Glyphosate-Resistant EPSP Synthase Variants
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Solution Overview
Problem
Current herbicides like glyphosate are toxic to both plant and bacterial cells due to their inhibition of the enzyme EPSP synthase, and existing resistance genes do not effectively predict or confer resistance across all sequences, necessitating the development of novel genes for herbicide resistance in plants and bacteria.
Innovation Solution
The introduction of novel nucleic acid molecules encoding glyphosate-resistant EPSP synthase proteins, such as the GRG-1 protein, which can be used to transform plants and bacteria, providing resistance to glyphosate toxicity by encoding proteins that tolerate higher concentrations of the herbicide.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing EPSP synthase sequences are used, then the enzyme can catalyze the conversion of PEP and 3-phosphoshikimic acid, but the enzyme is inhibited by glyphosate causing cell death
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the EPSP synthase enzyme through site-directed mutagenesis. Specific residues (such as Phe376, Phe378, and others in the glyphosate binding region) are mutated to create variants with altered binding characteristics that reduce glyphosate inhibition while maintaining catalytic activity. This directly changes the molecular parameters of the enzyme to achieve resistance.
Solution Approach 2:
The patent applies local quality by making targeted modifications only in the glyphosate binding region of the EPSP synthase enzyme, rather than changing the entire protein structure. The mutations are localized to specific residues that interact with glyphosate, allowing the enzyme to maintain its overall structure and function while gaining resistance properties in the specific region that binds the herbicide.
2Object-affected harmful factors
If bacterial EPSP synthase genes are introduced to confer resistance, then plant cells gain glyphosate tolerance, but the sequences do not predictably confer resistance across all variants
Solution Approach 1:
The patent establishes predictive parameters by systematically analyzing the relationship between specific amino acid sequences and glyphosate resistance levels. By identifying key residues in the binding region and their mutation effects, the patent creates a framework where the amino acid sequence can predict the level of resistance, enabling selection of variants with desired resistance characteristics before experimental testing.
Solution Approach 2:
The patent replaces empirical trial-and-error methods with a rational design approach based on sequence analysis and structure-function relationships. Instead of randomly testing bacterial EPSP synthase variants, the patent uses computational analysis of amino acid sequences to predict which variants will confer resistance, substituting mechanical experimentation with informational prediction based on sequence characteristics.
3Object-affected harmful factors
If novel EPSP synthase variants are created through mutagenesis, then glyphosate resistance is improved, but the catalytic activity may be reduced
Solution Approach 1:
The patent applies parameter changes by systematically varying amino acid residues in the binding region while monitoring both resistance and catalytic activity parameters. Through iterative mutagenesis and screening, the patent identifies variants that achieve an optimal balance between glyphosate resistance and enzyme activity, changing the molecular parameters to simultaneously improve resistance while maintaining sufficient catalytic function for plant growth.
Solution Approach 2:
The patent applies local quality by confining mutations to the glyphosate binding region while preserving the catalytic core of the enzyme. This localized modification strategy ensures that the active site responsible for catalysis remains intact and functional, while only the regions that interact with glyphosate are altered to provide resistance, thereby maintaining catalytic activity while gaining herbicide tolerance.
Data Source
AI summary
Compositions and methods for conferring herbicide resistance to plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a polypeptide that confers resistance or tolerance to glyphosate herbicides are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants. Compositions also comprise transformed plants, plant cells, tissues, and seeds. In particular, isolated nucleic acid molecules encoding glyphosate resistance proteins are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:2 or the nucleotide sequence set forth in SEQ ID NO:1.