ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2012 12 28 12 1141 ~ 1146 1 2 3 * 2 2 2 2 * 1 044099 2 430074 3 430064 Taxus x media Z58 10 2 min M + Na + = 876 1 H-NMR 18S rdna Hypocrea sp Z58 2 5 ~ 3 0 μg / g / 18S rdna Q93 R73 Isolation and Identification of a Taxol-producing Endophytic Fungus Z58 MIAO Li-Yun 1 2 ZHANG Peng 3 * LIU Bo 2 XU Man 2 ZHOU Peng-Peng 2 YU Long-Jiang 2 * 1 Department of Life Sciences Yuncheng University Yuncheng 044099 Shanxi China 2 Department of Biotechnology Institute of Resource Biology and Biotechnology College of Life Science and Technology Huazhong University of Science and Technology Wuhan 430074 China 3 Institute of Industrial Crops Hubei Academy of Agricultural Sciences Wuhan 430064 China Abstract A taxol-producing endophytic fungus was isolated from the inner bark of Taxus x media and named as Z58 The fungal extracts from Z58 were analyzed for taxol with high performance liquid chromatography HPLC mass spectrometry MS and nuclear magnetic resonance NMR spectroscopy The results showed that the Z58 produced taxol peaked at about the same retention time 10 2 minutes as authentic taxol in chromatography and with an identical signature of M + Na + = 876 in mass spectrum as well as an identical 1 H-NMR spectrum as the authentic taxol Z58 was later identified to belong to Hypocrea sp based on its morphology and conserved sequence of 18S rdna Hypocrea sp Z58 was able to produce 2 5 ~ 3 0 μg / g taxol / dry weight of mycelium Our finding of Hypocrea sp Z58 may be used as a promising strain for larger scale production of taxol in fermentation Key words Taxus taxol endophytic fungus Hypocrea sp 18S rdna taxol 2012-10-18 2012-11-14 1-3 1993 Taxomyces andreanae 3 No 20776058 20906036 No 20100471183 No 20060025 * Tel 86-27-87792264 E-mail zhangpenghust @ 126 com yulongjiang@ mail hust edu cn Received October 18 2012 Accepted November 14 2012 Supported by National Natural Science Foundation of China No 20776058 20906036 China Postdoctoral Science Foundation 1 2 No 20100471183 Natural Science Foundation of Yuncheng University No 20060025 1 2 * Corresponding auther Tel 86-027-87792264 E-mail zhangpenghust@ 126 com yulongjiang@ mail hust edu cn
1142 28 1200 HPLC System C18 5 mm 300 mm Waters H 2 O 65 35 V / V 1 ml / min 227 nm Z58 high 25 10 μl performance liquid chromatography HPLC mass spectrum MS nuclear 3 magnetic resonance spectroscopy NMR Agilent 1100 LC / MSD trap 4 18S rdna 100% deuterated methanol Bruker 400 MHz instrument 1 1 1 20 Taxus x media 1 ~ 3 3 ~ 5 cm Z58 PDA potato dextrose agar PDL potato dextrose liquid 4 Sigma YES 1 5 18S rdna 1 2 28 150 r / min 3 ~ 5 d 4 5 Sp fungal DNA extract Kit 30 ~ 50 s 70% V / V DNA 18S-F1 5'- 30 ~ 50 s GATCCTGCCAGTAGTCATATGC-3' 18S-ITS2 5'- 50 μg / ml GCTGCGTTCATCGATGC-3' 18S rdna ampcilin PDA 25 ~ 28 30 ~ 50 ng DNA 2 5 μl 10 Taq 1 0 μl 10 mmol / L dntps 0 8 μl Taq 10 U 0 5 μl PDA 25 ~ 28 7 ~ 10 d 50 mmol / L 18S-F1 0 5 μl 50 mmol / L ITS2 ddh 2 O 25 μl 95 6 min 4 1 3 72 10 min 6 7 1 Z58 300 ml GenBank Blast http / / blast ncbi YES 28 200 r / min 10 ~ nlm nih gov / Blast 12 d 3 45 2 1 g 6 ml / 1 1 V / V 3 3 50 2 1 Z58 HPLC 1 ml 100% 10 0 45 μm HPLC 1 4 Z58 PDA 28 10 ~ 15 d Z58 30 ml PDL 94 50 s 55 50 s 72 1 5 min 30 12 HPLC-MS 2 HPLC Agilent 300 ml YES 28 200 r / min
12 1143 10 d Fig 1 HPLC 10 2 min Z58 Z58 Fig 1 HPLC analysis of authentic taxol A and the fungal taxol B from the fungus Z58 One gram dried mycelia of the fungus Z58 was crushed and extracted with 6 ml methanol / chloroform 1 1 V / V 3 times and the extracts were concentrated under reduced pressure and dissolved in 1 ml 100% methanol 10 μl of authentic taxol A or fungal taxol B were identified using Agilent 1200 HPLC system with a C18 column 5 300 mm Waters A variable wavelength recorder set at 227 nm was used to detect compounds eluting from the column 2 2 Fig 3 Fig 2 Na + 1 H-NMR Z58 Z58 M + Na + = 876 3 2 3 NMR Z58 HPLC MS NMR Z58 300 ml YES 28 200 r / min 10 2 min 12 d 2 5 ~ 3 0 μg / g / 0 5 ml 100% NMR Fig 2 Electrospray mass spectra of authentic taxol A and fungal taxol of Z58 B Electrospray mass spectrum were obtained on authentic taxol A and the fungal taxol of Z58 B using the electrospray with an Agilent 1100 LC / MSD trap 10 μl of sample in 100% methanol was injected with spray flow of 2 μl / min and a spray voltage of 2 2 kv by the loop injection method The arrows showed the identical electrospray mass peak with M + Na + = 876 of the authentic taxol and the fungal taxol
