Enhancing the production of tropane alkaloids in transgenic Anisodus acutangulus hairy root cultures by over-expressing tropinone reductase I and hyoscyamine-6β-hydroxylase

Abstract

Tropane alkaloids (TA) including hyoscyamine, anisodamine, scopolamine and anisodine, are used medicinally as anticholinergic agents with increasing market demand, so it is very important to improve TA production by metabolic engineering strategy. Here, we report the simultaneous introduction of genes encoding the branch-controlling enzyme tropinone reductase I (TRI, EU424321) and the downstream rate-limiting enzyme hyoscyamine-6β-hydroxylase (H6H, EF187826) involved in TA biosynthesis into Anisodus acutangulus hairy roots by Agrobacterium-mediated gene transfer technology. Transgenic hairy root lines expressing both TRI and H6H (TH lines) produced significantly higher (P < 0.05) levels of TA compared with the control and single gene transformed lines (T or H lines). The best double gene transformed line (TH53) produced 4.293 mg g(-1) TA, which was about 4.49-fold higher than that of the control lines (0.96 mg g(-1)). As far as it is known, this is the first report on simultaneous introduction of TRI and H6H genes into TA-producing plant by biotechnological approaches. Besides, the content of anisodine was also greatly improved in A. acutangulus by over-expression of AaTRI and AaH6H genes. The average content of anisodine in TH lines was 0.984 mg g(-1) dw, about 18.57-fold of BC lines (0.053 mg g(-1) dw). This is the first time that this phenomenon has been found in TA-producing plants.

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.…”

Influence of nano‐zinc oxide on tropane alkaloid production, h6h gene transcription and antioxidant enzyme activity in Hyoscyamus reticulatus L. hairy roots

“…In addition to producing defensive responses, elicitation can induce the accumulation of secondary metabolites, including tropane alkaloids [44]. Elicitation with ZnO caused an increase in hyoscyamine and scopolamine content at all exposure times compared to the control hairy roots, although the maximum level of alkaloids was detected with 100 mg/L ZnO.…”

Biochemical characterization reveals the functional divergence of two tropinone reductases from Przewalskia tangutica

“…Suppression of SlTRII has been used as an alternative method to enhance the hyoscyamine production in Solanaceae species. It has been demonstrated that overexpression of TRI can increase the content of hyoscyamine and scopolamine , suggesting that PtTRI is a potential candidate for modifying the TAs biosynthetic pathway on TRI to obtain high yielding scopolamine medicinal plants.…”