Paclitaxel (taxol) has long been used as a potent anticancer agent for the treatment of many cancers. Ever since the fungal species Taxomyces andreanae was first shown to produce taxol in 1993, many endophytic fungal species have been recognized as taxol accumulators. In this study, we analyzed the ...
Paclitaxel (taxol) has long been used as a potent anticancer agent for the treatment of many cancers. Ever since the fungal species Taxomyces andreanae was first shown to produce taxol in 1993, many endophytic fungal species have been recognized as taxol accumulators. In this study, we analyzed the taxol-producing capacity of different Colletotrichum spp. to determine the distribution of a taxol biosynthetic gene within this genus. Distribution of the taxadiene synthase (TS) gene, which cyclizes geranylgeranyl diphosphate to produce taxadiene, was analyzed in 12 Colletotrichum spp., of which 8 were found to contain the unique skeletal core structure of paclitaxel. However, distribution of the gene was not limited to closely related species. The production of taxol by Colletotrichum dematium, which causes pepper anthracnose, depended on the method in which the fungus was stored, with the highest production being in samples stored under mineral oil. Based on its distribution among Colletotrichum spp., the TS gene was either integrated into or deleted from the bacterial genome in a species-specific manner. In addition to their taxol-producing capacity, the simple genome structure and easy gene manipulation of these endophytic fungal species make them valuable resources for identifying genes in the taxol biosynthetic pathway.
Paclitaxel (taxol) has long been used as a potent anticancer agent for the treatment of many cancers. Ever since the fungal species Taxomyces andreanae was first shown to produce taxol in 1993, many endophytic fungal species have been recognized as taxol accumulators. In this study, we analyzed the taxol-producing capacity of different Colletotrichum spp. to determine the distribution of a taxol biosynthetic gene within this genus. Distribution of the taxadiene synthase (TS) gene, which cyclizes geranylgeranyl diphosphate to produce taxadiene, was analyzed in 12 Colletotrichum spp., of which 8 were found to contain the unique skeletal core structure of paclitaxel. However, distribution of the gene was not limited to closely related species. The production of taxol by Colletotrichum dematium, which causes pepper anthracnose, depended on the method in which the fungus was stored, with the highest production being in samples stored under mineral oil. Based on its distribution among Colletotrichum spp., the TS gene was either integrated into or deleted from the bacterial genome in a species-specific manner. In addition to their taxol-producing capacity, the simple genome structure and easy gene manipulation of these endophytic fungal species make them valuable resources for identifying genes in the taxol biosynthetic pathway.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
Characterization of the morphological differences among 12 Colletotrichum spp. (11 from the KACC and 1 from the CFGR) was conducted by incubating each species on PDA and then evaluating the colony growth and shape over 1 week (Fig. 1A~L). Eight of the 12 species developed white colonies, but 3 of them soon developed orange conidial masses near the inoculum point (Fig.
In summary, we have developed a rapid and simple method to screen taxol-producing Colletotrichum species using the TS gene as a molecular marker. After screening several PCR primer pairs, we identified the most sensitive TS-gene-specific primer set as TS-2F and TS-2R. In addition to determining the taxonomic distance of 12 different Colletotrichum spp.
After testing several primer pairs for specificity for the taxadiene synthase (TS) gene, the candidate fungi for taxol production were screened by PCR amplification using the TS primer set TS-2F (5'- GTCGAATTGAGAAGCGTGGT-3') and TS-2R (5'- TCCCATCTCTTTACTCCCTCA-3').
DNA analyses were conducted by sequencing the PCR products of the internal transcribed spacer (ITS) region of the 18S rRNA gene using the primer set ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3').
2A). However, the background peaks surrounding the C. dematium taxol peak were relatively high (Fig. 2B), and therefore, further purification of the extract was carried out using solid-phase extraction. The resultant HPLC 13.
The presence and quantification of taxol were analyzed using a liquid chromatography-mass spectrometry (LC/ MS) system composed of an HPLC apparatus (model 2695; Waters, Milford, MA, USA) equipped with a pentafluorophenyl column (Luna, 3 × 150 mm, 3 µm; Phenomenex, Torrance, CA, USA) and a mass spectrometer (model 3100; Waters).
