Bioproduction of 3,4-methylenedioxymethamphetamine and derivatives

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a recreational drug under clinical investigation for the treatment of central nervous system disorders, including post-traumatic stress disorder. Chemical synthesis of MDMA mainly relies on the use of safrole and piperonal as starting materials. We report a novel strategy integrating bioconversion and biocatalysis in the bioproduction of MDMA and other methamphetamine derivatives. For the initial step, a yeast-based bioconversion system was used to produce phenylacetylcarbinol (PAC) derivatives from ring-substituted benzaldehyde precursors, including piperonal, using variants of pyruvate decarboxylase (PDC). Among seven wildtype enzymes tested, <i>Candida tropicalis</i> PDC (CtPDC) showed the highest yield from piperonal, and a CtPDC1-I479A mutant further improved the titer. Five of sixteen ring-substituted benzaldehyde analogs (i.e., piperonal, 6-chloropiperonal, 4-acetylbenzaldehyde, 2-fluoro-4-methoxybenzaldehyde, and 2-fluoro-4-propoxybenzaldehyde) yielded corresponding PAC derivatives with yields between 20 and 70 ​%, which allowed the purification of multiple milligram quantities of each product. Three stereoselective ω-transaminases were evaluated for their ability to catalyze the transamination of PAC derivatives, with the (<i>R</i>)-selective enzyme ATA-117-Rd11 able to convert all five isolated PAC derivatives. Isolated transamination products were subsequently <i>N</i>-methylated using human phenylethanolamine <i>N</i>-methyltransferase. Chemical reduction facilitated the final production of MDMA and its analog 6-chloro-MDMA. Our work represents the first reported bioproduction method leading to MDMA and other methamphetamine derivatives, suitable for future pathway and strain optimization.