Appl Biochem Biotechnol DOI 10.1007/s12010-014-1254-y
Greater Taxol Yield of Fungus Pestalotiopsis hainanensis from Dermatitic Scurf of the Giant Panda (Ailuropoda melanoleuca) Yu Gu & Yanlin Wang & Xiaoping Ma & Chengdong Wang & Guizhou Yue & Yuetian Zhang & Yunyan Zhang & Shanshan Li & Shanshan Ling & Xiaomin Liu & Xintian Wen & Sanjie Cao & Xiaobo Huang & Junliang Deng & Zhicai Zuo & Shumin Yu & Liuhong Shen & Rui Wu
Received: 1 July 2014 / Accepted: 10 September 2014 # Springer Science+Business Media New York 2014
Abstract While taxol yields of fungi from non-animal sources are still low, whether Pestalotiopsis hainanensis isolated from the scurf of a dermatitic giant panda, Ailuropoda melanoleuca, provides a greater taxol yield remains unknown. The objective of the study was to determine the corresponding taxol yield. The structure of the taxol produced by the fungus was evaluated by thin layer chromatography (TLC), ultraviolet (UV) spectroscopy, highperformance liquid chromatography (HPLC), 1H and 13C nuclear magnetic resonance spectroscopy (1H-NMR and 13C-NMR), and time-of-flight mass spectrometry (TOF-MS), with standard taxol as a control. The results demonstrated that the P. hainanensis fungus produced taxol, which had the same structure as the standard taxol and yield of 1,466.87 μg/L. This fungal taxol yield from the dermatitic giant panda was significantly greater than those of Xiaoping Ma and Chengdong Wang contributed equally to this work, tied for Xiaoping Ma. Yu Gu and Yanlin Wang contributed equally to this work and should be considered as joint first authors, tied for Yu Gu. Y. Gu College of Life Sciences, Sichuan Agricultural University, Ya’an 625014, China Y. Wang : G. Yue College of Sciences, Sichuan Agricultural University, Ya’an 625014, China X. Ma : Y. Zhang : Y. Zhang : S. Li : X. Wen : S. Cao : X. Huang : J. Deng : Z. Zuo : S. Yu : L. Shen : R. Wu College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China X. Ma (*) College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected] C. Wang (*) : S. Ling : X. Liu China Conservation and Research Center for Giant Panda, Wolong 623006, China e-mail: [email protected]
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fungus from non-animal sources. The taxol-producing fungus may be a potential candidate for the production of taxol on an industrial scale. Keywords Pestalotiopsis hainanensis . Animal surface fungi . Ailuropoda melanoleuca . Taxol . Higher yield
Introduction Taxol, a type of diterpene, was first isolated from the bark of the Pacific Yew tree (Taxus brevifolia) in the late 1960s by Wani [25]. Taxol has been found to exhibit antitumor and antimicrobial activity and is thus being used to treat breast, ovarian, lung and head cancers, and Kaposi’s sarcoma [9]. While paclitaxel has been successfully produced on a commercial scale at a yield of more than 350 mg/L using Taxus cell culture by Phyton Biotech (Germany) and Samyagn Genex (South Korea) since 2000, because Taxus spp. grows extremely slow but is the main source of taxol, a large amount of Taxus spp. has to be used, which will run out resource of Taxus spp. to endanger ecosystems. Taxol has been successfully synthesized in the laboratory, but its production of chemical synthesis on a large scale in the factory is still not feasible [3, 7]. In 1993, Stierle separated a taxol-producing endophytic fungus Taxomyces andreanae from the phloem of T. brevifolia [19]. Since then, many taxol-producing plant endophytic fungi have been isolated from Taxus spp. and its close species [11, 12, 24, 17]. More than 30 fungi species are found to produce taxol [15]. Pestalotiopsis genus, isolated from the Taxus spp. host, is the most common and dominant endophyte fungi. However, Pestalotiopsis yields low levels of taxol [20, 21, 10, 8, 18, 23, 1, 4], with the highest yield only up to 500 μg/L from Pestalotiopsis versicolor [18], 557.8 μg/L from Aspergillus fumigates [22], and 846.1 μg/L from Metarhisium anisopliae [14]. In 2011, a Pestalotiopsis hainanensis strain was isolated from the scurf of a dermatitis giant panda (Ailuropoda melanoleuca) in the Chinese Conservation and Research Center for the Giant Panda in Ya’an, Sichuan Province, China [5]. P. hainanensis is an endophyte isolated from Podocarpus macrophyllus [13] and Vitex negundo L. var. cannabifolia [6]. To the best of our knowledge, this is the first report of P. hainanensis found in an animal; all other taxolproducing Pestalotiopsis species are isolated from plants of Taxus [13–20]. It is unknown whether the taxol-producing level of P. hainanensis from the animal host source is greater than those from non-animal host sources, which are still low. In this study, we investigated the taxol yield of P. hainanensis from the scurf of a dermatitic giant panda.
