Heterologous Expression of CbFAD3 in Yeast

The coding region of CbFAD3 was cloned into pYES2.0 (Invitrogen, USA) using specific primers (P13 and P14; Supplementary Table S1), to construct the expression plasmid pYES2-CbFAD3. pYES2-CbFAD3 and pYES2.0 were transformed into Saccharomyces cerevisiae strain INVSc1 (Invitrogen, USA) using S. cerevisiae EasyComp transformation kit (Invitrogen, USA). The yeast transformants were selected and cultured according to the method of Román et al. (2012) (link). When the OD₆₀₀ of the culture reached 0.2–0.3, gene expression was induced by adding 2% (w/v) galactose. Yeast cells were harvested by centrifugation at 1500 g for 5 min at 4 °C and washed with distilled water. The extraction and SDS-PAGE of total yeast proteins were performed as described by Horvath and Riezman (1994) (link). The production of C18:3 was induced by adding 2% (w/v) galactose, 50 μM C18:2 (Sigma-Aldrich, USA) and 0.1% (w/v) NP-40, and was measured after growth at 20 °C for 3 d.