Isolation and characterization of a new highly effective 17β-estradiol-degrading Gordonia sp. strain R9

Abstract

In this report, Gordonia sp. strain R9 isolated from an enrichment culture of chicken leachate was confirmed to degrade 17β-estradiol (E2), which can also use other estrogens (estrone, estriol, and 17α-ethynylestradiol) and testosterone as sole carbon and energy sources. Optimization of growth conditions showed that Gordonia sp. strain R9 can tolerate a very wide range of temperature (4–40 °C) and pH (1.0–11.0), and is sensitive to antibiotics including kanamycin, ampicillin, chloramphenicol, and carbenicillin. Optimal culture conditions for E2 degradation were 30 °C and pH 7.0 with almost 100% degradation of E2 concentrations ranging from 50 µg/L to 5 mg/L within 24 h. The E2 intermediates so generated included estrone (E1), estratriol (E3), (3Z)-3-(3-hydroxy-3a-methyl-7-oxododecahydro-6H-cyclopenta[a]naphthalen-6-ylidene) propanoic acid and 3-hydroxy-3a-methyl-7-oxododecahydro-1H-cyclopenta[a]naphthalene-6-carboxylic acid. These results indicate that the highly effective E2-degradative ability of Gordonia sp. strain R9 merits further investigation as a candidate for large-scale estrogen biodegradation.

Introduction

Estrogens are a group of related hormones including estrone (E1), 17β-estradiol (E2) and estratriol (E3) that are produced largely by female invertebrates. Synthetic estrogens, such as 17α-ethinylestradiol (EE2), are also widely used for preventing pregnancy, and related compounds are used in agriculture, especially in the breeding industry. Consequently, effluents from wastewater treatment plants (WWTPs) and concentrated animal feeding operations (CAFOs) are important sources of estrogens, which are continuously discharged into rivers, lakes, oceans, and even groundwater worldwide (Combalbert and Hernandez-Raquet 2010; Bartelt-Hunt et al. 2011; Griffith et al. 2016; Duong et al. 2010; Liu et al. 2015). Levels of released E1, E2 and EE2 from the effluent of WWTPs have been estimated to be up to 670, 150 and 70 ng/L, respectively (Khanal et al. 2006; Wu et al. 2017), with 30–2500 ng/L estradiol in animal wastes (Chen et al. 2010). Estrogens have the highest estrogenic activity among all endocrine-disrupting compounds (EDCs), especially E2 and EE2 (Combalbert and Hernandez-Raquet 2010), the latest predicted-no-effect concentrations (PNECs) are 0.1 ng/L for EE2, 2 ng/L for E2, 6 ng/L for E1 and 60 ng/L for E3, respectively (Caldwell et al. 2012). Long-term exposure to estrogen-contaminated water can cause alterations in development, growth, and reproduction of all animals (Wang et al. 2019a, b). Increasing populations and more intensive farming have resulted in estrogen pollution becoming one of the major challenges of the new age (Whitman 2017).

Biodegradation using microorganisms is considered one of the most efficient strategies to remove estrogens, and bacterial strains capable of degrading estrogens such as Sphingomonas, Novosphingobium, Rhodococcus, Acinetobacter, Pseudomonas, and Nocardioides have been isolated from soil, rivers, oceans, and other environments (Yu et al. 2013). In general, these reported bacteria required several days to degrade estrogens at the mg/L level. For example, Xiong et al. (2018) reported that Deinococcus actinosclerus SJTR1 could degrade nearly 90% of 10 mg/L E2 in 5 days. Sphingobacterium sp. KC8 could completely degrade 3 mg/L of E2 in 5 days, while Bacillus spp. E2Y1 needed 6 days to degrade 1 mg/L (Roh and Chu 2010). Currently, however, application of the above strains for enhancing estrogen removal faces several obstacles: (1) most of the described organisms metabolize E2 at the mg/L level, but are not effective at ng/L or μg/L levels, the ones commonly detected in actual wastewater (Caldwell et al. 2012); (2) some reported organisms could not completely degrade estrogens, with E1 accumulating as an intermediate (Yu et al. 2013; Combalbert and Hernandez-Raquet 2010); (3) reported biodegradation of estrogens by heterotrophic bacteria is very slow, with several days needed to degrade half of the pollutants under aerobic conditions and a longer time under anaerobic conditions (Yu et al. 2013; Wang et al. 2018). Hence, this study aimed at isolation of E2-degrading bacteria from livestock leachate or manure which could effectively degrade estrogens at ng/L or μg/L levels for treatment of estrogen contamination in animal feeding operations or wastewater treatment plants. We report here the isolation and optimal growth conditions of a highly effective E2-degrading Gordonia sp. strain R9. In addition, some E2 metabolites generated by Gordonia sp. strain R9 were also characterized by high-performance liquid chromatography with fluorescence detection (HPLC-FD) and GC-MS.

