New Microbiological Transformations of Steroids by Streptomyces

Technol. , 2009, 43 (15), pp 5967–5974. DOI: 10.1021/es900585w. Publication Date (Web): July 6, 2009 ... East China University of Science and Techno...
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Environ. Sci. Technol. 2009, 43, 5967–5974

New Microbiological Transformations of Steroids by Streptomyces virginiae IBL-14 FENG-QING WANG,† CHENG-GANG ZHANG,† BO LI,† DONG-ZHI WEI,† AND W A N G - Y U T O N G * ,†,‡ State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China, and Integrated Biotechnology Laboratory, School of Life Science, Anhui University, Hefei 230601, China

Received February 24, 2009. Revised manuscript received May 14, 2009. Accepted June 17, 2009.

A bacterium Streptomyces virginiae IBL-14 capable of effective degradation of diosgenin was isolated from activated sludge for treatment of waste from a steroidal drug factory. From the culture broth of diosgenin degradation, 11 compounds were purified and then identified, eight of which were previously unidentified compounds including 1-dehydroisonuatigenone [VI], nuatigenone [VIII], 1-dehydronuatigenone [X], 26acetyl-nuatigenone [XII], 6-methoxy-6-dehydrodiosgenone [XIII], 6-methoxy-6-dehydroisonuatigenone [XIV], 6-methoxy-6dehydronuatigenone [XV], and 6-dimethoxy-7R-hydroxyldiosgenone [XVI]. Additionally, two important microbial transformations of diosgenin (6-methoxylation and C25-tertiary carbon hydroxylation) were found. Two valuable chemical reactions of the steroids (structural rearrangement and esterification) were also confirmed. As a result, a new metabolic pathway of diosgenin metabolism was postulated.

Introduction Diosgenin is a highly important natural product for its important pharmacological attributes and its key role in the steroid pharmaceutical industry as the primary precursor for the commercial synthesis of cortisone, pregnenolone, progesterone, and many other steroid drugs (1, 2). Diosgenin and its derivates have important physiological activities, such as anticancer, antagonistic effect on rheumatoid arthritis, hyperlipidemia, and cardiovascular diseases (3-5). The physiological activities of diosgenin have attracted scientists’ attention to study its derivatives and structural analogues for their potential pharmacological and practical application (4-8). As well-known, both natural and synthetic steroids are powerful endocrine disruptors which can cause serious impairment to the development and reproduction of wildlife and humans when their concentrations accumulated to certain levels (9-11). For example, 17β-estradiol (E2) and 17R-ethinylestradiol (EE2) at concentrations higher than 1 ng /L and 4.0 ng/L can cause vitellogenin production in male fish and a failure to develop normal secondary sexual * Corresponding autho phone: +86-021-64253156; fax: +86-02164250068; e-mail: [email protected]. † East China University of Science and Technology. ‡ Anhui University. 10.1021/es900585w CCC: $40.75

Published on Web 07/06/2009

 2009 American Chemical Society

characteristics as well as a lack of sexual differentiation in males, respectively (10, 12, 13). Consequently, a lot of effort has been expended to investigate their impacts and to solve the environmental issue (11, 14, 15). Also, a lot of reports on microbial transformation and degradation of steroids are published (16, 17). The degradation pathway of testosterone via the metacleavage pathway by Comamonas testosteroni (formerly Pseudomonas testosteroni) was reported by Paul Talalay et al. over 40 years ago (18, 19). Comamonas testosteroni which can utilize certain C19 and C21 steroids as the sole carbon source has been well studied (17-22) and its genes related to the steroid degradation were reported (17, 20, 23, 24). To produce the useful derivatives of natural products, microbial transformation of diosgenin has been well studied due to the region- and stereoselectivity of many microbial enzymes. Diosgenone and 1-dehyrodiosgenone are the most common derivatives of diosgenin transformed by microorganisms (2, 25-27). Other microbial transformations such as hydroxylation, dehydrogenation and epoxidation of diosgenin have also been reported (28, 29). Xenobioticmetabolizing enzymes for digestion of exogenous compounds in microorganisms are usually inducible. For example, Comamonas testosteroni contains many steroid-inducible enzymes for the degradation of steroids (19-21). The 3βhydroxysteroid dehydrogenase (3β-HSD), responsible for the formation of diosgenone (II) from diosgenin, is considered to be the first key inducible enzyme in the degradation of testosterone by C. testosteroni (21). However, to our knowledge, carbon atoms on the rings E and F of steroidal sapogenins have not been found to be attacked by any microorganisms but Streptomyces virginiae (30). In this report, a novel metabolic pathway of diosgenin discovered from a newly isolated actinomyces, S. virginiae IBL-14 is presented. Among the metabolites of the pathway, 11 have been purified and identified and eight are new compounds. In addition, two important microbial transformations of steroidal sapogenins (6-methoxylation and C25tertiary carbon hydroxylation) and two valuable chemical reactions of the steroids (structural rearrangement and esterification) are also presented.