대상 데이터
kr). The 12 species tested were C. coccodes CBP1 (KACC 40009), C. dematium CBP2 (KACC 40013), C. acutatum CBP3 (KACC40042), C. higginsianum CBP4 (KACC 40806), C. truncatum CBP5 (KACC40810), C. boninense CBP7 (KACC 40893), C. liliacerum CBP8 (KACC 40981), C. caudatum CBP10 (KACC 41028), C. lindemuthianum CBP11 (KACC 42433), C. musae CBP18 (KACC 40947), and C. orbiculare CBP17 (KACC40809) from the KACC, and C. falcatum CBP42 (2007-112-00023) from the CFGR.
Colletotrichum spp. were obtained from the Korean Agricultural Culture Collection (KACC, Gene Resources Center, Rural Development Administration, Republic of Korea; http://www.genebank.go.kr) and Center for Fungal Genetic Resources (CFGR, Republic of Korea; http:// genebank.snu.ac.kr). The 12 species tested were C.
이론/모형
All 12 species were cultured on potato dextrose agar (PDA) medium at 25℃. Genomic DNA was isolated from 4-day-old mycelia cultured on PDA medium, using the standard quick method [21]. DNA analyses were conducted by sequencing the PCR products of the internal transcribed spacer (ITS) region of the 18S rRNA gene using the primer set ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3').
DNA analyses were conducted by sequencing the PCR products of the internal transcribed spacer (ITS) region of the 18S rRNA gene using the primer set ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3'). Phylogenetic analysis was carried out by the neighbor-joining method using MEGA ver. 6.0 software (The Biodesign Institute, Tempe, AZ, USA) [22]. Bootstrap analysis of each dataset was conducted with 1,000 replicates.
성능/효과
truncatum grew gray-olive colonies. The mycelial growth rates were quite different, with C. coccodes and C. caudatum growing the fastest, and C. lindemuthianum and C. orbiculare growing slowly. The phylogenetic relationship among the species was identified by comparing their 18S rRNA sequence homology (Fig.
참고문헌 (38)
1 Charles AG Han TY Liu YY Hansen N Giuliano AE Cabot MC Taxol-induced ceramide generation and apoptosis in human breast cancer cells Cancer Chemother Pharmacol 2001 47 444 450 11391861
2 Holton RA Kim HB Somoza C Liang F Biediger RJ Boatman PD Shindo M Smith CC Kim S Nadizadeh H First total synthesis of taxol. 2. Completion of the C and D rings J Am Chem Soc 1994 116 1599 1600
4 Expósito O Bonfill M Onrubia M Jané A Moyano E Cusidó RM Palazón J Piñol MT Effect of taxol feeding on taxol and related taxane production in Taxus baccata suspension cultures N Biotechnol 2008 25 252 259 19101660
5 Kim JA Baek KH Son YM Son SH Shin H Hairy root cultures of Taxus cuspidata for enhanced production of paclitaxel J Korean Soc Appl Biol Chem 2009 52 144 150
6 Stierle A Strobel G Stierle D Taxol and taxane production by Taxomyces andreanae , an endophytic fungus of Pacific yew Science 1993 260 214 216 8097061
7 Strobel G Yang X Sears J Kramer R Sidhu RS Hess WM Taxol from Pestalotiopsis microspora , an endophytic fungus of Taxus wallachiana Microbiology 1996 142 Pt 2 435 440 8932715
8 Shrestha K Strobel GA Shrivastava SP Gewali MB Evidence for paclitaxel from three new endophytic fungi of Himalayan yew of Nepal Planta Med 2001 67 374 376 11458463
9 Guo BH Wang YC Zhou XW Hu K Tan F Miao ZQ Tang KX An endophytic taxol-producing fungus BT2 isolated from Taxus chinensis var. mairei Afr J Biotechnol 2006 5 875 877
10 Gangadevi V Muthumary J Isolation of Colletotrichum gloeosporioides , a novel endophytic taxol-producing fungus from the leaves of a medicinal plant, Justicia gendarussa Mycol Balc 2008 5 1 4
11 Zhou X Zhu H Liu L Lin J Tang K A review: recent advances and future prospects of taxol-producing endophytic fungi Appl Microbiol Biotechnol 2010 86 1707 1717 20358192
12 Perfect SE Hughes HB O'Connell RJ Green JR Colletotrichum : a model genus for studies on pathology and fungal-plant interactions Fungal Genet Biol 1999 27 186 198 10441444
13 Freeman S Katan T Shabi E Characterization of Colletotrichum species responsible for anthracnose diseases of various fruits Plant Dis 1998 82 596 605
