Bacillus subtilis aprX Gene Deletion for Protease Control
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Solution Overview
Problem
Microorganisms used for industrial protein production face challenges due to proteolytic enzyme activity, which degrades desired proteins, reducing yield and efficiency, particularly in Bacillus subtilis strains where existing gene deletions do not fully prevent proteolytic enzyme activity.
Innovation Solution
A recombinant microorganism is created by deleting or knocking out the Bacillus subtilis aprX gene, which encodes an intracellular serine protease, to prevent proteolytic enzyme activity and enhance protein production by using a method such as SOE-PCR for gene deletion and introducing a drug resistance marker gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If multiple protease genes (aprE, nprE, etc.) are deleted to prevent protein degradation, then proteolytic enzyme activity is reduced, but proteolytic enzyme activity remains detectable and protein degradation continues
Solution Approach 1:
The invention extracts and removes the specific aprX gene responsible for intracellular serine protease activity from the Bacillus subtilis genome. By targeting and eliminating this specific gene through homologous recombination, the patent removes the source of harmful proteolytic activity that was not eliminated by previous deletions of extracellular protease genes, thereby preventing protein degradation and improving production yield.
Solution Approach 2:
The invention performs preliminary genetic modification by deleting the aprX gene before protein production occurs. This preliminary action creates a host strain that lacks proteolytic enzyme activity, preventing degradation of the target protein during the production process. The gene deletion is accomplished through construction of a deletion cassette and homologous recombination, establishing a stable strain with improved properties before scaling up production.
2Reliability
If the aprX gene is deleted to eliminate intracellular serine protease activity, then protein degradation is prevented, but additional genetic manipulation steps are required
Solution Approach 1:
The invention uses a deletion cassette as an intermediary tool to achieve gene deletion. The cassette contains antibiotic resistance genes (spectinomycin and chloramphenicol resistance) that serve as selectable markers. This intermediary construct facilitates the homologous recombination process by providing a mechanism to select for successful transformants and verify gene deletion, thereby simplifying the overall genetic manipulation process despite the multiple steps involved.
3Productivity
If existing protease gene deletions are used as host strains, then some proteolytic activity is reduced, but residual protease activity still degrades desired proteins
Solution Approach 1:
The invention applies local quality modification by specifically targeting the intracellular compartment where the aprX gene operates. While previous deletions addressed extracellular proteases, this invention focuses on the intracellular serine protease activity that processes and degrades target proteins during secretion. By locally modifying the proteolytic environment within the cell, the patent eliminates the specific harmful activity that persists despite extracellular protease deletions.
Data Source
AI summary
A host microorganism capable of increasing productivity of a protein or polypeptide, a recombinant microorganism obtained by introducing a gene encoding a protein or polypeptide into the host microorganism, and a method for producing a protein or polypeptide using the recombinant microorganism are provided.Also provided is a recombinant microorganism obtained by introducing into a host microorganism a gene encoding a heterologous protein or polypeptide, wherein in said host microorganism the Bacillus subtilis aprX gene or a gene corresponding to the aprX gene has been deleted or knocked out, and a method for producing a protein or polypeptide using the recombinant microorganism.