MetA Mutant Engineering for Stable O-Succinyl Homoserine Yield
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Solution Overview
Problem
Existing methods for producing O-succinyl homoserine suffer from low yield and stability issues due to feedback inhibition by methionine, leading to insufficient production of O-succinyl homoserine in microorganisms, which is crucial for economical methionine production.
Innovation Solution
A mutated O-succinyl homoserine transferase polypeptide with specific amino acid substitutions at positions 313 and 176, derived from Corynebacterium glutamicum, is used to enhance the conversion activity of O-succinyl homoserine, overcoming feedback inhibition and improving stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If feedback inhibition of metA is removed to increase O-succinyl homoserine production, then production yield is improved, but enzymatic stability deteriorates
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the MetA enzyme structure (positions 176 and 313) to alter its regulatory properties. The mutations R176K/E313K or R176K/E313Q change the enzyme's feedback inhibition characteristics and stability parameters, enabling high production yield while maintaining enzymatic stability through precise structural modification rather than complete removal of regulation.
2Productivity
If MetA activity is increased to overcome feedback inhibition, then O-succinyl homoserine production is improved, but protein stability further deteriorates
Solution Approach 1:
The patent resolves this contradiction by making precise parameter changes to the enzyme structure through site-directed mutagenesis at positions 176 and 313. These specific amino acid substitutions optimize the balance between catalytic activity and protein stability, allowing the enzyme to maintain high activity for O-succinyl homoserine production while preserving structural integrity and stability.
3Quantity of substance
If conventional MetA is used in methionine-producing strains, then methionine synthesis is maintained, but O-succinyl homoserine accumulation is limited due to feedback inhibition
Solution Approach 1:
The patent applies parameter changes by introducing specific mutations (R176K/E313K or R176K/E313Q) that alter the feedback inhibition parameters of MetA. This enables the enzyme to maintain high catalytic activity even in the presence of methionine, allowing O-succinyl homoserine to accumulate to high concentrations while the strain continues to produce methionine effectively.
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 mutated polypeptide significantly increases O-succinyl homoserine production, making it suitable for high-yield production of methionine as an alternative to conventional chemical synthesis pathways.
Implementation Method 1
homoserine O-succinyl transferase (MetA), as an enzyme involved in the synthesis of O-succinyl homoserine, by binding a succinyl group of succinyl-CoA to homoserine
Data Source
AI summary
The present disclosure relates to an O-succinyl homoserine transferase mutant, a polynucleotide encoding the same, a microorganism including the mutant, and a method of producing O-succinyl homoserine using the microorganism.