Materials and Methods The fungus P. hainanensis was isolated from the scurf of a dermatitis giant panda in the Chinese Conservation and Research Center for the Giant Panda in Ya’an, Sichuan Province, China in 2011 [5] and stored in the fungal laboratory of Sichuan Agricultural University. Fungus Culture The fungus was cultured in potato dextrose agar (PDA) media purchased from AOBOX (Aoboxing Bio-tech Co., Ltd., Beijing, China) at 25±1 °C until spores were generated. Then,
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four mycelia and spore agar plugs (0.5 cm diameter) were inoculated into 3-L Erlenmeyer flasks containing 1 L modified MID liquid medium according to Kathirava [8] supplemented with 1 g soytone and incubated in an oscillating incubator with shaking at 180 rpm and 28± 1 °C for 28 days for taxol production. Extraction and Isolation of Taxol Taxol extraction was performed according to Strobel [21, 20]. Briefly, the culture filtrate was passed through four layers of cheesecloth, and the supernatant and the mycelia were collected. The supernatant was extracted with an equal volume of methylene chloride three times every 4 h. To avoid fatty acid contamination of taxol, Na2CO3 (0.5 g in 1,000 ml) was added to the supernatant. Mycelia were extracted with methylene chloride through soxhlet at 35 °C for 8 h. The two parts of the organic phases were collected and evaporated to dryness under reduced pressure at 35 °C using a rotary vacuum evaporator. In the whole extraction process, assumed taxol was preliminarily tested by thin layer chromatography (TLC), and the concentration of taxol in crude extraction was tested by high-performance liquid chromatography (HPLC). Column Chromatography of Taxol by Gravity Flow The silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) was dissolved in an equal volume methylene chloride to activate the gel and added to a chromatography column (25×400 mm, Shanghai Zhi Sun Instrument Co., Ltd., Shanghai, China). Then, methylene chloride was flowed through the column at 1.0 ml/min to equilibrate the column. The sample was dissolved in 5 ml methylene chloride and was eluted at 1.0 ml/min with 20:1 (v/v) methylene chloride/methanol. Every 20 ml of eluate was stepwise collected with an automatic fraction collector and tested by TLC. The taxol eluates were combined and concentrated and purified by the same column chromatography process twice. Finally, the sample was evaporated at 35 °C until dry. A taxol standard (paclitaxel; Sigma, St. Louis, MO, USA) was used as a reference. All solvents used for analyses were of HPLC grade. TLC Analysis of Taxol TLC analysis was carried out on a 5×20-cm GF254 silica thin layer plate (Qingdao Haiyang Chemical Co., Ltd, Qingdao, China), and the solvent system was methylene chloride/methanol (20:1). Taxol was confirmed with 1 % (w/v) vanillin/sulfuric acid reagent by an appearance of a bluish spot under 254 nm ultraviolet (UV) light after gentle heating [23] and fading of the spot to dark gray in 24 h. UV Spectroscopic Analysis of Taxol The purified sample was dissolved in 100 % methanol and analyzed by UV absorption (UV1750, Shimadzu, Kyoto, Japan), using the standard taxol as a control. The scanning range was 190 to 400 nm, and the absorption peak was observed. High-Performance Liquid Chromatography (HPLC) Analysis of Taxol An HPLC study was conducted using an Agilent 1260 instrument (Agilent, Santa Clara, USA). Chromatographic conditions were as follows: C18 reverse phase
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chromatographic column (Agilent, Santa Clara, USA), 150×4.6×5 μm; column temperature, 30 °C; mobile phase methanol/water, 65:35 (v/v); UV detection wavelength, 227 nm; flow rate, 1.0 ml/min; and sample quantity, 20 μl filtered through a 0.2 μm PVDF membrane. The yield of taxol in P. hainanensis fermentation was calculated by the area under the peak. Nuclear Magnetic Resonance (NMR) Analysis of Taxol The samples were dissolved in CDCl3. The NMR spectroscopic analysis was performed using a Bruker Avance 600 Spectrometer (Bruker BioSpin GmbH, Stuttgart, Germany) at 26.85 °C and 600.13 MHz, with a 13C-1H DUL probe. Time-of-Flight Mass Spectrometry (TOF-MS) Analysis of Taxol A MicrOTOF-Q II electrospray ionization (ESI)-Qq-TOF mass spectrometer (Bruker Daltonic, Stuttgart, Germany) was used to test the sample mass and molecular formula. ESI was performed as the source type. The samples were tested with a nebulizer at 0.4 bar, capillary at 4,000 V, dry heater at 180 °C, and dry gas at 3.0 l/min, with ion polarity being negative.
Results and Discussion TLC TLC analysis results are shown in Fig. 1. The Rf value of the extraction solution, final purified sample, and taxol standard was 0.42. The samples showed up as bluish spots under 254 nm UV light after treatment with 1 % (w/v) vanillin/sulfuric acid reagent, indicating that P. hainanensis secreted taxol or taxol analogues.
