A Pull‐Block Metabolic Engineering Strategy for High‐Yield and High‐Purity Hyoscyamine Production in Atropa belladonna

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.