Initial step in the catabolism of cholesterol by Mycobacterium smegmatis mc²155

Abstract

The first step in the catabolism of cholesterol, i.e. the transformation of cholesterol into cholestenone, has been investigated in Mycobacterium smegmatis. In silico analysis identified the MSMEG_1604 gene encoding a putative protein similar to the ChoD cholesterol oxidase of M. tuberculosis H37Rv (Rv3409c) and the MSMEG_5228 gene coding for a protein similar to the NAD(P)-dependent cholesterol dehydrogenase/isomerase of Nocardia sp. The expression of the MSMEG_5228 gene was inducible by cholesterol whereas the expression of MSMEG_1604 gene was constitutive. When both genes were expressed in Escherichia coli only the MSMEG_5228 protein was active on cholesterol. The function of ChoD-like MSMEG_1604 protein remains to be elucidated, but it does not appear to play a critical role in the mineralization of cholesterol as a MSMEG_1604(-) mutant was not affected in the production of cholestenone. However, a MSMEG_5228(-) mutant showed a drastic reduction in the synthesis of cholestenone. The finding that this mutant was still able to grow in cholesterol, allowed us to demonstrate that the cholesterol-inducible MSMEG_5233 gene encodes an additional cholesterol dehydrogenase/isomerase similar to the AcmA dehydrogenase of Sterolibacterium denitrificans. The observation that the double MSMEG_5228-5233(-) mutant was able to grow in cholesterol suggests that in addition to these enzymes other dehydrogenase/isomerases can also catalyse the first reaction of the cholesterol degradation pathway in M. smegmatis, which is not the limiting step of the process.

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

“…Apparently, this reductase activity is not redundant in M. smegmatis and thus, it cannot be replaced by similar enzymes, as it has been demonstrated for other key enzymes of the pathway (Uhía et al ., 2011). In addition, the kshB1 gene does not appear to be critical for the degradation of the side‐chain of sterols in M. smegmatis , as suggested in Rhodococcus (van der Geize et al ., 2002a), and therefore, its absence does not impair an efficient transformation of sterols into AD or ADD in the mutant strain.…”

“…S1). M. smegmatis genomic DNA extraction was performed as described (Uhía et al ., 2011). The two fragments generated were digested with the corresponding enzymes and cloned into the plasmid pJQ200x using E. coli DH10B competent cells as described (Uhía et al ., 2011).…”

“…M. smegmatis genomic DNA extraction was performed as described (Uhía et al ., 2011). The two fragments generated were digested with the corresponding enzymes and cloned into the plasmid pJQ200x using E. coli DH10B competent cells as described (Uhía et al ., 2011). Plasmid DNA from E. coli DH10B recombinant strains was extracted using the High Pure Plasmid Purification Kit (Roche, Basel, Switzerland), according to the manufacturer's instructions.…”

Characterization of the KstR-dependent promoter of the gene for the first step of the cholesterol degradative pathway in Mycobacterium smegmatis

“…1a). Moreover, we have previously demonstrated that this gene is induced when M. smegmatis grows with cholesterol as the sole carbon and energy source, but it has a low but significant basal expression in a glycerol-containing medium (Uhía et al, 2011). The MSMEG_5228 gene is preceded by the MSMEG_5225-5226-5227 genes, which appear to form an operon encoding an unknown protein and the large and small subunit of the exoDNase VII (XseAB), respectively.…”

“…To study the regulation mediated by KstR from M. smegmatis (KstR Msm ) we selected the region preceding the MSMEG_5228 gene, one of the genes belonging to the kstR regulon, because it catalyses the first step in the catabolism of cholesterol (Uhía et al, 2011). The promoter of this gene (hereafter named P 5228 ) contains a putative KstR operator sequence (Fig.…”