Unraveling the 17β-Estradiol Degradation Pathway in Novosphingobium tardaugens NBRC 16725

Abstract

We have analyzed the catabolism of estrogens in Novosphingobium tardaugens NBRC 16725, which is able to use endocrine disruptors such as 17β-estradiol, estrone, and estriol as sole carbon and energy sources. A transcriptomic analysis enabled the identification of a cluster of catabolic genes (edc cluster) organized in two divergent operons that are involved in estrogen degradation. We have developed genetic tools for this estrogen-degrading bacterium, allowing us to delete by site-directed mutagenesis some of the genes of the edc cluster and complement them by using expression plasmids to better characterize their precise role in the estrogen catabolism. Based on these results, a catabolic pathway is proposed. The first enzyme of the pathway (17β-hydroxysteroid dehydrogenase) used to transform 17β-estradiol into estrone is encoded out of the cluster. A CYP450 encoded by the edcA gene performs the second metabolic step, i.e., the 4-hydroxylation of estrone in this strain. The edcB gene encodes a 4-hydroxyestrone-4,5-dioxygenase that opens ring A after 4-hydroxylation. The initial steps of the catabolism of estrogens and cholate proceed through different pathways. However, the degradation of estrogens converges with the degradation of testosterone in the final steps of the lower catabolic pathway used to degrade the common intermediate 3aα-H-4α(3′-propanoate)7a-β-methylhexahydro-1,5-indanedione (HIP). The TonB-dependent receptor protein EdcT appears to be involved in estrogen uptake, being the first time that this kind of proteins has been involved in steroid transport.

Experimental and Genomic Evaluation of the Oestrogen Degrading Bacterium Rhodococcus equi ATCC13557

“…Comparably, the gene oecA of Sphingomonas strain KC8 and the gene of N. tardaugens NBRC 16725 encoding 17β-hydroxysteroid dehydrogenase were both outside of the main gene cluster, like the gene in this study (Chen et al, 2017;Ibero et al, 2020). Phylogenetic trees were constructed to confirm the evolutionary relationship of these gene sequences to those identified as functionally involved in oestrogen metabolism within other bacteria.…”

“…Cytochrome P450 monooxygenase may therefore be responsible for conversion of E2 to 4-OH-E2, additionally, a gene encoding cytochrome P450 monooxygenase in the genome of Novosphingobium sp. ES2-1 was expressed in the presence of E2 (Whirl-Carrillo et al, 2012;Hsiao et al, 2020;Ibero et al, 2020;Li et al, 2021). However, gene expression or gene knockout experiments are required to provide conclusive Hu et al, 2011;Liang et al, 2012;Zhang Y. et al, 2012;Ye et al, 2017;Xiong et al, 2018Xiong et al, , 2020Zhao et al, 2018;Qiu et al, 2019;Ibero et al, 2020;Pratush et al, 2020;Tian et al, 2020; Zhao et al, 2018;Hsiao et al, 2020;Ibero et al, 2020;Tian et al, 2020;Xiong et al, 2020;Li et al, 2021 evidence that these genes are responsible for the metabolism of E2 by R. equi ATCC13557.…”

“…strain AXB, N. tardaugens NBRC 16725, R. equi DSSKP-R-001, Rhodococcus sp. strain B50, and Sphingomonas strain KC8 (Hu et al, 2011;Chen et al, 2017;Zhao et al, 2018;Ibero et al, 2020;Qin et al, 2020;Tian et al, 2020), revealed that there (Avram et al, 2019) showing the ORF count per genome (A), and orthologous groups shared between the genomes of oestrogen degrading bacteria (B).…”

“…There is growing evidence that these gene clusters are evolutionarily conserved within the genomes of putative oestrogen degraders. Indeed, clusters similar to clusters I and II within the genomes of Novosphingobium tardaugens and Altererythrobacter estronivorus , and homologous open reading frames (ORFs) within cluster III were identified within the genomes of C. testosteroni, A. estronivorus , and N. tardaugens ( Chen et al, 2017 ; Ibero et al, 2020 ). Additionally, those genes involved in the 4, 5- seco pathway ( oecB and oecC ) were identified in the genomes of 12 bacteria belonging to the alphaproteobacteria or gammaproteobacteria (>40% amino acid sequence identity) ( Chen et al, 2017 ).…”

“…More recent studies have confirmed both the existence of orthologues for the above clusters and the genes on them, in addition to their function (through gene knock-out experiments) or upregulation in transcriptomic studies. Thus, orthologues of 17β-hydroxysteroid dehydrogenase ( oecA ), which encodes an enzyme that transforms E2 to E1, have been identified in N. tardaugens NBRC16725 ( Ibero et al, 2020 ), Deinococcus actinosclerus SJTR1 ( Xiong et al, 2018 ), and the actinobacteria, Rhodococcus sp. strain DSSKP-R-001 ( Zhao et al, 2018 ; Tian et al, 2020 ) and Rhodococcus sp.…”