Materials and Methods Materials. All of the steroids (purity g 95%) were kindly donated by Hei-Qing Wang, Zhejiang University, Hangzhou, Zhejiang, China and Gang Zhang, Tianjin Research Institute of Industrial Microbiology, Tianjin, China. Analytic solvents were chromatographically pure and other reagents and solvents were of analytical grade or higher. Strain Isolation and Cultivation. In order to solve increasing environmental pollution caused by steroidal pharmaceutical factories and to seek new promising steroidal drugs from derivatives of diosgenin, Streptomyces virginiae IBL-14, an effective steroidal sapogenins-degrading strain, was isolated from activated sludge (pH 5.5, CODCR 1500) of a factory for steroidal drugs production in China (Xianju Pharmaceutical Co., Ltd., Zhejiang province). More details about the isolation, identification, cultivation of the S. virginiae IBL-14 and the media were reported in our earlier paper (30). Product Extraction and Isolation. All of the fermentation cultures were extracted twice with chloroform (chloroform to fermentation culture at a volume ratio of 1:2). The chloroform layer was treated with anhydrous Na2SO4 for dehydration and then in a vacuum hood for chloroform volatilization. The extract was redissolved in methanol and VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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then analyzed by TLC and HPLC. Analytical thin layer chromatography (TLC) was performed with cyclohexane/ ethyl acetate (6:4 v/v) on a preparative TLC plate purchased from the Qingdao Marine Chemical Factory, China and the visualization was done by spraying with 20% H2SO4 and heating in an oven at 100 °C for about 5 min until the colors developed. Analytical high performance liquid chromatography (HPLC) was carried out with an Agilent 1100 instrument. The chromatographic conditions were: an agilent XDBC18 column (4.6 × 250 mm) with a constant temperature of 40 °C, a mobile phase of methanol/water (binary gradient: water content held 40% in 0.01-15 min, decreased from 40 to 30% in 15-35 min, further decreased from 30 to 5% in 35-55 min, and held 5% in 55-70 min), a flow rate of 1 mL/min, and an internal time of 10 min between two samples. Analyses were performed with UV detection simultaneously at 211 and 240 nm. After extraction, a total of 4.0 g yellow residue was obtained from the fermentation broth of the S. virginiae IBL-14 and then redissolved in 400 mL methanol (10 mg/mL). For each run, 4 mL of the solution was loaded onto a waters 600E HPLC system with a Sunfire C18 column (19 × 150 mm). The elution was carried out by using a linear gradient from 3:7 (water/methanol) to 100% methanol from time 0 to 32 min at a flow rate of 15 mL/min at room temperature. Five fractions (that is 0-12 min, 12-14 min, 14-25 min, 25-28 min, 28-32 min) corresponding to five obvious peaks were collected respectively and then most of the methanol was removed in a vacuum hood and subsequently extracted twice with the same volume of ethyl acetate. The five preisolated fractions were reisolated on preparative TLC (using a gradient mixture of ethyl acetate-cyclohexane from 6:4 to 9:1 as the mobile phase) respectively. Under ultraviolet radiation the fluorescent bands were collected and eluted with ethyl acetate. Finally the fractions with high purities (Supporting Information (SI) Figure S34A) were crystallized, and as a result, about 460 mg II, 262 mg III, 43 mg V, 32 mg VI, 56 mg VIII, 31 mg X, 23 mg XII, 21 mg XIII, 16 mg XIV and XV, and 9 mg XVI were obtained (total 953 mg). The total yield for the isolation and purification of the eleven metabolites was about 24% (953 mg:4000 mg). Identification and Structure Analysis of Products. To identify the eleven compounds, to assign 1H and 13C chemical shifts of the nine new compounds unambiguously and to establish their structures, 1H NMR, 13C NMR, 1H-1H COSY, HMBC, and HMQC spectra were measured, and also their melting points were determined as below. Melting points (mp) were determined with an XT4A micromelting apparatus and were uncorrected. Infrared (IR) spectra were recorded on a Magna-IR 550 Nicolet FTIR spectrometer. Mass (MS) spectra were obtained by using a Micromass GCT instrument via electron impact (EI) at 70 eV. Ultraviolet (UV) spectra were recorded on a Varian Cary 500 UV/vis spectrophotometer. The 1H and 13C nuclear magnetic resonance (NMR) spectra were obtained using a Bru ¨ ker DRX500 spectrometer at 500 and 125 MHz, respectively, with tetramethylsilane (TMS) as the internal standard in CDCl3. Chemical shifts (δ) are given in parts per million (ppm) relative to TMS. Coupling constants (J) are given in hertz (Hz). Distortionless enhancement by polarization transfer (DEPT), nuclear overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), 1H-1H chemical shift correlation spectroscopy (COSY) and heteronuclear multiple bond coherence (HMBC) experiments were performed with standard pulse sequences provided by the spectrometer manufacturer. The analysis of HPLC in combination with mass spectrometry (LC/MS) was performed using a LC/TOF MS instrument (an Agilent Technologies Series 1100 LC/MSD SL system with an Agilent XDB-C18, 4.6 × 250 mm column, Agilent Technologies, Palo 5968

