Accepted Manuscript Baeyer-Villiger oxidation of progesterone by Aspergillus sojae PTCC 5196 Mehri Javid, Bahman Nickavar, Hossein Vahidi, Mohammad Ali Faramarzi PII: DOI: Reference:
S0039-128X(18)30131-4 https://doi.org/10.1016/j.steroids.2018.07.008 STE 8292
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Received Date: Revised Date: Accepted Date:
10 February 2018 13 July 2018 18 July 2018
Please cite this article as: Javid, M., Nickavar, B., Vahidi, H., Faramarzi, M.A., Baeyer-Villiger oxidation of progesterone by Aspergillus sojae PTCC 5196, Steroids (2018), doi: https://doi.org/10.1016/j.steroids.2018.07.008
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Baeyer-Villiger oxidation of progesterone by Aspergillus sojae PTCC 5196
Mehri Javida, Bahman Nickavara, Hossein Vahidi*,a, and Mohammad Ali Faramarzib
Department of Pharmaceutical Biotechnology and Pharmacognosy, Faculty of Pharmacy,
Shahid Beheshti University of Medical Sciences, P.O. Box 14155-6153, Tehran, Iran b
Department of Pharmaceutical Biotechnology and Biotechnology Research Center, Faculty
of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155–6451, Tehran 1417614411, Iran ------------------------------*Corresponding author: Tel: +98-21-88200100; Fax: +98–21–88209620; E-mail: [email protected]
Abstract Microbial transformations are capable of producing steroid substances difficult to synthesize by chemical methods. Strains belonging to the genus Aspergillus are effective facilitators of microbial biotransformations due to their enzymatic diversity. In this study, the biotransformation of progesterone by the fungus Aspergillus sojae (A. sojae) PTCC 5196 was examined. Analysis of the bioconversion process revealed that progesterone was converted to testololactone through a three-step pathway (17β-acetyl side chain cleavage, 17β-hydroxyl oxidation, and oxygenative lactonization of 17-ketone), indicating the presence of BaeyerVilliger monooxygenase (BVMO) activity in the fungal strain. GC analysis confirmed the production of testololactone with a yield of 99% in 24 h. Faster testololactone production was induced in the presence of both C-21 (progesterone) and C-19 (androstenedione, testosterone, and dehydroepiandrosterone [DHEA]) steroid substances. Due to the high biotransformation rate observed in the present study, A. sojae may be a novel and promising candidate in the production of testololactone. Keywords: Biotransformation, Aspergillus sojae, Progesterone, Testololactone, BaeyerVilliger oxidation, Steroids
1. Introduction Of all biologically important compounds, steroids are among the most widely used in the pharmaceutical industry for therapeutic purposes [1, 2]. In addition to their widespread application as sex hormones, steroids have been utilized as anabolic, anti-allergic, antiandrogenic, antibacterial, anti-estrogenic, antifungal, anti-inflammatory, antitumor, antiviral, diuretic, immunomodulatory, and sedative agents [1, 3–5]. This lends particular exigence to the ongoing search for new and stronger steroid analogues [6–8]. Biotransformation technology, a wide and developing branch of biotechnology, has played and continues to play a significant role in the discovery and production of such active pharmaceutical ingredients [1–3]. In comparison to traditional chemical synthesis, microbial transformations via elective chemical reaction feature remarkable regio- and stereoselectivity, affordable and effective processes, mild temperature and pressure operations, green chemistry ideology actualization, and environmental compatibility [4–8]. Steroid biotransformation prepares about 5,000 potential compounds with different functional groups on the steroidal hydrocarbon skeleton [8, 9]. Moreover, current research and development efforts in the steroid biotransformation industry have resulted in new microbial strain identification techniques with potential industrial uses [9, 10], improvements on existing biocatalysts , yield enhancements, and key intermediate production difficult to synthesize by chemical methods [10, 11]. Among various microorganisms, fungi with high enzymatic diversity are among the strongest tools in steroid biotransformation studies [10, 12–14]. Several species of the genus Aspergillus have been found to possess diverse enzymatic activities useful in the transformation of progesterone to its valuable derivatives. Hydroxylation, oxidation, reduction, hydrolysis, degradation, and double bond formation are typical examples of chemical reactions performed on steroidal compounds by this genus. Particular attention,
