Protease Pro Region Mutations for Enhanced Bacterial Secretion
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Solution Overview
Problem
Current methods for producing proteases in bacterial host cells, such as Bacillus species, face inefficiencies in extracellular protein secretion, limiting the production of enzymes for industrial applications like cleaning and textile processing.
Innovation Solution
The use of modified polynucleotides encoding proteases with mutations in the pro region, specifically combinations of amino acid substitutions at positions 6, 30, and 32, to enhance the production of mature proteases in bacterial host cells, including Bacillus subtilis, through expression cassettes and vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type protease secretion system is used, then natural protein secretion occurs, but production efficiency of mature protease is limited
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions 6, 30, and 32 of the pro region (e.g., E6R, A32K, E30G) to optimize the protease precursor structure. These parameter modifications enhance the efficiency of proteolytic processing and mature protease secretion, directly resolving the contradiction between limited natural secretion and desired high productivity.
Solution Approach 2:
The invention applies local quality by making targeted amino acid substitutions at specific positions (6, 30, and 32) within the pro region rather than modifying the entire protein structure. This localized modification approach optimizes the processing efficiency and secretion capability of the protease precursor while maintaining the overall protein function, thereby improving mature protease production without compromising secretion reliability.
2Productivity
If pro region is modified with amino acid substitutions, then processing and secretion of mature protease is enhanced, but protein structure complexity increases
Solution Approach 1:
The patent modifies only specific positions (6, 30, and 32) within the pro region through targeted amino acid substitutions, rather than redesigning the entire pro region structure. This localized approach enhances mature protease secretion while minimizing structural complexity increases, as the modifications are confined to three specific residues out of the entire pro region sequence.
Solution Approach 2:
The invention changes specific parameters (amino acid identities at positions 6, 30, and 32) of the pro region to optimize processing and secretion. By modifying only these discrete parameters rather than the overall pro region architecture, the patent achieves enhanced productivity with controlled structural complexity.
Data Source
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AI summary
The present invention provides methods and compositions for the production of mature proteases in bacterial host cells. The compositions include modified polynucleotides that encode modified proteases, which have at least one mutation in the pro region; the modified serine proteases encoded by the modified polynucleotides; expression cassettes, DNA constructs, and vectors comprising the modified polynucleotides that encode the modified proteases; and the bacterial host cells transformed with the vectors of the invention. The methods include methods for enhancing the production of mature proteases in bacterial host cells e.g. Bacillus sp. host cells. The produced proteases find use in the industrial production of enzymes, suitable for use in various industries, including but not limited to the cleaning, animal feed and textile processing industry.