Study on the Preparation and Regeneration of Protoplast from Taxol-producing Fungus Nodulisporium sylviforme

The effects of some factors on the preparation and regeneration of protoplasts from the taxol-producing fungus Nodulisporium sylviforme were discussed in this paper, including combination of various enzymes, digesting time and temperature, and pH value, osmotic stabilizers, pretreation, culture medium and culture method. Under the condition of the mixed enzymes in osmotic pressure stabilizer (0.7 mol/L NaCl) combination of 3% lywallzyme, 2% snailase and 1% lysozyme, pH 5.5~6.0, digesting time 9h and digesting temperature 30°C, the results showed that the highest preparation frequency of protoplasts is a concentration of 2.0×10 protoplasts per microlitre. After the obtained protoplasts were purified, they would be regenerated by bilayer plate culture method in PDA regenerative medium. This study laid the foundation to construct the engineering taxol-producing strains by protoplast mutagensis, transformation, fusion, and turn to a new page of research of producing anti-tumor drug taxol by microbiological fermentation. [Nature and Science, 2004,2(2):52-59] Keyword: taxol; Nodulisporium sylviforme; protoplast; preparation; regeneration