however, has been given to its capacity to form steroidal D-ring lactones due to their potential use as anticarcinogen, antiandrogen, and antibacterial agents [1, 5, 6, 14, 15]. Testololactone as an aromatase inhibitor and estrogen synthesis reducer is a case in point [7, 16]. This compound can be produced from a number of steroids via either chemical or microbial Baeyer-Villiger reactions. Chemical methods, however, can produce dangerous peracid oxidants with explosive features and shock sensitivity as well as various by-product generations, including regioisomeric lactones, epoxy lactones, and epoxy ketones. Microbial enzymatic conversion using Baeyer-Villiger monooxygenase (BVMO) is thus a preferable candidate for production of testololactone. Indeed, BVMO enzymes can be observed in a variety of fungi, although they have been often found to exhibit a low level of productivity as a result of slow biotransformation and undesired by-products [7, 17–19]. The present study investigated the use of Aspergillus sojae (A. sojae) PTCC 5196 in the conversion of progesterone, testosterone, and androstenedione, determining plausible metabolic pathways and inducer effects in the biotransformation of progesterone.
2. Experimental 2.1. Chemicals, instruments, and analytical methods Progesterone, testosterone, androstenedione, and dehydroepiandrosterone (DHEA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Culture media and organic solvents were acquired from Merck (Darmstadt, Germany). All other reagents were of commercially available and analytical grade. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded using a Bruker DRX (Avance 500) spectrometer (Rheinstetten, Germany) 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. The coupling constant (J) is given in Hertz (Hz). EI-MS spectra were obtained using an Agilent 6410 Triple
Quad mass spectrometer. Infra-red (IR) data were collected using a Perkin-Elmer 843 spectrometer with KBr as a diluent. Thin layer chromatography (TLC) was conducted on 0.25-mm thick layers of silica gel G (Kieselgel 60 HF254+366, Merck) using acetone/chloroform/n-hexane (2:2:6; v/v/v) as an eluent solvent; observation of the plates was carried out under a UV lamp (Strstedt-Gruppe HP-UVIS) at 254 nm. GC-FID analysis was performed using an Agilent GC 7890A gas chromatograph (GC) with an FID and an HP-5 capillary column (30 m × 0.25 mm, 0.25 μm film thickness).
2.2. Fungal cultivation and growth curve The fungal strain of A. sojae used was taken from the Persian Type Culture Collection (Iranian Research Organization for Science and Technology, Tehran, Iran). Stock cultures were maintained on Sabouraud Dextrose Agar (SDA) slants at 4 °C and freshly sub-cultured before use in the biotransformation experiments. An Erlenmeyer flask (1000 ml) containing 250 ml of Sabouraud Dextrose Broth (SDB) medium was inoculated with freshly obtained spores from slant cultures (5 ×104 CFU/ml as the final concentration) and incubated at 25 °C in a rotary shaker (170 rpm). Sampling was carried out every 24 h. The content of each sample was then filtered and dried at 60 °C. Fungal growth was measured by dry weight estimation; the process was repeated until relative stabilization of biomass weight was achieved. All experiments were in triplicate. Growth curve of the fungus was plotted based on cell weight measurements against cultivation time.
2.3. Biotransformation conditions and product extraction 250-ml Erlenmeyer flasks, each containing 50 ml of the cultivation medium, were inoculated with a suspension of fresh spores and then incubated at 25 °C and 170 rpm. After 24 h, 50 mg of progesterone dissolved in 1 ml of methanol were aseptically added to each
flask. Incubation was continued for 48 h and the progress of the reaction was monitored by TLC. A sterile control was similarly processed. After detecting the transformation and distinction of appropriate time by TLC, a semi-preparative scale was carried out using a biotransformation volume of 2000 ml under the same procedures described above. Following incubation, the reaction mixture was filtered, extracted (×3 with CHCl3), and then concentrated using a rotary evaporator under vacuum pressure.
