Anchoring mechanism-inspired discovery of a bacterial P450 gene conferring resistance to auxin herbicides

Herbicides in combination with genetically modified herbicide-resistant crops have revolutionized modern weed management, increased crop yields, and facilitated farming practices. However, rapid evolution of herbicide-resistant weeds necessitates new resistance traits to sustain control efficacy. Here, we introduce a terminal carboxyl anchoring mechanism-inspired approach for precise discovery of P450 herbicide resistance genes, by which a number of bacterial P450 peroxygenases are predicted and confirmed to degrade auxin herbicides. Upon enzyme engineering, the optimal mutant P450_BSβ-F46A can efficiently degrade diverse auxin herbicides and other carboxyl-containing herbicides. Mechanistic studies reveal that Compound I-mediated hydroxylation initiates the C‒O bond cleavage, followed by aromatic ring hydroxylation, thus forming a unique two-step degradation pathway. Transgenic rice expressing P450_BSβ-F46A-CPR confers significant resistance to a recently commercialized auxin herbicide fluchloraminopyr. This work demonstrates the potential of the mechanism-driven strategy in directed discovery of broad-spectrum resistance genes for herbicide-resistant crop engineering.