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  • Anoxic Androgen Degradation by the Denitrifying Bacterium Sterolibacterium denitrificans via the 2,3-seco Pathway

    Po-Hsiang Wang,a Chang-Ping Yu,b Tzong-Huei Lee,c Ching-Wen Lin,a Wael Ismail,d Shiaw-Pyng Wey,e An-Ti Kuo,a Yin-Ru Chianga

    Biodiversity Research Center, Academia Sinica, Taipei, Taiwana; Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, Chinab; Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwanc; Biotechnology Program, College of Graduate Studies, Arabian Gulf University, Manama, Kingdom of Bahraind; Department of Medical Imaging and Radiological Sciences, College of Medicine, Chang-Gung University, Kweishan, Taiwane

    The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C19 andro- gens. By using a 13C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the iden- tification of a C17 intermediate, we propose that the A-ring cleavage may be followed by the removal of a C2 side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (

  • tected androgens (for example, 80% of detected androsta-1,4- diene-3,17-dione [ADD]) were eliminated by microbial activities in the anaerobic tanks (22).

    In addition to the environmental concerns, microbial steroid biodegradation has also potential applications in pharmacy and medicine (36, 37). Numerous studies have reported the microbial production of androst-4-en-3,17-dione (AD) and ADD from in- expensive cholesterol and phytosterols (27, 38). Additional inter- est is due to the potential applications of steroid-transforming enzymes with high regio- and stereo-specificity in the industrial synthesis of steroids (37).

    We recently proposed an anaerobic testosterone degradation pathway (the 2,3-seco pathway) adopted by a gammaproteobacte- rium, Steroidobacter denitrificans (Sdo. denitrificans) DSMZ 18526 (39). The oxygenase-independent mechanism of the steroidal A- ring cleavage is highly comparable to that operating in anoxic cyclohexanol catabolism by Alicycliphilus denitrificans DSMZ 14773 (39). However, there is a dearth of information concerning the carbon removal mechanisms involved in anoxic testosterone catabolism. Furthermore, the androgenic activity of the interme- diates needs to be determined. The distribution of the 2,3-seco pathway among testosterone-degrading anaerobes also remains unclear. Prior to this study, Sdo. denitrificans DSMZ 18526 was the only known testosterone-degrading anaerobe (40–42).

    BLAST analysis has indicated that Sterolibacterium strains can be detected in the natural environment and engineered systems (see Fig. S1 in the supplemental material). Among them, Steroli- bacterium denitrificans (S. denitrificans) DSMZ 13999, a betapro- teobacterium, was reported to degrade cholesterol both under oxic and anoxic conditions (43–46). In this study, the ability of S. denitrificans to degrade C18 and C19 steroid hormones under an- oxic conditions was investigated. We applied a 13C-metabolomic approach to show that S. denitrificans uses the 2,3-seco pathway to anaerobically degrade androgens. The androgenic activities of the bacterial culture and the identified intermediates were then as- sessed.

    MATERIALS AND METHODS Chemicals and bacterial strains. [4-14C]testosterone was obtained from Perkin-Elmer. [2,3,4-13C]testosterone was purchased from Isosciences. The chemicals used were of analytical grade and were purchased from Mallinckrodt Baker, Merck, or Sigma-Aldrich. 3,17-Dihydroxy-9,10- seco-androsta-1,3,5(10)-triene-9-one, 3-hydroxy-9,10-seco-androsta- 1,3,5(10)-triene-9,17-dione, 1-testosterone, androst-1-en-3,17-di- one, 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (2,3-SAOA), and 1,17-dioxo-2,3-seco-androstan-3-oic acid were produced as described else- where (39, 47). Sterolibacterium denitrificans DSMZ 13999 was purchased from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. Comamonas testosteroni ATCC 11996 was obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

