4.4. Yeast Extraction Procedures

2 mL of the final yeast cultures was used for metabolite extraction and analysis: 2 mL culture was transferred to 2 mL screw cap microcentrifuge tubes and centrifuged at high speed (Centrifuge 5424, Eppendorf) for 4 min. Supernatants were discarded and pellets were dissolved in 1 mL 80% ethanol. The samples were then heated to 80 °C and agitated for 10 min followed by 4 min of high speed centrifugation. Supernatants were transferred to 14 mL glass vials and extracted: miltiradiene and dehydroabietadiene diterpenes were extracted with 1:1 n-hexane (AR, Biosolve, Dieuze, France) while DHA was extracted with 1:1 ethyl acetate (HPLC grade, Biosolve). Extracted samples were transferred to 1.5 mL glass vials after 10 seconds of vortexing to be ready for analysis. In the case of samples putatively containing DHA, 100 µL absolute methanol (HPLC grade, Biosolve) and 120 µL trimethylsilyl-diazomethane (TMSD in 2M diethyl ether, Sigma Aldrich, Buchs, Switzerland) were added for derivatization. The samples were then agitated at room temperature for 20 min prior to GC-MS analysis.

To verify identity and determine product levels, authentic and relative standards were used (dehydroabietic acid, derivatized as above for detection and quantification, Glentham Life Sciences, Corsham, England; ent-Kaurene, Evolva).