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Alto, CA) by electrospray ionization (ESI) with source temperature 100 °C, desolvation temperature 300 °C, source cone 30 V, desolvation 500 L nitrogen/h). Compound VI. Amorphous solid (ethyl acetate); mp 56-58 °C; molecular weight (MW) 426; chemical formula C27H38O4; IR (coat) cm-1: 3454, 2927, 2864, 2846, 1740, 1710, 1665, 1620, 1450, 1380, 1240, 1178, 1055, 1030, 980, 940, 890, 805; ESI-MS: m/z 427.2; EI-MS: m/z 427.3, 155.1 (the base peak). The molecular weight (MW) and chemical formula of VI were 426 and C27H38O4, as deduced from its ion peaks of ESI-MS at m/z 427.2 ([M+H]+) and EI-MS at m/z 427.3 ([M+H]+), supported by the 13C NMR and 1H NMR spectra of VI. In the EI-MS, the signal of m/z 155.1 (m/z 139, a diagnostic peak of ion fragment of diosgenin F-ring) was a peak of diagnostic fragment (m/z 139 + 16) containing F-ring of compound VI, suggesting that one oxygen atom was inserted into the fragment. The IR spectrum of VI showed absorption bands characteristic of hydroxyl group (3454 cm-1) and carbonyl group (1740 cm-1). Compared with the corresponding data of isonuatigenone (III) (30), metabolite VI should have the same structure as isonuatigenone (III) except for their A-rings (particularly the signal differences between III and VI at δC1 and δC2 in their 13C NMR spectra). After comprehensive analysis of the corresponding data of VI, compound VI should have a carbon-carbon double bond at C1-C2, which was supported by the diagnostic experimental result (red color) with sulphuric acid reaction on a thin-layer chromatographic plate. Hence, the structure of compound VI, denominated as 1-dehydroisonuatigenone, was established as (20S, 22R, 25S)-spirost-1, 4-diene-25-ol3-one. Compound VIII. Colorless needle (ethyl acetate); mp 199-200 °C; molecular weight (MW) 428; chemical formula C27H40O4; IR (KBr) cm-1: 3454, 2932, 2852, 1733, 1667, 1621, 1456, 1379, 1261, 1172, 1055, 1000, 922, 879, 867, 805; ESIMS: m/z 429.4; EI-MS: m/z 429.3, 155.1 (the base peak). The molecular weight (MW) and chemical formula of VIII were 428 and C27H40O4, as deduced from its ion peaks of ESI-MS at m/z 429.4 ([M+H]+) and EI-MS at m/z 429.3 ([M+H]+), supported by the 13C NMR and 1H NMR spectra. In the EIMS, the diagnostic fragments of VIII at m/z 340.3, 298.3, and 269.2 suggested the metabolite VIII should have the same structure of A, B, C, and D rings as diosgenone (II) (31) and isonuatigenone (III), supported by their 13C NMR and 1H NMR spectra as well as the long-range correlation of C3 with C4 in the HMBC spectrum of VIII (a choles-4-en-3-one). The absorption band characteristic of hydroxyl group (3454 cm-1) in the IR spectrum of VIII and the diagnostic signals (δC22120.9 and δC25 86.7 ppm) of furostanol saponins in the 13C NMR spectrum of VIII indicated that metabolite VIII is the F-ring rearrangement product of isonuatigenone III, which was further supported by the DEPT spectrum of VIII (C25, a quaternary carbon atom) and the 1H NMR [δ1.09, (3H, s, H-27)] spectrum of VIII as well as chemically experimental results (Figure 2A). After further analysis of the COSY, HMQC, and HMBC spectra of VIII, Compound VIII was determined as nuatigenone [VIII, (20S, 22S, 25S)-22, 25-epoxy-furost-4en-26-ol-3-one]. Compound X. Amorphous solid (ethyl acetate); mp 51-52 °C; molecular weight (MW) 426; chemical formula C27H38O4; IR (coat) cm-1: 3467, 2927, 2875, 2846, 1733, 1710, 1665, 1620, 1450, 1378, 1260, 1240, 1170, 1055, 920, 890, 867, 805; ESIMS: m/z 427.2; EI-MS: m/z 427.3, 155.1 (the base peak). The molecular weight (MW) and chemical formula of X were 426 and C27H38O4, as deduced from its ESI-MS and EI-MS ion peaks at m/z 427.2 ([M+H]+) and 427.3 ([M+H]+), supported by the 13C NMR and 1H NMR spectra. Compared with the corresponding data of nuatigenone (VIII), metabolite X should have the same structure as nuatigenone (VIII) except for their A-rings. Further compared with the corresponding data of

