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Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6β-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Atropa belladonna is one of the most important plant sources for producing pharmaceutical tropane alkaloids (TAs). T1 progeny of transgenic A. belladonna, in which putrescine N-methyltransferase (EC. 2.1.1.53) from Nicotiana tabacum (NtPMT) and hyoscyamine 6β-hydroxylase (EC. 1.14.11.14) from Hyoscyamus niger (HnH6H) were overexpressed, were established to investigate TA biosynthesis and distribution in ripe fruits, leaves, stems, primary roots and secondary roots under field conditions. Both NtPMT and HnH6H were detected at the transcriptional level in transgenic plants, whereas they were not detected in wild-type plants. The transgenes did not influence the root-specific expression patterns of endogenous TA biosynthetic genes in A. belladonna. All four endogenous TA biosynthetic genes (AbPMT, AbTRI, AbCYP80F1 and AbH6H) had the highest/exclusive expression levels in secondary roots, suggesting that TAs were mainly synthesized in secondary roots. T1 progeny of transgenic A. belladonna showed an impressive scopolamine-rich chemotype that greatly improved the pharmaceutical value of A. belladonna. The higher efficiency of hyoscyamine conversion was found in aerial than in underground parts. In aerial parts of transgenic plants, hyoscyamine was totally converted to downstream alkaloids, especially scopolamine. Hyoscyamine, anisodamine and scopolamine were detected in underground parts, but scopolamine and anisodamine were more abundant than hyoscyamine. The exclusively higher levels of anisodamine in roots suggested that it might be difficult for its translocation from root to aerial organs. T1 progeny of transgenic A. belladonna, which produces scopolamine at very high levels (2.94-5.13 mg g(-1)) in field conditions, can provide more valuable plant materials for scopolamine production., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Atropa belladonna is officially deemed as the commercial plant to produce scopolamine in China. In this study we report the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which encode the upstream key enzyme putrescine N-methyltransferase (PMT) and the downstream key enzyme hyoscyamine 6β-hydroxylase (H6H), respectively, in transgenic herbal plants Atropa belladonna. Analysis of gene expression profile indicated that both pmt and h6h were expressed at a higher level in transgenic lines, which would be favorable for biosynthesis of scopolamine. High-performance liquid chromatography result suggested that transgenic lines could produce higher accumulation of scopolamine at different levels compared with wild-type lines. Scopolamine content increased to 7.3-fold in transgenic line D9 compared with control lines. This study not only confirms that co-overexpression of pmt and h6h is an ideal method to improve the biosynthetic capacity of scopolamine but also successfully cultivates the transgenic line D9, which significantly enhanced the scopolamine accumulation. Our research can serve as an alternative choice to provide scopolamine resources for relative industry, which is more competitive than conventional market., (Copyright © Physiologia Plantarum 2011.)

Tropane alkaloids (TAs) are large secondary metabolite alkaloids that find extensive applications in the synthesis of antidotes, anesthetics, antiemetics, motion sickness drugs, and antispasmodics. The current production method primarily depends on extraction from medicinal plants of the Solanaceae family. Elicitation, as a highly effective biotechnological approach, offers significant advantages in augmenting the synthesis of secondary metabolites. The advantages include its simplicity of operation, low cost, and reduced risk of contamination. This review focuses on the impact of elicitation on the biosynthesis of TAs from three aspects: single-elicitor treatment, multiple-elicitor treatment, and the combination of elicitation strategy with other strategies. Some potential reasons are also proposed. Plant hormones and growth regulators, such as jasmonic acid (JA), salicylic acid (SA), and their derivatives, have been extensively employed in the separate elicitation processes. In recent years, novel elicitors represented by magnetic nanoparticles have emerged as significant factors in the investigation of yield enhancement in TAs. This approach shows promising potential for further development. The current utilization of multi-elicitor treatment is constrained, primarily relying on the combination of only two elicitors for induction. Some of these combinations have been found to exhibit synergistic amplification effects. However, the underlying molecular mechanism responsible for this phenomenon remains largely unknown. The literature concerning the integration of elicitation strategy with other strategies is limited, and several research gaps require further investigation. In conclusion, the impact of various elicitors on the accumulation of TAs is well-documented. However, further research is necessary to effectively implement elicitation strategies in commercial production. This includes the development of stable bioreactors, the elucidation of regulatory mechanisms, and the identification of more potent elicitors. [ABSTRACT FROM AUTHOR]

