caa a22 4500 605501319 CHVBK 20210128100600.0 cr unu---uuuuu 210128e20150901xx s 000 0 eng 10.1007/s00253-015-6496-2 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6496-2 Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis [Elektronische Daten] [Naoki Kitaoka, Yisheng Wu, Meimei Xu, Reuben Peters] 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. Springer-Verlag Berlin Heidelberg, 2015 Metabolic engineering nationallicence Natural products nationallicence Labdane-related diterpenoids nationallicence Phytoalexin nationallicence Antibiotic nationallicence Evolution nationallicence Kitaoka Naoki Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut Wu Yisheng Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut Xu Meimei Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut Peters Reuben Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/18(2015-09-01), 7549-7558 0175-7598 99:18<7549 2015 99 253 https://doi.org/10.1007/s00253-015-6496-2 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6496-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kitaoka Naoki Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut NATIONALLICENCE 700 1- Wu Yisheng Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut NATIONALLICENCE 700 1- Xu Meimei Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut NATIONALLICENCE 700 1- Peters Reuben Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 50011, Ames, IA, USA aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/18(2015-09-01), 7549-7558 0175-7598 99:18<7549 2015 99 253