Cryptococcus Mutant Strain Reducing Polysaccharide Viscosity
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Solution Overview
Problem
Basidiomycetous yeasts, such as Cryptococcus sp., produce large amounts of extracellular polysaccharides during heterologous protein production, leading to increased culture liquid viscosity and membrane clogging, making large-scale protein purification inefficient and costly.
Innovation Solution
A UV mutant strain, Cryptococcus sp. S-2 D11, is developed, which significantly reduces extracellular polysaccharide production, allowing for easier protein separation and purification by suppressing polysaccharide secretion and maintaining protein productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If basidiomycetous yeast is used as a host for heterologous protein production, then protein productivity is improved, but extracellular polysaccharide production increases causing viscosity increase and purification difficulties
Solution Approach 1:
The invention extracts and removes the harmful extracellular polysaccharide production capability from the basidiomycetous yeast host through genetic modification, specifically creating a mutant strain where the polysaccharide-producing genes are deleted or inactivated, thereby separating the desirable protein production function from the harmful polysaccharide production function
Solution Approach 2:
The invention changes the genetic parameters of the basidiomycetous yeast by introducing mutations that alter the expression levels or complete elimination of polysaccharide synthesis genes, transforming the strain from high polysaccharide producer to low or non-producer while maintaining protein production capability
2Object-generated harmful factors
If extracellular polysaccharides are removed by freezing and melting, then polysaccharide aggregation is achieved, but scaling up becomes difficult due to operational complexity
Solution Approach 1:
The invention performs preliminary action by genetically modifying the yeast strain beforehand to prevent extracellular polysaccharide production from the start, eliminating the need for subsequent polysaccharide removal steps and their associated operational complexities during scale-up
Solution Approach 2:
The invention converts the harmful effect of polysaccharide production into a benefit by using genetic engineering to eliminate the problem at its source, transforming a process that required complex removal steps into a straightforward production process with no polysaccharide interference
3Object-generated harmful factors
If cold acetone is added to aggregate polysaccharides, then polysaccharide removal is achieved, but safety concerns and inefficiency arise from large amounts of chemical usage
Solution Approach 1:
The invention extracts and eliminates the need for chemical aggregation agents like cold acetone by genetically modifying the yeast to not produce extracellular polysaccharides in the first place, thereby removing the source of the problem rather than treating it with chemicals
Solution Approach 2:
The genetically modified yeast strain serves itself by naturally producing minimal or no extracellular polysaccharides, eliminating the need for external chemical interventions and their associated safety and waste management issues
4Object-generated harmful factors
If polyethylene glycol is used to precipitate polysaccharides, then polysaccharide aggregation is achieved, but ultrafiltration membrane deterioration and waste treatment problems occur
Solution Approach 1:
The invention performs preliminary genetic modification to prevent extracellular polysaccharide production before cultivation, thereby preventing the need for polyethylene glycol precipitation and protecting ultrafiltration membranes from deterioration
Solution Approach 2:
The invention converts the harmful interaction between polysaccharides and ultrafiltration membranes into a beneficial situation by eliminating polysaccharide production at the genetic level, thereby preserving membrane integrity and simplifying waste treatment
Data Source
AI summary
The present invention provides a host for genetic recombination useful in the production of heterologous proteins in a large scale, by suppressing the production of extracellular polysaccharides in the basidiomycetous yeasts. Cryptococcus sp. S-2 D11 strain (FERM BP-11482) which is characterized in that the production of extracellular polysaccharides is suppressed as compared with the parent strain.