Fei Qiu

Tropane alkaloids (TA) constitute a class of plant specialized metabolites with important pharmaceutical applications, including the anticholinergic agents hyoscyamine and scopolamine and the local anesthetic cocaine. Over the past decade, advances in genomics, structural biology, and synthetic biology have substantially revised our understanding of TA biosynthesis, leading to the identification of numerous key biosynthetic enzymes and evolutionary mechanisms. This review comprehensively summarizes current knowledge of TA biosynthesis from precursor formation to structurally diverse end products. We describe the pathway from putrescine to tropinone, the stereoselective metabolic branching mediated by Tropinone Reductases, and the downstream biosynthesis of medicinal tropane alkaloids, calystegines, and cocaine. Particular emphasis is placed on recent discoveries concerning catalytic mechanisms, structural determinants of substrate specificity, metabolic compartmentalization, and the convergent evolution of TA biosynthesis in Solanaceae and Erythroxylaceae. We further integrate advances in genomics, evolutionary biology, and metabolic engineering to highlight emerging strategies for microbial production and pathway redesign. By providing a comprehensive synthesis of recent progress and critical perspectives on unresolved questions, this review offers an updated framework for understanding TA biosynthesis and supports future research in plant specialized metabolism, synthetic biology, and natural product engineering.

Anisodus luridus, a perennial herb belonging to the genus Anisodus of the Solanaceae family, is an important Tibetan medicinal plant that produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine. Its high yield of hyoscyamine makes A. luridus a valuable plant source for commercially producing TAs. In this study, we conduct homologous gene research across transcriptome data of different tissues together with functionally tested sequences in Atropa belladonna as a reference and identify 13 candidate genes for TAs biosynthesis in A. luridus. The results show that these 13 TAs biosynthesis genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to A. belladonna, suggesting that the two species may share the same biosynthetic pathway for TAs biosynthesis. Furthermore, scopolamine was detected in Nicotiana benthamiana leaves when these 13 enzymes were co-expressed in N. benthamiana, which confirmed that these 13 TAs biosynthesis genes are involved in the biosynthesis of TAs. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also realize the de novo synthesis of TAs in N. benthamiana for the first time. It is now possible to make N. benthamiana a potential source for TAs production through synthetic biology techniques.

Anthocyanins are water-soluble flavonoid pigments that play a crucial role in plant growth and metabolism. They serve as attractants for animals by providing plants with red, blue, and purple pigments, facilitating pollination and seed dispersal. The fruits of solanaceous plants, tomato (Solanum lycopersicum) and eggplant (Solanum melongena), primarily accumulate anthocyanins in the fruit peels, while the ripe fruits of Atropa belladonna (Ab) have a dark purple flesh due to anthocyanin accumulation. In this study, an R2R3-MYB transcription factor (TF), AbMYB1, was identified through association analysis of gene expression and anthocyanin accumulation in different tissues of A. belladonna. Its role in regulating anthocyanin biosynthesis was investigated through gene overexpression and RNA interference (RNAi). Overexpression of AbMYB1 significantly enhanced the expression of anthocyanin biosynthesis genes, such as AbF3H, AbF3′5′H, AbDFR, AbANS, and Ab3GT, leading to increased anthocyanin production. Conversely, RNAi-mediated suppression of AbMYB1 resulted in decreased expression of most anthocyanin biosynthesis genes, as well as reduced anthocyanin contents in A. belladonna. Overall, AbMYB1 was identified as a fruit-expressed R2R3-MYB TF that positively regulated anthocyanin biosynthesis in A. belladonna. This study provides valuable insights into the regulation of anthocyanin biosynthesis in Solanaceae plants, laying the foundation for understanding anthocyanin accumulation especially in the whole fruits of solanaceous plants.

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.

Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis.

Rhodiola crenulata is a Tibetan native herbal plant belonging to the family of Crassulaceae, which produces the pharmaceutical icariside D2 with the activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. In this study, we functionally characterized a novel UDP-glycosyltransferase (RcUGT1) that converted tyrosol to specifically produce icariside D2 from R. crenulata at molecular and biochemical levels. RcUGT1 was highly expressed in flowers and roots, while the icariside D2 content was much higher in stems than that in other organs, suggesting the potential translocation of icariside D2 from flowers and roots to stems. The high production of icariside D2 in stems provided a reasonable suggestion to farmers to harvest stems instead of roots for icariside D2 production. Enzymatic assays of recombinant RcUGT1 indicated that it converted tyrosol to specifically form icariside D2, with the values of Km 0.97±0.10 mM, Vmax 286±8.26 pKat/mg, Kcat 0.01552 s⁻¹, and Kcat/Km 159.55 s⁻¹ M⁻¹. Functional identification of RcUGT1 facilitated the icariside D2 production through metabolic engineering in plants or synthetic biology in microbes.