Materials and methods

Chemicals, enzymes, and kits

E2 (> 98% purity), E1 (> 98% purity), E3 (> 98% purity), EE2 (> 98% purity), and testosterone (T, > 99% purity) were purchased from J&K Scientific Co. (Beijing, China). Ethanol, acetonitrile, ethyl acetate, and chloroform, all of HPLC grade, were purchased from Thermo Fisher Scientific (USA). Stock solutions of E1, E2, and E3 were prepared in ethanol and stored at 4 °C. Lysozyme and enzymatic lysis buffer, tris-saturated phenol, SOC broth, agar powder, Petri dishes, 96-well plates, and other chemicals were obtained from Sangon Biotech (Shanghai, China). Taq DNA polymerase was from New England Biolabs.

Enrichment, isolation, and identification of E2-degrading bacteria

To enrich E2-degrading bacteria, 10 mL leachate of chicken manure collected from a laying hen farm in Shenyang, Liaoning province, China, was added to a 250-mL flask containing 100 mL sterile M9-G medium (without glucose) supplemented with 5 mg/L E2 as the sole carbon source. The ingredients of M9-G medium in 1 L Milli-Q water were as follows: NaHPO₄·7H₂O, 12.8 g; KH₂PO₄, 3.0 g; NaCl, 0.5 g; NH₄Cl, 1.0 g; MgSO₄, 0.24 g; CaCl₂, 0.011 g; and 2 mL trace elements, composed of 0.063 g CuSO₄·5H₂O, 0.1 g H₃BO₃, 0.012 g NaMO₄·2H₂O, 0.0112 g MnSO₄·H₂O and 0.0534 g ZnSO₄·7H₂O in 1 L Milli-Q water. The enrichment culture was shaken at 150 rpm in an orbital shaker for 2 weeks at 30 °C, then transferred to fresh M9-G + E2 medium and incubated for one more week. The resulting culture was spread onto M9-G + E2 plates and incubated at 30 °C for 2 days. Colonies were selected and inoculated into new fresh M9-G + E2 medium, which were further spread onto agar plates and then to M9-G + E2 medium. This process was repeated several times to obtain pure isolates. More than 30 colonies with different morphologies and colors on the plates were tested for their utilization of E2 as the sole carbon source, and the one with the fastest growth rate was selected and designated R9 (R = red colony).

The antibiotic resistance of strain R9 was tested by incubation in SOC medium with the following antibiotics: 100 μg/mL ampicillin, 30 μg/mL kanamycin, 25 μg/mL chloramphenicol, 300 μg/mL carbenicillin, 50 μg/mL erythromycin and 50 μg/mL streptomycin. Overnight cultures were diluted 1:5 with distilled water prior to OD₆₀₀ determination. Utilization of strain R9 with E1, E3, EE2, and T, respectively, as the sole carbon source was also tested by overnight incubation of 1 mL strain R9 in 30 mL M9-G medium supplemented with the individual steroids at 30 °C and pH 7.0, following which the OD₆₀₀s were determined. To further characterize this novel E2-degrading strain R9, the morphology of a single colony, following incubation in SOC medium at 30 °C, was observed by electron microscopy (Hitachi H-7650, Japan). Gram staining was performed according to the manufacturer’s instructions (Sangon Biotech, China).