2.4. Product purification and structure identification Final residue of the dried extract was dissolved in CHCl3 and subjected to repeated preparative TLC using a solvent system of acetone/chloroform/n-hexane (2:2:6; v/v/v). Steroids were purified and the purified metabolite was analyzed and structurally identified by 1
H-NMR, 13C-NMR, EI-MS, and IR.
2.5. Time course experiment and GC analysis 2.5.1. Time course study of the biotransformation of progesterone A time course study was performed to investigate the metabolic pathways used in the biotransformation of progesterone. The reaction conditions were similar to those mentioned in section 2.3. After adding the substrate, sampling and extraction were conducted on all experiments at 4, 8, 16, 24, and 48 h. Reaction mixtures were concentrated and analyzed by GC. The initial oven temperature was held at 50 °C for 2 min before being increased to 100 °C at a heating rate of 3 °C/min; the column temperature was then programmed from 100 °C to 290 °C at a heating rate of 6 °C/min and held at this temperature for 6 min. The carrier gas was N2 with a flow rate of 2 ml/min. The injector and detector temperature were adjusted to 300 °C. The sample size was 1.0 μl with a split ratio of 1:5. The retention time of each
metabolite was determined using the co-injection method. The entire procedure was performed in triplicate for each analytical determination.
2.5.2. Time course study of the biotransformation of testosterone and androstenedione To further evaluate the biocatalytic proficiency of A. sojae in D-lactonization, testosterone and androstenedione, two intermediate steroid metabolites, were examined as starting materials. Biotransformation experiments, sampling, and GC analysis conditions were identical to the procedures described in section 2.5.1.
2.5.3. Time course study of the biotransformation of progesterone using a substrate-induced culture After inoculation in 500-ml Erlenmeyer flasks containing 100 ml of the cultivation medium, 10 mg of four steroids susceptible to BVMO transformation (progesterone, androstenedione, testosterone, and DHEA) were separately added to the cultures as inducers; incubation were carried out under the conditions mentioned above. After 24 h, 100 mg of progesterone was aseptically added to each flask and transformations were continued for a further 48 h. The samples (5 ml) were extracted from all environments at 4, 8, 16, 24, and 48 h and subsequently analyzed by GC.
3. Results and discussion 3.1. Biotransformation of progesterone by Aspergillus sojae Analytical TLC resulted from the biotransformation process are shown in Figure 1. According to the presented pattern, 16 h after the beginning of the transformation served as an appropriate extraction time in the semipreparative scale. Analyses of the substrate bioconversion process showed that progesterone (I) was converted to three major metabolites
(II–IV) by A. sojae. No transformation occurred in the control media. The chemical structures of the products were primarily determined based on analytical data described in the following sections (Figure 2).
3.2. Structure elucidation of the isolated metabolites Androst-4-en-3,17-dione (II): White solid; Rf in acetone/chloroform/hexane (2:2:6; v/v/v): 0.45; IR νmax (cm-1): 1734, 1667, 1609; MS (EI) m/z (%): 286 (M+, C19H26O2; 92 ), 244 (62), 201 (20), 148 (64), 124 (100), 97 (40), 69 (10). 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) were assigned to Table 1 and Table 2, respectively. The molecular formula of compound II was deduced as C19H26O2 on the EI-MS spectrum. It was found to be 286, which indicated a decrease of 28 units compared to the substrate. The IR spectrum indicated absorbance at 1734 cm-1 for the 17-carbonyl group and at 1667 cm-1 for 3-ketone conjugated with the double bond on C-4. On the 1H NMR spectrum, the absence of the characteristic H-21 singlet at δ 2.12 and H-17 triplet at δ 2.53, along with the large downshift of H-18 in progesterone from δ 0.67 to δ 0.93, indicated a change near C-18. These data were supported with 13C NMR, which indicated that δ 63.5 had been replaced by δ 220.0 for C-17 and identified the absence of signals for C-20 and C-21. These results showed that the side chain of progesterone had been cleaved; the substrate was then converted to androst4-en-3,17-dione (androstenedione). These findings were consistent with the data reported in existing literature [20, 21]. 17β-Hydroxyandrost-4-en-3-one (III): White solid; Rf in acetone/chloroform/hexane (2:2:6; v/v/v): 0.37; IR νmax (cm-1): 3432, 2934, 1664, 1609; MS (EI) m/z (%): 288 (M+, C19H28O2; 32), 246 (29), 228 (12), 203 (20), 164 (22), 147 (33), 124 (100), 109 (25), 81 (20). 1
H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) were assigned to Table 1 and
Table 2, respectively.
