Yeast transformation

The transgenic yeast strains generated in this study are listed in Table 2. The parent yeast strain for all transformations in this study was S. cerevisiae CBS 8340 (syn. CEN.PK 113-7D; genotype MATa MAL2-8^c SUC2), which was purchased from Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands). The selection agents G418 disulfate and hygromycin B were purchased from Formedium Ltd (Norfolk, UK). An aqueous stock solution of hygromycin B was prepared to a final concentration of 50 g/L, sterilized by filtration and stored at 4 °C. An aqueous stock solution of G418 disulfate was prepared to a final concentration of 100 g/L, sterilized by filtration, and stored as aliquots at − 20 °C.

Transgenic yeast strains used in this study

The transformation protocol used in this study is a simplified version of the standard lithium acetate protocol. A S. cerevisiae pre-culture was diluted to a final OD₆₀₀ of 0.1 in 50-mL fresh YM broth (3-g/L yeast extract, 3-g/L malt extract, 5-g/L peptone, and 10-g/L glucose) supplemented with 75-mg/L carbenicillin and incubated at 30 °C with shaking at 200 rpm until OD₆₀₀ reached 0.5–0.6. Cells were collected by centrifugation (2500×g, 5 min) and resuspended in 1.5-mL sterile water. The washed cells were collected by centrifugation (6900×g, 3 min), the supernatant removed and the pellet resuspended in 0.4-mL 100-mM lithium acetate. The resuspended cells were incubated at 30 °C for 15 min and then divided into 50-μL aliquots. 10-μL purified SwaI-digested integration plasmid was added to each aliquot of cells, which were then incubated at 30 °C for 15 min. 0.3 mL of a 100 mM lithium acetate/40% (w/v) PEG3350 mixture was added to each sample, and mixed and incubated for a further 15 min at 30 °C. Samples were then incubated for 15 min at 42 °C followed by immediate centrifugation (6900×g, 3 min) and removal of the supernatant. 0.5 mL of fresh YM broth was added to each sample, which was left to stand for 5 min at room temperature before being fully resuspended. Each sample of 0.5-mL resuspended cells were transferred to a fresh tube containing 2.5-mL fresh YM broth and incubated at 30 °C for 1 h in a rotary shaker set to 200 rpm. Samples were then centrifuged (6900×g, 3 min) and the supernatant discarded. The cells in each sample were resuspended in 0.2-mL fresh YM broth and spread on solid YM medium (20 g/L agar) containing either 400-mg/L G418 disulfate or 200-mg/L hygromycin B as specified in the “Results and discussion” section.

Correct chromosomal integration and the deletion of the PUT1 locus were confirmed by PCR analysis of purified genomic DNA from each strain (Fig. 2a). Successful integration at the PUT1 locus was assayed using primers pFA6a ctrl fwd and ScPUT1 5′ ctrl rev, which produce no product in the CBS 8340 parent strain, a 417-bp amplification product in strains TLSC001–TLSC009, and a 1980-bp amplification product TLSC010 due to the insertion of the hphEC6 cassette at site II (Fig. 2b). Successful deletion of the PUT1 coding sequence was assayed using primers ScPUT1 3′ ctrl fwd and ScPUT1 ctrl rev, which produce a 1063-bp amplification product in the CBS 8340 parent strain and no product in strains TLSC001–TLSC010 (Fig. 2c).