Modified Bacillus Promoter Resolving Catabolite Repression
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Solution Overview
Problem
Current promoters for gene expression in microorganisms like Bacillus subtilis face challenges in enhancing productivity due to catabolite repression, which suppresses enzyme production when glucose is abundant, leading to reduced industrial production efficiency.
Innovation Solution
A modified promoter is developed by deleting or modifying cre-like sequences in the nucleotide sequences of alkaline cellulase gene promoters from Bacillus strains, such as KSM-64 and KSM-S237, to enhance gene transcription and productivity, using techniques like SOE-PCR for site-directed mutagenesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the original promoter with cre-like sequences is used, then the microorganism can respond to catabolite repression signals and regulate gene expression, but the gene transcription and productivity are suppressed when glucose is abundant
Solution Approach 1:
The invention extracts and removes the cre-like sequences (specific sequences represented by SEQ ID NO:1 and SEQ ID NO:2) from the promoter region of the alkaline cellulase gene. By taking out these specific regulatory elements that mediate catabolite repression, the promoter loses its ability to respond to glucose repression signals while gaining constitutive high-level expression capability, thereby resolving the contradiction between productivity and adaptability to repression signals
Solution Approach 2:
The invention changes the regulatory parameter of the promoter by deleting the cre-like sequences, transforming the promoter from a repressible type (responsive to catabolite signals) to a constitutive type (always active). This parameter change in the promoter structure enables continuous high-level gene transcription regardless of glucose presence, achieving high productivity while eliminating the adaptive response to repression signals
2Productivity
If cre-like sequences are present in the promoter, then the microorganism can regulate enzyme production in response to carbon source availability, but industrial production efficiency is reduced due to suppressed enzyme production when glucose is abundant
Solution Approach 1:
The invention converts the harmful effect of catabolite repression into a benefit by removing the cre-like sequences that mediate this repression. The original repression mechanism, which harmed industrial production by suppressing enzyme production during glucose availability, is eliminated. The resulting constitutive promoter transforms what was a harmful regulatory response into a beneficial continuous expression system that maintains high enzyme production efficiency regardless of carbon source conditions
Data Source
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AI summary
The present invention provides a modified promoter, an expression vector and a transformant each containing the promoter, and a method for producing a gene product of interest using the transformant. The invention provides a modified promoter, including a nucleotide sequence of a promoter derived from bacterium belonging to the genus Bacillus in which at least one nucleotide sequence selected from the following has been modified: a nucleotide sequence represented by SEQ ID NO: 1; a nucleotide sequence equivalent to the nucleotide sequence represented by SEQ ID NO: 1, except that one or a plurality of bases therein are substituted, deleted, added or inserted; and a nucleotide sequence having a sequence identity of 70% or more with respect to the nucleotide sequence represented by SEQ ID NO: 1.