Fig. 1 Taxol test by thin layer chromatography with GF254 silica thin layer plate. One percent (w/v) vanillin/ sulfuric acid reagent was sprayed, and bluish spots appeared under 254 nm UV light. a Taxol standard, b extracted intermediate products from cultured P. hainanensis, c final purified sample. Solvent system was methylene chloride/methanol (20:1 (v/v))
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UV Absorbance The taxol standard and the sample had the same maximum absorption peak at 227 nm, with an absorbance value of 1.93 and 0.75, respectively, indicating the existence of the taxol molecule in P. hainanensis (Fig. 2). HPLC The taxol standard retention time was 11.086 min, with an area (mAU*s) of 2,188.3 (Fig. 3a). The crude extraction solution retention time was 11.048 min, with an area (mAU*s) of 1,604.98 (Fig. 3b). The final purified sample retention time was 11.302 min, with an area (mAU*s) of 13,595.7 (Fig. 3c). The taxol concentration of the fermented liquid of P. hainanensis was 1,466.87 μg/L. When sample was purified by column chromatography, the concentration of taxol was 1,242.58 μg/L because of the loss in chromatography. NMR The characteristic chemical shifts of the standard taxol and sample taxol were obtained according to reference [2] and test results (Tables 1 and 2). The 1H and 13C-NMR spectra and assignments of the sample taxol were identical with those of the standard taxol.
Fig. 2 UV absorption spectrum and absorbance. a Taxol standard. b Sample
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Fig. 3 HPLC analysis of taxol. a Taxol standard. b Crude extraction solution from cultured P. hainanensis. c Final purified sample from cultured fungus P. hainanensis. The mobile phase was methanol/water 65:35 (v/v), the flow rate was 1.0 ml/min, and column temperature was 30 °C. The retention time and area were obtained at 227 nm by UV absorbance
Appl Biochem Biotechnol Table 1 1H-NMR chemical shifts for taxol and sample in CDCl3 (600 MHz, ppm) Assignment
Taxol
Sample
Assignment
Taxol
Sample
o-BZ2
8.13
8.16
H2′
4.79
4.81
o-NHBZ
7.74
7.
H7
4.30
4.33
p-BZ2
7.61
4.88
H20α
4.21
4.24
p-NHBZ m-BZ2
7.52 7.50
2.49 1.83
H20β H3
3.80 3.61
3.83 3.73
m-Ph3′
7.47
6.20
H6
2.48
2.48
o-Ph3′
7.41
6.18
OAc4
2.38
2.41
m-NHBZ
7.40
2.30
H14
2.23
2.26
p-Ph3′
7.35
2.24
OAc10
1.92
1.89
NH
7.00
7.02
H6
1.80
1.82
H10
6.27
6.30
Me18
1.68
1.71
H13 H3′
6.23 5.79
6.26 5.82
Me19 Me17
1.64 1.24
1.64 1.27
H2
5.68
5.71
Me16
1.14
1.17
H5
4.94
4.97
In the 1H-NMR spectroscopic analysis, nearly all signals were well-resolved and distributed in the range from 1.0 to 8.0 ppm (Figs. 4a and 5b). The main proton signals caused by the methyl and acetyl groups lied in the 1.0- to 2.5-ppm range. Most of the protons in the taxane skeleton and the side chain were from 2.5 to 7.0 ppm, and the aromatic proton groups Table 2
13
C-NMR chemical shifts for taxol and sample in CDCl3 (600 MHz, ppm)
Carbon
Taxol
Sample
Carbon
Taxol
Sample
C-1
77.65
77.81
C-20
75.65
75.77
C-2
73.64
73.76
C-1′
171.39
171.51
C-3
44.29
44.41
C-2′
73.41
73.42
C-4
79.85
79.97
C-3′
53.69
53.81
C-5 C-6
83.05 28.32
83.18 28.45
OAc4 OAc10
169.01 169.87
169.13 170.00
C-7
70.80
70.94
Me
19.47
19.60
C-8
57.28
57.42
C=O
165.77
165.76
C-9
202.26
202.38
q-Ph1
129.44
129.44
C-10
74.22
74.35
o-Ph1
128.85
128.98
C-11
136.64
136.77
o-Ph3
125.68
125.81
C-12
140.57
140.71
p-Ph1
132.34
132.47
C-13 C-14
71.82 34.28
71.97 34.40
q-Ph2 o-Ph2
133.92 127.35
133.93 127.48
C-15
41.84
41.97
p-Ph2
130.62
130.74
C-16
21.26
21.39
q-Ph3
138.22
138.23
C-17
25.51
25.64
m-Ph3
129.26
129.27
C-18
13.45
13.59
p-Ph3
128.60
128.60
C-19
8.21
8.34
Appl Biochem Biotechnol
exhibited from 7.0 to 8.0 ppm. The structures of the sample and standard taxol obtained from the tested spectra were identical (Fig. 4a, b). In the 13C-NMR spectroscopic analysis, the signals were distributed from 10 to 205 ppm (Fig. 5a, b). The main carbon skeleton existed in the 10- to 90-ppm range, the aromatic proton mainly exhibited near 130 ppm, and methyl showed up at 22 ppm. In comparison with the standard taxol, the taxol sample produced the same spectrum and thereby the same structure (Fig. 5a, b). TOF-MS ESI-Qq-TOF mass spectrometry was used to test the taxol sample from the cultured fungus (Fig. 6). The (M+H)+ peaks at mass-to-charge (m/z) 854.34 and the (M+Na)+ peaks at m/z 876.32 corresponding to the molecular formula of C47H52NO14 and C47H51NNaO14, respectively, were obtained. The peaks were analogous to the standard taxol exhibiting m/z ratios of the molecular ions (M+H)+ and (M+Na)+ (854.34 and 876.32, respectively) [2]. These results indicated that P. hainanensis produced taxol with a molecular formula of C47H51NO14. Therefore, based on the results above, we demonstrated that the fungal culture of P. hainanensis produced taxol with a yield of 1,466.87 μg/L. At present, about 100 taxol-producing species have been isolated and identified, among which, more than 80 % are fungi, and the remaining are bacteria. Except for Pestalotia
