Yeast Transformation Efficiency Evaluation

Competent cells of each yeast strain were prepared by EZ-yeast Transformation kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions, and their transformation efficiency was assessed for each introduced plasmid. Equal amounts of yeast transformants that were adjusted according to the transformation efficiency were directly suspended in 4 mL SDC+ 5 × U liquid medium and cultured at 250 rpm and 30 °C for 48 h. The harvested cells were washed with 4 mL of PBS (10 mM Na₂HPO₄, 1.76 mM NaH₂PO₄, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) twice and then diluted to 0.2 of OD₆₀₀ in 200 μL SDC liquid medium on the 96-well Polystyrene Microplates (353072; Corning, Corning, NY, USA). During the incubation at 30 °C without shaking, the cell growth curve was monitored by measuring absorbance at 600 nm of cell culture with the plate reader (Tecan Infinite 200 Pro F Plex; Tecan Ltd., Männedorf, Switzerland). The plates were sealed tightly by the transparent sealing tape (232698; Thermo Fisher Scientific), and the moat was filled by 70 μL of sterilized 0.1% agarose (Nacalai tesque) to reduce evaporation of the medium. The maximum specific growth rate was calculated by using Curveball 0.2.16 [39 (link)].