Eighteen endophytic fungi were isolated from various tissues of Datura metel and genes encoding for putrescine N-methyltransferase (PMT), tropinone reductase 1 (TR1) and hyoscyamine 6β-hydroxylase (H6H) were used as molecular markers for PCR-based screening approach for tropane alkaloids (TAs) producing endophytic fungi. These fungi were identified taxonomically by sequence analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) and also based on morphological characteristics of the fungal spore as Colletotrichum boninense, Phomopsis sp., Fusarium solani, Colletotrichum incarnatum, Colletotrichum siamense and Colletotrichum gloeosporioides. The production of TAs hyoscyamine and scopolamine by the fungi has been ascertained using chromatography and spectroscopy methods by comparison with the standards. Among the fungi, the highest yields of hyoscyamine (3.9 mg/L) and scopolamine (4.1 mg/L) were found in C. incarnatum culture. This is the first report of endophytic fungi possess the PMT, TR1 and H6H genes and produces TAs. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for TAs economically.
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Acknowledgements This work was supported by a grant BT/ PR14760/NDB/52/188/2010 to CJB from the Department of Biotechnology (DBT), Government of India, New Delhi, India. VSC and MC thank the Department of Biotechnology and UGC, New Delhi, India for Post-Doctoral fellowship awards. SK thanks DST-SERB New Delhi, India for the young scientist award. We also thank DBT-IISC Partnership Programme, DST-FIST sponsored by GOI New Delhi, India and UGC special Assistance programme for financial support and providing facilities. We also thank emeritus Prof. T. Ramasarma and Prof. R. Manjunath for critical review of the manuscript.