malR-Knockout Bacillus licheniformis for Bacitracin Yield
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Solution Overview
Problem
Current methods for increasing Bacitracin yield in Bacillus licheniformis focus on amino acid supply rather than genetic modification, with limited understanding of which genes affect yield and effective genetic modification techniques.
Innovation Solution
A method for constructing a malR-knockout Bacillus licheniformis strain by PCR amplification, restriction enzyme digestion, and plasmid transformation to knockout the malR gene, a carbon metabolism transcription factor, resulting in a strain with increased Bacitracin production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If genetic modification methods are used to increase Bacitracin yield, then productivity is improved, but device complexity and manufacturing difficulty increase due to limited understanding of effective genetic modification techniques
Solution Approach 1:
The invention extracts and removes the malR gene from the Bacillus licheniformis genome through targeted knockout. By specifically eliminating this single transcriptional regulator gene rather than attempting complex multi-gene modifications, the method simplifies the genetic engineering process while achieving significant Bacitracin yield improvement of over 23%.
Solution Approach 2:
The invention changes the genetic parameter by knocking out the malR gene, which regulates carbon metabolism. This parameter change in the bacterial genome directly leads to improved Bacitracin production, demonstrating that modifying specific genetic parameters can enhance antibiotic yield without requiring complex modification systems.
2Productivity
If transcriptional regulators are modified to increase Bacitracin yield, then productivity is improved, but manufacturing precision is worsened due to lack of knowledge on which genes to target
Solution Approach 1:
The invention identifies and extracts the specific malR gene as the target for knockout. By focusing on this single transcriptional regulator rather than attempting to modify multiple unknown genes, the method achieves both high productivity improvement and precise genetic modification, eliminating the uncertainty about which genes to target.
3Productivity
If amino acid supply is increased to boost Bacitracin production, then productivity is improved, but loss of substance increases due to resource allocation to precursor synthesis
Solution Approach 1:
The invention converts the harmful effect of amino acid depletion into a benefit by knocking out the malR gene, which regulates carbon metabolism. This genetic modification redirects metabolic flux toward Bacitracin synthesis, improving yield without requiring additional amino acid supplementation and actually reducing the loss of precursor substances.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The malR-knockout strain achieves a yield increase of over 23% in Bacitracin compared to the wild-type strain, demonstrating the effectiveness of genetic modification in enhancing antibiotic production.
Implementation Method 1
using genomic DNA of Bacillus licheniformis DW2 as a template, obtaining an upstream homology arm of the malR gene and a downstream homology arm of the malR gene by PCR (polymerase chain reaction) amplification
Implementation Method 2
performing double digests of the target gene segment by XbaI and BamHI restriction enzymes to obtain a digested gene segment
Implementation Method 3
ligating the digested gene segment obtained in step (3) and the linear plasmid segment obtained in step (4) by DNA ligase to obtain a knockout plasmid T2(2)-ΔmalR
Implementation Method 4
transforming the knockout plasmid T2(2)-ΔmalR into Bacillus licheniformis DW2, and screening to obtain a positive transformant using kanacillin as a screening marker
Implementation Method 5
after transferring and culturing the positive transformant for several times at 45° C., performing colony PCR to obtain positive single crossover binder strains
Data Source
AI summary
Provided are malR-knockout Bacillus licheniformis strain, a construction method and a use. Using a genetic engineering method, gene, namely, the malR gene, which is in charge of conducting the transcription of the carbon metabolism transcription factor MalR, in the genome of Bacillus licheniformis DW2 is knocked out, thereby successfully obtaining Bacillus licheniformis DW2ΔmalR, from which the malR gene has been deleted. In comparison with Bacillus licheniformis DW2, the strain obtained by the construction can improve the Bacitracin yield in a fermentation broth during Bacitracin fermentation by at least 23%.