Amplification, sequencing and phylogenetic analysis of 16S rRNA gene

To classify the newly isolated strain R9, chromosomal DNA was extracted from an overnight culture to serve as the template for 16S rRNA amplification by PCR using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) and Taq DNA polymerase. The PCR cycles were performed as follows: denaturation at 98 °C for 5 min; 35 cycles of predenaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 1.5 min; final elongation at 72 °C for 10 min. The obtained amplicons were sequenced by Genecore Biotech Crop. Shanghai, and the resulting 16S rRNA gene sequence was aligned with reference sequences through BLAST. Sequence identity of 16S rRNA sequences between R9 and reference strains was performed with DNASTAR. A phylogenetic tree based on the 16S rRNAs was constructed with MEGA v7.0 (Center for Evolutionary Functional Genomics, Tempe, AZ) after alignment with Clustal W, and the maximum likelihood method with a bootstrap of 1000 was used.

HPLC-FD and GC–MS

The efficiency of E2 degradation by strain R9 was determined from the reduction of E2. Two mL of strain R9 were inoculated into 50 mL M9-G medium containing concentrations of E2 ranging from 50 ng/L to 5 mg/L in 100-mL flasks at 30 °C in a rotary shaker. The tolerance of strain R9 to temperatures (4–40 °C) and pH (1.0–12.0) was determined by measuring the OD₆₀₀ value of samples collected at various time points up to 168 h post-incubation. The collected samples were stored at − 20 °C until further use. E2 and its products in the collected samples (2 mL) were extracted by shaking with three successive aliquots ethyl acetate (2 mL) and dried by vacuum evaporation. The dried product was then dissolved in 1 mL acetonitrile: water (45:55 v/v), and analyzed by HPLC coupled with a fluorescent detector (HPLC-FD) (Shimadzu, Kyoto, Japan) (Liu et al. 2015).

The intermediate degradation products of E2 was analyzed by GC–MS (6890-5973 N, Angilent, USA). An R9 culture was first extracted with ethyl acetate/dichloromethane (1/1, v/v), then with 10 mL ethyl acetate/dichloromethane (1/1, v/v) at pH 2.0 (acidified with HCl) and pH 12.0 (alkalized with NaOH) separately. The combined extracts were dehydrated with anhydrous NaSO₄ and dried in argon, then finally dissolved in 1 mL CH₂Cl₂. A GC–MS equipped with a HP-5 MS capillary column (30.0 m × 250 mm × 0.25 μm) was used to analyze the extracted E2 intermediates using full-scan mode. Helium was used as the carrier gas at a flow rate of 1 mL/min, and 1 μL of sample was injected in the splitless mode. The inlet temperature was maintained at 200 °C with the oven temperature programmed as follows: 70 °C kept for 2 min, 70 °C to 180 °C at 3 °C/min, 180 °C to 240 °C at 1 °C/min, 240 °C to 270 °C at 3 °C/min, 270 °C to 300 °C at 1.5 °C/min kept for 2 min. The chemical structures of the intermediates were identified through comparison of their mass spectra with built-in GC–MS libraries [US National Institute of Standard Technology (NIST98) and Wiley 275 library].

Results

Isolation and identification of E2-degrading Gordonia sp. strain R9

Colonies of the bacterial strain R9 were circular, red, smooth surfaced and wetting on a M9-G + E2 nutrient agar plate, and spherical upon observation byelectron microscopy (Fig. 1a). Analysis of 16S rRNA gene sequences, which have been deposited in GenBank under accession number MN900528, showed that strain R9 was identical with Gordonia polyisoprenivorans W8130, and clustered together with other Gordonia sp. strains (Fig. 1b), but branched away from other steroid-degrading bacteria. Strain R9, therefore, appears to be a species of genus Gordonia, designated as Gordonia sp. R9, and is the first reported Gordonia strain that can degrade estrogens. In addition, Gordonia sp. R9 was found to be sensitive to all tested antibiotics: kanamycin, ampicillin, chloramphenicol, and carbenicillin (Fig. 2).

Fig. 1.

Identification of E2-degrading Gordonia sp. strain R9. a Observation of strain R9 by electron microscopy. b Phylogenetic tree based on 16S rRNA nucleotide sequences, was constructed with Mega v7.0 with a bootstrap value of 1000. The reference strains are steroid-degrading bacteria except the Gordonia sp. strains. Black triangle: Gordonia sp. strain R9

Fig. 2.

