Abstract
The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.
Highlights
Fatty acids, which is initiated by a terminal carboxylation by Cyp12520,21
Hsd4AMN, was estimated to be 30.9 kDa and shares high similarities with its counterparts, such as 97%, 71% and 67% amino acid identities with the Hsd4As from Mycobacterium sp.VKM Ac-1815D, M. tuberculosis H37Rv and R. jostii RHA1, respectively (Fig. 2b)
Hsd4AMN and its genetic organization are highly conserved in some mycobacteria and rhodococci (Fig. 2b), indicating the conserved function of Hsd4A in these strains for sterol catabolism
Summary
Fatty acids, which is initiated by a terminal carboxylation by Cyp12520,21. The side-chain degradation is proposed to comprise three β -oxidation-like cycles, which each cycle containing successive enzymatic steps catalyzed by acyl-CoA ligases, acyl-CoA dehydrogenases, enoyl-CoA hydratases, β -hydroxyacyl-CoA-dehydrogenases and acyl-CoA thiolases[1,18]. Hsd4A was further proposed to be a β -hydroxyacyl-CoA dehydrogenase in the degradation of the sterol side chain because the N-terminal domain of 17β HSD4 could act as a D-3-hydroxyacyl-CoA dehydrogenase in degrading branched fatty acids and bile acids[15,23,24] These two possible biochemical functions have been assigned to Hsd4A, neither have been validated[1]. We attempted to investigate the physiological role of Hsd4A and its possible mechanism in the conversion of sterols to C22 steroids, based on which we tried to establish a rational strategy to modify hsd4A and other key genes to develop attractive HBCs producing strains
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