, thereby simplifying the overall process despite the initial mutagenesis work.
3Manufacturing precision
If endogenous cellulase and protease secretion is reduced to below 20 % of wild type levels, then specific enzyme production purity improves, but the host's natural enzyme secretion capability is compromised
Solution Approach 1:
The patent applies parameter changes by modifying the secretion levels of specific enzymes (proteases and cellulases) to be below 20 % of wild type levels through mutant isolation. This parameter modification creates a more favorable secretion profile for producing specific heterologous enzymes at high purity while maintaining the host's overall secretory functionality.
Data Source
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AI summary
The present invention provides a new fungal production system comprising a fungal host strain of Chrysosporium lucknowense wherein the endogenous cellulase secretion is less than 20 % of the endogenous cellulase secretion of Chrysosporium lucknowense strain UV 18-25. Preferably, also the secretion of endogenous protease, endogenous β-glucanase and endogenous cellobiohydrolase is less than 20 % of the secretion of Chrysosporium lucknowense strain UV 18-25. Furthermore, fungal host strains are provided wherein several genes have been disrupted. According to another aspect of the invention a method for homologous and/or heterologous production of a pure protein with a purity of higher than 75 %, comprising expressing a gene encoding said protein in the strains according to the invention have been described. Furthermore, a method for production of artificial protein mixes comprising expressing a gene encoding each of said proteins in a strain according to the invention have been disclosed. Finally a method for simplified screening of strains functionally expressing a desired enzyme by application of said strains have been provided.