Sensitivity of Gordonia sp. strain R9 to antibiotics. Strain R9 was cultured overnight at 30 °C in SOC medium containing ampicillin (100 μg/mL), tetracycline (10 μg/mL), carbenicillin (300 μg/mL), chloramphenicol (25 μg/mL), gentamicin (50 μg/mL), streptomycin (50 μg/mL) or kanamycin (30 μg/mL). Bacterial growth was determined at OD₆₀₀

Gordonia sp. R9 can utilize different steroids as sole carbon and energy sources

Previous studies have reported that most E2-degrading strains cannot completely degrade estrogens, with accumulation of E1 as an intermediate. It was, therefore, necessary to determine the bioavailability of estrogens other than E2 following treatment with Gordonia sp. R9. As shown in Fig. 3, Gordonia sp. R9 grew well in M9-G medium supplemented with individual steroids. The OD₆₀₀ value for each steroid was 0.3–0.6, the optimal growth of strain R9 being obtained upon incubation with E3 (Fig. 4a), showing differences in the effective utilization of steroids by strain R9. Furthermore, the plateau phase of Gordonia sp. R9 was observed to be persistent for at least 5 days upon incubation with the steroids (Fig. 3).

Fig. 3.

Growth curve of strain R9 in M9-G minimal medium with different substrates (E1, estrone; E2, 17 β-estradiol; E3, estriol; EE2, 17 α-ethynylestradiol; T, testosterone; Control, no steroids). Cells were cultured for 168 h and the OD₆₀₀ of samples were determined at different time points

Fig. 4.

Effect of temperature and pH on the degradation of E2 by Gordonia sp. strain R9. a E2 degradation; b degradation of intermediates E1; c degradation of intermediates E3; d effect of pH on E2 degradation

E2 can be degraded by Gordonia sp. R9 over a wide range of temperature and pH

To test the effect of temperature and pH on E2 degradation by Gordonia sp. R9, cultures were incubated in M9-G medium supplemented with 5 mg/L E2 at 4–40 °C and pH 1.0-12.0. According to the degradation curves of E2 and its intermediates E1 and E3 (Fig. 4), Gordonia sp. R9 could exist at the extreme ranges tested, with a 95% removal efficiency for E2 (Fig. 4a–c) between 10 °C and 40 °C, with > 80% E2 being degraded even at 4 °C. Optimal growth temperature was about 30 °C. Most of the degradation of E2 was effected within the first 24 h with accumulation of E1 and E3 (major and minor intermediates) which, in turn, were completely degraded by 48 h (Fig. 4). These results show that Gordonia sp. R9 can degrade E1, E2 and E3 and use them as carbon and energy sources over a very wide range of temperatures.

The degradation efficiency of E2 by Gordonia sp. R9 at different initial pHs showed that it can tolerate a very wide range of pH but grows best at pH 7.0 with almost complete degradation being achieved between pH 4.0 and pH 9.0 (Fig. 4). Gordonia sp. R9 can also degrade E2 efficiently between pH 1.0–3.0 and pH 10.0–11.0, but the degradation efficiency dropped markedly at pH 12.0. To test if it could degrade trace amounts of E2, concentrations of E2 ranging from 50 ng/L to 5 mg/L were incubated with the bacterium under the optimal conditions of 30 °C and pH 7.0. Results showed that initial E2 concentrations ranging from 5 × 10⁻² mg/L to 5 mg/L were almost 100% degraded after incubation for 24 h, while a much longer time (168 h) was needed to degrade E2 with an initial concentration of 50 ng/L (Fig. 5).

Fig. 5.

