Tryptophan

pYC2/CT plasmids were transformed into S. cerevisiae BJ5465 fex:GSHU fex2Δ using the lithium-acetate/PEG method (Gietz, 2014 (link)). Transformants were selected on SD-URA plates (20 g/L D-glucose, 6.7 g/L yeast nitrogen base (Becton, Dickinson & Co., Sparks, MD, USA), 5 g/L casamino acids (Becton, Dickinson & Co., Sparks, MD, USA), 40 mg/L Tryptophan, 40 mg/L Adenine, 16.25 g/L sodium citrate dihydrate, 4.2 g/L citric acid monohydrate, 20 g/L agar (Becton, Dickinson & Co., Sparks, MD, USA)). Genes were expressed essentially as described in Drew et al., with the following modifications (Drew et al., 2008 (link)). Briefly, individual colonies were used to inoculate 3 mL overnight cultures in SD-URA medium (20 g/L D-glucose, 6.7 g/L yeast nitrogen base (Becton, Dickinson & Co., Sparks, MD, USA), 5 g/L casamino acids (Becton, Dickinson & Co., Sparks, MD, USA), 40 mg/L Tryptophan, 40 mg/L Adenine, 16.25 g/L sodium citrate dihydrate, 4.2 g/L citric acid monohydrate) and grown at 30 °C, 225 rpm, for ∼16 h. The overnight cultures were used to seed an expression culture in SR-URA medium (same composition as SD-URA, except with 20 g/L raffinose instead of glucose) to a starting OD₆₀₀ of 0.12. The culture was grown at 30 °C, 225 rpm for 6–7 h or until OD₆₀₀ ∼0.6–1, when gene expression was induced with 2% w/v galactose for 22–24 h (30 °C, 225 rpm).

Plasmids were extracted from the evolved yeast strains using a Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research (Irvine, CA)). Isolated plasmids were propagated and extracted from E. coli DH5α and verified by Sanger sequencing (Genewiz, South Plainfield, NJ, USA). In order to cure evolved yeast strains of plasmid, cells were plated onto SD plates (20 g/L D-glucose, 6.7 g/L yeast nitrogen base (Becton, Dickinson & Co., Sparks, MD, USA), 5 g/L casamino acids (Becton, Dickinson & Co., Sparks, MD, USA), 40 mg/L Tryptophan, 40 mg/L Adenine, 40 mg/L uracil, 16.25 g/L sodium citrate dihydrate, 4.2 g/L citric acid monohydrate, 20 g/L agar (Becton, Dickinson & Co., Sparks, MD, USA)) containing the auxotrophic marker (uracil) encoded in the pYC plasmid backbone. Colonies were replica-plated onto SD and SD-URA plates to identify those unable to grow in the absence of uracil due to loss of plasmid. The Ura^‒ phenotype was verified by confirming growth or lack of growth after overnight incubation in 3 mL SD and SD-URA cultures, respectively.