Recombinant Bacillus subtilis Surfactin Production via Multi-Gene Synergy
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Wild-type B. subtilis 168 lacks the ability to synthesize surfactin, limiting its industrial application potential, and existing methods for surfactin production primarily rely on single gene or pathway modifications, which are insufficient for improving yield.
Innovation Solution
A multi-gene synergy approach is employed by replacing the sfp genes of B. subtilis with those from B. amyloliquefaciens, knocking out gene clusters associated with biofilm formation and negative regulatory factors, and overexpressing surfactin efflux and resistance genes, as well as those involved in the branched chain fatty acid synthesis and glycolytic pathways, to enhance surfactin production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If single gene or single pathway modification is used for surfactin production, then the transformation method is simple, but the surfactin yield is insufficient
Solution Approach 1:
The patent combines multiple gene modifications including sfp gene replacement, biofilm formation gene cluster knockout, and overexpression of efflux pumps and regulatory genes into a single integrated transformation strategy, achieving synergistic enhancement of surfactin production while maintaining manageable complexity
2Reliability
If wild type B. subtilis 168 is used as host, then the strain is safe (GRAS certified), but it lacks ability to synthesize surfactin
Solution Approach 1:
The patent introduces sfp genes from B. amyloliquefaciens as a functional intermediary to confer surfactin synthesis capability onto the safe B. subtilis 168 host, while additional gene modifications serve as mediators to overcome regulatory barriers and enhance production
3Productivity
If multi-gene transformation is performed to improve surfactin yield, then the surfactin production increases to 12.8 g/L, but the device complexity increases
Solution Approach 1:
The patent segments the complex multi-gene transformation into distinct functional modules: sfp gene replacement module, biofilm formation gene knockout module, efflux pump overexpression module, and regulatory gene modification module, allowing systematic construction and optimization
Data Source
AI summary
The present invention discloses a method for promoting B. subtilis to synthesize surfactin based on multi-gene synergy, and belongs to the field of genetic engineering. Firstly, B. subtilis is enabled to obtain the ability to synthesize surfactin by integrant expression of sfp protein derived from a high-yield strain, on this basis, by knocking out genes associated with a competitive pathway, overexpressing genes associated with the surfactin tolerance of B. subtilis, strengthening genes associated with a branched chain fatty acid synthesis pathway or improving the intracellular srfA gene transcription level, the synergy among genes is realized, and systemic metabolic engineering transformation is performed on the B. subtilis, thereby greatly improving the ability of B. subtilis genetically engineered bacteria to synthesize surfactin. Compared with a starting strain B. subtilis 168, the amount of extracellular accumulation of surfactin of the B. subtilis genetically engineered bacteria increases from 0 g/L to 12.8 g/L. The present invention obtains the recombinant B. subtilis with significantly improved ability to synthesize surfactin by multi-gene cooperative transformation of B. subtilis, and has a good application prospect.