ORIGINAL ABSTRACT

Hyoscyamine, an active tropane alkaloid primarily extracted from the medicinal plant Atropa belladonna, exhibits analgesic and sedative effects and is widely used as a precursor in pharmaceutical formulations. However, the low yield of hyoscyamine and the complex composition of tropane alkaloids in wild-type A. belladonna plants pose significant challenges for industrial production, making it imperative to modify the metabolic pathway to enhance hyoscyamine yield and purity. In this study, a pull-block metabolic engineering strategy, which enhanced hyoscyamine biosynthesis by co-overexpressing AbPYKS, AbCYP82M3, AbUGT1, AbLS, and AbHDH genes and blocked the metabolic diversion of hyoscyamine by deleting the AbH6H gene, was employed. During this process, the glyphosate resistance gene G2-EPSPS was introduced into A. belladonna. Ultimately, the engineered plants with glyphosate resistance were obtained, which showed significantly enhanced hyoscyamine yield (6.511 vs. 0.989 mg/g DW in leaves) and purity (the absence of anisodamine and scopolamine) relative to the wild-type. These results not only enrich the germplasm resources of A. belladonna but also hold promise for advancing the production of the hyoscyamine-based pharmaceuticals. Next, we will evaluate these engineered plants under field conditions for both hyoscyamine production and growth/development.