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Nootkatone is a valuable sesquiterpene widely used in the food, fragrance, and flavor industries. Its price is very high due to its limited production in grapefruit peels or Alaska cypress heartwoods. Chemical synthesis of nootkatone uses heavy metals, highly flammable compounds, and strong oxidants, which cause severe damage to the environment. In this study, nootkatone is synthesized in Artemisia annua , using synthetic biology methods. Engineered Artemisia annua coexpressing valencene synthase (VS) and valencene oxidase (VO) in the cytosol produced nootkatone ranging from 0.89 to 8.52 μg/g fresh weight (FW). Furthermore, transgenic Artemisia annua coexpressing farnesyl diphosphate synthase (FPS), VS, and VO in plastids produced nootkatone ranging from 12.11 to 47.80 μg/g FW. These results indicated that engineering nootkatone biosynthesis in plastids was superior to that in the cytosol. Meanwhile, artemisinin production was unaltered in nootkatone-producing Artemisia annua . Our study developed a green approach for producing nootkatone in Artemisia annua with great market potential.

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 6 β -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.

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 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.)

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]

Artemisia annua , commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A . annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A . annua , which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A . annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A . annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A . annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin. [ABSTRACT FROM AUTHOR]

N -methylputrescine is the precursor of nicotine and pharmaceutical tropane alkaloids such as hyoscyamine. Putrescine N -methyltransferase (PMT) catalyzes the N -methylation of putrescine to form N -methylputrescine. While the role of PMT in nicotine biosynthesis is clear, knowledge of PMT in the biosynthesis of tropane alkaloids (TAs) and the regulation of polyamines remains limited. We characterized a PMT gene from Hyoscyamus niger , designated HnPMT that was specifically expressed in roots, especially in the secondary roots and dramatically induced by methyl jasmonate (MeJA). The GUS gene was specifically expressed in Arabidopsis roots or in the vascular tissues, including pericycles and endodermis, of the H. niger hairy root cultures, when it was driven by the 5′-flanking promoter region of HnPMT . The recombinant HnPMT was purified for enzymatic assays. HnPMT converted putrescine to form N -methylputrescine, as confirmed by LC-MS. The kinetics analysis revealed that HnPMT had high affinity with putrescine but low catalytic activity, suggesting that it was a rate-limiting enzyme. When HnPMT was suppressed in the H. niger plants by using the VIGS approach, the contents of N -methylputrescine and hyoscyamine were markedly decreased, but the contents of putrescine, spermidine and a mixture of spermine and thermospermine were significantly increased; this suggested that HnPMT was involved in the biosynthesis of tropane alkaloids and played a competent role in regulating the biosynthesis of polyamines. Functional identification of HnPMT facilitated the understanding of TA biosynthesis and thus implied that the HnPMT-catalyzed step might be a target for metabolic engineering of the TA production in H. niger . [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • A tyrosine aminotransferase gene (RcTAT) was cloned from Rhodiola crenulata. • RcTAT catalyzes the formation of 4-hydroxyphenylpyruvate from tyrosine. • Suppression of RcTAT significantly enhances the salidroside production. Rhodiola crenulata is a traditional Tibetan medicinal plant mainly distributed in high altitude areas of Tibet. As the main extract of R. crenulata , salidroside has important medicinal activities in enhancing the body's immunity and resisting microwave radiation by influencing cell metabolism. Tyrosine is the starting amino acid precursor for salidroside biosynthesis. As an aromatic amino acid, tyrosine participates not only in salidroside biosynthesis, but also in other metabolite biosynthesis which might compete against salidroside biosynthetic pathway. In this study, we functionally identified a tyrosine aminotransferase in R. crenulata (RcTAT) that could convert tyrosine into 4-hydroxyphenylpyruvate (4-HPP). The Km and Vmax values of RcTAT for tyrosine were respectively 0.21 mM and 0.17 μmol/min.mg under 9.0 pH and 39 °C. RNAi-mediated suppression of RcTAT expression markedly increased salidroside production in hairy root culture of R. crenulata. In summary, RcTAT is a key gene involved in the metabolic pathway that competes against salidroside biosynthesis, and suppressing its expression is a promising way to elevate salidroside production in planta. [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]