Mutant Isopropyl Malate Synthase Resolving Feedback Inhibition
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Solution Overview
Problem
Current industrial methods for producing L-leucine, such as fermentation, face issues like nutritional deficiencies, slow growth, unstable fermentation performance, and low yield due to feedback inhibition by L-leucine in wild-type isopropyl malate synthases, leading to inefficient production processes.
Innovation Solution
A genetically engineered bacterium is developed by introducing a mutant isopropyl malate synthase (LEUAM) and other genes to relieve feedback inhibition, allowing for overexpression and efficient production of L-leucine, using CRISPR/Cas9 gene editing and specific genetic modifications to enhance fermentation performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type isopropyl malate synthase is used in L-leucine production, then the enzyme provides basic catalytic function, but it is subject to feedback inhibition by L-leucine resulting in low production yield
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of isopropyl malate synthase through site-directed mutagenesis. Specific amino acid residues are changed to alter the enzyme's properties, particularly its sensitivity to L-leucine feedback inhibition. This allows the enzyme to maintain catalytic activity while producing high concentrations of L-leucine, thereby resolving the contradiction between productivity and reliability.
2Productivity
If existing L-leucine production strains are used, then they can produce L-leucine through fermentation, but they exhibit slow growth, nutritional deficiency, and unstable fermentation performance
Solution Approach 1:
The patent modifies physiological parameters of the host strain by introducing mutated isopropyl malate synthase genes and optimizing cultural conditions. The mutated enzyme maintains activity at higher L-leucine concentrations, enabling stable fermentation performance. Additionally, the patent optimizes nutritional parameters including carbon source, nitrogen source, and mineral composition to support both rapid growth and stable L-leucine production.
3Productivity
If conventional fermentation methods are used for L-leucine production, then the process can proceed with standard conditions, but it results in long fermentation period and low conversion rate
Solution Approach 1:
The patent changes kinetic parameters by introducing mutated isopropyl malate synthase with improved catalytic properties. The mutated enzyme maintains higher activity levels throughout the fermentation process, enabling faster L-leucine accumulation. Combined with optimized cultural parameters such as temperature, pH, and aeration, this reduces the fermentation period while increasing conversion rate and yield.
Data Source
AI summary
The invention relates to a 2-isopropyl malate synthase, a genetically engineered bacterium for producing L-leucine and application thereof and belongs to the field of metabolic engineering. The genetically engineered bacterium is obtained by overexpressing an isopropyl malate synthase coding gene leuAM for relieving feedback inhibition by L-leucine, an acetohydroxy acid synthase coding gene ilvBNM for relieving feedback inhibition by L-isoleucine, a 3-isopropyl malate dehydrogenase coding gene leuB and a 3-isopropyl malate dehydratase coding gene leuCD in host cells. The genetically engineered bacterium for producing the L-leucine is free from nutritional deficiency, rapid in growth, short in fermentation period, high in yield and high in conversion rate.