Continuous Flow Synthesis of Epinephrine through Modularized Multienzyme Cascades

Abstract

Heavily used as an emergency medicine, the natural hormone epinephrine is still primarily produced via chemical synthesis followed by chiral resolution, with efficient biomanufacturing yet to be developed. Here, we designed an in vitro multienzyme route for epinephrine synthesis with high chiral purity. The route comprises four enzymes arranged in two modules. The first module for norepinephrine synthesis consists of an well-engineered L-threonine aldolase achieving the reported highest diastereoselectivity (97.3% diastereomeric excess) for droxidopa synthesis, and a tyrosine decarboxylase that drives the reversible aldol reaction to completion. The second module for epinephrine synthesis comprises a phenylethanolamine N-methyltransferase and a halogen methyltransferase, enabling efficient N-methylation of norepinephrine and in situ S-adenosylmethionine regeneration. These enzymes were immobilized on commercial resin carriers and packed into modular reactors for continuous-flow synthesis, achieving epinephrine production with a space-time yield of 2.17 g L⁻¹ h⁻¹ and 97.1% enantiomeric excess.