Fig. 4 1H-NMR spectrum of taxol in CDCl3 at 600 MHz. a Taxol standard. b Fungal taxol from cultured P. hainanensis. The structure of taxol is shown as an inset in a and b according to the corresponding spectrum
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Fig. 5 13C-NMR spectrum of taxol in CDCl3 at 600 MHz. a Taxol standard. b Fungal taxol from cultured P. hainanensis. The structure of taxol is shown as an inset in a and b according to the corresponding spectrum
heterocornis, which is from the soil of Taxus chinensis forest, the rest of the taxol-producing fungi species come from the endophyte plants [16]. The fungal strain P. hainanensis from the
Fig. 6 Spectral peak of P. hainanensis samples using ESI-Qq-TOF mass spectrometry. The peak of mass-tocharge (m/z) ratios to the molecular ions (M+H)+ and (M+Na)+ are 854 and 876, respectively, and the molecular formula was C47H51NO14, which was the same as that of the taxol standard
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dermatitic giant panda nidus might be a saprophytic fungus or opportunistic pathogen of the giant panda [5]. P. hainanensis is also an endophyte fungus of P. macrophyllus [13] and V. negundo L. var. cannabifolia [6] in China. Thus, P. hainanensis may have a wide range of nutritional adaptation and can grow in a variety of environments. In this study, the P. hainanensis culture produced a high yield of taxol (1,466.87 μg/L), while the taxol yields from other endophytic Pestalotiopsis fungi from plants are 211.1 μg/L from Pestalotiopsis terminaliae [4], 208.6 μg/L from Pestalotiopsis pauciseta [23], 1.71 μg/L from Pestalotiopsis microspora [20, 10], 0.485 μg/L from Pestalotiopsis guepinii [21], 478 μg/L from P. versicolor [18], 186 μg/L from Pestalotiopsis malicola [1], and 64 μg/L from Pestalotiopsis breviseta [8]. The taxol yields range from 0.1 to 500 μg/L from other species of different genus, such as Nodulisporium, Fusarium, Alternaria, Phomopsis, Tubercularia, and Periconia [15]. For example, the taxol yield is 557.8 μg/L from A. fumigates [22] and 846.1 μg/L from M. anisopliae [14]. We hypothesize that the possibility of lesion and its surrounding cells becoming cancerous increases when the giant panda skin disease occurs; therefore, to prevent the cells from becoming cancerous, the P. hainanensis in the panda produces more anticancer substance, the taxol. The nutrients for P. hainanensis from the giant panda body skin are extremely different from those of plant or soil environment [26]; the nutrients from the animal skin maybe help fungi produce more taxol. In conclusion, a greater taxol yield was observed from P. hainanensis of an animal source in the current study than all previous known taxol yields in the literature. To the best of our knowledge, this is the first report of such high taxol yield. The fungus P. hainanensis from the giant panda provides a potential candidate for the industrial production of taxol. However, the fermentation cycle of P. hainanensis is long; further study is necessary to optimize the fermentation conditions in order to make it a valuable taxol-producing fungus. Acknowledgments This study was supported by the Program for Youth Fund of Sichuan Provincial Education Department (12ZB093); National Key Technology R&D Program of China (2012BAC01B06); The giant panda international cooperation project funds (AD1415); and Applied Basic Research Project in Sichuan Province (2013TY0175). This study is a part of undergraduate graduation thesis breeding project of Sichuan Agricultural University. Examinations of taxol and samples of HPLC, 1H and 13C-NMR, and TOF-MS tests are performed in the Chengdu Institute of Biology, Chinese Academy of Sciences. Thanks for them. All authors have agreed to submit this manuscript to the Applied Biochemistry and Biotechnology.
