Identification of novel catabolic genes involved in 17β‐estradiol degradation by Novosphingobium sp. ES2‐1

Abstract

Summary Novosphingobium sp. ES2‐1 is an efficient 17β‐estradiol (E2)‐degrading bacterium, which can convert E2 to estrone (E1), then to 4‐hydroxyestrone (4‐OH‐E1) for subsequent oxidative cracking. In this study, the molecular bases for this process were elucidated. Two novel monooxygenase systems EstP and EstO were shown to catalyse the oxygenation of E1 and 4‐OH‐E1, respectively. EstP was a three‐component cytochrome P450 monooxygenase system consisting of EstP1 (P450 monooxygenase), EstP2 (ferredoxin) and EstP3 (ferredoxin reductase). Ultraperformance liquid chromatography‐high resolution mass spectrometry (UPLC‐HRMS) analysis revealed that EstP catalysed the 4‐hydroxylation of E1 to produce 4‐OH‐E1. The resultant 4‐OH‐E1 was further oxidized by a two‐component monooxygenase system EstO consisting of EstO1 (flavin‐dependent monooxygenases) and EstO2 (flavin reductase). UPLC‐HRMS combined with 1H‐nuclear magnetic resonance analysis demonstrated that EstO catalysed the breakage of C9‐C10 to yield a ring B‐cleavage product. In addition, the oxygenase component genes estP1 and estO1 exhibited contrary inductive behaviours when exposed to different steroids, suggesting that EstP1‐mediated 4‐hydroxylation was E2‐specific, whereas EstO1‐mediated monooxygenation might be involved in the degradation of testosterone, androstenedione, progesterone and pregnenolone. This also implied that the mechanisms of the catabolism of different steroids by the same microorganism might be partially interlinked.

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

“…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.…”

“…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 evidence that these genes are responsible for the metabolism of E2 by R. equi ATCC13557.…”

“…More recently, gene expression experiments combined with identification of the enzymatic products of degradation revealed two monooxygenase systems EstP and EstO within the genome of Novosphingobium sp. ES2-1 ( Li et al, 2021 ). EstP is a three-component cytochrome P450 monooxygenase that catalyses the oxidation of E1, whilst EstO is a two-component monooxygenase system that catalyses the oxidation of 4-hydroxyestrone ( Li et al, 2021 ).…”

“…ES2-1 ( Li et al, 2021 ). EstP is a three-component cytochrome P450 monooxygenase that catalyses the oxidation of E1, whilst EstO is a two-component monooxygenase system that catalyses the oxidation of 4-hydroxyestrone ( Li et al, 2021 ). Further, degradation of different steroids may be interlinked as gene EstO1 which encodes EstO monooxygenase may be involved in E2, testosterone, androstenedione, progesterone, and pregnenolone degradation, however, gene EstP1 encoding EstP monooxygenase is only involved in E2 degradation ( Li et al, 2021 ).…”

“…DSSKY-A-001, Novosphingobium sp. ES2-1, and L. sphaericus DH-B01, the first step in the degradation of E2 appears to be the oxidation of the C-17 hydroxyl position by an SDR such as 17β-hydroxysteroid dehydrogenase to produce E1 ( Hu et al, 2011 ; Qin et al, 2016 ; Zheng et al, 2016 ; Chen et al, 2017 ; Ye et al, 2017 ; Xiong et al, 2018 , 2020 ; Qiu et al, 2019 ; Wang et al, 2019 ; Wu et al, 2019 ; Wang Y. et al, 2020 ; Li et al, 2021 ). However, there are examples of different E2 degradation pathways reported in bacteria, such as hydroxylation at different positions within different saturated rings (B, C, or D) to produce 4-hydroxyestradiol (4-OH-E2), keto-E1, keto-E2, or E3 ( Xuan et al, 2008 ; Kurisu et al, 2010 ; Yu et al, 2013 ).…”