Fig. 2

Screening of the putative transformants and molecular identification of S. cerevisiae ERG5ΔERG4ΔERG3Δ. A Construction strategy of four engineered S. cerevisiae strains; B Screening of four putative transformants on solid plates containing two antibiotics, while the wild-type strain could not grow on the screening plate; C Screening of vector loss of S. cerevisiae ERG5Δ transformants for further transformation of ERG4. The cell proliferation of S. cerevisiae ERG5Δ transformant after transformation of ERG5-gRNA-trp-HyB and Cas9-NTC was carried out via the liquid fermentation approach. The proliferative colonies were screened and cultured on YPD plate a for 48 h at 30 °C. Then, the colonies on plate a were transferred to the corresponding positions on plates b and c. The media in a, b, and c were YPD medium, YPD medium containing 80 μg/mL of nourseothricin (NTC), and YPD medium containing 300 μg/mL of hygromycin B (HyB), respectively. The colonies (arrow) could not grow on both b and c, which meant that the corresponding colony on plate a had lost both ERG5-gRNA-trp-HyB and Cas9-NTC; D S. cerevisiae ERG5ΔERG4ΔERG3Δ identification by amplification of XYNA (lane 2, 1129 bp), CEL (lane 3, 880 bp), and MFC (lane 4, 628 bp). Lane M and 1 represented DNA Marker and the control, respectively