Bacillus ykf Operon Modification for Protein Expression
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Solution Overview
Problem
There is a need for new recombinant strains of Gram-positive bacterial cells that express increased levels of a protein of interest, as existing technologies have limitations in enhancing protein production in bacterial host cells.
Innovation Solution
Genetically altered Gram-positive microorganisms, such as Bacillus species, with modifications in the ykf operon, specifically altering amino acids at positions corresponding to 252 and 253, result in increased expression of a protein of interest, including enzymes like subtilisin, by modifying the activity of proteins encoded by the ykf operon.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If genetic engineering is used to improve protein production in microorganisms, then productivity is increased, but the complexity of the system increases
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid positions (252 and 253) in the YkfA protein sequence to alter its peptidase activity. This targeted parameter modification enables enhanced protein production through controlled changes in enzyme function rather than complex genetic restructuring, thereby improving productivity while limiting system complexity
Solution Approach 2:
The invention segments the approach to protein production enhancement by focusing on specific functional domains of the YkfA protein rather than modifying the entire genetic system. By targeting particular amino acid positions and their corresponding peptidase activities, the patent achieves productivity improvement through localized genetic modifications rather than system-wide complexity
2Productivity
If the activity of YkfA protein is increased to enhance protein expression, then productivity improves, but this may lead to unwanted side effects from excessive peptidase activity
Solution Approach 1:
The patent applies local quality by creating YkfA protein variants with modified peptidase activity specifically tailored for enhanced protein expression. By adjusting the catalytic properties of YkfA through amino acid substitutions at positions 252 and 253, the invention achieves localized optimization of protein expression capability while controlling peptidase activity to prevent harmful effects
Solution Approach 2:
The invention implements feedback control through the use of YkfA variants with modified peptidase activity that responds to cellular conditions. The altered enzyme activity provides a feedback mechanism that enhances protein expression while automatically regulating peptidase function to prevent excessive activity and associated harmful effects
Data Source
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AI summary
The present invention relates in general to bacterial cells having a genetic alteration that results in increased expression of a protein of interest and methods of making and using such cells. Aspects of the present invention include Gram positive microorganisms, such as Bacillus species, having a genetic alteration that modifies activity of a protein encoded by the ykf operon and results in enhanced expression of a protein of interest.