Efficient One-Step Biocatalytic Multienzyme Cascade Strategy for Direct Conversion of Phytosterol to C-17-Hydroxylated Steroids

Abstract

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- ayr1 S.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.

Whole genome sequencing and analysis of selenite-reducing bacteria Bacillus paralicheniformis SR14 in response to different sugar supplements

“…Fructokinase, a key enzyme involved in the process of carbohydrate degradation of Bacillus subtilis (Nocek et al 2011 ), Corynebacterium glutamicum (Peng et al 2011 ), and Zobellia galactanivorans (Groisillier et al 2015 ), turned out to be significantly upregulated in glucose-supplement group of SR14. In addition, glucose-6-phosphate 1-dehydrogenase participated in in NADPH-related glucose metabolism in prokaryotes, such as Escherichia coli (Xia et al 2015 ), Mycolicibacterium neoaurum (Tang et al 2021 ), and Cyanobacteria (Tiruveedula and Wangikar 2017 ). Glucose-6-phosphate 1-dehydrogenase and fructokinase mutant strain of SR14 would be constructed for further experiment.…”

One-pot biosynthesis of 7β-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum

“…However, it was not practical to apply the above catalytic reaction to industrial production due to the expensive substrates. Mycolicibacterium can produce steroid precurosrs via the nucleus oxidation and the side chain degradation of PS, a cheap and available industrial waste [ 36 , 37 ]. The biotransformation from PS to AD was carried out by multi-catabolic enzymes, and the accumulation of AD in Mycolicibacterium can be realized by the method of metabolic engineering [ 30 , 38 ].…”

One-Step Biosynthesis Of 7β-Hydroxy-Androst-4-Ene-3,17-Dione From Phytosterols By Cofactor Regeneration System In Engineered Mycolicibacterium Neoaurum

“…However, it was not practical to apply the above catalytic reaction to industrial production due to the expensive substrates. Mycolicibacterium can produce steroid precurosrs via the nucleus oxidation and the side chain degradation of PS, a cheap and available industrial waste [36,37]. The biotransformation from PS to AD was carried out by multi-catabolic enzymes, and the accumulation of AD in Mycolicibacterium can be realized by the method of metabolic engineering [30,38].…”