title: Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.
abstract: Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes.
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Su
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
authors.forename: Yuru
authors.initials: Y
authors.lastname: Tong
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.|State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau, China.
authors.forename: Qiqing
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-41296 Göteborg, Sweden.
authors.forename: Yating
authors.initials: Y
authors.lastname: Hu
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhang
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, 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, China.
authors.forename: Zhongqiu
authors.initials: Z
authors.lastname: Teng
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
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, China.
authors.forename: Luqi
authors.initials: L
authors.lastname: Huang
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways / Q000235:genetics; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005875:Gibberellins / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D027561:Salvia miltiorrhiza / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015003:Yeasts / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D005875:Gibberellins; D010940:Plant Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C066398:ent-kaurene oxidase; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A; C021459:kaurene synthetase