title: Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.
abstract: Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.
authors.affiliation: †School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan.
authors.forename: Yan-Liang
authors.initials: YL
authors.lastname: Lin
authors.affiliation: †School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan.
authors.forename: Yi-Ru
authors.initials: YR
authors.lastname: Lee
authors.affiliation: ‡Department of Forestry, National Chung-Hsing University, Taichun, Taiwan.
authors.forename: Nai-Wen
authors.initials: NW
authors.lastname: Tsao
authors.affiliation: ‡Department of Forestry, National Chung-Hsing University, Taichun, Taiwan.|§Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.|⊥Agricultural Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan.
authors.forename: Sheng-Yang
authors.initials: SY
authors.lastname: Wang
authors.affiliation: ∥Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan.
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
authors.affiliation: †School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan.|#Experimental Forest, National Taiwan University, Nan-Tou, Taiwan.
authors.forename: Fang-Hua
authors.initials: FH
authors.lastname: Chu
mesh_terms: D055491:Antrodia / Q000737:chemistry / Q000235:genetics*; D048690:Fruiting Bodies, Fungal / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D019751:Intramolecular Transferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015394:Molecular Structure; D025282:Mycelium; D013185:Squalene / Q000031:analogs & derivatives / Q000737:chemistry; D013624:Taiwan; D014315:Triterpenes / Q000378:metabolism*
chemical_list: C002821:2,3-oxidosqualene; D014315:Triterpenes; D013185:Squalene; D019751:Intramolecular Transferases; C021924:lanosterol synthase