Tropane alkaloids (TAs) are widely distributed in the Solanaceae, while some important medicinal tropane alkaloids (mTAs), such as hyoscyamine and scopolamine, are restricted to certain species/tribes in this family. Little is known about the genomic basis and evolution of TAs biosynthesis and specialization in the Solanaceae. Here, we present chromosome-level genomes of two representative mTAs-producing species: Atropa belladonna and Datura stramonium. Our results reveal that the two species employ a conserved biosynthetic pathway to produce mTAs despite being distantly related within the nightshade family. A conserved gene cluster combined with gene duplication underlies the wide distribution of TAs in this family. We also provide evidence that branching genes leading to mTAs likely have evolved in early ancestral Solanaceae species but have been lost in most of the lineages, with A. belladonna and D. stramonium being exceptions. Furthermore, we identify a cytochrome P450 that modifies hyoscyamine into norhyoscyamine. Our results provide a genomic basis for evolutionary insights into the biosynthesis of TAs in the Solanaceae and will be useful for biotechnological production of mTAs via synthetic biology approaches.

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.

Artemisinin is biosynthesized in Artemisia annua and widely used for the treatment of malaria. Abscisic acid (ABA)‐responsive kinase 1 (AaAPK1), a member of the SnRK2 family, is involved in the regulation of artemisinin biosynthesis through the phosphorylation of AabZIP1, which directly transactivates genes involved in artemisinin biosynthesis. Through diverse assays – including yeast two‐hybrid and bimolecular fluorescence complementation assays – we report that the ABA‐responsive protein phosphatase AaPP2C1 physically interacts with AaAPK1. In addition, phos‐tag mobility shift assays indicate that AaPP2C1 dephosphorylates AaAPK1. Moreover, dual‐luciferase assays demonstrate that the presence of AaPP2C1 reduces the transactivation of artemisinin biosynthesis genes by AabZIP1. These results further refine the post‐translational regulatory network of artemisinin biosynthesis, showing that AaPP2C1 is negatively involved through dephosphorylation of AaAPK1.