14 Hong JK Hwang BK Kim CH Induction of local and systemic resistance to Colletotrichum coccodes in pepper plants by DL-β-amino-n-butyric acid J Phytopathol 1999 147 193 198
15 Ji C Kúc J Non-host resistance to Colletotrichum lagenarium in pumpkin and squash is not primarily associated with β-1,3-glucanase and chitinase activities Physiol Mol Plant Pathol 1997 50 361 370
16 Irieda H Maeda H Akiyama K Hagiwara A Saitoh H Uemura A Terauchi R Takano Y Colletotrichum orbiculare secretes virulence effectors to a biotrophic interface at the primary hyphal neck via exocytosis coupled with SEC22-mediated traffic Plant Cell 2014 26 2265 2281 24850852
17 Subhani MN Chaudhry MA Khaliq A Muhammad F Efficacy of various fungicides against sugarcane red rot ( Colletotrichum falcatum ) Int J Agric Biol 2008 10 725 727
18 Ye F Albarouki E Lingam B Deising HB von Wirén N An adequate Fe nutritional status of maize suppresses infection and biotrophic growth of Colletotrichum graminicola Physiol Plant 2014 151 280 292 24512386
19 Narusaka M Abe H Kobayashi M Kubo Y Narusaka Y Comparative analysis of expression profiles of counterpart gene sets between Brassica rapa and Arabidopsis thaliana during fungal pathogen Colletotrichum higginsianum infection Plant Biotechnol 2006 23 503 508
20 Khan M Sinclair JB Pathogenicity of sclerotia- and nonsclerotiaforming isolates of Colletotrichum truncatum on soybean plants and roots Phytopathology 1992 82 314 319
21 Chi MH Park SY Lee YH A quick and safe method for fungal DNA extraction Plant Pathol J 2009 25 108 111
22 Tamura K Stecher G Peterson D Filipski A Kumar S MEGA6: molecular evolutionary genetics analysis version 6.0 Mol Biol Evol 2013 30 2725 2729 24132122
23 Walji AM MacMillan DW Strategies to bypass the taxol problem: enantioselective cascade catalysis, a new approach for the efficient construction of molecular complexity Synlett 2007 10 1477 1489
24 Wildung MR Croteau R A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis J Biol Chem 1996 271 9201 9204 8621577
25 Li JY Strobel G Sidhu R Hess WM Ford EJ Endophytic taxol-producing fungi from bald cypress, Taxodium distichum Microbiology 1996 142 Pt 8 2223 2226 8760934
26 Nithya K Muthumary J Growth studies of Colletotrichum gloeosporioides (Penz.) Sacc.: a taxol producing endophytic fungus from Plumeria acutifolia Indian J Sci Technol 2009 2 14 19
27 Senthilkumar N Murugesan S Mohan V Muthumary J Taxol producing fungal endophyte, Colletotrichum gleospoiroides (Penz.) from Tectona grandis L Curr Biotica 2013 7 8 15
28 Kumaran RS Jung H Kim HJ In vitro screening of taxol, an anticancer drug produced by the fungus, Colletotrichum capsici Eng Life Sci 2011 11 264 271
29 Ahn IP Uhm KH Kim S Lee YH Signaling pathways involved in preinfection development of Colletotrichum gloeosporioides , C. coccodes , and C. dematium pathogenic on red pepper Physiol Mol Plant Pathol 2003 63 281 289
33 Russo VM Pappelis AJ Observations of Colletotrichum dematium f. circinans on Allium cepa : halo formation and penetration of epidermal walls Physiol Plant Pathol 1981 19 127 136
34 Washington WS Irvine G Aldaoud R DeAlwis S Edwards J Pascoe IG First record of anthracnose of spinach caused by Colletotrichum dematium in Australia Australas Plant Pathol 2006 35 89 91
35 Sato T Muta T Imamura Y Nojima H Moriwaki J Yaguchi Y Anthracnose of Japanese radish caused by Colletotrichum dematium J Gen Plant Pathol 2005 71 380 383
36 Chitkara S Singh T Singh D Histopathology of Colletotrichum dematium infected chilli seeds Acta Bot Indica 1990 18 226 230
37 Shovan LR Bhuiyan MK Begum JA Pervez Z In vitro control of Colletotrichum dematium causing anthracnose of soybean by fungicides, plant extracts and Trichoderma harzianum Int J Sustain Crop Prod 2008 3 10 17
38 Li JY Sidhu RS Ford EJ Long DM Hess WM Strobel GA The induction of taxol production in the endophytic fungus: Periconia sp from Torreya grandifolia J Ind Microbiol Biotechnol 1998 20 259 264
※ AI-Helper는 부적절한 답변을 할 수 있습니다.