Gene constructs, yeast transformation, and microsomal preparation

Camelina (C.sativa) PDCT and yeast vector were designed as described by Lager et al. (2020). Castor bean PDCT and DGAT2 were S.cerevisiae codon-optimized and synthesized synthetically. Both genes were processed utilizing Gateway Cloning and finally expressed in binary vector Gateway pYES-DEST52 under the GAL1 (galactokinase, YBR020W) promoter in plasmids containing URA3 (orotidine-5′-phosphate decarboxylase, YEL021W) marker for yeast selection. Yeast transformations of TAG-deficient H1246 yeast strain (MATα are1-Δ::HIS3 are2-Δ::LEU2 dga1-Δ::KanMX4 Iro-Δ::TRP1 ADE2 ura3) (Sandager et al., 2002) and subsequent microsomal preparations were carried out as described by Jeppson et al. (2020). Yeast mutated in ale1 and overexpressing GPC1 and microsomal preparations of this yeast were produced as described by Glab et al. (2016). Microsomal preparations from developing castor bean endosperm were done as described by Bafor et al. (1991).