Aconitase Gene Modification for L-Lysine Fermentation Yield
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Solution Overview
Problem
Current methods for producing L-lysine by fermentation do not effectively utilize the regulatory elements of the aconitase gene, leading to suboptimal yields and potential bacterial growth issues when attempting to modify or knockout the aconitase gene in L-lysine-producing bacteria like Escherichia coli and Corynebacterium.
Innovation Solution
Modifying the aconitase gene and its regulatory elements in bacteria by techniques such as site-directed mutation and homologous recombination to reduce but not eliminate aconitase enzyme activity and expression, specifically through substitutions, deletions, and additions of nucleotides in the nucleotide sequences of the aconitase gene and its promoter, allowing for increased L-lysine fermentation yields without interfering with existing modification sites in chromosomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the aconitase gene (acnA) is knocked out to eliminate aconitase activity, then L-lysine production yield is improved, but bacterial growth becomes slow and practical use becomes difficult
Solution Approach 1:
Instead of completely eliminating aconitase activity through gene knockout, the patent applies partial action by reducing aconitase activity to a controlled level (5-50% of wild type). This is achieved through specific mutations in the aconitase gene (acnA) such as acnA(D135N), acnA(D135A), or acnA(E136K), which partially inhibit enzyme activity while preserving enough function for bacterial growth. This partial reduction resolves the contradiction by maintaining both improved L-lysine yield and acceptable bacterial growth.
Solution Approach 2:
The patent changes the functional parameter of aconitase from complete activity to reduced activity by introducing specific point mutations in the enzyme's active site or regulatory regions. These parameter changes in enzyme activity allow the system to achieve optimal L-lysine production while maintaining bacterial viability, resolving the trade-off between yield improvement and growth maintenance.
2Productivity
If the aconitase gene is completely eliminated by knockout, then L-lysine yield increases, but the method becomes unsuitable for practical industrial production
Solution Approach 1:
The patent applies partial action by reducing aconitase activity to a controlled level (5-50% of wild type) rather than complete elimination. This is achieved through specific mutations in the aconitase gene (acnA) such as acnA(D135N), acnA(D135A), or acnA(E136K), which partially inhibit enzyme activity while preserving enough function for bacterial growth. This partial reduction resolves the contradiction by maintaining both improved L-lysine yield and acceptable bacterial growth.
3Productivity
If existing modification sites in bacterial chromosomes are interfered with to increase L-lysine yield, then production efficiency improves, but the modifications cannot be applied to a great variety of existing bacterial strains
Solution Approach 1:
The patent applies local quality by making targeted modifications specifically at the aconitase gene (acnA) and its regulatory elements, rather than requiring widespread changes across the entire bacterial genome. The specific mutations (acnA(D135N), acnA(D135A), acnA(E136K)) are localized to the enzyme's active site or regulatory regions, allowing the modification to be applied independently to various existing strains without interfering with other chromosomal modification sites used in different strains.
Solution Approach 2:
The patent achieves universality by developing a modification approach that can be applied across multiple bacterial species (Escherichia coli, Corynebacterium glutamicum, Corynebacterium pekinense) and is compatible with existing strain modifications. The regulatory element modifications (promoter regions, operon structures) are designed to work with various chromosomal backgrounds, enabling the same aconitase reduction strategy to be implemented in diverse existing L-lysine producing strains without requiring strain-specific redesign.
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
This approach results in a significant increase in L-lysine yield, maintaining bacterial viability and cost-effectiveness, and can be applied to both high and low-yielding bacteria strains, making the process more efficient and practical for industrial production.
Implementation Method 1
An aconitase is an enzyme of the tricarboxylic acid cycle, which catalyzes two-step chemical reactions: the transformation of citric acid to aconitic acid and the transformation of aconitic acid of isocitric acid
Implementation Method 2
producing L-lysine by fermentation with the bacterium obtained by the modification of step (1)
Data Source
AI summary
Provided is a method of producing L-lysine by fermentation, which comprises the steps of modifying an aconitase gene and/or regulatory element thereof in a chromosome of a bacterium so that the activity and/or the expression amount of the aconitase of the bacterium are reduced but not eliminated; and producing L-lysine by the fermentation of the modified bacterium. In addition, also provided are methods and uses derived from the method as well as bacteria used in these methods and uses.