ADE2 Auxotrophic Marker for Starmerella Gene Recombination
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Solution Overview
Problem
The existing gene recombination techniques for enhancing sophorolipid productivity in Starmerella bombicola face challenges due to the risk of horizontally transferring foreign genes, which are not inherently present in the organism, and the need for a novel endogenous selection marker that does not violate regulations on living modified organisms.
Innovation Solution
Development of a Starmerella bombicola mutant strain with suppressed or inactivated phosphoribosyl-glycinamide formyltransferase-like protein expression, utilizing adenine auxotrophy as an endogenous selection marker for efficient transformation and glycolipid production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If foreign antibiotic resistance genes are used as selection markers for gene recombination in Starmerella bombicola, then transformation efficiency and selection capability are improved, but the risk of horizontal gene transfer increases and regulatory compliance deteriorates
Solution Approach 1:
The invention extracts and eliminates foreign antibiotic resistance genes from the selection marker system. Instead, it utilizes the endogenous ADE2 gene and its auxotrophic mutant phenotype as a selection marker, thereby removing the harmful element (foreign gene) while preserving the useful function (selection capability).
Solution Approach 2:
The invention employs the microorganism's own endogenous gene (ADE2) and its natural auxotrophic mutation as the selection marker. This self-service approach uses the organism's intrinsic genetic characteristics rather than introducing foreign markers, thereby eliminating horizontal gene transfer risk while maintaining selection efficiency.
2Object-affected harmful factors
If endogenous genes are used as selection markers to comply with regulations, then horizontal gene transfer risk is reduced, but the availability of functional markers deteriorates due to difficulty in establishing host strains
Solution Approach 1:
The invention performs preliminary action by pre-establishing the ADE2 auxotrophic mutant host strain before the gene recombination experiment. This pre-prepared host strain with known auxotrophic characteristics simplifies subsequent transformation and selection processes, making the system readily available for use.
Solution Approach 2:
The invention changes the selection parameter from antibiotic resistance (foreign gene-based) to auxotrophic phenotype (endogenous gene-based). By utilizing the ADE2 gene's role in adenine biosynthesis and the resulting auxotrophic growth requirement, it creates a functional selection marker that is both regulatory-compliant and experimentally useful.
3Adaptability or versatility
If only ura3 gene is used as endogenous selection marker, then self-cloning compliance is achieved, but marker versatility and selection options are limited
Solution Approach 1:
The invention demonstrates that endogenous genes can serve as versatile selection markers by utilizing the ADE2 gene's auxotrophic function. This approach provides a universal selection system that can be applied to various gene recombination experiments in Starmerella bombicola, not limited to specific applications, thereby achieving marker versatility comparable to foreign gene markers.
Data Source
Figure 1(A)~1(B)
AI summary
Provided are a novel marker for gene recombination of Starmerella bombicola and a method for using the same. The present invention provides an adenine auxotrophic selection marker consisting of a gene encoding a phosphoribosyl-glycinamide formyltransferase-like protein or a gene equivalent thereto. The present invention also provides a Starmerella bombicola mutant strain in which expression of a phosphoribosyl-glycinamide formyltransferase-like protein or a protein equivalent thereto is suppressed or the protein is inactivated.