Metabolite III (C19H28O2) exhibited a molecular ion peak at m/z 288 on its EI-MS. The IR spectrum revealed absorption bands for 3-ketone (1664 cm-1) conjugated with a double bond at the C-4 position (1609 cm-1) and the hydroxyl group (3432 cm-1). The 1H NMR spectrum included signals for two methyl groups (δ 0.79, H-18, and δ 1.20, H-19; each as singlet) and a singlet at δ 5.72 for H-4. Cleavage of the side chain including C-20 and C-21 was indicated by the absence of characteristic C-20 and C-21 on the 13C NMR spectrum. The 17β-hydroxyl group was confirmed through the replacement of δ 63.5 with δ 81.6 by C-17 as well as by the appearance of a distinctly visible triplet at δ 3.65 (J = 8.5 Hz). The comparison of spectral data from the present study with that in the literature [20, 21] confirmed the presence of 17βhydroxyandrost-4-en-3-one (testosterone). D-Homo-17a-oxaandrost-4-en-3,17-dione (IV): White crystalline solid; Rf in acetone/chloroform/hexane (2:2:6; v/v/v): 0.23; IR νmax (cm-1): 2953, 1732, 1667, 1611; MS (EI) m/z (%): 302 (M+, C19H26O3; 56), 287 (12), 260 (90), 246 (12), 230 (14), 187 (19), 133 (31), 124 (39), 108 (32), 97 (22). 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) were assigned to Table 1 and Table 2, respectively. For compound IV (C19H26O3), a molecular ion was displayed at m/z 302 on the EI-MS spectrum. The IR spectrum showed absorption frequencies at 1732, 1667, and 1611 cm-1 for lactone carbonyl, 3-ketone conjugated with 4-ene, and a double bond at C-4, respectively. Neither 17-H nor 21-H signals were detected. There was, however, a notable downfield shift from δ 0.67 to δ 1.36 for the H-18 in progesterone on the 1H NMR spectrum. This suggested that there would be an alteration near the position of C-13 on the 13C NMR spectrum. The absence of C-20 and C-21 signals further indicated the existence of 19C-steroid, which featured significant differences in positions C-13 to C-18 compared to the substrate. The most significant changes were observed for C-13 (from δ 43.9 to δ 82.6) and C-17 (from δ 63.5 to δ 170.9). This evidence, in good accordance with the literature [9, 20, 21], indicated D-lactone
formation via the insertion of an oxygen atom adjacent to the position of C-13 on ring D, thus supporting the proposed structure of compound IV as D-homo-17a-oxaandrost-4-en-3,17dione (testololactone).
