Directed evolution of HIS1-Like proteins to enhance herbicide metabolism in crops

HPPD (4-hydroxyphenylpyruvate dioxygenase) inhibitor are widely used in agriculture due to their high efficacy and environmental friendliness. However, many important crops, such as rice, wheat, and soybean, are naturally sensitive to these herbicides. In this study, we employed a directed evolution strategy to enhance the metabolic capacity of Os HSL2, Os HSL4, Os HSL6, and Sb HSL1 proteins toward HPPD inhibitors, providing a new technological approach as well as theoretical foundation for molecular breeding of herbicide-resistant crops. By combining AlphaFold 3 protein models with crystal structures, we systematically redesigned key residues to resemble the active residues found in HIS1. Catalytic activity assays demonstrated that specific mutations significantly improved the metabolic activity of HSLs proteins toward various HPPD inhibitors. Notably, the Os HSL2-M4 mutant exhibited enhanced metabolic activity for BBC-OH and methyl-benquitrione, while the Os HSL4-M5 mutant completely metabolized BBC-OH and topramezone. Additionally, the Sb HSL1-M4 mutant showed significant improvement in the metabolism of BBC-OH and several other herbicides, providing strong evidence to support the use of structure-guided HSL mutations to enhance crop resistance to HPPD inhibitors.