De Novo Synthesis of Anticholinergic Hyoscyamine and Scopolamine in Nicotiana benthamiana Based on Elucidating Tropane Alkaloid Biosynthetic Pathway of Anisodus luridus

Abstract

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.