Alpha-isopropylmalate Synthase Mutations for Leucine Production
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Solution Overview
Problem
Current methods for producing ketoisocaproate and L-leucine through fermentation by Corynebacterium glutamicum strains face challenges in yield and productivity, particularly due to feedback inhibition of the α-isopropylmalate synthase enzyme, which reduces enzyme activity when exposed to high intracellular leucine concentrations.
Innovation Solution
A mutation at position 553 of the α-isopropylmalate synthase gene, replacing tyrosine with aspartic acid, reduces feedback inhibition, leading to increased production of ketoisocaproate and L-leucine by decreasing the enzyme's sensitivity to product inhibition, thereby enhancing yield and productivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the α-isopropylmalate synthase enzyme is used in wild-type form, then the feedback regulation mechanism is maintained for metabolic control, but the enzyme activity is reduced when exposed to high intracellular leucine concentrations, limiting production yield
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the α-isopropylmalate synthase enzyme at position 553, replacing tyrosine with aspartic acid. This specific parameter change in the enzyme's primary structure alters its feedback inhibition characteristics, reducing sensitivity to leucine while maintaining catalytic function. The mutation changes the enzyme's interaction with the feedback inhibitor without completely abolishing regulatory control.
2Productivity
If the enzyme sensitivity to product inhibition is high, then feedback control is effective for metabolic homeostasis, but the production yield and productivity are limited due to reduced enzyme activity at high leucine concentrations
Solution Approach 1:
The patent modifies the enzyme's sensitivity parameter by introducing a point mutation at position 553. This changes the inhibition constant (Ki) for leucine, effectively altering the concentration at which 50% inhibition occurs. The aspartic acid substitution reduces the enzyme's sensitivity to product inhibition, allowing higher productivity while maintaining some level of feedback control.
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 mutation results in a significant increase in the production of ketoisocaproate and L-leucine, with yields improved by up to 122% compared to the starting strain, as demonstrated in fermentation experiments, indicating reduced feedback inhibition and improved enzyme activity.
Implementation Method 1
The α-isopropylmalate synthase (IPMS, EC=2.3.3.13) catalyses the condensation of the acetyl group of acetyl-CoA with 3-methyl-2-oxobutanoate (2-oxoisovalerate, ketoisovalerate) for the formation of 3-carboxy-3-hydroxy-4-methylpentanoate (2-isopropylmalate)
Implementation Method 2
Fine chemicals, which include, in particular, amino acids, organic acids, vitamins, nucleosides and nucleotides, are used in human medicine, in the pharmaceuticals industry, in cosmetics, in the food industry and in animal feeding. A great number of these compounds are produced by fermentation of strains of coryneform bacteria
Data Source
Figure 1
AI summary
The invention relates to an isolated nucleotide sequence encoding an amino acid sequence that is at least ≥ 90%, ≥ 92%, ≥ 94%, ≥ 96%, ≥ 97%, ≥ 98%, ≥ 99% or 100%, preferably ≥ 97%, particularly preferably ≥ 98%, very particularly preferably ≥ 99%, and extremely preferably 0%, identical to the amino acid sequence of SEQ ID NO:2, wherein SEQ ID NO:2, at position 553, or at a corresponding position of the amino acid sequence, has a proteinogenic amino acid other than L-tyrosine, to a microorganism comprising the nucleotide sequence and also to a process for producing fine chemicals using this microorganism.