Unravelling a new catabolic pathway of C-19 steroids in Mycobacterium smegmatis

In this work, we have characterized the C-19+ gene cluster (MSMEG_2851 to MSMEG_2901) of Mycobacterium smegmatis. By in silico analysis, we have identified the genes encoding enzymes involved in the modification of the A/B steroid rings during the catabolism of C-19 steroids in certain M. smegmatis mutants mapped in the PadR-like regulator (MSMEG_2868), that constitutively express the C-19+ gene cluster. By using gene complementation assays, resting-cell biotransformations and deletion mutants, we have characterized the most critical genes of the cluster, that is, kstD2, kstD3, kshA2, kshB2, hsaA2, hsaC2 and hsaD2. These results have allowed us to propose a new catabolic route named C-19+ pathway for the mineralization of C-19 steroids in M. smegmatis. Our data suggest that the deletion of the C-19+ gene cluster may be useful to engineer more robust and efficient M. smegmatis strains to produce C-19 steroids from sterols. Moreover, the new KshA2, KshB2, KstD2 and KstD3 isoenzymes may be useful to design new microbial cell factories for the 9α-hydroxylation and/or Δ1-dehydrogenation of 3-ketosteroids.