| Rice (Oryza sativa) is an important food crop and produces more than fifteen labdane-related diterpenoid natural products as antibiotics against plant pathogens, for example, against the devastating blast disease pathogen Magnaprothe grisea. The biosynthesis of these labdane-related diterpenoid phytoalexins has been partially clarified, as early enzymatic genes involved in their biosynthesis have been identified, but the downstream pathways from the inactive diterpene olefin precursors to bioactive diterpenoid natural products have not been identified, which limits our understanding and ability to engineer production of these important natural products. Previous work defined two functional diterpenoid biosynthetic gene clusters in rice that include cytochromes P450. Focusing on these P450s, we have been working to identify their biochemical functions using our metabolic engineering system. So far, we have found 10 P450s that exhibit catalytic activity against various rice diterpenes, and 17 novel diterpenoids have been produced and identified. Enzymatic characterization and rice plant metabolite analysis indicate physiological relevance for the identified P450 activity. Through these efforts we have characterized the initial oxidation steps of three groups of diterpenoids, including elucidation of the production of the bioactive Oryzalexin E. Some P450s exhibited activity against a range of diterpene substrates, and these are candidates for further enzymatic investigations. In conclusion, our study is significant because we are elucidating the enzymatic machinery involved in the biosynthesis of rice diterpenoids with important antibiotic activities. |
