Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3β-tigloyloxytropane

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3 α - and 3 β -tropanol. Although littorine synthase is revealed to be responsible for 3 α -tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3 β -tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna , 3 β -tigloyloxytropane synthase (TS), catalyzes 3 β -tropanol and tigloyl-CoA to form 3 β -tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3 β -tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3 β -tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3 β -tigloyloxytropane from tiglic acid and 3 β -tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3 β -tigloyloxytropane.