title: Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.
abstract: Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis.
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Guo
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.|Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Ma
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.|Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Cai
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Ma
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Zhilai
authors.initials: Z
authors.lastname: Zhan
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, China.
authors.forename: Yongjin J
authors.initials: YJ
authors.lastname: Zhou
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, China.
authors.forename: Wujun
authors.initials: W
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China.
authors.forename: Mengxin
authors.initials: M
authors.lastname: Guan
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Guanghong
authors.initials: G
authors.lastname: Cui
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Kang
authors.affiliation: Plant Science Research Center, Shanghai Chenshan Botanical Garden, Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai, 201602, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Shen
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Jinfu
authors.initials: J
authors.lastname: Tang
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Huixin
authors.initials: H
authors.lastname: Lin
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Ma
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Baolong
authors.initials: B
authors.lastname: Jin
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China.
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Reuben J
authors.initials: RJ
authors.lastname: Peters
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, China.
authors.forename: Zongbao K
authors.initials: ZK
authors.lastname: Zhao
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Luqi
authors.initials: L
authors.lastname: Huang
mesh_terms: D045784:Abietanes / Q000737:chemistry / Q000378:metabolism*; D053898:Biosynthetic Pathways; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering; D013058:Mass Spectrometry; D062105:Molecular Docking Simulation; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D027561:Salvia miltiorrhiza / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein
chemical_list: D045784:Abietanes; D010940:Plant Proteins; C021751:tanshinone; D003577:Cytochrome P-450 Enzyme System
keywords: Salvia miltiorrhiza Bunge; cytochrome P450 (CYP) monooxygenases; diterpenoid biosynthesis; enzymatic promiscuity; metabolic pathways; synthetic biology