SummaryA 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 mc2155 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 mc2155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (KshAB) and a ketosteroid Δ1‐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 MS6039 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 MS6039‐5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_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 mc2155 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.

The 4S pathway is the most studied bioprocess for the removal of the recalcitrant sulfur of aromatic heterocycles present in fuels. It consists of three sequential functional units, encoded by the dszABCD genes, through which the model compound dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) molecule. In this work, a set of synthetic dsz cassettes were implanted in Pseudomonas putida KT2440, a model bacterial "chassis" for metabolic engineering studies. The complete dszB1A1C1-D1 cassette behaved as an attractive alternative -to the previously constructed recombinant dsz cassettes -for the conversion of DBT into 2HBP. Refactoring the 4S pathway by the use of synthetic dsz modules encoding individual 4S pathway reactions revealed unanticipated traits, e.g., the 4S intermediate 2HBP-sulfinate (HBPS) behaves as an inhibitor of the Dsz monooxygenases, and oncesecreted from the cells it cannot be further taken up. That issue should be addressed for the rational design of more efficient biocatalysts for DBT bioconversions. In this sense, the construction of synthetic bacterial consortia to compartmentalize the 4S pathway into different cell factories for individual optimization was shown to enhance the conversion of DBT into 2HBP, overcome the inhibition of the Dsz enzymes by the 4S intermediates, and enable efficient production of unattainable high added value intermediates, e.g., HBPS, that are difficult to obtain using the current monocultures. IntroductionCrude oils contain undesirable contaminant molecules, such as thiophenic aromatics compounds, which have a negative impact on oil processing and pose serious environmental threats (Soleimani et al., 2007). A wide spectrum of desulfurization technologies have been developed to remove sulfur mainly from finished refinery products (Stanislaus et al., 2010). Hydrodesulfurization (HDS) treatment has proved to be the common technology of choice to reduce the level of sulfur in crude oil products.Significant environmental, technical and economic limitations have been reported in applying the HDS process (Babich and Moulijn, 2003).During the past 30 years, research to develop alternative desulfurization technologies resulted in a biotechnological strategy to eliminate sulfur from thiophenic compounds (biodesulfurization (BDS)) via serial reactions known as the 4S pathway (Gray et al., 2003;Gupta et al., 2005;Kilbane, 2006;Monticello, 2000;Nuhu, 2013; Xu et al., 2009). This pathway was firstly reported in the gram-positive bacteriumRhodococcus erythropolis IGTS8 (Gallagher et al., 1993), but the 4S pathway has been also found in other bacteria (Duarte et al., 2001;Kilbane, 2006;Mohebali and Ball, 2008).The 4S pathway provides a nondestructive oxidative process used by the cells to obtain the sulfur required for growth, which involves the transformation of dibenzothiophene (DBT), the model compound for sulfur heterocycles present in oil and refractory to HDS, into 2-hydroxybiphenyl (2HBP) and sulfite ( Fig. 1A) (Gallagher et al...

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