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Greater Taxol Yield of Fungus Pestalotiopsis hainanensis from Dermatitic Scurf of the Giant Panda (Ailuropoda melanoleuca) Yu Gu & Yanlin Wang & Xiaoping Ma & Chengdong Wang & Guizhou Yue & Yuetian Zhang & Yunyan Zhang & Shanshan Li & Shanshan Ling & Xiaomin Liu & Xintian Wen & Sanjie Cao & Xiaobo Huang & Junliang Deng & Zhicai Zuo & Shumin Yu & Liuhong Shen & Rui Wu
Received: 1 July 2014 / Accepted: 10 September 2014 # Springer Science+Business Media New York 2014
Abstract While taxol yields of fungi from non-animal sources are still low, whether Pestalotiopsis hainanensis isolated from the scurf of a dermatitic giant panda, Ailuropoda melanoleuca, provides a greater taxol yield remains unknown. The objective of the study was to determine the corresponding taxol yield. The structure of the taxol produced by the fungus was evaluated by thin layer chromatography (TLC), ultraviolet (UV) spectroscopy, highperformance liquid chromatography (HPLC), 1H and 13C nuclear magnetic resonance spectroscopy (1H-NMR and 13C-NMR), and time-of-flight mass spectrometry (TOF-MS), with standard taxol as a control. The results demonstrated that the P. hainanensis fungus produced taxol, which had the same structure as the standard taxol and yield of 1,466.87 μg/L. This fungal taxol yield from the dermatitic giant panda was significantly greater than those of Xiaoping Ma and Chengdong Wang contributed equally to this work, tied for Xiaoping Ma. Yu Gu and Yanlin Wang contributed equally to this work and should be considered as joint first authors, tied for Yu Gu. Y. Gu College of Life Sciences, Sichuan Agricultural University, Ya’an 625014, China Y. Wang : G. Yue College of Sciences, Sichuan Agricultural University, Ya’an 625014, China X. Ma : Y. Zhang : Y. Zhang : S. Li : X. Wen : S. Cao : X. Huang : J. Deng : Z. Zuo : S. Yu : L. Shen : R. Wu College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China X. Ma (*) College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China e-mail: [email protected] C. Wang (*) : S. Ling : X. Liu China Conservation and Research Center for Giant Panda, Wolong 623006, China e-mail: [email protected]
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fungus from non-animal sources. The taxol-producing fungus may be a potential candidate for the production of taxol on an industrial scale. Keywords Pestalotiopsis hainanensis . Animal surface fungi . Ailuropoda melanoleuca . Taxol . Higher yield
Introduction Taxol, a type of diterpene, was first isolated from the bark of the Pacific Yew tree (Taxus brevifolia) in the late 1960s by Wani [25]. Taxol has been found to exhibit antitumor and antimicrobial activity and is thus being used to treat breast, ovarian, lung and head cancers, and Kaposi’s sarcoma [9]. While paclitaxel has been successfully produced on a commercial scale at a yield of more than 350 mg/L using Taxus cell culture by Phyton Biotech (Germany) and Samyagn Genex (South Korea) since 2000, because Taxus spp. grows extremely slow but is the main source of taxol, a large amount of Taxus spp. has to be used, which will run out resource of Taxus spp. to endanger ecosystems. Taxol has been successfully synthesized in the laboratory, but its production of chemical synthesis on a large scale in the factory is still not feasible [3, 7]. In 1993, Stierle separated a taxol-producing endophytic fungus Taxomyces andreanae from the phloem of T. brevifolia [19]. Since then, many taxol-producing plant endophytic fungi have been isolated from Taxus spp. and its close species [11, 12, 24, 17]. More than 30 fungi species are found to produce taxol [15]. Pestalotiopsis genus, isolated from the Taxus spp. host, is the most common and dominant endophyte fungi. However, Pestalotiopsis yields low levels of taxol [20, 21, 10, 8, 18, 23, 1, 4], with the highest yield only up to 500 μg/L from Pestalotiopsis versicolor [18], 557.8 μg/L from Aspergillus fumigates [22], and 846.1 μg/L from Metarhisium anisopliae [14]. In 2011, a Pestalotiopsis hainanensis strain was isolated from the scurf of a dermatitis giant panda (Ailuropoda melanoleuca) in the Chinese Conservation and Research Center for the Giant Panda in Ya’an, Sichuan Province, China [5]. P. hainanensis is an endophyte isolated from Podocarpus macrophyllus [13] and Vitex negundo L. var. cannabifolia [6]. To the best of our knowledge, this is the first report of P. hainanensis found in an animal; all other taxolproducing Pestalotiopsis species are isolated from plants of Taxus [13–20]. It is unknown whether the taxol-producing level of P. hainanensis from the animal host source is greater than those from non-animal host sources, which are still low. In this study, we investigated the taxol yield of P. hainanensis from the scurf of a dermatitic giant panda.
