Production of 11α‐hydroxysteroids from sterols in a single fermentation step by Mycolicibacterium smegmatis

11α‐hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α‐hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α‐hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4‐androstene‐3,17‐dione (AD), 1,4‐androstadiene‐3,17‐dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH‐AD and 11αOH‐ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α‐hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end‐products.