Batch and continuous flow asymmetric synthesis of anabolic-androgenic steroids via a single-cell biocatalytic Δ1-dehydrogenation and C17β-carbonyl reduction cascade

Abstract

Chemoenzymatic asymmetric synthesis of an anabolic-androgenic steroid (+)-boldenone (3) and its prodrug (+)-boldenone undecylenate (4) was accomplished starting from commercially available 4-androstene-3,17-dione (4-AD, 1) under both batch and continuous flow conditions. The key feature of the current synthesis is the construction of an enzymatic cascade process in a single Escherichia coli cell for straightforward synthesis of (+)-boldenone (3), enabled by the combined action of ReM2 (I51L/I350T), an engineered 3-ketosteroid-Δ¹-dehydrogenase (Δ¹-KstD) possessing 5-fold and 3-fold higher Δ¹-dehydrogenation activity towards 4-AD and testosterone (2b) relative to the wild-type Δ¹-KstD, respectively, and 17β-CR, a newly mined carbonyl reductase from Empedobacter stercoris showing strong C17-carbonyl reduction activity. With the optimal reaction conditions established for mutual tolerance between ReM2 and 17β-CR, complete conversion of 4-AD into (+)-boldenone was first realized in a conventional batch mode with a space-time yield (STY) of 1.09 g L⁻¹ h⁻¹. Furthermore, this single cell-catalyzed synthesis of (+)-boldenone was successfully implemented in continuous flow, achieving an order of magnitude higher STY (10.83 g L⁻¹ h⁻¹) than that for batch synthesis, which also represents the highest record for the biocatalytic synthesis of (+)-boldenone reported to date. Finally, (+)-boldenone undecylenate (4) was produced in a fully continuous flow mode with an overall yield of 75%, through telescoping the newly developed biocatalytic Δ¹-dehydrogenation/17β-carbonyl reduction cascade with the follow-up esterification reaction. The present work not only provides a concise, efficient, and sustainable avenue for the asymmetric synthesis of (+)-boldenone and (+)-boldenone undecylenate, but also showcases the effectiveness and great potential of flow biocatalysis in the production of value-added compounds.