Abstract

Phoma betae, an endophytic fungus, was isolated from the healthy leaves of Ginkgo biloba. The fungus was screened for the production of taxol on a modified liquid medium for the first time. The fungal species were identified by their characteristic culture morphology and molecular analysis. The presence of taxol was confirmed by spectroscopic: ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR),liquid chromatography–mass spectrometry (LC-MS); and chromatographic: thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods of analysis. The taxol production was quantified by HPLC analysis. The maximum amount of taxol production was recorded as 795 µg/L. The production rate was 15,900-fold more than that found in the culture broth of earlier reported fungus,Taxomyces andreanae. The extracted fungal taxol demonstrate a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay. This indicates that the increase in taxol concentration induced an increased cell death. Apolymerase chain reaction (PCR) based screening for taxadiene synthase (ts), a unique gene in the formation of the taxane skeleton was confirmed as a molecular blueprint for taxol biosynthesis. These results designate that the fungus, P. betae is an excellent candidate for taxol supply and can serve as a potential species for genetic engineering to enhance the production of taxol to a higher level.

Key words: Taxol production, Phoma betae, analytical methods, cytotoxicity assay, taxadiene synthase.

Abbreviation

UV, Ultraviolet; IR, infrared; NMR, nuclear magnetic resonance; LC-MS, liquid chromatography-mass spectrometry; TLC, thin layer chromatography;HPLC, high performance liquid chromatography; PCR, polymerase chain reaction.