1144 28 Fig 3 1 H-NMR spectra of authentic taxol A and the fungal taxol B from Z58 Nuclear magnetic resonance spectroscopy were performed on authentic taxol A dissolved in 100% deuterated methanol as control and the fungal taxol B in 100% deuterated methanol with a Bruker 400 MHz instrument The sample was subjected to 2048 scans with a sweep width of 6024 and 8 K real points And the fungal taxol of the fungus Z58 had an identical 1 H-NMR spectrum to that of authentic taxol 2 4 Z58 Z58 PDA 28 Fig 4 C Fig 4 D 3 d 3 8 cm Z58 8 Fig 4A B Fig 4 Morphological observation of the fungus Z58 The colonies and colony reverse of the fungus Z58 on PDA medium were showed in Fig 4A and B respectively The light microscopic photographs of hyphae and conididia were showed in Fig 4C and 4D with magnification 400 and 1 000 respectively 2 5 Z58 18S rdna Z58 18S rdna 3 DNA Fig 5 A PCR 18S rdna 1 2 0 kb Fig 5 B Z58 18S rdna 1 725 bp Hypocrea koningii JH GenBank No EU722404 1 Hypocrea koningii 18S rdna GenBank No AJ301990 1 Hypocrea rufa 18S rdna Hypocrea sp 9 1-4 GenBank No AJ301991 1 95% 10-13 20 70 1 18S rdna 2
12 1145 Fig 5 Analysis of fungus Z58 genomic DNA and 18S rdna PCR product The fungus Z58 was incubated into 30 ml PDL medium at 150 r / min and 28 for 5 days and harvested by centrifugation at 12 000 g for 10 min 0 5 ~ 1 mg mycelia were ground into powder in liquid nitrogen and then extracted using Sp fungal DNA extract Kit 1 μl genomic DNA was analysed with agarose gel electrophoresis showed in lanes 1 ~ 2 in Fig 5A The 18S rdna sequence was amplified from Z58 in a PTC-100 Peltier Thermal Cycler Bio-Rad using the primers 18S-F1 and 18S-ITS2 in a typical 25 μl reaction mixture containing 8U Taq DNA polymerase Tiangen The PCR product of the 18S rdna sequence was analysed with agarose gel electrophoresis showed in lanes 1 ~ 2 in Fig 5B M in Fig 5A λ DNA / Hind Ⅲ M in Fig 5B DNA Marker Ⅲ 11 6 Zhao C F Yu L J Li L Q et al 1 2 5 ~ 3 0 μg / g Z58 J Curr Microbiol 2009 59 3 227-232 8 M Shanghai Shanghai Science and Technology Press 1982 1 J fermentation J Microbiology China 2 565 10 J Kang Ji-Chuan 14-16 et al 3 taxol J Mycosystema 2011 30 2 168-179 17-19 11 J Z58 Z58 References 1 Ji Y Bi J N Yan B et al Taxol-producing fungi a new approach to industrial production of taxol J Chin J Biotechnol 2006 22 1 1-6 2 Zhou X Zhu H Liu L et al A review recent advances and future prospects of taxol-producing endophytic fungi J Appl Microbiol Biotechnol 2010 86 6 1707-1717 3 Stierle A Stroble G Stierle D Taxol and taxane production by Taxomyces andreanae endophytic fungus of Pacific yew J Science 1993 260 5105 214-216 4 YN6 J Zhang Peng Liu Bo Zhou Peng-Peng et al Isolation and identification of a taxol-producing endophytic fungus YN6 J Chin J Biochem Mol Biol 2011 27 10 961-967 5 Zhang P Zhou PP Jiang C et al Screening of taxol-producing fungi based on PCR amplification from Taxus J Biotechnol Lett 2008 30 12 2119-2123 Simultaneous identification and determination of major taxoids from extracts of Taxus chinenesis cell cultures J Z Naturforsch C 2007 62 1-2 1-10 7 Zhang P Zhou P P Yu L J An endophytic taxol-producing fungus from Taxus media Cladosporium cladosporioides MD2 Wei Jing-Chao A Manual for Fungi Identification M 9 Wang Shi-Wei Ma Xi Ping Wen- Xiang et al Research advances on taxol production by microbe Wei et al 2007 34 3 561- Jin Rui Lei Bang-Xing Recent research advances on endophytic fungi producing Taxol Ma Yu-Chao Zhao Kai Wang Shi- Biological diversity of taxol-producing endophytic fungi J J Fungal Res 2003 1 1 28-32 12 Wang Y Tang K A new endophytic taxol-and baccatin Ⅲ-
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