Efficient production of 14α-OH-AD by engineered Mycolicibacterium neoaurum via coupled cofactor and reconstructed electron transport system

14α-hydroxy-androst-4-ene-3,17-dione (14α-OH-AD) is an important precursor for the synthesis of steroid drugs with anti-cancer and carcinolytic activity. Initially, 14α-OH-AD was mostly synthesized by whole-cell fermentation of mold fungi using androstenedione (AD) as a substrate, which had difficulties in product isolation and purification as well as problems of high production cost. In this study, the source of the 14α-hydroxylase gene was expanded. And 14α-hydroxylase genes were heterologously expressed in Mycolicibacterium neoaurum (MNR) M3ΔksdD, which enabled the one-step biotransformation from the cheap substrate phytosterols (PS) to 14α-OH-AD, reducing the difficulty of product purification and production cost. What is more, to alleviate the problem of poor activity of 14α-hydroxylase, the 14α-hydroxylase gene was co-expressed with the electron transport chain element genes and the coenzyme regeneration genes, and a superior engineered strain MNR M3ΔksdD/pMV261-14α-G6PDH was obtained. Finally, the transformation conditions were optimized for the transformation of PS by the engineered strain. The molar yield of 14α-OH-AD reached to 60.4 ± 2.3% (about 0.22 g/L productivity). This study investigated for the first time the effects of the tandem electron transport chain element genes and the tandem coenzyme regeneration genes on the 14α-hydroxylation reaction, providing a theoretical basis for the industrial production of 14α-OH-AD.