HPPD Polypeptide Mutations for Broad Herbicide Tolerance
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Solution Overview
Problem
Current methods for providing plants with tolerance to hydroxyphenyl pyruvate dioxygenase (HPPD) herbicides are inadequate, as existing mutations do not confer commercially significant benefits and are often specific to certain herbicides, lacking broad tolerance and effective kinetic improvements.
Innovation Solution
Development of novel HPPD polypeptides with specific amino acid sequences that exhibit enhanced tolerance to HPPD herbicides by modifying key positions, such as those found in Alopecurus species, which are used to create mutant HPPD enzymes with improved k_off and k_cat/K_m values, allowing for increased resistance or tolerance when expressed in plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing HPPD mutations are used to provide herbicide tolerance, then some measure of tolerance is achieved, but the tolerance is not commercially significant and lacks broad coverage across different herbicide classes
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues at positions 422, 423, and/or 424 in the HPPD enzyme sequence. These point mutations alter the enzyme's kinetic parameters (k_off and k_cat/K_m values) to achieve superior tolerance across multiple HPPD-inhibiting herbicide classes, resolving the contradiction between achieving tolerance and achieving broad commercial significance
Solution Approach 2:
The mutated HPPD enzyme provides universal tolerance across multiple herbicide classes (triketones, pyridine-2,5-diones, and other HPPD inhibitors) rather than being specific to a single herbicide type. This multi-functional tolerance allows a single enzyme variant to confer resistance to diverse herbicide mechanisms, addressing the versatility requirement
2Reliability
If HPPD enzyme is overexpressed to provide herbicide tolerance, then sufficient functional enzyme is available to thrive despite herbicide presence, but the method does not confer commercial-level tolerance to desirable herbicides
Solution Approach 1:
Instead of increasing enzyme quantity through overexpression, the patent changes the enzyme's kinetic parameters by introducing point mutations at positions 422, 423, and/or 424. These mutations improve the enzyme's affinity for substrate (lower K_m) and reduce herbicide binding (higher k_off), achieving superior commercial-level tolerance without requiring excessive enzyme production
3Reliability
If mutated HPPD enzymes from cool-climate grasses are developed, then improved resistance to triketone herbicides is achieved, but such mutants have not yet been reported and remain desirable
Solution Approach 1:
The patent successfully generates and characterizes mutated HPPD enzymes with improved kinetic parameters (k_off and k_cat/K_m) that confer resistance to triketone and other HPPD-inhibiting herbicides. The mutations at positions 422, 423, and/or 424 create functional variants that can be produced through standard molecular biology techniques, making the desired mutant enzyme readily available for agricultural application
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 novel HPPD polypeptides demonstrate superior tolerance to HPPD herbicides, characterized by increased k_off values and catalytic efficiency, leading to improved resistance or tolerance in transgenic plants, enabling effective herbicide resistance across various classes of HPPD-inhibiting herbicides.
Implementation Method 1
The hydroxyphenylpyruvate dioxygenases (HPPDs) are enzymes that catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate
Implementation Method 2
This reaction takes place in the presence of enzyme-bound iron (Fe 2+via the photo-generation of singlet oxygen
Data Source
Figure 1
Figure 2A~2B
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AI summary
Novel hydroxyphenyl pyruvate dioxygenase (HPPD) polypeptides, variants and fragments thereof, as as well as polynucleotides encoding the same, capable of conferring commercial levels of conferring HPPD herbicide resistance or tolerance to plants. Compositions include amino acid sequences, and variants and fragments thereof, for HPPD polypeptides, as well as polynucleotides encoding the same. Methods for the production and use of HPPD herbicide resistant plants that express these novel HPPD polypeptides, methods for selectively controlling weeds in a field at a crop locus, and method for the assay, characterization, identification and selection of these novel HPPDs are also provided.