RESUMEN
Oleanane-type ginsenosides are highly biologically active substances in Panax ginseng, a popular Chinese dietary plant. Lack of key enzymes for glycosylation reactions has hindered de novo synthesis of these bioactive molecules. We mined candidate glycosyltransferases (GTs) of the ginseng database by combining key metabolites and transcriptome coexpression analyses and verified their function using in vitro enzymatic assays. The PgCSyGT1, a cellulose synthase-like GT rather than a UDP-dependent glucuronosyltransferase (UGT), was verified as the key enzyme for transferring a glucuronosyl moiety to the free C3-OH of oleanolic acid to synthesize calenduloside E. Two UGTs (PgUGT18 and PgUGT8) were first identified as, respectively, catalyzing the glycosylation reaction of the second sugar moiety of C3 and the C28 in the oleanane-type ginsenoside biosynthetic pathway. Then, we integrated these GTs in combinations into Saccharomyces cerevisiae genome and realized de novo biosynthesis of oleanane-type ginsenosides with a yield of 1.41 µg/L ginsenoside Ro in shake flasks. This report provides a basis for effective biosynthesis of diverse oleanane-type ginsenosides in microbial cell factories.
Asunto(s)
Ginsenósidos , Ácido Oleanólico , Panax , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
To enhance production of Antrodia cinnamomea triterpenoids (ACTs) from mycelia in solid-state culture, α-terpineol was added to the medium as an elicitor at an optimal concentration of 0.05 mL L-1. Multi-stage solvent extraction and HPLC analysis were performed, and the compositions of ACTs-E (from culture with elicitor) and ACTs-NE (from culture without elicitor) were found to be quite different. In assays of in vitro antitumor activity, ACTs-E, in comparison with ACTs-NE, produced stronger viability reduction in several tumor cell lines and stronger apoptosis induction in HeLa in a dose-dependent manner. Several related proteins involved in the mitochondrial pathway of apoptosis (p53, Bax, caspase-3) did not show expression upregulation by ACTs-E, suggesting that apoptosis induction occurred through a p53-independent process. Further analysis revealed that ACTs-E strongly inhibited synthesis of topoisomerase I (TOP1) and tyrosyl-DNA phosphodiesterase I (TDP1), which are involved in DNA repair, at both transcriptional and protein levels. Our findings suggest that ACTs-E have potential for applications in the pharmaceutical, clinical, and functional food industries, as a novel antitumor agent and a dual TOP1/TDP1 inhibitor.