Corynebacterium glutamicum aspB Promoter Base Substitution
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Solution Overview
Problem
Current methods for enhancing lysine production in Coryneform bacteria do not effectively improve the expression rate of the aspartate aminotransferase gene, which is crucial for lysine biosynthesis, as the promoter of the aspB gene has not been adequately optimized.
Innovation Solution
Development of a nucleic acid molecule with enhanced promoter activity derived from Corynebacterium glutamicum, specifically designed to increase the expression of the aspB gene by introducing modified promoter sequences through base substitution, leading to higher aspartate aminotransferase activity and improved lysine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the promoter of the aspB gene is not optimized, then the expression rate of aspartate aminotransferase remains low, but the promoter structure remains simple and unmodified
Solution Approach 1:
The promoter sequence of the aspB gene was modified by changing specific nucleotide bases (parameter change at molecular level) to create enhanced promoter variants (aspBP1, aspBP2, aspBP3) with higher transcriptional activity. This involved substituting bases at positions -35 and -10 regions of the promoter to match consensus sequences, thereby increasing aspartate aminotransferase expression and lysine production without fundamentally changing the promoter's overall structure
Solution Approach 2:
Specific regions of the promoter were selectively optimized rather than the entire promoter sequence. The -35 box (TTGACA) and -10 box (TATAAT) regions were targeted for base substitution to improve RNA polymerase binding affinity locally, while the rest of the promoter structure remained unchanged. This local optimization achieved enhanced expression with minimal structural modification
2Reliability
If base substitution is introduced to enhance promoter activity, then aspartate aminotransferase activity increases 2.1 to 2.6-fold, but the genetic modification process becomes more complex
Solution Approach 1:
Specific nucleotide parameters at critical positions in the promoter were changed (base substitution) to enhance promoter strength. The modifications were made at positions -35 and -10 relative to the transcription start site, changing the DNA sequence parameters to match consensus promoter sequences, which reliably increased aspartate aminotransferase activity by 2.1 to 2.6-fold
Solution Approach 2:
The enhanced promoter sequences (aspBP1, aspBP2, aspBP3) were created as modified copies of the wild-type aspB promoter. These promoter copies contained the optimized base sequences that were then introduced into the aspB gene locus through homologous recombination, replacing the original promoter while maintaining the overall gene structure
Data Source
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AI summary
The present invention provides a nucleic acidmolecule having enhanced promoter activity, which is operably linked to a gene encoding aspartate aminotransferase and derived from Corynebacterium glutamicum, a vector comprising the nucleic acid molecule, a transformant transformed with the vector, and a method for producing L-lysine using the transformant.