ALS Enzyme Mutations for Herbicide Tolerance and Substrate Preference
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Solution Overview
Problem
Current methods for achieving tolerance to ALS-inhibiting herbicides in crops often result in altered substrate preferences for the acetolactate synthase enzyme, leading to potential detrimental effects on the organism, and there is a need for mutations that confer insensitivity while maintaining kinetic behavior similar to wild-type ALS enzymes.
Innovation Solution
Development of polynucleotides and polypeptides with acetolactate synthase activity that are tolerant to ALS inhibitors, exhibiting an increased preference for 2-ketobutyrate similar to native ALS enzymes, while retaining insensitivity to these inhibitors, thereby minimizing adverse effects on the organism.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If point mutations are introduced in ALS genes to confer tolerance to ALS-inhibiting herbicides, then insensitivity to herbicides is achieved, but substrate preference of the enzyme is altered leading to detrimental effects on the organism
Solution Approach 1:
The patent applies parameter changes by introducing specific point mutations (P178S, A103T, P207S, D357E) in the ALS gene that modify the enzyme's kinetic parameters. These mutations change the substrate preference ratio (Rf value) to maintain it within a specific range (greater than 3 and less than 150) while simultaneously conferring herbicide insensitivity. The mutations alter the enzyme's interaction with substrates 2-ketobutyrate and pyruvate, ensuring proper metabolic function is preserved while achieving the desired herbicide tolerance.
2Adaptability or versatility
If mutations in ALS are introduced to confer tolerance to different ALS herbicides, then broad spectrum herbicide resistance is achieved, but kinetic behavior of the enzyme deviates from wild type ALS
Solution Approach 1:
The patent employs parameter changes by carefully selecting specific amino acid substitutions that modify the ALS enzyme's kinetic properties. The mutations are designed to achieve a substrate preference ratio (Rf) within the range of greater than 3 and less than 150, which maintains kinetic behavior similar to wild-type ALS. This approach allows the enzyme to function properly while conferring tolerance to multiple ALS-inhibiting herbicide groups including sulfonylureas, imidazolinones, and triazolopyrimidines.
3Reliability
If ALS mutations are introduced to achieve herbicide insensitivity, then crop tolerance to ALS-inhibiting herbicides is achieved, but downstream effects on the organism occur due to altered substrate preference
Solution Approach 1:
The patent applies parameter changes by introducing specific point mutations (P178S, A103T, P207S, D357E) in the ALS gene that modify the enzyme's kinetic parameters. These mutations change the substrate preference ratio (Rf value) to maintain it within a specific range (greater than 3 and less than 150) while simultaneously conferring herbicide insensitivity. The mutations alter the enzyme's interaction with substrates 2-ketobutyrate and pyruvate, ensuring proper metabolic function is preserved while achieving the desired herbicide tolerance.
Solution Approach 2:
The patent converts the potential harm of altered substrate preference into a benefit by selecting mutations that maintain the substrate preference ratio within an optimal range. The altered kinetics are directed toward achieving herbicide insensitivity while preserving essential metabolic functions. The specific mutations create an enzyme variant that is both herbicide-tolerant and metabolically functional, turning what could be a detrimental alteration into a beneficial trait for crop protection.
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 ALS inhibitor-tolerant polypeptides and nucleic acid constructs enable the production of crops that are resistant to ALS-inhibiting herbicides, allowing tolerant crops to thrive while damaging weeds, with minimal impact on the enzyme's kinetic behavior and substrate preference.
Implementation Method 1
Acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS), catalyzes the biosynthesis of the branched chain amino acids valine, leucine and isoleucine
Data Source
AI summary
Compositions and methods comprising polynucleotides and polypeptides having ALS activity and tolerance to at least one ALS inhibitor are provided. In specific embodiments, the sequence has an increased preference for 2-ketobutyrate, when compared to an appropriate control, such as for example, HRA, and/or a preference for 2-ketobutyrate similar to a native ALS. Further provided are nucleic acid constructs, plants, plant cells, explants, seeds and grain having the ALS inhibitor tolerant sequences. Various methods of employing the ALS inhibitor tolerant sequences are provided. Such methods include methods for producing an ALS inhibitor 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.