Pullulanase Expression in Bacillus subtilis via Protease Knockout
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Solution Overview
Problem
The low fermentation activity and high production cost of pullulanase-producing strains limit the industrial application of pullulanase, primarily due to protease degradation, lack of suitable promoters and signal peptides, and plasmid instability during expression in Bacillus subtilis.
Innovation Solution
Gene knockout technology is used to delete alkaline and neutral protease genes in Bacillus subtilis, combined with an optimized expression vector containing specific promoters and signal peptides from other Bacillus species, to enhance pullulanase expression and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If pullulanase is expressed in Bacillus subtilis using conventional expression systems, then the enzyme can be produced, but the enzyme activity is low due to protease degradation
Solution Approach 1:
The patent removes the harmful protease genes (alkaline protease gene and neutral protease gene) from the Bacillus subtilis genome through gene knockout technology. This extraction of harmful elements eliminates the protease degradation that was suppressing pullulanase activity, thereby resolving the contradiction between producing the enzyme and maintaining its activity.
Solution Approach 2:
The patent converts the harmful protease activity into a benefit by using protease genes as tools for gene knockout. The alkaline protease gene and neutral protease gene, which originally degraded pullulanase, are utilized as selectable markers and tools for creating mutant strains that lack these harmful proteases, thereby protecting pullulanase from degradation.
2Productivity
If conventional promoters and signal peptides are used for pullulanase expression, then the expression system can be established, but the expression efficiency is low
Solution Approach 1:
The patent changes the parameters of the expression system by optimizing the promoter and signal peptide sequences. Multiple promoters (including alkaline protease promoter, neutral protease promoter, and others) and signal peptides are tested and combined to create an optimized expression system that significantly increases pullulanase expression efficiency and enzyme activity.
Solution Approach 2:
The patent creates a composite expression system by combining multiple promoters and signal peptides into a single optimized expression vector. This composite approach integrates the strengths of different regulatory elements to achieve high-level expression of pullulanase, resolving the contradiction between establishing the expression system and achieving high expression efficiency.
3Ease of manufacture
If plasmid-based expression is used in Bacillus subtilis, then protein secretion is enabled, but the plasmid loses stability during passage
Solution Approach 1:
The patent extracts the plasmid-based expression system and replaces it with a chromosomal integration approach. By deleting the protease genes and integrating the pullulanase expression cassette into the bacterial chromosome, the system eliminates plasmid instability while maintaining protein secretion capability, thereby resolving the contradiction between ease of manufacture and stability.
4Reliability
If protease genes are deleted from Bacillus subtilis, then pullulanase stability is improved, but the strain complexity increases
Solution Approach 1:
The patent segments the protease gene deletion into distinct steps: first deleting the alkaline protease gene, then deleting the neutral protease gene. This segmented approach allows for systematic removal of harmful elements while maintaining trackability of the strain modifications, reducing the practical complexity despite the multiple deletions required.
Data Source
AI summary
The present invention relates to the field of genetic engineering, particularly to a method for efficiently expressing pullulanase in Bacillus subtilis and recombinant Bacillus subtilis. said method includes steps of constructing modified Bacillus subtilis strain with deletion of alkaline protease gene and neutral protease gene, constructing expression vector including an optimized combination of promoter and signal peptide and pullulanase gene, and transforming said modified Bacillus subtilis strain with by said expression vector. A series of combinations of promoter and signal peptide are optimized to obtain the combination for efficiently expressing pullulanase, provide an industrial application basis.