3.3. Determination of plausible metabolic pathways and the effect of inducers Time course of the production of steroid metabolites was investigated by GC analysis (Figure 3) and the results are summarized in Table 3 and Figure 4. Progesterone was converted to androstenedione and testosterone within 4 h of incubation by the fungus. The reaction mixture contained a larger percentage of androstenedione and testosterone after 8 h of incubation; however, after 24 h, these metabolites were nonexistent and the amount of testololactone had considerably increased in the medium. It can thus be inferred that androstenedione and testosterone are intermediate metabolites in the production of testololactone. A separate experiment confirmed that androstenedione and testosterones as primary substrates were converted into testololactone and accumulated in a culture of A. sojae (Table 3). Androstenedione was detected when testosterone was applied as a substrate; testosterone was also found when androstenedione was used as the starting material. The results of the present study suggest that the side chain was oxidized by BVMO with the insertion of molecular oxygen into a progesterone C17-C20 bond, yielding testosterone acetate that fast hydrolyzed to testosterone and, following oxidation, yielded androstenedione. These two intermediates were in equilibrium via a reversible redox reaction in the 17-carbonyl group. Finally, ring-D Baeyer-Villiger oxidation was performed by the insertion of oxygen into the androstenedione C13-C17 bond, yielding testololactone (Figure 2) [19, 22]. Time course analysis of the transformation of substrates indicated that BVMOs acted as steroid-induced enzymes. The reaction mixture contained 1.72% androstenedione and
1.33% testosterone after 4 h of the biotransformation of progesterone. Testololactone was initially identified after 8 h of reaction time in the mixture; its content increased in intervals of 8 h to 70.35% and 98.8%, indicating that enzyme-catalyzed oxidation reactions were inducible in the presence of progesterone as a substrate. The rapid increase in the amount of testololactone after 16 h of incubation with progesterone and androstenedione acting as substrates shows good induction of the enzymes responsible for D-lactonization of C17ketones. Further, the higher amount of progesterone transformation after 24 h, along with the more quickly obtained maximum amount of testololactone in samples resulting from progesterone conversion (Table 3), support the presumption that progesterone is a more effective inducer than androstenedione. A separate experiment confirmed that testololactone production in the presence of progesterone as an inducer was faster than in the presence of androstenedione as an inducer (Figure 5). These results are in keeping with a number of previous studies that have found progesterone and androstenedione to be effective inducers for BVMOs [6, 19, 23, 24]. However, while testosterone was found to be the intermediate metabolite in the proposed reaction pathway, the yield of testololactone from testosterone after 48 h was much lower than the yield from progesterone and androstenedione. There are three potential explanations for this unexpected phenomenon. First, the higher concentration of testosterone substrate at the beginning of the transformation may have inhibited oxygenative lactonization of 17-ketosteroid, however, it acts similar to androstenedione as the inducer (Figure 5). Second, the reversible reaction resulted in androstenedione production from testosterone might be rate-determining step that postponed testololactone production. Last but not least, progesterone and androstenedione as substrates might be more effective inducers for BVMO production than testosterone. A similar observation was reported for the biotransformation of progesterone to ∆1-testololactone by Septomyxa affinis, in which androst-1,4-diene-3,17-dione
(ADD) as a suggested intermediate could not be transformed to ∆1-testololactone when used as a starting substrate . The composition of mixtures formed during the transformation of progesterone using a substrate-induced culture indicated that the BVMOs of A. sojae can carry out effective progesterone oxidation in the presence of steroids susceptible to BVMO transformation (Figure 5). Time lag (~ 8 h) in the transformation of progesterone was eliminated using both 4-en-3-oxo compounds, including progesterone, androstenedione, and testosterones, and 3βhydroxyl-5-ene (DHEA) as BVMO inducers. This phenomenon is dominant at the presence of progesterone (as a C-21-inducer) in comparison to androstenedione, testosterone, and DHEA (as C-19-inducers). C-19 compounds accelerate the production of testololactone as a result of induction of the BVMO responsible for D-ring lactonization. In contrast, BVMOs induced in the presence of C-21 compounds is responsible for 17β-acetyl chain cleavage. This facilitated progesterone conversion, leading to faster production of intermediate metabolites with the potential for BVMO induction. This also resulted in faster production of testololactone in the presence of progesterone as an inducer. The production of testololactone from progesterone, testosterone, or androstenedione has been performed using various microbial strains and BVMOs, yielding results with diverse catalytic features. As can be seen in Table 4, the transformation process is performed within 1–5 days with testololactone isolated in yields of 10.8-98%. Two major disadvantages of using Penicillium citreo-viride , Penicillium notatum , Penicillium lilacinum , Penicillium camemberti , Rhizopus stolonifer , Aspergillus terreus , and Aspergillus tamari  are their long reaction time and/or low substrate concentration. Furthermore, industrial production of testololactone is restricted in order to the undesired byproducts accumulated in the reaction mixture of some species, which can require additional steps in the purification of final product. In the current study, BVMOs from A. sojae
facilitated the biotransformation of progesterone and androstenedione to testololactone within 24 h with negligible by-products. The time lag of the bioconversion could be eliminated by the BVMO inducers. Testololactone was isolated in a yield of 99% and 98.6% for 1 g/l of progesterone and androstenedione, respectively.
