Mutant EPSPS Protein Glyphosate Tolerance via Targeted Amino Acid Substitution
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Solution Overview
Problem
Existing methods for creating herbicide-tolerant plants using mutated EPSPS genes often result in enzymes with lower catalytic efficiency and require high overproduction or transgenic approaches, leading to complications such as low expression and abnormal growth.
Innovation Solution
Employing recombinagenic oligonucleobases to introduce targeted mutations in the EPSPS gene, specifically at amino acid positions Thr179 and Pro183, to produce a non-transgenic plant with a mutant EPSPS protein that maintains normal catalytic activity and confers resistance to glyphosate without the need for transgenic modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a mutated EPSPS gene is introduced to increase glyphosate tolerance, then herbicide resistance is improved, but catalytic efficiency decreases due to higher Km for PEP
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the EPSPS protein sequence (such as positions 178 and 182 in Arabidopsis EPSPS) to alter the enzyme's kinetic parameters. These targeted mutations change the protein's structure to reduce glyphosate binding affinity while maintaining substrate binding capability, thereby improving herbicide tolerance without significantly compromising catalytic efficiency
Solution Approach 2:
The patent applies local quality by making specific localized mutations at particular amino acid positions in the EPSPS protein rather than global changes. The mutations are concentrated in regions that interact with glyphosate (such as the substrate binding pocket), leaving other functional regions intact to maintain normal catalytic activity and PEP binding
2Reliability
If high levels of overproduction of variant EPSPS enzyme are used to maintain normal catalytic activity, then glyphosate tolerance is improved, but device complexity and manufacturing difficulty increase
Solution Approach 1:
The patent uses parameter changes to optimize the balance between enzyme quantity and quality. By carefully selecting amino acid mutations that preserve catalytic efficiency, the patent reduces the need for high-level overexpression. The modified enzyme maintains sufficient activity at near-wild-type expression levels, simplifying the genetic transformation and regulatory requirements
3Reliability
If mutations are introduced to increase glyphosate resistance, then herbicide tolerance is improved, but plant growth and development become abnormal
Solution Approach 1:
The patent applies parameter changes by selecting specific amino acid substitutions that maintain the enzyme's ability to perform its essential metabolic function. The mutations are designed to specifically affect glyphosate binding while preserving substrate binding and catalytic activity, ensuring that the shikimate pathway continues to function normally for the synthesis of aromatic amino acids and other essential compounds
Solution Approach 2:
The patent converts the harmful effect of glyphosate binding into a beneficial selective advantage. By introducing mutations that reduce glyphosate affinity, the enzyme becomes resistant to the herbicide's inhibitory effect while maintaining its physiological function. The mutation that would normally be harmful (reducing enzyme-substrate interaction) is converted into a benefit (reducing enzyme-herbicide interaction) through precise positioning in the protein structure
Data Source
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AI summary
The present invention relates to the production of a non-transgenic plant resistant or tolerant to a herbicide of the phosphonomethylglycine family, e.g., glyphosate. The present invention also relates to the use of a recombinagenic oligonucleobase to make a desired mutation in the chromosomal or episomal sequences of a plant in the gene encoding for 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS). The mutated protein, which substantially maintains the catalytic activity of the wild-type protein, allows for increased resistance or tolerance of the plant to a herbicide of the phosphonomethylglycine family, and allows for the substantially normal growth or development of the plant, its organs, tissues or cells as compared to the wild-type plant irrespective of the presence or absence of the herbicide. Additionally the present invention relates to mutant E. coli cells that contain mutated EPSPS genes.