1-dehydroisonuatigenone (VI), the metabolite should have a carbon-carbon double bond at C1-C2 [δC1 156.2 and δC2 127.9 in the 13C NMR spectrum of X; δ: 6.97 (1H, d, J ) 10.2 Hz, H-2) and 6.16 (1H, d, J ) 10.1 Hz, H-1)], which was supported by the diagnostic experimental result (red color) with sulphuric acid reaction on a TLC plate. From these results, the structure of compound X, named as 1-dehydronuatigenone, was established as (20S, 22S, 25S)-22, 25epoxy-furost-1, 4-diene-26-ol-3-one. Compound XII. Amorphous powder (ethyl acetate); mp 73-75 °C; molecular weight (MW) 470; chemical formula C29H42O5; IR (coat) cm-1: 2922, 2847, 2846, 1744, 1678, 1612, 1457, 1382, 1245, 1138, 1034; EI-MS: m/z 471.3, 43.0 (the base peak). The molecular weight (MW) and chemical formula of compound XII were 470 and C29H42O5, as deduced from its EI-MS ion peak of XII at m/z 471.3 ([M+H]+), supported by the 13C NMR and 1H NMR spectra. Compound XII could be produced by chemical reaction of nuatigenone (VIII) with anhydrous acetic acid, suggesting that compound XII may be an esterified product of nuatigenone (VIII). Compared with the corresponding spectrum data of nuatigenone (VIII), compound XII should have the same structure as nuatigenone (VIII) but C26 hydroxyl group disappears (δC28 171.9 in the 13 C NMR spectrum of XII and no adsorption peak of hydroxyl group from 3400 to 3700 cm-1 in its IR spectrum). Therefore, compound XII was determined as 26-acetyl-nuatigenone [XII, (20S, 22S, 25S)-22, 25-epoxy-26-hydroxy furost-4-ene-3-one26-acetate]. Compound XIII. Amorphous powder (ethyl acetate); mp 41-43 °C; molecular weight (MW) 440; chemical formula C28H40O4; IR (coat) cm-1: 2933, 2867, 1733, 1661, 1583, 1452, 1378, 1231, 1078, 1052, 980, 900; ESI-MS: m/z 441.5, 903.4; EI-MS: m/z 440.3, 139.1 (the base peak). The molecular weight (MW) and chemical formula of compound XIII were 440 and C28H40O4, as deduced from its ion peaks of ESI-MS at m/z 441.5 ([M+H]+), 903.4 ([2M+Na]+) and EI-MS at m/z 440.3 [M]+, supported by the 13C NMR and 1H NMR spectra. The signal at m/z 139.1 in the EI-MS of XIII suggested that the metabolite XIII should have the same structure of F-ring as diosgenone (II). The prominent peak signals at m/z 297.2 (269 + 28), 326.3 (298 + 28) and 440.3 (412 + 28) suggested that metabolite XIII might have the similar structure of steroid nucleus with diosgenone (II) (diagnostic fragments of diosgenone: m/z 269 and 298). The hydrogen signal [δ 3.59 (3H, s, H-28) ] of methoxyl group in the 1H NMR spectrum of XIII and the signal of δC4 121.2 and δC5 160.8 (shift 3 ppm and 11 ppm toward high field respectively) in the 13C NMR spectrum of XIII indicated that the methoxyl group and the carbon-carbon double bond, respectively, located at C6 and C6-C7, which was supported by the DEPT spectrum of XIII (C6, a quaternary carbon atom; C7 a tertiary carbon atom) and the HMBC spectrum of XIII (the long-range correlations of the H-4 with C2, C5, C6 and C10, and H-7 with C5, C6, C8, C9, C14). Hence, compound XIII was determined as 6-methoxy-6-dehydrodiosgenone [XIII, (25R)-spirost-4, 6-diene-6methoxy-3-one]. Compounds XIV and XV. Amorphous powder (ethyl acetate); mp 62-64 °C; molecular weight (MW) 456; chemical formula C28H40O5; IR (coat) cm-1: 3466, 2929, 2867, 1733, 1766, 1661, 1455, 1378, 1233, 1050, 978, 981, 755; ESI-MS: XIV: m/z 457.4, XV: m/z 457.5, 935.4; EI-MS: m/z 456.4, 155.1 (the base peak). A homogeneous sample of about 16 mg was isolated and purified by preparative TLC. Further analyses of the spectroscopic data indicated the sample was a mixture containing compounds XIV and XV, which was supported by the experimental result of the F-ring rearrangement of the mixture between six membered pyran ring and five membered furan ring through pH adjustment. They have the identical molecular weights (MW) 456 and chemical formula C28H40O5, as deduced from their ion peaks of ESI-MS at m/z

457.4 ([M+H]+) (XIV), 457.5 ([M+H]+) (XV), 935.4 ([2M+Na]+) and EI-MS at m/z 456.4[M]+, supported by their 13C NMR and 1H NMR spectra. Compared with the corresponding data of isonuatigenone (III) and 6-methoxy-6-dehydrodiosgenone (XIII), metabolite XIV should have the same F-ring structure as isonuatigenone (III) (IR spectrum: 3466 cm-1) and the same B-ring structure as 6-methoxy-6-dehydrodiosgenone (XIII) [1H NMR spectrum: δ 3.60 (3H, s, H-28, XIV) and 3.59 (3H, s, H-28, XV)]. Also, compared with the corresponding data of nuatigenone (VIII) and 6-methoxy-6-dehydrodiosgenone (XIII), metabolite XV should have the same F-ring structure as nuatigenone (VIII) and the same B-ring structure as 6-methoxy-6-dehydrodiosgenone (XIII). Hence, compounds XIV and XV were determined as 6-methoxy-6-dehydroisonuatigenone (XIV) [(20S, 22R, 25S)-spirost-4, 6-diene-6methoxy-25-ol-3-one] and 6-methoxy-6-dehydronuatigenone (XV) [(20S, 22S, 25S)-furost-4, 6-diene-6-methoxy-26-ol-3one], respectively. Compound XVI. Amorphous solid (ethyl acetate); mp 56-57 °C; molecular weight (MW) 488; chemical formula C29H44O6; IR (coat) cm-1: 3466, 2931, 2850, 1731, 1666, 1454, 1378, 1261, 1050, 980, 898, 802, 756; ESI-MS: m/z 489.3, 999.3; EI-MS: 488.4, 139.1 (the base peak). The molecular weight (MW) and chemical formula of compound XVI were 488 and C29H44O6, as deduced from its ion peaks of ESI-MS at m/z 489.3 ([M+H]+), 999.3 ([2M+Na]+) and EI-MS at 488.4 [M]+, supported by the 13C NMR and 1H NMR spectra of XVI. Compared with the corresponding data of 6-methoxy-6dehydrodiosgenone (XIII), metabolite XVI should have the same F-ring structure as 6-methoxy-6-dehydrodiosgenone (XIII). The hydrogen signals [δ 3.22 (3H, s, H-28)] and [δ 2.96 (3H, s, H-29)] of two methoxyl groups, the correlation of δC6 99.3 with the two signals and the long-range correlation of C6 with H-4 in the HMBC spectrum of XVI indicated the two methoxyl groups should be located at the same carbon atom C6. The absorption bands characteristic of hydroxyl group (3466 cm-1) in IR spectrum of XVI, the low-field signal (C-O) of δC7 69.6 in the HMQC spectrum of XVI and no correlation (C7R-OH) between H-7 and H-8β in COSY spectrum of XVI suggested that metabolite XVI should have one hydroxyl group positioning at C7 in the R configuration, which was supported by the data of the DEPT spectrum of XVI (C6, a quaternary carbon atom; C7 a tertiary carbon atom). Hence, compound XVI was determined as 6-dimethoxy-7R-hydroxyldiosgenone [XVI, (25R)-spirost-6-dimethoxy-4-en-7Rol-3-one].