4. Conclusion Progesterone, testosterone, and androstenedione were converted to testololactone in a culture of the fungus A. sojae. The biotransformation pathway represents that the fungal strain produces BVMOs, which can carry out both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids. The time lag of the bioconversion is eliminated by both C-21 and C-19 BVMO inducers. These conditions produced significant amounts of testololactone within 24 h. The high rate of D-ring lactonization exhibited in the present study suggests that this fungal strain may have potential commercial applications in the production of testololactone.
Acknowledgements This study was financially supported by a grant from Shahid Beheshti University of Medical Sciences, Tehran, Iran. The results were extracted from the Pharm. D. Thesis of Mehri Javid (Faulty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran).
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Figures legends Fig. 1. TLC chromatogram of progesterone (I) biotransformation by Aspergillus sojae (A. sojae). Lanes 1–6 indicate fungal transformation at 0, 4, 8, 16, 24, and 48 h, respectively. Fig. 2. Plausible D-lactonization pathway of progesterone (I) by A. sojae. Fig. 3. GC chromatogram of progesterone (I) biotransformation by A. sojae. A 16 hincubation sample was analyzed. Fig. 4. Time course profile of cell growth and the progesterone (I) biotransformation by A. sojae at 25 °C for 48 h (mean + SD; n = 3). Fig. 5. Comparison of testololactone percentage obtained after progesterone transformation using non-induced and substrate-induced cultures of A. sojae.
Table 1. 1H NMR signals of substrate (I) and metabolites (II-IV) [δ in ppm downfield from TMS, in CDCl3 (see Fig. 2)]. Hydrogen atom
5.75 (1H, s)
5.72 (1H, s)
5.75 (1H, s)
2.53 (1H, t, J =
3.65 (1H, t, J =
0.92 (3H, s)
0.79 (3H, s)
1.36 (3H, s)
1.22 (3H, s)
1.20 (3H, s)
1.17 (3H, s)
2.12 (3H, s)
Table 2. 13C NMR signals of substrate (I) and metabolites (II-IV) [δ in ppm downfield from TMS, in CDCl3 (see Fig. 2)]. Carbon atom
Table 3. GC analysis of crude mixture obtained from transformation of progesterone by A. sojae. Substrate
Steroid present in
the mixture (%)
Time of transformation (h)
Steroid present in
the mixture (%)
Time of transformation (h)
Table 4. Microbial production of testololactone by various fungal strains.
tion (g/l) Penicillium citreo-
viride ACCC 0402 Progesteron e Penicillium notatum
ostenedione, 17a- oxa-D homo-5αandrostan3,17-dione Progesteron
e Testosteron e Penicillium
mum WY134-2 29
e Testosteron e Penicillium lilacinum
i AM83 Progesteron e Rhizopus
androstan1,6-dione, 11αhydroxyandr ost-4-en3,17-dione, 11αhydroxytesto lactone
MRC 200365 Testosteron
e Aspergillus tamari
MRC 72400 Testosteron e Determined by a HPLC and b GC analyses
Progesterone converted by the fungus Aspergillus sojae PTCC 5196 produced testololactone.
The production of testololactone indicated fungal Baeyer-Villiger monooxygenase (BVMO) activity.
Substrate-induced cultures have a decisive impact on the metabolism of progesterone.
Progesterone, a C-21 steroidal compound, induced 17β-acetyl side chain cleavage.
Androstenedione, testosterone, and DHEA, C-19 steroidal substances, induced ring-D oxidation.