Yeast transformations

Yeast strains were chemically transformed using the Frozen-EZ Yeast Transformation II Kit (Zymo Research). Individual colonies were inoculated into YPD media and grown overnight at 30 °C and 250 rpm. Saturated cultures were back-diluted into three new cultures at 1:5, 1:10, and 1:20 dilutions in YPD media and grown for an additional 5–7 h to reach exponential phase. For each transformation, 1 mL aliquots from each back-diluted culture were pelleted by centrifugation at 500 × g for 4 min (successively pelleting aliquots from each different dilution into a single pellet in a 1.5 mL microcentrifuge tube) and then washed twice by resuspending the pellet in 1 mL of 50 mM Tris-HCl buffer, pH 8.5. Washed pellets were resuspended in 50 μL of EZ2 solution per transformation and mixed with 100–600 ng of total DNA and 500 μL of the EZ3 solution. The yeast suspensions were incubated at 30 °C with gentle inversion for one hour. For plasmid transformations, the transformed yeast was directly plated onto YNB-Ura agar plates. For Cas9-mediated gene integrations, the yeast suspensions in the EZ3 solution were first mixed with 1 mL YPD media, pelleted by centrifugation at 500 × g for 4 min, and then resuspended in 250 μL of fresh YPD media. The suspensions were incubated at 30 °C with gentle inversion for an additional 90 min to allow production of G418 resistance proteins and then spread onto YPD plates containing 400 mg/L G418 sulfate. For all transformations, plates were incubated at 30 °C for 72 h before being used to inoculate cultures for metabolite assays.