Hyoscyamine and scopolamine, belonging to medicinal tropane alkaloids (MTAs), are potent anticholinergic drugs. Their industrial production relies on medicinal plants, but the levels of the two alkaloids are very low in planta. Engineering the MTA's production is an everlasting hot topic for pharmaceutical industry. With understanding the MTA's biosynthesis, biotechnological approaches are established to produce hyoscyamine and scopolamine in an efficient manner. Great advances have been obtained in engineering MTA's production in planta. In this review, we summarize the advances on the biosynthesis of MTAs and engineering the MTA's production in hairy root cultures, as well in plants. The problems and perspectives on engineering the MTA's production are also discussed. [ABSTRACT FROM AUTHOR]

Summary: Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood.Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP‐glycosyltransferase (UGT1) and littorine synthase (LS).UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His‐tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co‐expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added.This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology. [ABSTRACT FROM AUTHOR]

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6β-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost. [ABSTRACT FROM AUTHOR]

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6 β -hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer. • Biosynthesis of TAs was improved under low nitrogen (LN) condition. • LN treatment induced the expression of LNIR, a bHLH transcription factor in Atropa belladonna. • LNIR positively regulated the biosynthesis of TAs by promoting the expression of TAs biosynthesis genes. [ABSTRACT FROM AUTHOR]

Atropa belladonna is widely utilized for producing anticholinergic tropane alkaloids (TAs). Of them, scopolamine is the most valuable component due to its potent pharmacological effects and minimal side effects. However, the content of scopolamine in planta is extremely low, emphasizing the need to cultivate high-yield varieties of A. belladonna. Here it is reported that the genes encoding the rate-limiting upstream enzyme ornithine decarboxylase from A. belladonna (AbODC) and downstream enzyme hyoscyamine 6 β -hydroxylase from Hyoscyamus niger (HnH6H) were simultaneously overexpressed to genetically modify A. belladonna plants. Metabolite analysis in the AbODC overexpressing plants showed that the levels of hyoscyamine and anisodamine in both the roots and leaves of A. belladonna were significantly increased. However, there was no significant effect on the accumulation of scopolamine. On the other hand, overexpressing HnH6H alone was able to increase the contents of scopolamine to some extent. When AbODC and HnH6H were co-expressed, there was a greater increase in the contents of scopolamine in both roots and leaves, especially in the leaves, where the average content of scopolamine reached 6.67 mg/g dry weight (DW). Moreover, the increasing expression levels of ODC in transgenic plants greatly enhanced the biosynthesis of polyamines, thereby enhancing the cold tolerance trait of A. belladonna plants. These findings highlight the importance of co-overexpressing AbODC and HnH6H in A. belladonna and provide an effective promising approach to developing new varieties of A. belladonna with not only a high yield of scopolamine but also a high tolerance of cold stress. • AbODC overexpression boosts hyoscyamine yield and cold tolerance in A. belladonna. • HnH6H overexpression promotes scopolamine yield in A. belladonna. • Plants co-expressing ODC and H6H show cold tolerance and higher scopolamine yield. [ABSTRACT FROM AUTHOR]

