Pyes2 ct

maFACR was digested from pUC57-maFACR with HindIII/XhoI and cloned into pYES2/CT (Thermo Fisher, Singapore) to obtain pmaFACR.

The vector pYES2/CT (ThermoFisher Scientific) was digested with HindIII-HF and BamHI-HF (New England Biolabs). The MtVTL8 gene and its eight different mutant versions were amplified from pJYC16-23 using primers JYC10.FOR and JYC10.REV and assembled with the pYES2/CT-HindIII/BamH1 fragment using the Gibson assembly method to form pJYC10 (MtVTL8-wt) and eight different Mtvtl8-mut versions (pYJYC16-23).

The candidate genes for ELO and DES were extracted from the Parietichytrium draft genome database by local BLAST using previously known ELO/DES genes as query sequences. The ORFs of the putative ELO (ELO-1, ELO-2, and ELO-3) and DES (DES-1, DES-2, DES-3, DES-4, DES-5, and DES-6) were amplified by PCR using cDNA or genomic DNA of Parietichytrium, and inserted into the MCS of pYES2/CT (Invitrogen). The ω3DES of S. diclina^{33 (link)} and M. alpina^{50 (link)} were obtained from genomic DNA and cDNA, respectively, by PCR, and inserted into pYES2/CT. The expression vectors were introduced into S. cerevisiae INVSc1 (Invitrogen) using the lithium acetate method^{51 (link)}. The transformants were selected by plating on synthetic agar plates lacking uracil (SC-ura). S. cerevisiae transformants harboring ELO and DES were cultured in SC-ura medium containing 2% glucose at 25 °C for 1 day, and then cultured for an additional 1 day in SC-ura medium containing 2% galactose and 50 μM fatty acids (substrate), as described in Supplementary Fig. S1a. The cells were collected by centrifugation at 2000 × g for 10 min and lyophilized. The fatty acid profiles were obtained by GC analysis. ELO or DES activity was expressed as follows: activity (%) = product area × 100/(substrate area + product area).

The open reading frames (ORFs) of SmPPS1 and SmPPS2 were inserted into the yeast expression vector pYES2-CT (Invitrogen, United States) using two restriction enzymes to give the final vectors pYES2-SmPPS1 (BamH I/Xba I) and pYES2-SmPPS2 (Kpn I/Not I). The empty vector pYES2-CT and the resulting constructs were separately introduced into the S. cerevisiae coq1 mutant (strain 3138) using the yeast marker transformation kit (TaKaRa Bio, Japan). Functional complementation assay was performed as described previously (Ducluzeau et al., 2012 (link)). The yeast coq1 mutant with empty vector pYES2-CT and the wild type yeast strain BY4741 were used as controls.

Total RNA from C. zofingeinsis cells was extracted using the plant RNA extraction kit (Catalog No. 740949.50, TaKaRa, Tokyo, Japan) and reversely transcribed to cDNA with the Prime Script™ RT Master Mix (TaKaRa). The LD protein-encoding genes from C. zofingiensis were amplified using cDNA as template and cloned with C-terminal green fluorescence protein (GFP) into the vector pYES2-CT (Invitrogen, Carlsbad, CA). PCR primers used for cloning were listed in Table S1. The recombinant plasmids were confirmed by sequencing and then introduced into the Saccharomyces cerevisiae strain INVSc1 using the S.c. EasyComp Transformation Kit (Catalog No. K505001, Invitrogen). Transformants were selected on SD-uracil medium (Catalog No. 630416, TaKaRa). Single colonies carrying the plasmids were grown in SC-uracil medium containing 2% raffinose on an orbital shaker (250 rpm) at 30 °C for 24 h. To induce the expression of heterologous genes, yeast cells were cultured in SC-uracil medium containing 2% galactose, suspended to an initial OD₆₀₀ of 0.4, and allowed to grow for 48 h.

The endogenous AHA1 sequence was amplified using primers AHA1_BamHI and AHA1_XbaI containing cleavage sites for BamHI or XbaI at their 5′-ends. The PCR fragment was digested simultaneously with BamHI and XbaI subsequently cloned into the BamHI and XbaI sites of the expression vector pYES2/CT (Invitrogen). The wild-type AHA1 gene in the vector was mutagenized using the Phusion site-directed mutagenesis protocol (Thermo Scientific), to generate a series of clones with an amber stop codon at positions 59–66 (Table S1). The correct sequences of the corresponding plasmids (Table S3) were verified by sequencing.