2011; Chu et al. 2011). The existence of taxane gene clusters in fungi and plants raises intriguing questions about the SHP099 clinical trial origin and evolution of these highly-specialized biosynthetic pathways, and the potential for HGT from fungi to Taxus trees. However, HGT between distantly-related organisms is a rare evolutionary event which is also constrained by the amount of genetic information transferred and genetic barriers
involving incompatible regulation and codon usage. This contrasts sharply with the widespread observation of Taxol Ro-3306 cost biosynthesis in many different endophytic fungi (Kurland et al. 2003). Material and methods Isolation of endophytic fungi from Taxus spp. plant material Endophytic fungi were isolated as previously described by Guo et al. (2006). Bark segments (0.5 × 0.5 cm) were removed with a sterile scalpel and surface sterilized for 5 min in 70 % ethanol. The inner bark was separated from the outer layer and placed on PDA agar (Carl Roth GmbH, Karlsruhe, Germany) supplemented with 25 mg/L streptomycin. The plates were incubated at room temperature until fungal growth was visible. The mycelium was then transferred to fresh plates using the hyphal tip method. Cultivation of endophytic fungi
The isolated endophytic fungi were cultivated on solid media, PDA (Carl Roth GmbH) supplemented with streptomycin or on YM-6.3 agar (0.4 % (w/v) glucose, 0.4 % (w/v) yeast extract, 2 % (w/v) malt extract, pH 6.3, 1.5 % (w/v) agar-agar). The fungi were transferred to fresh Selleck Tucidinostat plates at weekly intervals by cutting out a piece of overgrown agar. In liquid culture, the fungi were grown in 0.6–10 L YM-6.3 medium (120 rpm in the dark) for 3 weeks or until no more glucose could be detected. The fungi were also cultivated in S7 medium as described for taxane-producing endophytes (Stierle et al. 1993). Taxoid extraction For taxane analysis, the Tangeritin fungal culture media were extracted twice with an equal volume of chloroform. The organic phase was then dried over magnesium sulfate, evaporated to dryness and the residue was redissolved
in 3–5 mL methanol. Plant material (30 g Taxus needles or tobacco leaf tissue) was lyophilized and extracted with 1:1 dichloromethane/methanol in a Soxhlet extractor. The organic solution was evaporated to dryness and redissolved in dichloromethane. After two rounds of extraction with water, the organic layer was dried over magnesium sulfate, evaporated to dryness and the residue was redissolved in methanol (Witherup et al. 1990). Anti-taxane immunoassay (competitive inhibition enzyme immunoassay, CIEIA) The anti-taxane immunoassay was carried out according to the manufacturer’s instructions (Cardax Pharmaceuticals, Hawaii). A standard curve for taxane quantitation was made using Taxol concentrations of 111, 37, 12.33, 4.11, 1.37, 0.46 and 1.15 ng/mL (Table S1). The samples were analyzed using three dilutions.