Efficient one-step biocatalytic multienzyme cascade strategy for direct conversion of phytosterol to C17-hydroxylated steroids.

Steroidal 17-carbonyl reduction is crucial to the production of natural bioactive steroid medicines, boldenone (BD) is one of the important C17-hydroxylated steroids. Although efforts have been made to produce BD through biotransformation, the challenge of complex transformation process, high substrates cost, and low catalytic efficiencies have yet to be mastered. Phytosterol (PS) is the most widely accepted substrate for the production of steroid medicines due to its similar foundational structure and ubiquitous sources. 17β-Hydroxysteroid dehydrogenase (17βHSD) and its native electron donor play significant roles in the 17β-carbonyl reduction reaction of steroids. In this study, we bridged 17βHSD with a cofactor regeneration strategy in Mycobacterium neoaurum to establish a one-step biocatalytic carbonyl reduction strategy for efficient biosynthesis of BD from PS for the first time. After investigating different intracellular electron transfer strategies, we rationally designed the engineered strain with co-expression of 17βhsd and glucose-6-phosphate dehydrogenase (G6PDH) gene in M. neoaurum. With establishment of an intracellular cofactor regeneration strategy, the ratio of [NADPH]/[NADP+] was maintained at a relatively high level, the yield of BD increased from 17% (in MNR M3M-ayr1S.c) to 78% (in MNR M3M-ayr1&g6p with glucose supplementation), and the productivity was increased by 6.5 times. Furthermore, under the optimal glucose supplementation condition, the yield of BD reached 82%, which is the highest yield reported by transformation from PS with one-step. This study demonstrated an excellent strategy for production of many other valuable carbonyl reduction steroidal products from natural cheap raw materials. Importance Steroid C17-carbonyl reduction is one of the important transformations for the production of valuable steroidal medicines or intermediates for further synthesis of steroidal medicines, but it remains a challenge through either chemical or biological synthesis. Phytosterol can be obtained from low-cost residue of waste natural materials, and it is preferred as the economical and applicable substrate for steroid medicines production by Mycobacterium. This study explored a green and efficient one-step biocatalytic carbonyl reduction strategy for direct conversion of phytosterol to C17-hydroxylated steroids by bridging 17β-Hydroxysteroid dehydrogenase with a cofactor regeneration strategy in Mycobacterium neoaurum. This work has practical value for the production of many valuable hydroxylated steroids from natural cheap raw materials.