Isolation and characterization of two imazamox-resistant anabolic acetohydroxyacid synthase enzymes from Paenibacillus sp. JY-9.

Imazamox targets anabolic acetohydroxyacid synthase (AHAS), and studying its resistant mutants is vital for developing herbicide-resistant transgenic crops. In this study, an imazamox-tolerant Paenibacillus sp. JY-9 was isolated, and two AHAS genes, PnAHASc1 and PnAHASc2, were cloned. Additionally, a highly resistant mutant, JYm56, harboring triple mutations (A37S+A65V+D66H) in PnAHASc1 (designated as PnAHASc1m) was obtained. PnAHASc1m exhibited 43.3% higher catalytic efficiency and a 1.36-fold increase in imazamox resistance compared with the wild-type PnAHASc1 enzyme. A37S was the primary resistance site that rescued the catalytic defect of D66H, a novel mutation pattern for bacterial anabolic AHAS. The A37S+D66H double mutant had the highest resistance, and the triple mutant PnAHASc1m achieved a favorable balance between catalytic activity and imazamox resistance. This study provides novel AHAS resources and a multi-mutation synergistic mechanism, supporting the formation of imazamox-resistant crops.