Novel alkylhydroperoxidase ahpd family core domain variant, and method for producing l-glutamic acid by using same
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Solution Overview
Problem
Existing methods for producing L-glutamic acid through microbial fermentation face challenges in enhancing the productivity of proteins involved in its biosynthetic pathways, necessitating further research on enzymes, transcription factors, and transport proteins.
Innovation Solution
Introduction of an alkylhydroperoxidase AhpD family core domain variant with a specific amino acid substitution, such as tryptophan to arginine at position 231, to enhance the activity of alkylhydroperoxidase, which is then expressed in a transformant to improve L-glutamic acid production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type strains or conventional mutant strains are used for L-glutamic acid production, then the production process is relatively simple, but the productivity is insufficient
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the AhpD protein sequence. The core domain variant with substituted residues (e.g., F201L, F201S, W231R) changes the biochemical parameters of the enzyme, leading to improved metabolic efficiency and oxidative stress response, thereby increasing L-glutamic acid productivity without requiring complex multi-gene modifications
Solution Approach 2:
The patent applies local quality by focusing mutations on specific critical regions of the AhpD protein, particularly the core domain. Rather than attempting to modify the entire protein or multiple unrelated genes, the invention targets specific local regions (residues 180-250) that are crucial for enzyme function and stress response, achieving improved productivity through localized modifications
2Productivity
If multiple genes and proteins are modified to improve L-glutamic acid production, then productivity may increase, but the complexity of strain development and metabolic pathway optimization increases significantly
Solution Approach 1:
The patent applies the extraction principle by isolating and modifying only the essential AhpD core domain gene rather than attempting to modify multiple genes simultaneously. By extracting the critical functional domain and focusing optimization efforts solely on this single gene's amino acid sequence, the invention simplifies the strain development process while still achieving significant productivity improvements
Solution Approach 2:
The AhpD core domain variant serves multiple functions: it maintains the enzyme's primary role in peroxide detoxification while simultaneously improving oxidative stress response and enhancing metabolic flux toward L-glutamic acid production. This multi-functionality allows a single gene modification to address multiple limiting factors in the production pathway
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 variant enhances the transformant's ability to produce L-glutamic acid by increasing its response to oxidative stress, resulting in higher yields, potentially up to 100% or more compared to parent strains.
Implementation Method 1
The alkylhydroperoxidase AhpD family core domain plays a role in removing intracellular peroxides and other reactive oxygen species
Implementation Method 2
L-glutamic acid is a typical amino acid that is produced by microbial fermentation
Implementation Method 3
glucose mainly undergoes the glycolytic pathway, but a portion thereof is metabolized into two pyruvic acid molecules through the pentose phosphate pathway
Implementation Method 4
oxaloacetic acid and citric acid enter the citric acid cycle (TCA cycle) to form α-ketoglutaric acid
Data Source
AI summary
The present invention relates to a novel alkylhydroperoxidase AhpD family core domain variant, and a method of producing L-glutamic acid using the same. The alkylhydroperoxidase AhpD family core domain variant is obtained by substituting one or more amino acids in the amino acid sequence constituting the alkylhydroperoxidase AhpD family core domain to change the activity of the protein, and L-glutamic acid may be efficiently produced from a recombinant microorganism expressing the variant.