Heterokonta DGAT2 Expression in Yeast

The ORFs of HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E were PCR-amplified using cDNA as a template and cloned into the yeast expression vector pYES2.0 (Invitrogen). After confirmation by restriction enzyme digestion and sequencing, the recombinant pYES2.0-HpDGAT2s plasmids were transformed into the S. cerevisiae TAG-producing strain INVSc1 or TAG-deficient quadruple mutant strain H1246 with the S.c. EasyComp Transformation Kit (Invitrogen) [20 (link)]. The expression of HpDGAT2 genes in the yeast strain was verified at the transcript level by qRT-PCR. For the feeding experiments, yeast cultures were induced as described above but in the presence of 1% (w/v) Tergitol NP-40 (Sigma Aldrich, St. Louis, MO, USA) in the medium. At the beginning of induction, the appropriate FAs (C18:2n6, C18:3n3, C18:3n6, and C18:4n3) were added to the culture to a final concentration of 100 μM. Samples at an OD600 of 2.5 were harvested for lipid extraction, separation by TLC and analysis by GC.