Ng the genes involved within the cholesterol uptake (mce4 genes; GMFMDNLD_02935 to 02949), steroidal side-chain degradation (GMFMDNLD_02968 to 02992 and GMFMDNLD_03076 to 03082), androgenic A/B-ring degradation (GMFMDNLD _03002 to 03014 and GMFMDNLD _03061 to 03069) and C/BCRP Storage & Stability D-ring degradation (GMFMDNLD _03019 to 03022 and GMFMDNLD _03039 to 03047) (Dataset S1). Amongst them, we identified the ipdAB [GMFMDNLD_03020 (ipdA) and _03021 (ipdB)] and echA20 (GMFMDNLD_03019) accountable for steroidal C- and D-rings degradation respectively (Fig. two). Moreover, the observation in the temporary HIP production and subsequent depletion within the E1-fed strain B50 cultures is consistent with the presence of HIP-CoA ligase gene fadD3 (GMFMDNLD_03043) responsible for the HIP activation in the strain B50 chromosome. Functional validation of actinobacterial aedA and aedB in oestrogenic A-ring degradation Subsequent, we aimed to confirm the function of the putative oxygenase genes aedA and aedB involved indegradation pathway in strain B50, strain 50 resting cells ( 109 cells ml) were aerobically incubated with E1 (10 mg l), sampled hourly and extracted making use of ethyl acetate, as well as the metabolite profile was analysed via UPLC PCI RMS. The metabolite profile evaluation revealed at least 4 E1-derived metabolites, which includes PEA and HIP in the established 4,5-seco pathway (Table S2). The retention time with the detected metabolites in the UPLC and their HRMS behaviours was identical to those on the corresponding genuine standards (Fig. 1B and Table S2), suggesting that strain B50 adopts the 4,5-seco pathway to degrade oestrogens. Moreover, we observed the accumulation of each PEA and HIP within the supernatants of strain B50 cultures within a dose-dependent JAK MedChemExpress manner according to added E1 (Fig. 1C). Identification in the oestrogen-degrading genes via comparative genomic analysis Metabolite profile analysis suggested that strain B50 degrades oestrogens through the 4,5-seco pathway established in proteobacteria. Nonetheless, the homologous genes involved in the proteobacterial four,5-seco pathway had been not annotated in the strain B50 genome, most likely as a consequence of distant phylogeny involving proteobacteria and actinobacteria. Thus, we compared the strain B50 genome for the genomes from the reported oestrogen-degrading actinobacteria within the database. By way of the comparative genomic evaluation, we identified a putative oestrogen-degrading gene cluster (GMFMDNLD _05329 to 05349; Dataset S1) on a circular genetic element (i.e., megaplasmid; GMFMDNLD three) of strain B50 (accession no.: WPAG00000000.1), that is also present in the genome of oestrogen-degrading Rhodococcus sp. strain DSSKP-R-001 (Zhao et al., 2018), but not in other Rhodococcus members incapable of degrading oestrogen. Additionally, the two homologous oestrogen-degrading gene clusters are each situated on their megaplasmids (Fig. two; Dataset S1). Amongst them, the gene cluster (aed, actinobacterial oestrogen degradation) of strain B50 is surrounded by a transcriptional regulator and a transposase gene (GMFMDNLD _05329 and 05330). Inside the putative oestrogen-degrading gene cluster, GMFMDNLD _05338 encodes a putative meta-cleavage enzyme, which most likely functions because the 4-hydroxyestrone four,5-dioxygenase (AedB). In addition, GMFMDNLD_05336 encodes a member with the cytochrome P450 protein family members and therefore most likely functions as an oxygen-dependent oestrone 4hydroxylase (AedA). The nucleotide sequences of 16S rRNA, and the aedA and aedB genes of strain B50 are shown in Appendic.