Degradation efficiency of strain R9 at different initial E2 concentrations. Strain R9 was incubated in the M9-G medium with 5 × 10⁻⁵ mg/L, 5 × 10⁻² mg/L, 1 mg/L, 3 mg/L, 5 mg/L of E2, respectively, at 30 °C and pH 7.0, and samples were collected at different time points followed by HPLC-FD detection

Identification of E2 metabolites generated by Gordonia sp. R9

Analysis of the metabolites generated by Gordonia sp. R9 during E2 biodegradation was effected by HPLC-FD and GC–MS. As shown in Fig. 6, most of E2 was degraded after incubation for 48 h and intermediates E1 and E3 were detected (Fig. 6c). After incubation for 96 h, E2 had almost disappeared and E1 and E3 were also degraded (Fig. 6d). After 144 h incubation, most of the metabolites had also disappeared (Fig. 6e). GC–MS revealed four peaks after subtracting the signal of the mock samples (Table 1). The presence of E2 (m/z 271, 26.07 min, 26.11 min) reflected incomplete reaction. The compounds with m/z 270 and 288 at retention time of 26.15 (26.58) min and 26.83 (27.02) min were verified to be E1 and E3 through comparison of their spectra with those of E1 and E3 standards. Moreover, two new peaks were observed at retention times of 27.56 min and 27.29 min, which were identified as (3Z)-3-(3-hydroxy-3a-methyl-7-oxododecahydro-6H-cyclopenta[a]naphthalen-6-ylidene) propanoic acid (E2 metabolite 1) and 3-hydroxy-3a-methyl-7-oxododecahydro-1H-cyclopenta[a]aphthalene-6-carboxylic acid (E2 metabolite 2), respectively (Table 1). In general, estrogens were metabolized by the 9,10-seco pathway with B-ring cleavage first (Combalbert and Hernandez-Raquet 2010; Horinouchi et al. 2012), or in the 4,5-seco pathway with A- or D-ring cleavage first (Combalbert and Hernandez-Raquet 2010; Wu et al. 2019; Yu et al. 2013). Two novel intermediates identified in this study were generated by first breakage of the A ring of E2, highlighting the existence of the 4,5-seco pathway in Gordonia sp. R9. Furthermore, E1 and E3 were also detected during E2 degradation. Altogether, the above results showed that at least two estrogen-degrading pathways exit in Gordonia sp. R9 (Fig. 7).

Fig. 6.

HPLC-FD detection of E2 degradation by strain R9. Gordonia sp. strain R9 was cultured with 5 mg/L E2 as the sole carbon source and samples were collected at different time points, and HPLC-FD was used to detect the removal of E2 and the production/degradation of both intermediates E1 and E3. a The standard of E1, E2 and E3; b E2 degradation without incubation (0 h); c–e E2 degradation over time after incubation with strain R9. The whole elution time was 10 min; the peak areas of E1, E2 and E3 were marked

Table 1.

Characterization of E2 metabolites generated in Gordonia sp. strain R9 by GC–MS

Parent compound	Retention time(min)	m/z	Suggested product
E2	26.11	272	E2
	26.58	270	E1
	27.02	288	E3
	27.56	294	(3Z)-3-(3-hydroxy-3a-methyl-7-oxododecahydro-6H-cyclopenta[a]naphthalen-6-ylidene)propanoic acid
	27.29	268	3-Hydroxy-3a-methyl-7-oxododecahydro-1H-cyclopenta[a]naphthalene-6-carboxylic acid

Fig. 7.

Proposed degradation pathways of estrogens by bacteria under aerobic (solid line), anoxic or anaerobic conditions (dashed line), by algae (dotted line), and by Gordonia sp. strain R9 in this study (red arrow) (Combalbert and Hernandez-Raquet 2010; Kurisu et al. 2010; Nakai et al. 2011; Yu et al. 2013; Blunt et al. 2017; Chen et al. 2018; Wu et al. 2019). E2 metabolite 1, (3Z)-3-(3-hydroxy-3a-methyl-7-oxododecahydro-6H-cyclopenta[a]naphthalen-6-ylidene) propanoic acid; E2 metabolite 2, 3-hydroxy-3a-methyl-7-oxododecahydro-1H-cyclopenta[a]aphthalene-6-carboxylic acid

Discussion

Estrogen pollution is currently a serious public concern, with biodegradation considered to be one of the most promising ways to tackle it. Hence, obtaining highly effective estrogen-degrading bacteria is still very important. In this study, the highly effective E2-degrading Gordonia sp. R9 was isolated from the chicken feces after enrichment with different medium and incubation conditions, which exhibits great degradation efficiency of estrogens because of the complete degradation of 50 ng/L–5 mg/L E2 in less than 48 h, and tolerates an outstanding environmental conditions of temperature (4–40 °C) and pH (1.0–11.0), which is very rare among all reported estrogen-degrading bacteria (Yu et al. 2013; Combalbert and Hernandez-Raquet 2010).

