title: Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis.
abstract: The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis, we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases, activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here, we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3β-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2α,3α-dihydroxy-ent-isokaurene via 2α-hydroxy-ent-isokaurene, and CYP71Z7 catalyzing the production of 3α-hydroxy-ent-cassadien-2-one via 2α-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively.
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kitaoka
authors.forename: Yisheng
authors.initials: Y
authors.lastname: Wu
authors.forename: Meimei
authors.initials: M
authors.lastname: Xu
authors.forename: Reuben J
authors.initials: RJ
authors.lastname: Peters
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004224:Diterpenes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System