Results Intermediate Accumulation and Isolation in Metabolic Pathway of Diosgenin. The biotransformation and degradation of diosgenin was done by the S. virginiae IBL-14 and a series of metabolites were accumulated via adjusting the addition time of diosgenin. Normally, metabolites II and III were easily obtained due to their relatively high accumulation compared to other metabolites. To obtain other metabolites in sufficient quantity for metabolic pathway analysis, the relationship between metabolite accumulation and growth conditions was first investigated. It was noted that the degradation level of diosgenin and the accumulation of intermediates were closely correlated to the cell age, or the time of adding diosgenin to the culture (for details, please see SI Figure S36). After the extraction of the broth mixture collected from both the cell culture and the resting cell culture, about 4 g of extract was obtained. From the 4 g of extract, 11 metabolites were isolated by preparative LC and TLC and then identified by various spectroscopic analyses (for details, please see Materials and Methods and the SI), among which both diosgenone (II) and 1-dehydrodiosgenone (V) are the products of C1 or/and C3-dehydrogenation of diosgenin (I), which VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. 13C-NMR Data (CDCl3) Are for Diosgenin and the Metabolites Produced from It by S. virginiae IBL-14a carbon no.

I

II

III

V

VI

VIII

X

XII

XIII

XIV

XV

XVI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28a 29b

36.7 31.7 71.8 42.3 140.8 121.4 33.7 31.5 50.1 37.2 21.1 39.8 40.3 56.5 31.6 80.8 62.1 16.5 12.4 41.6 14.5 109.3 31.4 28.8 30.3 66.9 17.1

36.4 34.6 200.1 124.6 171.8 33.5 32.8 35.9 54.5 39.4 21.5 40.4 41.1 56.4 32.4 81.3 62.8 17.0 18.1 42.4 15.2 109.9 32.1 29.5 31.0 67.6 17.8

36.4 34.6 200.1 124.6 171.6 33.4 32.8 35.9 54.4 39.4 21.5 40.3 41.1 56.4 32.3 81.9 62.5 17.0 18.1 42.3 15.1 109.8 33.4 27.7 67.3 69.8 25.4

156.3 127.9 186.8 124.2 169.7 33.2 34.2 35.6 52.8 44.1 23.2 39.9 41.1 55.7 32.3 80.9 62.5 16.8 19.2 42.1 14.9 109.7 31.8 29.2 30.7 67.3 17.5

156.2 127.9 186.8 124.3 169.5 33.2 34.1 35.6 52.8 44.0 23.1 39.9 41.1 55.7 32.2 81.5 62.2 16.8 19.2 42.0 14.8 109.5 33.1 27.4 67.1 69.4 25.2

36.4 34.6 200.0 124.6 171.6 33.4 32.8 35.9 54.4 39.3 21.5 40.2 41.3 56.2 32.4 81.7 62.5 16.8 18.1 38.6 15.4 120.9 34.6 31.2 86.7 69.4 24.6

156.2 127.9 186.8 124.3 169.5 33.2 34.1 35.5 52.8 44.0 23.1 39.8 41.3 55.5 32.3 81.3 62.2 16.6 19.1 38.3 15.1 120.6 34.4 30.8 86.4 69.1 24.3

36.4 34.6 200.2 124.6 171.7 33.4 32.8 35.9 54.4 39.4 21.5 40.3 41.2 56.3 32.4 81.2 62.3 16.9 18.1 39.0 15.3 120.8 33.7 33.5 83.1 71.1 24.6 171.9 21.7

35.9 35.3 201.5 121.2 160.8 151.8 110.2 36.8 52.9 38.2 22.2 41.3 43.0 55.9 33.1 82.2 63.9 18.0 18.2 43.3 16.2 111.0 33.1 30.5 31.9 68.6 18.8 56.4

33.8 33.2 199.3 119.2 158.5 149.8 107.8 34.6 50.7 36.0 20.0 39.1 41.1 53.8 31.0 80.6 61.5 16.0 16.7 40.9 14.0 108.7 32.3 26.6 66.3 68.6 24.3 54.3

