Pyes2 yeast expression vector

The sequences of MiFRS were subcloned to pYES2 yeast expression vector (Invitrogen) using BamHI and EcoRI (Thermo Scientific). Plasmids were transformed by heat shock using lithium acetate and single-stranded DNA into strain VZL1303 (genotype MATa, Δura3, Δhis3, Δleu2, ERG7-KanMX) and transformants were selected on SC medium (0.67% yeast nitrogen base, 2% glucose) without uracil (SC-ura). Yeast codon optimized KdFRS (Epoch Life Science, Missouri, TX, USA) was also cloned into pYES2 and transformed into the same strain. Cells were cultivated in SC-ura and heterologous expression was induced by adding 2% galactose, for 10 h at 30 °C with shaking. Friedelin production was accomplished by incubating cells in 0.1 M potassium phosphate, pH 7.0 with 3% glucose for 24 h at the same temperature [4 (link)]. Then, cells were collected, dried and about 30 mg were extracted with chloroform:methanol (2:1, v/v) [30 (link)] in an ultrasonic bath (2840D, Odontobrás, Ribeirão Preto, SP, Brazil) for 10 min. Organic phase was collected after addition of 0.73% NaCl and centrifugation. Extract was dried and resuspended in 200 µL acetonitrile.

Full-length cDNAs for Slc4a11A cDNA was subcloned into the pYES2 yeast expression vector (Invitrogen) and expressed in the Y01169 S. cerevisiae strain as described previously (22 (link)). To measure boron concentration, cells in the midexponential phase were harvested, washed, and dried. The boron concentration was measured using ICP-MS, as described previously.

OsSRO1a and OsRBD1 were amplified from rice cDNA using gene specific primers (Supplementary Table S1) and cloned into TOPO-TA vector (Invitrogen, USA). For localization assay, each of the OsSRO1a and OsRBD1 cDNA were cloned in pMBPII vector as done previously (Kumar et al., 2012 (link)) at BamH1 and XbaI/BamH1 sites, respectively. For in planta interaction studies, OsSRO1a and OsRBD1 cDNA were cloned in BiFC1 and BiFC2 vectors at Not1 and Nco1/Not1 sites, respectively. OsSRO1a and OsRBD1 full length cDNA were also cloned in pYES2 yeast expression vector (Invitrogen, USA) at BamH1 and HindIII/BamH1 for yeast expression studies.

PCR fragment corresponding to the ORF of the Elovl5 elongase was amplified from T. ovatus liver cDNA using primers that included HindIII and XhoI restriction sites (Supplementary Table S1). Subsequently, the DNA fragment was digested with the relevant restriction endonucleases (New England BioLabs, Herts, United Kingdom) and ligated into a coincident restricted pYES2 yeast expression vector (Invitrogen, Paisley, United Kingdom). The recombinant plasmid (pYES2-Elovl5) was then used to transform Saccharomyces cerevisiae competent cells (S.c. EasyComp Transformation Kit, Invitrogen). Transformation and selection of yeast with recombinant plasmids, and yeast culture were prepared according to previously described methods (Li et al., 2017 (link)). Fatty acids are: 18:3n-3 (α-linolenic acid), 18:3n-6 (γ-linolenic acid), 18:4n-3 (stearidonic acid), 20:4n-6 (arachidonic acid, ARA) and 20:5n-3 (eicosapentaenoic acid, EPA) were used as substrates for detecting the elongase activity of ToElovl5. Final concentrations of FA substrates varied according to their fatty acyl chain lengths, 0.5 mM (C18) and 0.75 mM (C20). Yeast cultures were incubated for 2 days at 30°C, and then were harvested, washed twice as described previously (Li et al., 2010 (link)). Under the same conditions, yeast transformed with pYES2 contain no insert was grown as a control.

Wild-type and TRBP phospho-mimic and phospho-defective point mutants were subcloned into the pYES3CT yeast expression plasmid (Invitrogen). Wild-type PKR was subcloned into the pYES2 yeast expression vector (Invitrogen) as previously described for galactose inducible PKR expression. The constructs were introduced into InvSc1 yeast cells (Invitrogen) using the Clontech Yeast Transformation Kit. Transformed yeast cells were grown to an OD₆₀₀ of 2 in YPD media (yeast extract, peptone, and dextrose). 500 μl of each culture was pelleted and resuspended in an appropriate amount of distilled water to yield an OD₆₀₀ of 10. Serial dilutions were then made to yield OD₆₀₀ values of 1, 0.1, and 0.01. 10 μl of each dilution was then spotted onto synthetic medium lacking uracil and tryptophan and containing either glucose or galactose as a carbon source (Clontech).