Improved taxol production in Nodulisporium sylviforme derived from inactivated protoplast fusion

Abstract

Inactivated protoplast fusion by UV irradiation and UV+LiCl mutation was conducted using Nodulisporium sylviforme strain UV_40-19 and UL_50-6 to breed a high taxol-producing fungus. Qualitative and quantitative analysis of taxol production was confirmed using thin-layer chromatography, high performance liquid chromatography and mass spectrometry. The protoplasts of UV_40-19 and UL_50-6 were fully inactivated by heating at 54°C for 5 min and by UV irradiation (30 w UV light and vertical distance 30 cm) for 85 s. The highest fusion rate (14.31 ± 1.13%) between UV_40-19 and UL_50-6 was obtained under the conditions of 35% PEG, 90 s fusion time and the addition of 0.01 mol/l CaCl₂. One high taxol production strain HDF-68 was obtained. The taxol production was up to 468.62 ± 37.49 μg/l, which was increased by 24.51 and 19.35% compared with the parental strain UV_40-19 and UL_50-6, respectively. This study provided a good basis for the application of this technique to the breeding of the strains with high taxol output.

Key words: Taxol, endophytic fungi, protoplast preparation, protoplast fusion.