33.8 33.2 199.3 119.2 158.5 149.8 107.7 34.6 50.7 36.0 20.0 39.0 41.1 53.7 31.0 80.4 61.5 16.0 16.7 37.4 14.2 119.8 33.5 29.9 85.7 68.2 23.5 54.3

37.8 34.5 200.2 129.3 162.0 99.3 69.6 36.0 44.5 39.9 23.4 39.9 41.1 50.5 32.6 81.4 62.7 19.9 18.5 42.4 15.2 109.9 32.1 29.5 31.0 67.5 17.8 48.2 49.9

a For compound XII, 28a and 29b, respectively, denote the carbon atoms of acetic moiety. For compounds XIII, XIV, and XV, 28a denotes the carbon atom of methoxyl group. For compound XVI, 28a and 29b denote the carbon atoms of two methoxyl groups at C6, respectively.

have been reported by Saunders and Saez (26, 31), and the other eight compounds of VI, VIII, X, XII, XIII, XIV, XV, and XVI are reported for the first time (for compound III, please see ref 31). The profiles of the nine new compounds in HPLC are shown in SI Figure S34, and the profiles of several volume compounds I, II, III, V, and VI on TLC were shown in SI Figure S34B. For convenience of understanding of these metabolites, the 13C NMR data of the eleven metabolites were listed in Table 1 (These data of compounds I and II are listed here only for comparison) (32). Metabolic Pathway of Diosgenin in S. virginiae IBL-14. To establish the relationship between metabolites after the isolation and identification of these metabolites, the experiments using metabolite II as substrate were conducted. The results showed that metabolite II was a more suitable substrate for degradation by the S virginiae IBL-14 than diosgenin. When 1.5 mM metabolite II instead of diosgenin was loaded to the culture after 6 h cultivation, metabolite II was almost totally depleted and no detectable intermediates were accumulated after 24 h cultivation. However when metabolite II was added to a 15 h culture, almost all intermediates including metabolites III, V, VI, VIII, X, XII, XIII, XIV, XV, and XVI found in the biotransformation of diosgenin could be detected, suggesting that metabolite II played a key precursor role in the further biotransformation and degradation of diosgenin (also showing that the C4dehydrogenation is the first step in the biotransformation of diosgenin). We noticed that when diosgenin was used as substrate, metabolite III could not be accumulated in abundance in resting cell mode (SI Figure S36C). In contrast, metabolite III accumulated at relatively high levels when metabolite II was used as substrate (30), which indicated that metabolite II is the precursor of metabolite III. In previous investigation for both the hydroxylation of 16,17R-epoxyprogesterone and dehydrogenation of 11β-hydroxy5970

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16,17R-epoxyprogesterone (33), we found that 16,17Repoxyprogesterone could be biotransformed to 1-dehydro11β-hydroxy-16,17R-epoxyprogesterone via both pathways; that is, hydroxylation first and then dehydrogenation or dehydrogenation first and then hydroxylation. Therefore, a presumed intermediate, isonuatigenin (IV), was postulated as the precursor of metabolite III despite the fact that metabolite IV was not detected by LC-MS. As a result, based on the experimental results and their rational relationship between metabolites described above, a conceivable new metabolic pathway of diosgenin in the isolated S. virginiae IBL-14 was established, as shown in Figure 1. In addition, experimental results showed that all of these metabolites could be further transformed and totally degraded. However, there is no evidence yet on how these metabolites degrade further. New Reactions of Derivatives of Diosgenin. Methoxylation is an important chemical reaction in in chemical synthesis and modification (34, 35) and often plays important roles in the activity modification of endogenous or exogenous compounds (36-38) as well as in the improvement of drug activity (37). 6-methoxylation is a novel microbial reaction for diosgenin and steroidal sapogenins and is reported in this paper for the first time. The compounds 6-methoxy6-dehydrodiosgenone (XIII), 6-methoxy-6-dehydroisonuatigenone (XIV), and 6-methoxy-6-dehydronuatigenone (XV) are, respectively, the methoxylation derivatives of metabolites II, III, and VIII. And 6-dimethoxy-7R-hydroxyldiosgenone (XVI) is a double methoxyl product of metabolite XIII. The methoxylation reactions by the S. virginiae IBL-14 are shown in Figure 1. Interestingly, the methoxylation of diosgenone is always concomitant with corresponding dehydrogenation (the conjugated double-bond carbon linkages), like the methoxylation of 4,4′-dibromodiphenyl ether (39) and apocynin (40). However the corresponding 6-dehydrogenation

