Abstract

EFY-21 (Ozonium sp.) is a newly isolated taxol-producing endophytic fungus fromTaxus chinensis var. mairei. In this study, an efficient PEG-mediated transformation of EFY-21 was established and conditions for transformation were evaluated. By the optimized enzyme system, mycelium age, digesting temperature and time, over 7 × 10⁷ ml protoplasts were obtained and protoplast regeneration frequency was more than 6%. Plasmid pV2 containing the hygromycin-B phosphotransferase gene driven by a fungal promoter (trpC) was used to transform EFY-21 and 50% PEG with 20 mM Ca²⁺ was found to be suitable for the transformation. Southern blot analysis revealed that the transforming DNA was successfully integrated into the EFY-21 genome. By the optimized procedure, over two transformants per μg DNA could be obtained. The establishment of efficient transformation system of taxol-producing endophytic fungus enables us to improve taxol production of the fungus by engineering the taxol biosynthetic pathway genes in the future.

Key words: Endophytic fungus, taxol, PEG-mediated transformation, protoplast, regeneration, Ozonium sp.