Materials and Methods The fungus P. hainanensis was isolated from the scurf of a dermatitis giant panda in the Chinese Conservation and Research Center for the Giant Panda in Ya’an, Sichuan Province, China in 2011 [5] and stored in the fungal laboratory of Sichuan Agricultural University. Fungus Culture The fungus was cultured in potato dextrose agar (PDA) media purchased from AOBOX (Aoboxing Bio-tech Co., Ltd., Beijing, China) at 25±1 °C until spores were generated. Then,
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four mycelia and spore agar plugs (0.5 cm diameter) were inoculated into 3-L Erlenmeyer flasks containing 1 L modified MID liquid medium according to Kathirava [8] supplemented with 1 g soytone and incubated in an oscillating incubator with shaking at 180 rpm and 28± 1 °C for 28 days for taxol production. Extraction and Isolation of Taxol Taxol extraction was performed according to Strobel [21, 20]. Briefly, the culture filtrate was passed through four layers of cheesecloth, and the supernatant and the mycelia were collected. The supernatant was extracted with an equal volume of methylene chloride three times every 4 h. To avoid fatty acid contamination of taxol, Na2CO3 (0.5 g in 1,000 ml) was added to the supernatant. Mycelia were extracted with methylene chloride through soxhlet at 35 °C for 8 h. The two parts of the organic phases were collected and evaporated to dryness under reduced pressure at 35 °C using a rotary vacuum evaporator. In the whole extraction process, assumed taxol was preliminarily tested by thin layer chromatography (TLC), and the concentration of taxol in crude extraction was tested by high-performance liquid chromatography (HPLC). Column Chromatography of Taxol by Gravity Flow The silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) was dissolved in an equal volume methylene chloride to activate the gel and added to a chromatography column (25×400 mm, Shanghai Zhi Sun Instrument Co., Ltd., Shanghai, China). Then, methylene chloride was flowed through the column at 1.0 ml/min to equilibrate the column. The sample was dissolved in 5 ml methylene chloride and was eluted at 1.0 ml/min with 20:1 (v/v) methylene chloride/methanol. Every 20 ml of eluate was stepwise collected with an automatic fraction collector and tested by TLC. The taxol eluates were combined and concentrated and purified by the same column chromatography process twice. Finally, the sample was evaporated at 35 °C until dry. A taxol standard (paclitaxel; Sigma, St. Louis, MO, USA) was used as a reference. All solvents used for analyses were of HPLC grade. TLC Analysis of Taxol TLC analysis was carried out on a 5×20-cm GF254 silica thin layer plate (Qingdao Haiyang Chemical Co., Ltd, Qingdao, China), and the solvent system was methylene chloride/methanol (20:1). Taxol was confirmed with 1 % (w/v) vanillin/sulfuric acid reagent by an appearance of a bluish spot under 254 nm ultraviolet (UV) light after gentle heating [23] and fading of the spot to dark gray in 24 h. UV Spectroscopic Analysis of Taxol The purified sample was dissolved in 100 % methanol and analyzed by UV absorption (UV1750, Shimadzu, Kyoto, Japan), using the standard taxol as a control. The scanning range was 190 to 400 nm, and the absorption peak was observed. High-Performance Liquid Chromatography (HPLC) Analysis of Taxol An HPLC study was conducted using an Agilent 1260 instrument (Agilent, Santa Clara, USA). Chromatographic conditions were as follows: C18 reverse phase
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chromatographic column (Agilent, Santa Clara, USA), 150×4.6×5 μm; column temperature, 30 °C; mobile phase methanol/water, 65:35 (v/v); UV detection wavelength, 227 nm; flow rate, 1.0 ml/min; and sample quantity, 20 μl filtered through a 0.2 μm PVDF membrane. The yield of taxol in P. hainanensis fermentation was calculated by the area under the peak. Nuclear Magnetic Resonance (NMR) Analysis of Taxol The samples were dissolved in CDCl3. The NMR spectroscopic analysis was performed using a Bruker Avance 600 Spectrometer (Bruker BioSpin GmbH, Stuttgart, Germany) at 26.85 °C and 600.13 MHz, with a 13C-1H DUL probe. Time-of-Flight Mass Spectrometry (TOF-MS) Analysis of Taxol A MicrOTOF-Q II electrospray ionization (ESI)-Qq-TOF mass spectrometer (Bruker Daltonic, Stuttgart, Germany) was used to test the sample mass and molecular formula. ESI was performed as the source type. The samples were tested with a nebulizer at 0.4 bar, capillary at 4,000 V, dry heater at 180 °C, and dry gas at 3.0 l/min, with ion polarity being negative.
Results and Discussion TLC TLC analysis results are shown in Fig. 1. The Rf value of the extraction solution, final purified sample, and taxol standard was 0.42. The samples showed up as bluish spots under 254 nm UV light after treatment with 1 % (w/v) vanillin/sulfuric acid reagent, indicating that P. hainanensis secreted taxol or taxol analogues.
