Co-overexpression of geraniol-10-hydroxylase and strictosidine synthase improves anti-cancer drug camptothecin accumulation in Ophiorrhiza pumila

Abstract

Camptothecin (CPT) belongs to a group of monoterpenoidindole alkaloids (TIAs) and its derivatives such as irinothecan and topothecan have been widely used worldwide for the treatment of cancer, giving rise to rapidly increasing market demands. Genes from Catharanthus roseus encoding strictosidine synthase (STR) and geraniol 10-hydroxylase (G10H), were separately and simultaneously introduced into Ophiorrhiza pumila hairy roots. Overexpression of individual G10H (G lines) significantly improved CPT production with respect to non-transgenic hairy root cultures (NC line) and single STR overexpressing lines (S lines), indicating that G10H plays a more important role in stimulating CPT accumulation than STR in O. pumila. Furthermore, co-overexpression of G10H and STR genes (SG Lines) caused a 56% increase on the yields of CPT compared to NC line and single gene transgenic lines, showed that simultaneous introduction of G10H and STR can produce a synergistic effect on CPT biosynthesis in O. pumila. The MTT assay results indicated that CPT extracted from different lines showed similar anti-tumor activity, suggesting that transgenic O. pumila hairy root lines could be an alternative approach to obtain CPT. To our knowledge, this is the first report on the enhancement of CPT production in O. pumila employing a metabolic engineering strategy.

Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica

“…There are a lot of successful cases in this field. In A. belladonna and A. acutangulus , biosynthesis of scopolamine was dramatically enhanced by overexpressing the committed enzymes, H6H and TRI in A. annua , the production of artemisinin was greatly promoted by overexpressing CYP71AV1 and aldehyde 11Δ(13) reductase ; in Catharanthus roseus , overexpressing strictosidine synthase led to the increased production of ajmalicine ; in Ophiorrhiza pumila , the anti‐cancer camptothecin was significantly improved by overexpressing geraniol‐10‐hydroxylase and strictosidine synthase ; in Salvia miltiorrhiza , biosynthesis of tanshinone was enhanced by different combination of the key enzymes . These cases strongly suggest that overexpression of key enzymes is a very potent tool for genetic improvement of metabolite biosynthesis.…”

Molecular Approaches to Screen Bioactive Compounds from Endophytic Fungi

“…Gene encoding STR has been cloned and characterized from hairy root cultures of Ophiorrhiza pumila (Yamazaki et al, 2003). Although much of the information regarding the steps in-between strictosidine and camptothecin is not available, Cui et al (2015) reported that the co-overexpression of geraniol-10-hydroxylase and strictosidine synthase increased camptothecin accumulation in O. pumila hairy root cultures.…”

Characterization of CYP154F1 from Thermobifida fusca YX and Extension of Its Substrate Spectrum by Site‐Directed Mutagenesis

“…In plants such as Catharanthus roseus , geraniol hydroxylation is catalyzed by CYP76B6. However, the low activity or expression level of CYP76B6 introduced in engineered recombinant microorganisms seem to be the bottlenecks in strictosidine synthesis . Brown et al.…”