CYP71Z18 exhibited plastic substrate specificity to catalyze oxidation of multiple rice diterpenes and elevated chemical defense against the blast fungus in transgenic rice. Diversified plant specialized metabolism relies on corresponding biosynthetic enzymes with differential substrate specificity. CYP71Z18 catalyzed formation of maize phytoalexins including zealexin A1, the sesquiterpenoid phytoalexin, and diterpenoid phytoalexin dolabralexin, indicating catalytic promiscuity on different terpene substrates. Here substrate specificity of CYP71Z18 was further explored through microbial metabolic engineering and it was identified to accept multiple rice diterpenes as substrates for oxidation. One CYP71Z18 enzymatic product derived from syn-pimaradiene was identified as 15,16-epoxy-syn-pimaradiene by NMR analysis, which was further elaborated by CYP99A3 to generate C19 hydroxylated product. 15,16-epoxy-syn-pimaradien-19-ol exhibited inhibitory effect on spore germination and appressorium formation of the blast pathogen Magnaporthe oryzae. Overexpression of CYP71Z18 in rice resulted in accumulation of several new diterpenoids, indicating promiscuous activity in planta. Transgenic rice also showed stronger resistance against M. oryzae infection, suggesting elevated chemical defense through changed diterpenoid metabolism by CYP71Z18 overexpression. This investigation sheds light on plant metabolic engineering using plastic substrate specificity of P450s to strengthen disease resistance and potentially provide abundant lead compounds.
Keywords: Disease resistance; Diterpenoid; P450; Rice; Substrate specificity.