We found that the plateau phase of the growth curve for Gordonia sp. R9 persists for at least 5 days, while other reported estrogen-degrading bacteria reached peak titers by 72 h post-incubation with subsequent decrease (Wang et al. 2019a, b). The much longer persistent plateau phase, therefore, indicates that Gordonia sp. R9 can tolerate a relatively harsh environment. In addition, we found that Gordonia sp. R9 resists degradation by lysozyme, indicating that its cell wall is resistant to damage (data not shown). When cultured below 10 °C, Gordonia sp. R9 was observed to coagulate into balls which would reduce the environmental impact of activated sludge.

Gordonia sp. are ubiquitous, slightly acid-fast nocardioform bacteria within the order Actinomycetales (Shtratnikova et al. 2016). They have been reported to be important components of wastewater treatment systems and are notable for their biodegradative abilities, acting on environmental pollutants, xenobiotics, hardly degradable natural polymers and cholesterol (Arenskötter et al. 2004; Drzyzga et al. 2009). Gordonia neofelifaecis NRRL B-59395 (Ge et al. 2011) and Gordonia cholesterolivorans (Drzyzga et al. 2011) are two representative strains for steroid degradation, and several steroid-degrading genes have been found in the genomes of these bacteria. To the best of our knowledge, however, the present report is the first that Gordonia sp. can use estrogens as sole carbon sources. Degradation of E2 by different estrogen-degrading bacteria is compared in Table 2, showing that, while most can degrade 0.1–20 mg/L E2 within 1–15 days (Ke et al. 2007; Yu et al. 2007; Roh and Chu 2010; Zeng et al. 2009; Li et al. 2012; Xiong et al. 2018; Wang et al. 2019a, b), Gordonia sp. R9 exhibits the highest degradation efficiency.

Table 2.

Comparison of E2 degradation efficiencies among estrogen-degrading bacteria

Bacteria	Source of isolates	Time for E2 degradation	E2 concentration (mg/L)	References
Acinetobacter sp. LHJ1	Artificial sandy aquifer	15 days	0.1	Ke et al. (2007)
Escherichia coli KC13	Activated sludge	7 days	3	Yu et al. (2007)
Pseudomonas aeruginosa TJ1	Activated sludge	> 4 days	20	Zeng et al. (2009)
Stenotrophomonas maltophilia ZL1	Activated sludge	100 h	3.3	Li et al. (2012)
Deinococcus actinosclerus SJTR1	Wastewater	> 5 days	10	Xiong et al. (2018)
Sphingobacterium sp. KC8	Wastewater treatment plants	5 days	3	Roh and Chu (2010)
Bacillus sp. E2Y1	Wastewater treatment plants	6 days	1	Roh and Chu (2010)
Pseudomonas putida SJTE1	Activated sludge	1 days	1	Wang et al. (2019a, b)
Gordonia sp. strain R9	Chicken leachate	1 days	5	This study

