RasP Gene Expression for Protein Secretion in Bacillus subtilis
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Solution Overview
Problem
Current methods for improving protein production and secretion in Gram-positive bacterial cells, such as Bacillus subtilis, are limited by the degradation of essential proteins like PrsA, which hampers efficient protein secretion and industrial use.
Innovation Solution
Modifying Gram-positive bacterial cells to over-express the rasP gene, encoding the regulating anti-sigma factor protease (RasP), by substituting the native promoter with a more active one like the spoVG or aprE promoter, or modifying the 5'-untranslated region, to enhance protein production and secretion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the native promoter is used to express rasP gene, then the cell maintains normal physiological function, but protein production remains limited
Solution Approach 1:
The patent applies parameter changes by substituting the native promoter with a stronger, more active promoter (such as spoVG or aprE promoters) to drive higher expression levels of the rasP gene. This increases the production of RasP protease, which in turn enhances protein secretion efficiency and overall productivity of the bacterial cell factory system.
2Productivity
If PrsA is overexpressed to improve protein folding, then secretion efficiency increases, but PrsA is degraded by WprA and other proteases, reducing its suitability for industrial use
Solution Approach 1:
The patent uses RasP protease as an intermediary to indirectly enhance protein secretion. Instead of directly overexpressing PrsA (which gets degraded), the system overexpresses RasP to regulate and optimize the secretion machinery, thereby improving protein export without the stability issues associated with PrsA overexpression.
3Productivity
If signal peptide composition is optimized to improve secretion, then protein export increases, but the complexity of optimization targets increases
Solution Approach 1:
The patent extracts and addresses the root cause limiting protein secretion by focusing on the rasP gene expression. Rather than optimizing multiple parameters simultaneously (signal peptides, SRP, translocation machinery, signal peptidases), the invention identifies and enhances the key regulatory factor (RasP) that controls the secretion efficiency, thereby simplifying the optimization process.
Data Source
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AI summary
The present disclosure is generally related to modified Gram-positive bacterial cells producing increased amounts of one or more protein(s) of interest. Certain embodiments of the instant disclosure are therefore directed to modified Gram-positive bacterial cells expressing an increased amount of a POI relative to unmodified (i.e., parental) Gram-positive bacterial cells, wherein the modified (i.e., daughter) bacterial cells comprise a modification which increases rasP gene expression. In certain other embodiments, the disclosure pertains to methods of modifying bacterial cells such that the modified (daughter) cells produce an increased level of a protein of interest. In other embodiments, the disclosure pertains to a protein of interest produced by fermenting a modified bacterial cell of the instant disclosure. Certain other embodiments of the disclosure are directed to one or more proteinaceous compositions comprising one or more protein(s) of interest thus made.