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 anticancer 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.

A streamlined high throughput screening method for the Mycobacterium neoaurum mutants with expected yield of biotransformation derivatives from sterols

Steroidal drugs have wide indications such as anti-inflammation, anti-tumor, endocrine regulation,fertility management. Phytosterol is the main starting materials for the industrial synthesis of steroid drugs. Microbial transformation of phytosterol is a simple and environmentally friendly process. Efficient microbial strains for industrial phytosterol transformation are critical for the commercial success. To this end, a 96-well plate based method was developed to discriminate the mixture of 4-androstene-3,17-dione, androsta-1,4-diene-3,17-dione, and bisnoraldehyde, with different ratio of the 3 components in the mixture, which mimics the sterol bioconversion products by using the Mycobacterium neoaurum. The M. neoaurum bioconversed broth test using phytosterol as substrate also found that the spectrum methodology can evaluate the relative content of different compounds. The method is practical, high throughput and can replace the conventional HPLC-based assay for rapid selection of desired microbial strains.