Bacillus subtilis Recombinant Protein Expression via PonA OppA SppA Co-expression
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Solution Overview
Problem
Current strategies for enhancing recombinant protein expression in Bacillus subtilis are limited by incomplete annotation of protein synthesis and secretion pathways, leading to one-sided modification approaches and restricted potential for B. subtilis as an expression host.
Innovation Solution
The method involves overexpressing class A penicillin-binding proteins PonA, truncated forms of PonA, the ABC pathway transporter OppA, or the signal peptide peptidase SppA from Bacillus species in B. subtilis to increase recombinant protein expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current modification strategies (overexpressing Sec pathway factors, knocking out proteases, increasing chaperone proteins) are used to enhance recombinant protein expression in B. subtilis, then protein expression level is improved, but the approach is limited by incomplete annotation of protein synthesis and secretion pathways, resulting in one-sided modifications and restricted potential
Solution Approach 1:
The patent segments the protein synthesis and secretion process into multiple independent pathways (Sec pathway, Tat pathway, ABC transporter system, signal peptide peptidase system) and identifies key regulatory factors in each pathway. By targeting multiple segmented pathways simultaneously with different enhancer factors, the patent overcomes the limitation of one-sided modifications and achieves comprehensive enhancement of protein expression capacity.
Solution Approach 2:
The patent employs multiple enhancer factors (PonA, OppA, SppA) that serve different functions but collectively enhance protein expression through multiple pathways. PonA enhances cell wall synthesis and protein secretion, OppA facilitates amino acid transport and protein folding, and SppA processes signal peptides. This multi-functional approach makes the modification strategy more versatile and adaptable to different protein expression needs.
2Productivity
If multiple pathways are targeted simultaneously with different enhancer factors, then comprehensive enhancement of protein expression is achieved, but system complexity increases
Solution Approach 1:
The patent merges multiple enhancer factor expression systems into a single integrated genetic construct. The plasmid contains multiple operons (ponA, oppA, sppA) that are co-expressed under the control of a single promoter (P43) and ribosome binding sites. This merging approach achieves comprehensive pathway enhancement while maintaining manageable system complexity through unified genetic control.
Solution Approach 2:
The patent uses a plasmid vector as an intermediary carrier to deliver and coordinate the expression of multiple enhancer factors. The plasmid serves as a mediator that organizes the complex system of multiple genes (ponA, oppA, sppA) into a single transferable and expressible unit, simplifying the introduction and regulation of multiple pathways simultaneously.
Data Source
AI summary
Disclosed is a method for increasing expression level of recombinant proteins in Bacillus subtilis through co-expressing Bacillus-derived enhancer factor, belonging to the technical field of enzyme engineering. According to the present disclosure, genes encoding recombinant proteins are respectively co-expressed with Bacillus-derived PonA, PonA truncated forms, OppA or SppA, so as to improve the expression level of the recombinant proteins. The results show that the method provided by the present disclosure can increase the expression level of ultra-high temperature amylase, medium temperature amylase or sucrose isomerase at the shake flask level to 3.96 times, 1.49 times and 2.26 times. The expression level of ultra-high temperature amylase can be increased by 26% in high-density fermentation of a 3L bioreactor. The B. subtilis provides a new developable target as an recombinant protein expression host, and provides technical support for the optimization and modification of the enhancer factors.