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.

An in vitro synthetic biology platform for emerging industrial biomanufacturing:Bottom-up pathway design

Although most in vitro(cell-free)synthetic biology projects are usually used for the purposes of fundamental research or the formation of high-value products,in vitro synthetic biology platform,which can implement complicated biochemical reactions by the in vitro assembly of numerous enzymes and coenzymes,has been proposed for low-cost biomanufacturing of bioenergy,food,biochemicals,and nutraceuticals.In addition to the most important advantage-high product yield,in vitro synthetic biology platform features several other biomanufacturing advantages,such as fast reaction rate,easy product separation,open process control,broad reaction condition,tolerance to toxic substrates or products,and so on.In this article,we present the basic bottom-up design principles of in vitro synthetic pathway from basic building blocks-BioBricks(thermoenzymes and/or immobilized enzymes)to building modules(e.g.,enzyme complexes or multiple enzymes as a module)with specific functions.With development in thermostable building blocks-BioBricks and modules,the in vitro synthetic biology platform would open a new biomanufacturing age for the cost-competitive production of biocommodities.