HPPD Polypeptide Variants for Broad Herbicide Tolerance
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Solution Overview
Problem
Current HPPD inhibitor herbicides exhibit slow-binding or slow, tight-binding properties, making it difficult to develop plants with full tolerance across various chemical classes, as mutations that reduce affinity do not completely overcome inhibition due to long-lasting binding.
Innovation Solution
Development of HPPD polypeptides with reduced affinity and increased dissociation rate, converting slow-binding inhibitors into fully reversible inhibitors, allowing for broad tolerance to multiple HPPD inhibitor herbicide classes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If HPPD polypeptides with reduced affinity to HPPD inhibitor herbicides are developed, then tolerance to herbicides is improved, but the slow-binding nature of the inhibitors causes long-lasting binding that prevents complete overcoming of inhibition
Solution Approach 1:
The patent applies parameter changes by modifying the dissociation rate constant (koff) of the HPPD polypeptide-inhibitor complex. Specific amino acid substitutions (e.g., Pro335Leu, Gly336Trp, Pro215Leu) are introduced to increase the dissociation rate, transforming the kinetic profile from slow-binding to fast-dissociating. This changes the fundamental binding parameters to achieve full reversibility and complete herbicide tolerance.
Solution Approach 2:
The invention introduces dynamic characteristics to the HPPD polypeptide by creating variants with altered kinetic properties. The mutant polypeptides exhibit fast association and fast dissociation rates, making the inhibitor binding transient and reversible. This dynamic behavior contrasts with the static, long-lasting binding of wild-type HPPD to slow-binding inhibitors, enabling the plant to recover from inhibitor exposure.
2Object-affected harmful factors
If mutations are introduced to reduce inhibitor affinity, then herbicide tolerance is achieved, but slow-binding inhibitors maintain inhibition due to long binding duration
Solution Approach 1:
The patent employs the 'skipping' principle by accelerating the dissociation phase of inhibitor binding. The mutant HPPD polypeptides facilitate rapid release of the inhibitor molecule, effectively skipping the prolonged inhibited state. This rush through the binding event minimizes the time the inhibitor remains bound, converting a potentially long-lasting inhibition into a brief, reversible interaction.
3Reliability
If HPPD polypeptides with increased dissociation rate are developed, then full reversibility of inhibitor binding is achieved, but developing such polypeptides requires extensive mutagenesis and screening
Solution Approach 1:
The patent applies preliminary action by using site-directed mutagenesis based on prior structural and kinetic knowledge of the HPPD-inhibitor complex. Specific residues known to be involved in inhibitor binding (positions 215, 335, 336, etc.) are targeted for mutation before functional testing. This pre-planned approach, guided by structural insights, reduces the randomness of screening and focuses efforts on mutations most likely to increase dissociation rates.
Solution Approach 2:
The invention systematically explores parameter changes by testing specific amino acid substitutions at key positions in the HPPD polypeptide. Rather than random mutagenesis, the approach focuses on changing specific parameters (amino acid identity at critical residues) that are predicted to affect dissociation kinetics, thereby efficiently navigating the sequence space to find functional variants.
Data Source
AI summary
In the present invention, HPPD polypeptides and plants containing them showing a full tolerance against one or more HPPD inhibitor herbicides belonging to various chemical classes are described. A set of mutant HPPD polypeptides have been designed which have either no or only a significantly reduced affinity to HPPD inhibitor herbicides and, at the same time, the rate of dissociation of the HPPD inhibitors of the mutant HPPD polypeptide is increased to such an extent that the HPPD inhibitors no longer act as slow-binding or slow, tight-binding inhibitors but, instead of this, have become fully reversible inhibitors. In particular, isolated polynucleotides encoding mutant HPPD polypeptides conferring tolerance to HPPD inhibitor herbicides belonging to various chemical classes are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed.