Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

Abstract Cytochrome P450s participate in the biosynthesis of natural active ingredients and catalyze the formation of hydroxyl, carboxyl, epoxy and other groups from C−H bonds. CYP728B70 from Tripterygium wilfordii oxidizes the miltiradiene‐C18 methyl group to produce dehydroabietic acid, a diterpenoid intermediate with anticancer, antibacterial and anti‐inflammatory activities. To elevate the carboxylation product output, we excavated serveral homologous CYP728Bs and an N‐terminal truncated CYP728B70 transcript from T. wilfordii genome, of which CYP728B79 possessed a weak carboxylation effect towards miltiradiene through heterologous expression in Saccharomyces cerevisiae . We employed RoseTTAFold method and molecular dynamic simulations to predict its protein structure and docking pose of CYP728B70, and obtained several mutants with increased yield using Funclib and site‐directed mutagenesis methods. Our study confirmed the influence of peripheral residues on protein activity, improved the catalytic activity of CYP728B70, and would provide ideas for related enzyme modification.