Degradation of C₁₉-Steroids and Effect of Androstenedione on Gene Expression in Nocardioides simplex

Molecular mechanisms of C₁₉-steroid core degradation have been intensively studied mostly in mycolic acid rich actinobacteria, mainly in the representatives of Mycobacterium, Mycolicibacterium and Rhodococcus genera, whilst much less data evidencing functionality of the 9(10)-seco pathway in other actinobacteria was reported. In this study, degradation of androstenedione (AD), androstadienedione (ADD), testosterone (T), Δ¹-dehydrotestosterone (DT) and 9α-hydroxyandrostenedione (9α-OH-AD) by the biotechnologically relevant actinobacterium of Nocardioides simplex VKM Ac-2033D was investigated. Key intermediates of 9α-OH-AD degradation were isolated and identified as 3-hydroxy-9,10-seco-androsta-1,3,5(10)-triene-9,17-dione (3-HSA) and 3,17β-dihydroxy-9,10-seco-androsta-1,3,5(10)-trien-9-one (3,17-DHSA). The structures of the compounds were confirmed by MS, ¹H- and ¹³C-NMR. Differential gene expression on medium with glycerol and yeast extract with and without AD addition was estimated. The expression of two sets of the genes related to the 9(10)-seco pathway was increased in the presence of AD. One set comprised the genes from the KstR2-regulons in the clusters A and C, while another set included the genes without the binding sites for KstR/KstR2 (Cluster D). These genes putatively encoded 3-oxosteroid-Δ¹-dehydrogenase and 3-oxosteroid 9α-hydroxylase, as well as a group of enzymes dealing with the ring B opening (HsaA3, HsaB3, HsaC3 and HsaD3). Process of degradation of exogenous C₁₉-steroids in N. simplex proceeds via the 9(10)-seco pathway and can be controlled not only by KstR2 regulator, but also by other transcriptional factors. The results contribute to the knowledge on steroid core degradation in actinobacteria and are of significance at the development of methods for production of valuable indane compounds.