title: CYP716A179 functions as a triterpene C-28 oxidase in tissue-cultured stolons of Glycyrrhiza uralensis.
abstract: CYP716A179, a cytochrome P450 monooxygenase expressed predominantly in tissue-cultured stolons of licorice ( Glycyrrhiza uralensis ), functions as a triterpene C-28 oxidase in the biosynthesis of oleanolic acid and betulinic acid. Cytochrome P450 monooxygenases (P450s) play key roles in the structural diversification of plant triterpenoids. Among these, the CYP716A subfamily, which functions mainly as a triterpene C-28 oxidase, is common in plants. Licorice (Glycyrrhiza uralensis) produces bioactive triterpenoids, such as glycyrrhizin and soyasaponins, and relevant P450s (CYP88D6, CYP72A154, and CYP93E3) have been identified; however, no CYP716A subfamily P450 has been isolated. Here, we identify CYP716A179, which functions as a triterpene C-28 oxidase, by RNA sequencing analysis of tissue-cultured stolons of G. uralensis. Heterologous expression of CYP716A179 in engineered yeast strains confirmed the production of oleanolic acid, ursolic acid, and betulinic acid from β-amyrin, α-amyrin, and lupeol, respectively. The transcript level of CYP716A179 was about 500 times higher in tissue-cultured stolons than in intact roots. Oleanolic acid and betulinic acid were consistently detected only in tissue-cultured stolons. The discovery of CYP716A179 helps increase our understanding of the mechanisms of tissue-type-dependent triterpenoid metabolism in licorice and provides an additional target gene for pathway engineering to increase the production of glycyrrhizin in licorice tissue cultures by disrupting competing pathways.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Tamura
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Seki
authors.affiliation: Department of Research and Development, Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba, 292-0818, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu, Hokkaido, 061-0293, Japan.
authors.forename: Mareshige
authors.initials: M
authors.lastname: Kojoma
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8675, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. muranaka@bio.eng.osaka-u.ac.jp.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Muranaka
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays; D008401:Gas Chromatography-Mass Spectrometry; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering; D029882:Glycyrrhiza uralensis / Q000033:anatomy & histology* / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*; D014315:Triterpenes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D012333:RNA, Messenger; D014315:Triterpenes; D003577:Cytochrome P-450 Enzyme System
keywords: Glycyrrhiza uralensis; Licorice; P450; RNA-seq; Triterpenoid