Construction and optimization of boldenone synthesis from androstenedione catalyzed by a dual-enzyme system

Boldenone is a protein-assimilating androgen steroid that can promote protein synthesis, support nitrogen storage, and enhance renal erythropoietin release. The industrial production of boldenone mainly relies on chemical synthesis, which has various problems, such as a complex conversion process, excessive byproducts, and serious environmental pollution. Therefore, it is of great significance to explore a new biosynthetic route. Recently, the enzymatic synthesis of steroid compounds has been performed more frequently than in the past. In this work, boldenone was produced from androstenedione (AD) in two steps by a dual-enzyme cascade of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and 3-sterone-Delta 1-dehydrogenase (KstD). The conversion efficiency of three isoenzymes of 17 beta-HSD from Mycobacterium sp. LY-1 for substrate AD was first analyzed. After that, the 17 beta-HSD2 with high selectivity and specificity for AD was screened and co-expressed with KstD3 in Escherichia coli BL21 to construct a dual-enzyme catalytic system. The results showed that the synthesis of boldenone from AD could be achieved by constructing the dual-enzyme expression system of 17 beta-HSD and KstD, as we determined that the concentration of boldenone reached 24.3 mg/L. To further improve the synthesis efficiency of boldenone, the expression conditions of the dual-enzyme system were optimized, and the concentration of boldenone reached 31.9 mg/L. The exploration of this route will provide a foundation for the efficient enzymatic synthesis of boldenone.