Example 18
The constructs pYes2_CpDGAT1, pYes2_CpDGAT1Ala, and pYes2_CpDGAT1trunc were transformed into S. cerevisiae strain H1246 (W303; MATα are1-Δ::HIS3 are2-Δ::LEU2 dga1::KanMX4 lro1-Δ::TRP1 ADE2 met ura3) (Sandager et al., 2002, J. Biol. Chem., 277:6478-82) using PEG/lithium acetate method (Gietz et al., 1995, Yeast, 11:355-60). The yeast cells harboring the empty pYes2 vector were used as negative control. Transformants were selected by uracil prototrophy on yeast synthetic medium (YSM) containing 2% (w/v) glucose and lacking uracil (Invitrogen, Carlsbad, Calif. USA). For functional expression YSM containing 2% (w/v) raffinose was inoculated with the yeast transformants and grown at 28° C. for 24 h in a shaker at 350 rpm. For induction, YSM containing 2% (w/v) galactose was inoculated with raffinose-grown cultures to obtain an OD of 0.2 at 600 nm and grown at 28° C. for 48 h. For fatty acid feeding experiments cultures were grown for 2.5 hs in YSM containing 2% galactose followed by addition of 1% (w/v) Tergitol-40 and 250 μM of the appropriate fatty acid substrate. Cells were harvested by centrifugation, washed twice with 0.1% NaHCO3, freeze-dried and used for fatty acid, TAG analysis and microsome isolation.