Fig. 1 Taxol test by thin layer chromatography with GF254 silica thin layer plate. One percent (w/v) vanillin/ sulfuric acid reagent was sprayed, and bluish spots appeared under 254 nm UV light. a Taxol standard, b extracted intermediate products from cultured P. hainanensis, c final purified sample. Solvent system was methylene chloride/methanol (20:1 (v/v))
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UV Absorbance The taxol standard and the sample had the same maximum absorption peak at 227 nm, with an absorbance value of 1.93 and 0.75, respectively, indicating the existence of the taxol molecule in P. hainanensis (Fig. 2). HPLC The taxol standard retention time was 11.086 min, with an area (mAU*s) of 2,188.3 (Fig. 3a). The crude extraction solution retention time was 11.048 min, with an area (mAU*s) of 1,604.98 (Fig. 3b). The final purified sample retention time was 11.302 min, with an area (mAU*s) of 13,595.7 (Fig. 3c). The taxol concentration of the fermented liquid of P. hainanensis was 1,466.87 μg/L. When sample was purified by column chromatography, the concentration of taxol was 1,242.58 μg/L because of the loss in chromatography. NMR The characteristic chemical shifts of the standard taxol and sample taxol were obtained according to reference [2] and test results (Tables 1 and 2). The 1H and 13C-NMR spectra and assignments of the sample taxol were identical with those of the standard taxol.
Fig. 2 UV absorption spectrum and absorbance. a Taxol standard. b Sample
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Fig. 3 HPLC analysis of taxol. a Taxol standard. b Crude extraction solution from cultured P. hainanensis. c Final purified sample from cultured fungus P. hainanensis. The mobile phase was methanol/water 65:35 (v/v), the flow rate was 1.0 ml/min, and column temperature was 30 °C. The retention time and area were obtained at 227 nm by UV absorbance
Appl Biochem Biotechnol Table 1 1H-NMR chemical shifts for taxol and sample in CDCl3 (600 MHz, ppm) Assignment
Taxol
Sample
Assignment
Taxol
Sample
o-BZ2
8.13
8.16
H2′
4.79
4.81
o-NHBZ
7.74
7.
H7
4.30
4.33
p-BZ2
7.61
4.88
H20α
4.21
4.24
p-NHBZ m-BZ2
7.52 7.50
2.49 1.83
H20β H3
3.80 3.61
3.83 3.73
m-Ph3′
7.47
6.20
H6
2.48
2.48
o-Ph3′
7.41
6.18
OAc4
2.38
2.41
m-NHBZ
7.40
2.30
H14
2.23
2.26
p-Ph3′
7.35
2.24
OAc10
1.92
1.89
NH
7.00
7.02
H6
1.80
1.82
H10
6.27
6.30
Me18
1.68
1.71
H13 H3′
6.23 5.79
6.26 5.82
Me19 Me17
1.64 1.24
1.64 1.27
H2
5.68
5.71
Me16
1.14
1.17
H5
4.94
4.97
In the 1H-NMR spectroscopic analysis, nearly all signals were well-resolved and distributed in the range from 1.0 to 8.0 ppm (Figs. 4a and 5b). The main proton signals caused by the methyl and acetyl groups lied in the 1.0- to 2.5-ppm range. Most of the protons in the taxane skeleton and the side chain were from 2.5 to 7.0 ppm, and the aromatic proton groups Table 2
13
C-NMR chemical shifts for taxol and sample in CDCl3 (600 MHz, ppm)
Carbon
Taxol
Sample
Carbon
Taxol
Sample
C-1
77.65
77.81
C-20
75.65
75.77
C-2
73.64
73.76
C-1′
171.39
171.51
C-3
44.29
44.41
C-2′
73.41
73.42
C-4
79.85
79.97
C-3′
53.69
53.81
C-5 C-6
83.05 28.32
83.18 28.45
OAc4 OAc10
169.01 169.87
169.13 170.00
C-7
70.80
70.94
Me
19.47
19.60
C-8
57.28
57.42
C=O
165.77
165.76
C-9
202.26
202.38
q-Ph1
129.44
129.44
C-10
74.22
74.35
o-Ph1
128.85
128.98
C-11
136.64
136.77
o-Ph3
125.68
125.81
C-12
140.57
140.71
p-Ph1
132.34
132.47
C-13 C-14
71.82 34.28
71.97 34.40
q-Ph2 o-Ph2
133.92 127.35
133.93 127.48
C-15
41.84
41.97
p-Ph2
130.62
130.74
C-16
21.26
21.39
q-Ph3
138.22
138.23
C-17
25.51
25.64
m-Ph3
129.26
129.27
C-18
13.45
13.59
p-Ph3
128.60
128.60
C-19
8.21
8.34
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exhibited from 7.0 to 8.0 ppm. The structures of the sample and standard taxol obtained from the tested spectra were identical (Fig. 4a, b). In the 13C-NMR spectroscopic analysis, the signals were distributed from 10 to 205 ppm (Fig. 5a, b). The main carbon skeleton existed in the 10- to 90-ppm range, the aromatic proton mainly exhibited near 130 ppm, and methyl showed up at 22 ppm. In comparison with the standard taxol, the taxol sample produced the same spectrum and thereby the same structure (Fig. 5a, b). TOF-MS ESI-Qq-TOF mass spectrometry was used to test the taxol sample from the cultured fungus (Fig. 6). The (M+H)+ peaks at mass-to-charge (m/z) 854.34 and the (M+Na)+ peaks at m/z 876.32 corresponding to the molecular formula of C47H52NO14 and C47H51NNaO14, respectively, were obtained. The peaks were analogous to the standard taxol exhibiting m/z ratios of the molecular ions (M+H)+ and (M+Na)+ (854.34 and 876.32, respectively) [2]. These results indicated that P. hainanensis produced taxol with a molecular formula of C47H51NO14. Therefore, based on the results above, we demonstrated that the fungal culture of P. hainanensis produced taxol with a yield of 1,466.87 μg/L. At present, about 100 taxol-producing species have been isolated and identified, among which, more than 80 % are fungi, and the remaining are bacteria. Except for Pestalotia
