Construction of the S. cerevisiae INVSc1[pYES2-PαXC-rDNA] recombinant strain

The PαXC expression cassette was ligated into pMD19-T and transformed into electrocompetent E. coli DH5α cells before being extracted and confirmed by sequencing. The extracted pMD19-T-PαXC plasmid and vector pYES2 were then digested with EcoRI and XbaI, respectively, and visualised by agarose gel electrophoresis. The target PαXC fragment was then recovered from the agarose gel and ligated into the linearised pYES2 vector using T4 ligase, generating plasmid pYES2-PαXC. The recombinant plasmid was then transformed into electrocompetent E. coli DH5α.

S. cerevisiae INVSc1 genomic DNA was used as a template to amplify the 2,300-bp core sequence of the rDNA unit using primers rDNA-F and rDNA-R, both of which introduced SnaBI restriction enzyme recognition sites into the resulting DNA fragment. pYES2-PαXC and pMD19-T-rDNA (constructed by TA cloning) were then digested with SnaBI and the target fragments recovered by gel purification. T4 ligase was used to ligate the target fragments, generating recombinant expression vector pYES2-PαXC-rDNA. pYES2-PαXC-rDNA was then linearised at the rDNA locus using SphI, and the LiAc/ssDNA method^12,13 was used to transform the linearised vector into electrocompetent S. cerevisiae INVSc1. Recombinants were selected by culture on SC-URA medium for 3–5 days.