EPSP Synthase Mutations for Glyphosate Tolerance
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Solution Overview
Problem
Current glyphosate-tolerant crops face challenges in maintaining adequate metabolic flux and catalytic efficiency due to limitations in plant EPSP synthase enzymes, which are sensitive to glyphosate inhibition.
Innovation Solution
Development of plant EPSP synthase (EPSPS) polynucleotides encoding polypeptides with specific amino acid mutations, such as G102A and others, to create glyphosate-tolerant plants through recombinant DNA constructs and CRISPR/Cas-mediated systems, ensuring the enzymes remain catalytically efficient in the presence of glyphosate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native plant EPSPS enzymes are used, then catalytic efficiency and metabolic flux are maintained, but glyphosate inhibition occurs reducing herbicide tolerance
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the EPSPS enzyme structure (e.g., positions 103, 107, 207, 210, 361, 445) to alter the enzyme's interaction with glyphosate while preserving its catalytic function. These point mutations change the physical-chemical parameters of the enzyme-substrate-inhibitor complex, enabling tolerance without sacrificing productivity
Solution Approach 2:
The invention applies local quality by introducing specific mutations only at critical positions within the EPSPS active site and glyphosate binding region, while leaving the rest of the enzyme structure unchanged. This localized modification approach ensures that only the necessary regions are altered to achieve glyphosate tolerance, maintaining overall catalytic efficiency
2Object-affected harmful factors
If EPSPS mutations are introduced to achieve glyphosate tolerance, then herbicide resistance improves, but catalytic capacity may be compromised
Solution Approach 1:
The patent systematically changes amino acid parameters at specific positions to reduce glyphosate binding affinity while maintaining substrate binding and catalytic parameters. The mutations are selected to preserve essential catalytic residues and structural integrity, ensuring reliable enzyme function under glyphosate exposure
Solution Approach 2:
The invention converts the harmful effect of glyphosate binding into a beneficial tolerance trait by mutating residues that normally contribute to glyphosate inhibition. The mutations at positions such as 103I/107S and 445G transform the enzyme's vulnerability into resistance, while screening ensures catalytic capacity is maintained or improved
3Reliability
If multiple amino acid mutations are combined in EPSPS, then glyphosate tolerance is enhanced, but enzyme complexity and potential off-target effects increase
Solution Approach 1:
The patent applies local quality by limiting mutations to specific, functionally critical positions within the EPSPS structure that directly interact with glyphosate. By concentrating changes at key residues (e.g., 103, 107, 445) rather than distributing them throughout the enzyme, the complexity is minimized while maximizing tolerance effectiveness
Solution Approach 2:
The invention creates composite enzyme structures by combining multiple point mutations within a single EPSPS polypeptide chain. These composite mutations work synergistically to provide enhanced glyphosate tolerance, with each mutation contributing a specific functional improvement while maintaining overall enzyme stability
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 modified EPSPS enzymes in plants exhibit enhanced catalytic capacity and tolerance to glyphosate, allowing for effective metabolic flux and resistance to herbicide applications, thereby improving crop tolerance and weed control efficacy.
Implementation Method 1
A method is provided for generating a plant with a modified endogenous EPSPS gene, wherein a guide RNA and one or more CRISPR/Cas9 endonucleases are provided to the plant cell, wherein the guide RNA and CRISPR/Cas9 endonuclease generate a double strand break at the endogenous EPSPS gene
Implementation Method 2
EPSP (5-enolpyruvylshikimate-3-phosphate) synthase is an enzyme that catalyzes the conversion of phosphoenolpyruvate and 3-phosphoshikimate to phosphate and 5-enolpyruvylshikimate-3-phosphate (EPSP), and it participates in the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan
Implementation Method 3
Glyphosate, the top selling herbicide in the world, acts a competitive inhibitor for phosphoenolpyruvate
Data Source
AI summary
Compositions and methods comprising polynucleotides and polypeptides having EPSP (5-enolpyruvylshikimate-3-phosphate) synthase (EPSPS) activity are provided. In specific embodiments, the sequence has an improved property, such as, but not limited to, improved catalytic capacity in the presence of the inhibitor, glyphosate. Further provided are nucleic acid constructs, plants, plant cells, explants, seeds and grain having the EPSPS sequences. Various methods of employing the EPSPS sequences are provided. Such methods include methods for producing a glyphosate tolerant plant, plant cell, explant or seed and methods of controlling weeds in a field containing a crop employing the plants and/or seeds disclosed herein.