Hyoscyamine and scopolamine are two pharmaceutical tropane alkaloids (TAs) whose biosynthesis is dependent on tropinone and littorine. The biosynthesis process of tropinone and littorine was elucidated only very recently, with the discoveries of four novel TAs biosynthesis genes, namely type III polyketide synthase (PYKS), tropinone synthase (CYP82M3), phenyllactate UDP-glycosyltransferase (UGT1), and littorine synthase (LS). Yet their values in engineering TAs production remain unknown. In this study, their roles in engineering TAs' production were studied via gene overexpression methods applied to root cultures of Atropa belladonna. PYKS or CYP82M3 overexpressed separately did not or slightly affected the biosynthesis of tropine, and had no effect on the accumulation of TAs. Co-overexpression of PYKS and CYP82M3 led to much higher levels of tropinone and tropine than those produced by the overexpression of either PYKS or CYP82M3. Overexpression of either UGT1 or LS significantly elevated the production of littorine, hyoscyamine, anisodamine, and scopolamine. Higher levels of these TAs were produced when UGT1 and LS were overexpressed together. Finally, when the four TA biosynthesis genes were overexpressed together, the highest production of TAs was obtained, indicating that synergistic effect did significantly augment TAs' production. [Display omitted] • Co-overexpressing PYKS and CYM82M3 increased tropinone and tropine contents. • Co-overexpressing UGT1 and LS increased hyoscyamine and scopolamine contents. • Four genes co-overexpressed, the highest levels of tropane alkaloids were obtained. • Tropinone and littorine biosynthesis are two rate-limiting steps. [ABSTRACT FROM AUTHOR]

• Glyphosate is used for screening transgenic plants of Atropa belladonna. • Homozygous transgenic plants overexpressing HnH6H and G2-EPSPS are obtained. • Homozygous transgenic plants have high-yield scopolamine and glyphosate resistance. Atropa belladonna , one of the most important medicinal plants in China, is used to produce anticholinergic tropane alkaloids. Weeds severely reduce the agricultural productivity of A. belladonna in fields, and the low content of high-value scopolamine in planta limits its economic value. In this study, homozygous lines of A. belladonna , with glyphosate resistance and high yield of scopolamine, were developed using metabolic engineering technology for the first time. Two homozygous transgenic lines (T2GH03 and T2GH06), harboring G2-EPSPS and HnH6H , were established and tested in the field. Glyphosate resistance assays demonstrated that the two homozygous transgenic lines could resist commercial recommended dose of glyphosate. Alkaloid analysis showed that the transgenic lines produced more scopolamine than wild-type plants. T2GH03 and T2GH06 produced scopolamine at the levels of 5.45 mg/g dry weight (DW) and 7.04 mg/g DW respectively in leaves, and 1.06 mg/g DW to 1.07 mg/g DW respectively in roots. Compared with wild-type plants, the scopolamine contents of homozygous transgenic lines increased by over 10 folds in leaves and nearly 5 folds in roots. To sum up, Atropa belladonna's new varieties with glyphosate resistance and high-yield scopolamine are developed, which are highly valuable for industrial production of scopolamine. [ABSTRACT FROM AUTHOR]

• A root-expressed SAUR gene (AbSAUR1) was cloned from Atropa belladonna. • Overexpression of AbSAUR1 significantly increased the biomass of Atropa belladonna. • Overexpression of AbSAUR1 did not reduce biosynthesis of tropane alkaloids. • The yield of TAs was significantly improved in AbSAUR1 -overexpressing plants. Tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are widely used as anticholinergic drugs in the clinic. At present, the production of TAs is completely dependent on extraction from medicinal plants of Solanaceae. Atropa belladonna , commonly known as deadly nightshade, is a major commercial source of TAs. Previous studies have focused on increasing TA content by overexpressing TA biosynthesis genes. However, research on increasing TA yield by increasing the biomass of A. belladonna has not been reported. In this study, a Small Auxin Up-regulated RNA (SAUR) gene from A. belladonna (AbSAUR1) had been cloned for the first time. Based on heat map and tissue expression analysis, it was demonstrated that AbSAUR1 was specifically expressed in roots. Compared to control plants, transgenic lines of A. belladonna overexpressing AbSAUR1 showed a number of phenotypic changes, including increased plant height, thickened stems, increased number of branches, leaves and roots. Meanwhile, the biomass of AbSAUR1 -overexpressing plants also increased significantly, with fresh weight and dry weight being 3.79- and 3.20-fold higher than control plants, respectively. On average, there was no significant change in TA content, but the production of hyoscyamine, anisodamine and scopolamine was slightly enhanced. The overall yield of TAs was significantly improved, reaching 3.55 times that of the control. In summary, the AbSAUR1 gene can be employed as an effective molecular tool to increase the yield of TAs by increasing the biomass of A. belladonna. [ABSTRACT FROM AUTHOR]