Fig. 4 1H-NMR spectrum of taxol in CDCl3 at 600 MHz. a Taxol standard. b Fungal taxol from cultured P. hainanensis. The structure of taxol is shown as an inset in a and b according to the corresponding spectrum
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Fig. 5 13C-NMR spectrum of taxol in CDCl3 at 600 MHz. a Taxol standard. b Fungal taxol from cultured P. hainanensis. The structure of taxol is shown as an inset in a and b according to the corresponding spectrum
heterocornis, which is from the soil of Taxus chinensis forest, the rest of the taxol-producing fungi species come from the endophyte plants [16]. The fungal strain P. hainanensis from the
Fig. 6 Spectral peak of P. hainanensis samples using ESI-Qq-TOF mass spectrometry. The peak of mass-tocharge (m/z) ratios to the molecular ions (M+H)+ and (M+Na)+ are 854 and 876, respectively, and the molecular formula was C47H51NO14, which was the same as that of the taxol standard
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dermatitic giant panda nidus might be a saprophytic fungus or opportunistic pathogen of the giant panda [5]. P. hainanensis is also an endophyte fungus of P. macrophyllus [13] and V. negundo L. var. cannabifolia [6] in China. Thus, P. hainanensis may have a wide range of nutritional adaptation and can grow in a variety of environments. In this study, the P. hainanensis culture produced a high yield of taxol (1,466.87 μg/L), while the taxol yields from other endophytic Pestalotiopsis fungi from plants are 211.1 μg/L from Pestalotiopsis terminaliae [4], 208.6 μg/L from Pestalotiopsis pauciseta [23], 1.71 μg/L from Pestalotiopsis microspora [20, 10], 0.485 μg/L from Pestalotiopsis guepinii [21], 478 μg/L from P. versicolor [18], 186 μg/L from Pestalotiopsis malicola [1], and 64 μg/L from Pestalotiopsis breviseta [8]. The taxol yields range from 0.1 to 500 μg/L from other species of different genus, such as Nodulisporium, Fusarium, Alternaria, Phomopsis, Tubercularia, and Periconia [15]. For example, the taxol yield is 557.8 μg/L from A. fumigates [22] and 846.1 μg/L from M. anisopliae [14]. We hypothesize that the possibility of lesion and its surrounding cells becoming cancerous increases when the giant panda skin disease occurs; therefore, to prevent the cells from becoming cancerous, the P. hainanensis in the panda produces more anticancer substance, the taxol. The nutrients for P. hainanensis from the giant panda body skin are extremely different from those of plant or soil environment [26]; the nutrients from the animal skin maybe help fungi produce more taxol. In conclusion, a greater taxol yield was observed from P. hainanensis of an animal source in the current study than all previous known taxol yields in the literature. To the best of our knowledge, this is the first report of such high taxol yield. The fungus P. hainanensis from the giant panda provides a potential candidate for the industrial production of taxol. However, the fermentation cycle of P. hainanensis is long; further study is necessary to optimize the fermentation conditions in order to make it a valuable taxol-producing fungus. Acknowledgments This study was supported by the Program for Youth Fund of Sichuan Provincial Education Department (12ZB093); National Key Technology R&D Program of China (2012BAC01B06); The giant panda international cooperation project funds (AD1415); and Applied Basic Research Project in Sichuan Province (2013TY0175). This study is a part of undergraduate graduation thesis breeding project of Sichuan Agricultural University. Examinations of taxol and samples of HPLC, 1H and 13C-NMR, and TOF-MS tests are performed in the Chengdu Institute of Biology, Chinese Academy of Sciences. Thanks for them. All authors have agreed to submit this manuscript to the Applied Biochemistry and Biotechnology.
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