Amino Acid Attenuator Modification for Fermentation Yield
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Solution Overview
Problem
Current microbial fermentation methods face challenges in efficiently producing amino acids like isoleucine, valine, tryptophan, histidine, phenylalanine, and others due to feedback repression mechanisms that inhibit gene expression and biosynthesis when the amino acids accumulate, leading to suboptimal yields.
Innovation Solution
Modifying the amino acid attenuator by removing the gene coding for a leader peptide and an anterior reverse complementary palindromic sequence in the terminator stem-loop structure, while maintaining a posterior reverse complementary palindromic sequence, to relieve attenuation regulation and enhance gene expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the amino acid accumulates in cells, then the concentration of amino acid increases, but feedback repression occurs which inhibits gene expression and biosynthesis
Solution Approach 1:
The patent removes the attenuator sequence from the amino acid operon, extracting the harmful feedback repression element. This deletion eliminates the ability of the attenuator to form termination structures that would otherwise block RNA polymerase, thereby preventing feedback repression and maintaining high gene expression levels even when amino acid concentration is high.
Solution Approach 2:
The patent inverts the normal feedback repression mechanism by deleting the attenuator entirely. Instead of modulating expression in response to amino acid levels, the system maintains constitutive high expression regardless of amino acid concentration, effectively reversing the regulatory logic from feedback inhibition to feedback insensitivity.
2Adaptability or versatility
If the attenuator regulation mechanism is intact, then the gene expression can be regulated, but the biosynthesis of amino acid is inhibited when amino acid concentration is high
Solution Approach 1:
The attenuator sequence is completely removed from the operon structure, eliminating the regulatory mechanism that causes productivity loss. This extraction of the harmful element allows the system to maintain high amino acid yield without the constraint of feedback-mediated transcription termination.
3Productivity
If the leader peptide and anterior reverse complementary palindromic sequence are removed, then the attenuation regulation is relieved and gene expression increases, but the structural complexity of the operon changes
Solution Approach 1:
The patent extracts and removes the leader peptide coding sequence and the anterior reverse complementary palindromic sequences that form the attenuator structure. This deletion simplifies the operon structure by eliminating complex regulatory elements while achieving the goal of increased gene expression through relief of transcription attenuation.
Data Source
AI summary
The present invention discloses a method for modifying an amino acid attenuator, a class of amino acid attenuator mutants, engineered bacteria created on the basis of the amino acid attenuator mutants, and use of the engineered bacteria. The present invention protects a method for relieving the attenuation regulation of an amino acid operon gene, which is modification of the amino acid operon gene by: removing a gene coding for a leader peptide and an anterior reverse complementary palindromic sequence in the terminator stem-loop structure, and maintaining a posterior reverse complementary palindromic sequence in the terminator. The amino acid operon particularly can be histidine operon, tryptophan operon, phenylalanine operon, alanine operon, threonine operon and etc. The present invention can be used for the production of amino acids and derivatives thereof in fermentation by bacteria, providing a novel method for improving the production of amino acids in fermentation.