    Androgen and estrogen utilization kinetics. The preparation of the denitrifying media and the anoxic in vivo assays were performed in an anaerobic chamber containing 95% nitrogen and 5% hydrogen gas. S. denitrificans (resting-cell assays; total proteins, 294 �g/ml) was incubated with individual androgens (ADD, androsterone [ADR], epiandros- terone [EADR], 19-nor-4-androsten-3,17-diol [NADL], or testoster- one) or estrogens (17�-estradiol, 17�-estradiol, estriol, estrone, or 17�- ethinylestradiol) at initial concentrations ranging from 5 to 50 mg/liter under denitrifying conditions. Hydroxypropyl-�-cyclodextrin (cyclodex- trin) (0.5% [wt/vol]) was added to the culture medium prepared accord- ing to a published procedure (46). Substrate utilization kinetics tests were

    performed in a series of 2.2-ml Eppendorf tubes containing 1 ml of resting cell suspension. The steroid stock solutions were prepared in dimethyl sulfoxide (DMSO), and the final DMSO concentration was adjusted to 0.5% (vol/vol). The resulting cell suspension was anaerobically incubated at 28°C with shaking (180 rpm) for 2 h. We measured the amount of residual steroids using high-pressure liquid chromatography (HPLC) and determined the protein content of the S. denitrificans cell suspension using the bicinchoninic acid (BCA) assay. The determination of Monod kinetic parameters was carried out as described before (48).

    Anaerobic growth of S. denitrificans on [4-14C]testosterone. In the following experiments involving steroid quantification, 17�-ethinylestra- diol (final concentration, 50 �M), which cannot be utilized by S. denitri- ficans, was added to bacterial cultures to serve as an internal control. In a denitrifying fed-batch culture (100 ml), S. denitrificans cells were incu- bated with 1 mM testosterone containing [4-14C]testosterone (1 � 108

    dpm). Nitrate (10 mM) was added to the culture when the nitrate added initially (10 mM NaNO3) was consumed. The fed-batch culture (initial total proteins, 32 �g/ml) was carried out in a 125-ml glass bottle sealed with a rubber stopper. The headspace (25 ml) of the culture was connected to a 125-ml glass bottle containing 100 ml of 5 M NaOH, which trapped 14CO2 produced by the S. denitrificans cells. A Tygon tube (2 mm in inner diameter and 25 cm long) was used for the connection. Every 12 h (0 to 60 h), samples (0.5 ml) were withdrawn from the 5 M NaOH solution. Ten minutes before each sampling, nitrogen gas (ca. 50 ml) was used as a carrier gas to expel the residual 14CO2 from the bacterial culture at a flow rate of 5 ml/min. At the same time intervals, samples (2.5 ml) were with- drawn from the bacterial culture. The culture samples (0.5-ml samples, 3 replicates) were extracted with the same volume of ethyl acetate three times to isolate the residual [4-14C]testosterone from the water fraction. The ethyl acetate was evaporated, and the residue was redissolved in 0.5 ml of ethanol. The water fraction, ethanol, and 5 M NaOH samples (0.1 ml) were added to 1.9 ml of Ultima Gold high-flash-point LSC scintillation cocktail reagent (Perkin-Elmer), and the amount of 14C was determined by liquid scintillation counting (Tri-Carb 2900 TR liquid scintillation analyzer [Perkin-Elmer]). The remaining ethanol samples (0.4-ml sam- ples, 3 replicates) were concentrated to 50 �l, and the testosterone in the ethanol samples was quantified by HPLC. The protein content and nitrate concentration in the samples were determined as described below. After 60 h of incubation, the bacterial cells were harvested twice by centrifuga- tion (10,000 � g for 20 min), and the remaining cells were recovered by passing the supernatant through a 0.22-�m nitrocellulose membrane fil- ter (Millipore). The S. denitrificans cells were lyophilized for 1 week, and the freeze-dried biomass was weighed.

    Anoxic transformation of [2,3,4-13C]testosterone by the batch cul- tures. In the following biotransformation assays, S. denitrificans was first anaerobically grown in fed-batch cultures (nitrate was continuously sup- plied). The amounts of residual substrates (testosterone, cholesterol, or palmitate [49]) in the denitrifying S. denitrificans cultures were monitored using HPLC. The utilization of last two substrates can avoid the HPLC detection of C19 catabolic intermediates in the precultures. We then used the log-phase S. denitrificans cultures to transform steroid substrates in a batch mode (no additional nitrate was given during the incubation).

    After the consumption of 2 mM testosterone, 10 ml of the anoxic S. denitrificans culture (200 ml) was transferred into two 12-ml glass bottles. The two cultures were subsequently fed with 2 mM testosterone (unla- beled testosterone and [2,3,4-13C]testosterone were mixed in a 1:1 molar ratio) under denitrifying conditions. From an S. denitrificans culture con- taining 5 mM nitrate, the sample (1 ml) was withdrawn after 4 h of incu- bation a

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