Chimeric ACCase Screening for Herbicide-Tolerant Rice Mutations
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Solution Overview
Problem
Current methods for developing herbicide-tolerant rice varieties are limited in generating and testing mutations, particularly for ACCase enzymes, as they are labor-intensive and inefficient in producing a high number of herbicide-tolerant variants.
Innovation Solution
A high-throughput method involving ACCase-deficient yeast cells with chimeric ACCase enzymes, comprising an N-terminal region from yeast or fungi and a C-terminal region from monocot plastidic ACCases, is used to screen for herbicide-tolerant variants through site-directed mutagenesis and selection in the presence of herbicides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to generate and test ACCase mutations in rice, then the process can be completed with standard laboratory equipment and procedures, but the productivity is low and only a limited number of mutations can be generated per month
Solution Approach 1:
The patent uses yeast cells as an intermediary model system to generate and screen ACCase mutations. Instead of directly working with rice plants, the invention transfers the ACCase gene to yeast, performs mutagenesis and selection in the simpler yeast system, and then applies the identified mutations to rice. This intermediary approach dramatically accelerates the mutation generation and screening process while maintaining relevance to the target crop.
Solution Approach 2:
The patent performs preliminary mutagenesis, selection, and characterization of ACCase variants in yeast before applying these mutations to rice plants. By conducting the time-consuming mutation generation and screening steps in advance using the faster-growing yeast system, the invention prepares a library of validated herbicide-tolerant ACCase mutations that can then be efficiently transferred to rice, significantly reducing the overall development timeline.
2Measurement precision
If site-directed mutagenesis is performed on each ACCase variant individually, then the mutations can be precisely controlled and characterized, but the process becomes labor-intensive and the throughput remains low
Solution Approach 1:
The patent employs a self-service selection system where yeast cells expressing mutated ACCase variants are automatically selected based on their ability to survive and grow in the presence of herbicides. The herbicide itself acts as the selection pressure, allowing only cells with functional or herbicide-tolerant ACCase variants to proliferate. This self-selecting mechanism eliminates the need for labor-intensive individual screening while maintaining precise identification of functional mutations through subsequent molecular characterization.
Solution Approach 2:
The patent systematically varies the herbicide concentration parameters during selection to enrich for progressively more tolerant ACCase variants. By implementing a gradient selection approach where the herbicide concentration is increased over successive generations or screening rounds, the system efficiently identifies variants with different levels of tolerance, thereby increasing throughput while maintaining precise characterization of mutation effects.
3Quantity of substance
If a high number of ACCase mutations are generated and tested, then more herbicide-tolerant variants can be identified, but the resource requirements and experimental complexity increase
Solution Approach 1:
The patent creates a universal yeast-based screening platform that can evaluate multiple ACCase mutations simultaneously using the same selection conditions. The yeast system serves multiple functions: it can perform mutagenesis, express the mutated ACCase genes, provide the necessary cellular machinery for protein folding and function, and enable herbicide-based selection. This multi-functional platform allows high-throughput screening without proportionally increasing system complexity, as the same basic infrastructure handles diverse mutation variants.
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
This approach enables the rapid identification and isolation of thousands of herbicide-tolerant ACCase variants per month, significantly surpassing the efficiency of previous methods, allowing for the generation of rice varieties with enhanced herbicide tolerance.
Implementation Method 1
ACCase is a biotinylated enzyme that converts acetyl-CoA to malonyl-CoA in a 2-step reversible reaction. The enzyme first carboxylates the biotin group and then the intrinsic carboxytranferase activity transfers the carboxyl group from carboxybiotin to acetyl-CoA
Implementation Method 2
The effects of the mutations are studied in rice after Agrobacterium-mediated transformation of the modified ACCase genes
Data Source
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Figure 2A
AI summary
The present invention relates to methods of producing, isolating, identifying and characterizing herbicide tolerant ACCase variants. In one embodiment, the invention encompasses a method of screening for an acetyle-CoA carboxylase (ACCase) enzyme which is tolerant to at least one herbicide, comprising: a) providing an ACCase-deficient yeast with a chimeric ACCase, said chimeric ACCase comprising: at least two regions that further comprise: i) an N-terminal region, said N-terminal region derived from yeast, fungi or monocot cytoplasmic ACCases, preferably a yeast ACCase; ii) a C-terminal region, said C-terminal region derived from monocot plastific ACCases and comprising an HSR; and iii) said N-terminal region comprises about 50% to about 60% of the chimeric ACCase; b) isolating herbicide tolerant yeast cells after culturing in the presence of at least one herbicide; and c) further comprising identifying the mutation(s) not present in chimeric ACCase prior to culturing, which confers tolerance to at least one herbicide.