Silent sinR Mutations Boost Bacterial Protein Expression
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Solution Overview
Problem
There is a need to develop new recombinant bacterial strains that express increased levels of a protein of interest, as existing methods for protein production in bacterial host cells are limited in achieving enhanced expression levels.
Innovation Solution
The development of recombinant Gram-positive bacterial cells with genetic alterations, specifically a silent mutation in the sinR gene that reduces its expression, leading to increased production of a protein of interest, such as proteases like subtilisin, through methods involving vector transformation and homologous recombination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional bacterial strains are used for protein production, then the production process is simple and well-established, but the expression levels of the protein of interest are limited
Solution Approach 1:
The invention changes the nucleotide sequence parameters of the sinR gene to achieve reduced expression. Specifically, it introduces silent mutations (nucleotide substitutions that do not change the amino acid sequence) at positions 330, 333, or 336 of the sinR gene, which alter the mRNA structure or stability to reduce SinR protein levels, thereby increasing protein of interest expression without changing the encoded amino acid sequence.
Solution Approach 2:
The invention extracts or removes the harmful effect of the sinR gene by reducing its expression through silent mutations. By targeting specific nucleotide positions in sinR that do not affect the amino acid sequence but do affect gene expression levels, the patent effectively takes out the repressive effect of SinR on protein of interest production while maintaining the normal function of the SinR protein sequence.
2Productivity
If silent mutations are introduced in the sinR gene to reduce its expression, then protein production increases by up to 10%, but the genetic modification process becomes more complex
Solution Approach 1:
The invention changes specific nucleotide parameters at positions 330, 333, or 336 of the sinR gene to achieve the desired effect. These are precise parameter changes (single nucleotide substitutions) that are relatively simple to implement through standard molecular biology techniques such as site-directed mutagenesis, while achieving the goal of increased protein production.
Solution Approach 2:
The invention uses silent mutations that do not affect the amino acid sequence, effectively creating a disposable or temporary genetic modification approach. The nucleotide changes are minimal and can be easily introduced and propagated without affecting the overall protein function, making the genetic modification process more economical and simpler compared to more complex genetic engineering approaches.
Data Source
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AI summary
The present invention relates in general to bacterial cells having genetic alterations that result in increased expression of a protein of interest and methods of making and using such cells. Aspects of the present invention include altered Gram positive microorganismshaving one or more a genetic alterations that reduce the expression of a gene in the sin operon, thereby resulting in the enhanced expression of one or more proteins of interest.