MetA Polypeptide Resistant to Methionine Feedback Inhibition
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Solution Overview
Problem
Current methods for producing O-succinylhomoserine face challenges due to feedback inhibition by methionine, which limits enzyme stability and productivity in microorganisms, as wild-type homoserine O-succinyltransferase has low stability and introducing mutations for feedback inhibition release worsens instability.
Innovation Solution
A novel isolated polypeptide with homoserine O-succinyltransferase activity and resistance to methionine feedback inhibition is developed, encoded by a specific polynucleotide sequence, and expressed in microorganisms like Escherichia sp. to produce O-succinylhomoserine with enhanced stability and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type homoserine O-succinyltransferase (metA) is used for O-succinylhomoserine production, then the enzyme can catalyze the reaction, but the enzyme stability is low and feedback inhibition by methionine limits productivity
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the homoserine O-succinyltransferase enzyme through site-directed mutagenesis. Specific amino acid residues are changed to alter the enzyme's properties, achieving both resistance to methionine feedback inhibition and improved thermal stability, thereby resolving the contradiction between productivity and reliability
Solution Approach 2:
The patent creates a modified copy of the wild-type metA gene with specific mutations introduced. This copied and modified enzyme retains the catalytic function while gaining resistance to feedback inhibition and improved stability, allowing high-yield production without sacrificing enzyme reliability
2Productivity
If mutations are introduced to release feedback inhibition of metA, then methionine feedback inhibition is reduced, but enzyme stability is aggravated and worsens
Solution Approach 1:
The patent simultaneously optimizes multiple parameters of the enzyme through rational design and site-directed mutagenesis. By carefully selecting specific amino acid substitutions, the enzyme achieves both feedback inhibition release and enhanced stability, turning the trade-off into a dual benefit
3Ease of operation
If L-methionine is added to the medium for strain cultivation, then the strain can grow, but homoserine O-succinyltransferase activity is inhibited by feedback inhibition
Solution Approach 1:
The patent introduces a modified metA gene that creates an enzyme copy resistant to methionine feedback inhibition. This allows the microorganism to grow in medium containing L-methionine while the modified enzyme continues to produce O-succinylhomoserine at high levels without being inhibited by the methionine present in the medium
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 microorganism expressing the novel polypeptide achieves high-yield production of O-succinylhomoserine, overcoming the limitations of feedback inhibition and enzyme instability, thus effectively utilizing O-succinylhomoserine as a precursor for methionine production.
Implementation Method 1
O-succinylhomoserine is produced by homoserine O-succinyltransferase (MetA) which conjugates the succinyl group of succinyl-coA to homoserine
Data Source
AI summary
The present invention relates to a polypeptide which is resistant to feedback inhibition by methionine and has an activity of homoserine O-succinyltransferase, a microorganism for producing O-succinylhomoserine which expresses the polypeptide, and a method for producing O-succinylhomoserine using the same.