Engineering the production of scopolamine and cold tolerance through overexpressing ODC and H6H in Atropa belladonna

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 beta-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.