Engineered Yeast for Resveratrol Biosynthesis

E. coli Trans 5α (TransGen Biotech, Beijing, China) was used for propagation of plasmids. Luria–Bertani (LB) broth medium (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl) containing 50 mg/L of kanamycin was used to culture E. coli carrying transformed plasmids. S. cerevisiae strain BY4742 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) was used as the parent strain for resveratrol biosynthesis. YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose, also marked as YPD-20G when needed) was used for routine cultivation of S. cerevisiae strains. YPD medium with 40 g/L glucose was also used for fermentation in shake flasks and marked as YPD-40G. Geneticin (G418, 100 mg/L) was supplemented in the YPD agar plate for the selection of engineered yeast strains edited by CRISPR/Cas9. SC agar plates (synthetic complete drop-out medium, 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, and 0.8 g/L dropout powder minus appropriate amino acids) was used for the selection of engineered yeast strains edited by homologous recombination using HIS3, LEU2, URA3 and LYS2 respectively as selective markers. Minimal medium used in our study was based on studies described previously [36 (link)]. The medium contained 20 g/L or 40 g/L glucose, 15 g/L (NH₄)₂SO₄, 8 g/L KH₂PO₄, 6.2 g/L MgSO₄∙7H₂O, 1.2% (v/v) vitamin solution, and 1% (v/v) trace metal solution. L-lysine (0.5 g/L) was added in minimal medium for the cultivation of strain BRT8 and BRT9. The trace metal solution contained 5.75 g/L ZnSO₄∙7H₂O, 0.32 g/L MnCl₂∙4H₂O, 0.47 g/L CoCl₂∙6H₂O, 0.48 g/L Na₂MoO₄∙2H₂O, 2.9 g/L CaCl₂∙2H₂O, 2.8 g/L FeSO₄∙7H₂O and 80 mL 0.5 M EDTA, pH 8.0. The vitamin solution contained 0.05 g/L biotin, 1 g/L calcium pantothenate, 1 g/L nicotinic acid, 25 g/L myo-inositol, 1 g/L thiamine HCl, 1 g/L pyridoxal HCl and 0.2 g/L p-aminobenzoic acid.