FIGURE 1. The new metabolic pathway of diosgenin discovered from S. virginiae IBL-14. Solid line arrows indicate the real reactions; dash line arrows represent the putative reactions. The structures in square brackets are presumed intermediates. intermediates VII, IX, and XI have not been isolated, perhaps due to the swift methoxylation of these conjugated doublebond compounds. The C25 tertiary carbon hydroxylation [isonuatigenone (III)] of diosgenin is the other newly observed microbial transformation reaction (30). To validate whether this hydroxylation were carried out by cytochrome P450 monooxygenase or not, the common inhibitors of hydroxylase (P450), 2,2′-bipyridyl, 8-hydroxyquinoline and menadione (0.3-0.6 mM) were separately added to the culture at 3 h before adding of diosgenin. No products of hydroxylation were detected, which suggested that this hydroxylation may be performed by a cytochrome P450 system. Besides microbial transformation, two chemical reactions could easily occur in new metabolites III and VIII due to the F-ring activation of these compounds after C25 tertiary carbon hydroxylation. Experiments validated that in acidic condition, compounds III and XIV could easily convert to compounds VIII and XV, and vice versa. These observations demonstrated that nuatigenone (VIII) and 6-methoxy-6dehydronuatigenone(XV) are the acidic derivatives of isonuatigenone (III) and 6-methoxy-6-dehydrisoonuatigenone. The putative mechanism is described in Figure 2A, in which carbocations XVII and XVIII are the presumed transitional intermediates from III to VIII and XIV to XV in the presence of hydrogen ion. 26-acetyl-nuatigenone (XII) as the esterified

product of compound VIII and acetic acid was found in the culture broth, and was also prepared with more than 95% yield simply by mixing compound III or VIII with anhydrous acetic acid and then incubating at 60 °C for 1 h. Based on the experimental results, the reaction formula between compounds VIII and XII was established as shown in Figure 2B. In addition, 1-dehydroisonuatigenone (VI) and 1-dehydronuatigenone (X) as the C1, 2 dehydrogenation derivatives of compounds III and VIII, respectively, were isolated and purified from the culture broth. The Degradation of Other Steroids by S. virginiae IBL14. In the steroid pharmaceutical industry, almost all of the steroidal drugs can be synthesized from diogenin, so, it is possible for S. virginiae IBL-14 as the diogenin-degrading strain to digest other steroidal drugs efficiently. Eight representative compounds were tested (for details, please see SI Figure S35). Among the eight steroids, six (oestrone, cholesterol, progesterone, isotestosterone, dihydrotestosterone, and hydrocortisone) were degraded at a significant level. However medroxyprogesterone acetate and cyproterone acetate could not be degraded by the S. virginiae IBL-14, perhaps due to the presence of C6 moieties, which implies that the 6-methoxylation in the biotransformation of diosgenin by the S. virginiae IBL-14 might play a key role in the degradation of the steroids XIII, XIV, XV, and XVI. Especially, we noticed that oestrone with the stable A-ring of alternating VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Postulated chemical reaction mechanism among compounds III, VIII, XII, XIV, and XV. A: Nucleophilic rearrangement mechanism between isonuatignenone (III) and nuatigenone (VIII) as well as 6-methoxy-6-dehydroisonuatigenone (XIV) and 6-methoxy-6-dehydronuatigenone (XV). B: The reaction formula between nuatigenone (VIII) and 26-acetyl-nuatigenone (XII). Solid line arrows indicated the detected reactions; dash line arrows denoted the putative reactions. double carbon bonds could be efficiently degraded by the S. virginiae IBL-14, which implies that the new isolated S. virginiae IBL-14 may have the potential ability to biodegrade other steroidal derivatives with the same A-ring structure.

Discussion Three dehydrogenated products, 1-dehydrodiosgenone (V), 1-dehydroisonuatigenone [VI], and 1-dehydronuatigenone [X] with the same A, B, C, and D-rings structure as androsta1,4-diene-3,17-dione, the precursor of metacleavage pathway of testosterone in Comamonas testosteroni reported by Paul Talalay et al. (19-21), were first obtained, suggesting that in the degradation of V, VI, and X by the S. virginiae IBL-14, there may exist the classical B-ring cleavage style similar to the degradation pathway of testosterone. However, oestrone with the stable A-ring of alternating double carbon bonds, which could not be attacked by C. testosteroni (19), can be completely degraded by the S. virginiae IBL-14 (please see SI Figure S35B), implying that there may be another novel pathway to hydrolyze other derivates of diosgenin and other steroids in the S. virginiae IBL-14. Like the testosterone degradation by C. testosterone (20, 21), the C3-dehydrogenation of diosgenin is the first step for the degradation of diosgenin by the S. virginiae IBL5972

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14, which was validated by our experimental results. However, the artificial diosgenin-3-acetate could not be transformed by the S. virginiae IBL-14, implying the conjugated double bond at C1, 3, and 4 may facilitate the B-ring cleavage (diosgenin derivates V, VI, and X). Furthermore, when metabolite II instead of diogenin as substrate was added to the culture, almost all of the metabolites (III, V, VI, VIII, X, XII, XIII, XIV, XV, and XVI) biotransformed from diosgenin were detected. This finding (in particular metabolites III and V) also substantiates that metabolite II is a first key precursor for further biotransformation of diosgenin. C1-dehydrogenation, another key step in testosterone degradation by C. testosteroni (20, 21), was also found in the diosgenin metabolism of the S. virginiae IBL-14 and the metabolites V, VI, and X are, respectively, the C1-dehydrogenated derivatives of metabolites II, III, and VIII (Figure 1). In addition, three 6-dehydrogenated metabolites XIII, XIV, and XV were isolated and purified. It has been reported that methoxylation of antibiotics plays an important role in resisting the corresponding enzymatic degradation (41, 42). Interestingly, we did not find further C1-dehydrogenated derivatives of methoxylated products XIII, XIV, XV, and XVI. Two explanations are possible: (1) methoxylation on the B-ring probably plays a role in inhibiting C1-dehydrogenation of metabolites

