Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots

Abstract

BackgroundTropane alkaloids (TA) including anisodamine, anisodine, hyoscyamine and scopolamine are a group of important anticholinergic drugs with rapidly increasing market demand, so it is significant to improve TA production by biotechnological approaches. Putrescine N-methyltransferase (PMT) was considered as the first rate-limiting upstream enzyme while tropinone reductase I (TRI) was an important branch-controlling enzyme involved in TA biosynthesis. However, there is no report on simultaneous introduction of PMT and TRI genes into any TA-producing plant including Anisodus acutangulus (A. acutangulus), which is a Solanaceous perennial plant that is endemic to China and is an attractive resource plant for production of TA.ResultsIn this study, 21 AaPMT and AaTRI double gene transformed lines (PT lines), 9 AaPMT single gene transformed lines (P lines) and 5 AaTRI single gene transformed lines (T lines) were generated. RT-PCR and real-time fluorescence quantitative analysis results revealed that total AaPMT (AaPMT T) and total AaTRI (AaTRI T) gene transcripts in transgenic PT, P and T lines showed higher expression levels than native AaPMT (AaPMT E) and AaTRI (AaTRI E) gene transcripts. As compared to the control and single gene transformed lines (P or T lines), PT transgenic hairy root lines produced significantly higher levels of TA. The highest yield of TA was detected as 8.104 mg/g dw in line PT18, which was 8.66, 4.04, and 3.11-times higher than those of the control (0.935 mg/g dw), P3 (highest in P lines, 2.004 mg/g dw) and T12 (highest in T lines, 2.604 mg/g dw), respectively. All the tested samples were found to possess strong radical scavenging capacity, which were similar to control.ConclusionIn the present study, the co-expression of AaPMT and AaTRI genes in A. acutangulus hairy roots significantly improved the yields of TA and showed higher antioxidant activity than control because of higher total TA content, which is the first report on simultaneous introduction of PMT and TRI genes into TA-producing plant by biotechnological approaches.

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.…”

Overexpression of artemisinic aldehyde Δ11 (13) reductase gene–enhanced artemisinin and its relative metabolite biosynthesis in transgenic Artemisia annua L.

“…DBR2 is the checkpoint enzyme catalyzing artemisinic aldehyde to form dihydroartemisinic aldehyde, which is directly involved in artemisinin biosynthesis . Overexpression of key enzymes usually leads to more accumulation of target compounds . According to the previously reported results, it was reasonable to predict that overexpression of DBR2 might facilitate metabolic flux forward to artemisinin biosynthesis.…”

Increasing the Pace of New Discoveries in Tropane Alkaloid Biosynthesis

“…A separate experiment, again using this same combination of genes, was able to increase hyoscyamine (41) levels to more than 24 times that found in wild-type A. belladonna . A different combination of genes encoding PMT and TRI to make transgenic hairy root cultures of Anisodus acutangulus resulted in an overall increase in TAs that was 3-8 times higher than in the control lines (Kai et al, 2011). Increases in both hyoscyamine (41) and scopolamine (44) were observed in addition to an increase in the growth rate of the roots when compared with the no-insert transgenic controls.…”