The degradation pathways of E2 have been studied in many bacteria and algae. Generally, E2 is firstly oxidized on the C-17 at ring D, producing E1 which is usually further degraded. In aerobic conditions, E1 can be degraded by D-ring cleavage by sewage bacteria (Combalbert and Hernandez-Raquet 2010; Yu et al. 2013) and B-ring cleavage by Sphingobacterium sp. JCR5 (Haiyan et al. 2007). In other conditions, other metabolic pathways also exist. For instance, two denitrifying bacteria Denitrosomonas oestradiolicum and Steroidobacter denitrificans, and two aerobic species, Rhoclococcus zopfii and Novosphingobium, can use E2 as the sole carbon source without E1 accumulation (Combalbert and Hernandez-Raquet 2010; Fahrbach et al. 2006, 2008; Fujii et al. 2002; Yoshimoto et al. 2004). Under sulfate-reducing conditions E2 is converted into E3 directly by some bacteria as an intermediate (Combalbert and Hernandez-Raquet 2010; Czajka and Londry 2006; Dytczak et al. 2008; Lai et al. 2002). In Nitrosomonas europaea strain NCIMB 11850, E2 is also degraded using estratetraenol (E0) as an intermediate (Nakai et al. 2011; Chen et al. 2018). In this study, we identified several metabolites of E2 following its incubation with Gordonia sp. R9, including E1, E3, (3Z)-3-(3-hydroxy-3a-methyl-7-oxododecahydro-6H-cyclopenta[a]naphthalen-6-ylidene) propanoic acid and 3-hydroxy-3a-methyl-7-oxododecahydro-1H-cyclopenta[a] naphthalene-6-carboxylic acid. These results indicate that several estrogen-degrading pathways exist in Gordonia sp. R9, including the 9,10-seco pathway (B-ring cleavage), the 4,5-seco pathway (A-ring cleavage), D-ring cleavage and the E3 pathway, a rare occurrence in a single bacterial strain. Hiessl et al. (2012) reported that certain Gordonia strains have strong anabolic and bioconversion capabilities that can degrade, transform, and synthesize a series of compounds. Therefore, Gordonia sp. R9 may be one of these powerful Gordonia strains, in which estrogens can be degraded, transformed and even synthesized (Schneider et al. 2008). To our knowledge, the present work is the first report of an A-ring cleavage pathway of E2 biodegradation, with two newly identified intermediates, by a Gordonia strain.

Although several estrogen-degrading strains have been isolated, the mechanisms underlying the degradation of estrogens remain unclear, with identification of only a few enzymes catalyzing the metabolism. In most bacteria, 17β-hydroxy-steroid dehydrogenase (17β-HSD) has been found to oxidize 17β-hydroxyl steroids such as 17β-estradiol to estrone (Ye et al. 2017), the reaction considered to be the first step in its transformation. It has also been found that 3-oxoacyl-(acyl-carrier-protein) reductase functions as 17β-hydroxy-steroid dehydrogenase in the estrogen-degrading Pseudomonas putida SJTE-1 (Wang et al. 2018). Until now, the mechanisms of steroid degradation have been based mainly on cholesterol and testosterone (Holert et al. 2018; Bergstrand et al. 2016). In testosterone-degrading Comamonas (Horinouchi et al. 2012), 3α-HSD and 3β-HSD catalyze the oxidoreduction at the C₃ site of steroid hormones, with three residues (Ser114, Tyr155, and Lys159) in 3α-HSD forming a triad essential for the catalysis. Other short-chain dehydrogenase/reductases (SDR) are also involved in the degradation of steroids. For instance, 3-ketosteroid-1-dehydrogenase, 3-hydroxy-9,10- secoandrosta-1,3,5(10)- triene-9,17- dione monooxygenase reductase, 3-hydroxy-9,10-seconandrosta-1,3,5(10)-trien-9,17-dione hydroxylase and aromatic ring-hydroxylating dioxygenase are required for A/B-ring degradation (Bergstrand et al. 2016). ABC transporter is involved in uptake and transportation of steroids (Xu et al. 2017). Transcriptional regulators TetR/LysR and other regulators regulate the expression of steroid-degrading enzymes in bacteria (Horinouchi et al. 2012; Gong et al. 2012; Göhler et al. 2008; Xiong et al. 2003, Xiong and Maser 2001). Since Gordonia sp. R9 is highly effective in degrading E2 it must harbor steroid-degrading enzymes and related regulators. Indeed, our work has indicated the presence of a number of = genes in Gordonia sp. R9 encoding transcriptional regulators, substrate transporters and catabolic enzymes, including those coding for 3α-HSD, 3-ketosteroid-1-dehydrogenase, 3β-HSD, 17β-HSD, aromatic ring-hydroxylating dioxygenase, sterol carrier protein, TetR/LysR transcriptional regulators. Future studies will focus on elucidation of the various metabolic pathways of E2 degradation and associated degradation mechanisms used by Gordonia sp. R9.

Author contributions

TZ designed the experiments and wrote the manuscript. NL performed the experiments. YS, JL and MZ assisted the experiments. All the authors discussed the results and commented on the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.