Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

Summary A number of pharmaceutical steroid synthons are currently produced through the microbial side‐chain cleavage of natural sterols as an alternative to multi‐step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc 2 155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc 2 155 we have demonstrated that the MSMEG _6039 ( kshB1 ) and MSMEG _5941 ( kstD1 ) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (Ksh AB ) and a ketosteroid Δ 1 ‐dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG _6039 ( kshB1 ) gene deletion mutant of M. smegmatis MS 6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4‐androstadiene‐3,17‐dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS 6039‐5941 [ Δ MSMEG _6039 ( ΔkshB1 ) and Δ MSMEG _5941 ( ΔkstD1 )] transforms natural sterols into 4‐androstene‐3,17‐dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc 2 155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.