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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ * * Taxus x media Z min M + Na + = H-NMR 18S rdna Hypocrea sp Z ~ 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 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 China 3 Institute of Industrial Crops Hubei Academy of Agricultural Sciences Wuhan 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 Taxomyces andreanae 3 No No No * Tel com yulongjiang@ mail hust edu cn Received October Accepted November Supported by National Natural Science Foundation of China No China Postdoctoral Science Foundation 1 2 No Natural Science Foundation of Yuncheng University No * Corresponding auther Tel zhangpenghust@ 126 com yulongjiang@ mail hust edu cn

2 HPLC System C18 5 mm 300 mm Waters H 2 O V / V 1 ml / min 227 nm Z58 high 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 Taxus x media 1 ~ 3 3 ~ 5 cm Z58 PDA potato dextrose agar PDL potato dextrose liquid 4 Sigma YES S rdna 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 ~ ~ 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 min Z ml GenBank Blast http / / blast ncbi YES r / min 10 ~ nlm nih gov / Blast 12 d g 6 ml / 1 1 V / V Z58 HPLC 1 ml 100% μm HPLC 1 4 Z58 PDA ~ 15 d Z58 30 ml PDL s s min HPLC-MS 2 HPLC Agilent 300 ml YES r / min

3 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 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 + = NMR Z58 HPLC MS NMR Z ml YES 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

4 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 respectively 2 5 Z58 18S rdna Z58 18S rdna 3 DNA Fig 5 A PCR 18S rdna kb Fig 5 B Z58 18S rdna bp Hypocrea koningii JH GenBank No EU Hypocrea koningii 18S rdna GenBank No AJ Hypocrea rufa 18S rdna Hypocrea sp GenBank No AJ % S rdna 2

5 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 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 ~ 3 0 μg / g Z58 J Curr Microbiol M Shanghai Shanghai Science and Technology Press J fermentation J Microbiology China J Kang Ji-Chuan et al 3 taxol J Mycosystema 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 Zhou X Zhu H Liu L et al A review recent advances and future prospects of taxol-producing endophytic fungi J Appl Microbiol Biotechnol Stierle A Stroble G Stierle D Taxol and taxane production by Taxomyces andreanae endophytic fungus of Pacific yew J Science 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 Zhang P Zhou PP Jiang C et al Screening of taxol-producing fungi based on PCR amplification from Taxus J Biotechnol Lett Simultaneous identification and determination of major taxoids from extracts of Taxus chinenesis cell cultures J Z Naturforsch C 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 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 Wang Y Tang K A new endophytic taxol-and baccatin Ⅲ-

6 producing fungus isolated from Taxus chinensis var mairei J Afr J Biotechnol Zhao K Ping W Li Q et al Aspergillus niger var taxi a new species variant of taxol-producing fungus isolated from Taxus cuspidate in China J J Appl Microbiol Wen-Xiang et al Study on breeding up high-yield strain of taxol J Zhao Kai Ping Wen-Xiang Zhou Dong-Po Recent advance and prospect on taxol production by endophytic fungus fermentation-a review J Acta Microbiol Sin Mycosystema Xu F Tao W Y Cheng L et al Strain improvement and optimization of the media of taxol-producing fungus Fusarium maire J Biochem Eng J J Zhao Kai Zhou Dong-Po Ping by protoplast mutagensis J Chin J Biotechnol Zhou D P Zhao K Ping W X et al Study on the mutagensis of protoplasts from taxol-producing fungus Nodulisporium sylviforme 15 J J Am Sci J Liu Xiao-Lan Zhou Dong-Po Sun Jian-Qiu et al A study on fermentation process 19 Zhao K Ping W X Zhang L N et al Screening and breeding of high taxol producing fungi by genome shuffling J Sci China for production of taxol by Nodulisporium sylviforme J Ser C Life Sci

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