or (2) the C1-dehydrogenation products of the methoxylated metabolites can easily be degraded so as to not be detected. However, further evidence is needed. Hydroxylation has been used to synthesize intermediates of many steroid drugs (43). However, C25-hydroxylation, a tertiary carbon hydroxylation, is a new microbial reaction that occurs on the F-ring of diosgenin to form isonuatigenone (III). To our knowledge, microbial reactions occurring on the ring E/F of steroidal sapogenins (off streroidal-nucleus) have not been reported, though several plant natural products with the similar structure to metabolite III have been documented (44, 45). Further research revealed that the C25 hydroxylation could activate the F-ring, which was substantiated by the interconversion of metabolites III and VIII (Figure 2). Additionally, one C7-hydroxylated metabolite XVI was also detected after C6 methoxylation. C6-methoxylation is a novel steroidal microbial transformation and the role of C5-methoxylation (chemical modification) in the improvement of anti-inflammatory activity of apocynin and its analogs in vitro has been reported (40). In our study, three C6 monomethoxylated metabolites XIII, XIV, and XV and one C6 double-methoxylated metabolite XVI have been isolated. Interestingly, the 6-methoxylation is always concomitant with 6-dehydrogenation. In view of the conceivable spatial hindrance of 6-methoxyl group and subsequently the formation of double-methoxylated metabolite XVI, we speculate that the 6-dehydrogenation occurs before the 6-methoxylation. Therefore, three 6-dehydrogenated intermediates VII, IX, and XI are postulated in Figure 1. Methoxylation enzyme systems in microorganisms can be divided into two classes, a double-enzyme system (38) and a two-protein-component system (36). The methoxylation enzyme system in the S. virginiae IBL-14 may belong to the two-protein-component system since no corresponding hydroxylated products before methylations were found in our research. This suggestion can be confirmed through further genetic analysis. Furthermore, the methoxylated metabolite XVI with three functional groups on the B-ring (two 6-methoxyl groups and a 7-hydroxyl group) is a potentially important derivate of diosgenin for the synthesis of many new steroidal drugs. Besides these microbial reactions discussed above, two important chemical reactions, structural rearrangement (nuatigenone VIII and 6-methoxy-6-dehydronautigenone XV) and esterification (26-acetyl-nuatigenone XII), were confirmed in our research due to the activation of the C25hydroxylation (isonuatignone III). Metabolite VIII is the F-ring rearrangement product of metabolite III, with a five membered furan ring instead of a six membered pyran ring, which easily occur under mild acidic condition (even in the acidic culture). Compound XII, another corresponding derivative of compound III, was also achieved by chemical synthesis (Figure 2B). These results manifested the compounds with the C25 or C26 hydroxyl group in diosgenin derivates are highly active. In fact, VIII is a rare furospirostane-type sapogenin mainly from avenacoside A and B in oat and aculeatisides A and B in Solanum aculeatissimum (45). So these new hydroxylated products of diosgenin (III, VI, VIII, X, XII, XIV, and XV) may become new materials to synthesize new active intermediates of steroids. Now, the consumption of diosgenin in the world is about 3000-3500 tons per year and the production in China was 2200 tons only in 2006 despite of falling demand in the last years (46). Normally, in China, extraction of one ton of diosgenin from the tubers of Dioscorea wild yam generates a loss of about 100 kg of steroids and results in release of about 250-1000 tons of wastewater to the environment (http://qkzz.net/magazine/1006-9739/2007/08/ 1964873.htm). To implement PRC South to North Water Project and to protect water quality in Han river, over 60

diosgenin factories were closed down (http://www.sxdaily.com.cn/data/bssnlw/20081014_9987152_11.htm). To control the wastewater pollution caused in industrial production of diosgenin has become an important task for Shangluo Environmental Protection Bureau in Shangluo City, China. In this research, eight representative steroids including oestrone, cholesterol, progesterone, isotestosterone, dihydrotestosterone, hydrocortisone, medroxyprogesterone acetate and cyproterone acetate were tested. Among them, the forenamed six were effectively degraded and the latter two could not be degraded by the S. virginiae IBL-14 probably due to the presence of C6 moieties and the ester formation compared with other steroids above. In particular, oestrone, the representative of estrogen which could not be degraded by the well-known steroids-degrading strain C. testosteroni, could be efficiently degraded by the S. virginiae IBL-14 (30), suggesting that the new isolated S. virginiae IBL-14 has potential for application in the biodegradation and environmental protection in the steroid drug industry.

Acknowledgments We are grateful to Hei-Qing Wang, Zhejiang University, Hangzhou, Zhejiang, China and Gang Zhang, Tianjin Research Institute of Industrial Microbiology, Tianjin, China, for a generous gift of steroidal materials. We are grateful to Michael L Shuler, Cornell University, and Xiang Dong, Anhui University, China, for suggestions to improve this manuscript.

Supporting Information Available The data for structural analysis of the metabolites produced from diosgenin by S. virginiae IBL-14, the profiles of the new metabolites in HPLC, the profiles of several compounds I, II, III, IV, and V on TLC, the degradation of several steroidal compounds by S. virginiae IBL-14 and the biotransformation and degradation of diosgenin by Streptomyces virginiae IBL14. This material is available free of charge via the Internet at http://pubs.acs.org.

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