ATP-PRT Variant Reduces Histidine Feedback Inhibition
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current ATP-phosphoribosyltransferase variants expressed from the E. coli hisG gene are susceptible to feedback inhibition by histidine, limiting histidine production in microorganisms, and no known variant has effectively reduced this inhibition.
Innovation Solution
An ATP-phosphoribosyltransferase variant with specific amino acid substitutions, such as lysine for glutamic acid at position 271, and additional mutations at other positions, maintains enzyme activity even at high histidine concentrations, reducing feedback inhibition and increasing histidine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type ATP-PRT is used for histidine biosynthesis, then the enzyme maintains normal catalytic activity, but the enzyme is strongly feedback-inhibited by histidine, limiting histidine production
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of ATP-PRT through site-directed mutagenesis. Specifically, it substitutes amino acids at positions 232, 250, 252, 271, and 288 to alter the enzyme's sensitivity to histidine feedback inhibition while maintaining its catalytic function. The E271K mutation is particularly critical as it directly reduces feedback inhibition by histidine, enabling increased histidine production.
2Productivity
If ATP-PRT variants with reduced feedback inhibition are created, then histidine production increases, but the enzyme may lose catalytic activity or stability
Solution Approach 1:
The patent carefully selects and combines multiple amino acid substitutions to achieve reduced feedback inhibition while preserving catalytic activity. The mutations at positions 232, 250, 252, 271, and 288 are designed to modify the enzyme's interaction with histidine without disrupting the active site. Combinatorial mutations allow fine-tuning of the balance between feedback resistance and catalytic function.
Solution Approach 2:
The modified ATP-PRT variant serves the dual function of maintaining catalytic activity for histidine biosynthesis while simultaneously providing resistance to feedback inhibition. The enzyme essentially serves itself by having built-in resistance to the product it helps synthesize, eliminating the need for external regulatory mechanisms.
3Productivity
If multiple amino acid substitutions are introduced to reduce feedback inhibition, then histidine production increases by up to 92%, but the protein structure and function may be compromised
Solution Approach 1:
The patent introduces multiple specific amino acid substitutions at positions 232, 250, 252, 271, and 288 to reduce feedback inhibition. These targeted changes modify the enzyme's properties without causing random structural degradation, achieving both reduced inhibition and maintained stability through rational design.
Solution Approach 2:
The patent creates a composite mutant enzyme by combining multiple amino acid substitutions into a single functional protein. This composite approach allows the enzyme to integrate multiple functional improvements (reduced feedback inhibition at multiple sites) while maintaining overall structural integrity through coordinated mutations.
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 variant strain expressing these mutations shows increased histidine production by up to 92% compared to wild-type strains, maintaining enzyme activity across a range of histidine concentrations from 5 mM to 25 mM.
Implementation Method 1
ATP-phosphoribosyltransferase (which hereinafter may be referred to as ATP-PRT) catalyzes the reaction 1-(5-phospho-D-ribosyl)-ATP+diphosphate⇄ATP+5-phospho-alpha-D-ribose 1-diphosphate which is the first step of histidine biosynthesis
Data Source
AI summary
The present invention relates to an E. coli hisG-derived ATP-phosphoribosyltransferase variant having a reduced feedback inhibition by histidine and a strain expressing the same. The variant may maintain its activity even at a high histidine concentration, thus increasing histidine production.