Bacillus prsA Cassette Integration at amyL Locus
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Solution Overview
Problem
Current methods for optimizing protein production in Bacillus host cells, such as Bacillus licheniformis, face challenges in achieving consistent and enhanced yields of proteins of interest, particularly due to unpredictable expression and secretion of heterologous proteins.
Innovation Solution
The introduction of a 2nd copy of a prsA gene expression cassette integrated at a defined genomic locus, specifically the amyL locus, in Bacillus licheniformis cells, which includes a prsA gene promoter sequence with at least 85% identity to SEQ ID NO: 29 and a prsA gene coding sequence with at least 80% identity to SEQ ID NO: 30, enhances protein production compared to integration at the catH locus.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a prsA gene expression cassette is integrated at the catH locus, then protein production is achieved, but productivity is suboptimal compared to integration at the amyL locus
Solution Approach 1:
The patent applies local quality by creating locus-specific integration strategies where the prsA gene cassette is integrated at particular genomic locations (amyL locus preferred over catH locus) to achieve different expression levels. This principle resolves the contradiction by making the integration location a quality attribute that determines productivity, thereby achieving both specificity and high yield simultaneously.
2Productivity
If heterologous protein expression is optimized, then protein yield increases, but expression becomes unpredictable
Solution Approach 1:
The patent applies parameter changes by systematically varying multiple parameters including integration locus (amyL vs catH), prsA gene copy number (1st copy vs 2nd copy), and promoter strength to achieve predictable high-yield expression. This resolves the contradiction by transforming the unpredictable expression into a controllable process where specific parameter combinations reliably produce high yields.
Solution Approach 2:
The patent employs feedback by using the amyL locus as a validated integration target that has been shown to provide consistent high-level expression. By establishing this locus-specific integration protocol based on prior experimental feedback, the system achieves both high productivity and reliable predictable expression outcomes.
3Productivity
If protein secretion is enhanced, then productivity increases, but complex optimization is required
Solution Approach 1:
The patent extracts the secretion enhancement function by utilizing the native amyL locus, which inherently possesses strong secretion capabilities. By integrating the prsA cassette at this pre-validated locus, the system leverages the locus's intrinsic secretion properties without requiring additional complex optimization elements, thereby achieving high productivity with reduced complexity.
Data Source
AI summary
Certain embodiments of the disclosure are related to recombinant Bacillus strains comprising enhanced protein productivity phenotypes, compositions and methods for constructing such recombinant Bacillus cells, and the like. The recombinant Bacillus strains described herein are particularly useful for the enhanced production of proteins of interest when grown/cultivated/fermented under suitable conditions.