Fig. 3: Application of the protein-based TAQing system, TAQing2.0, to the non-conventional yeast Cu.

a A schematic diagram describing a procedure of the TAQing2.0 system. b, c Reduction of cell viability by activation of TaqI (TAQed) introduced to Cu cells by the TAQing2.0 system. Cells subjected to the optimized TAQing2.0 with no protein, TaqI, or BSA were incubated at 30 or 38 °C for 90 min, and plated onto YPD plates. Plates were then incubated at 30 °C for a few days. b Representative images of plates (9 cm diameter) with formed colonies after the treatment. c Cell viabilities after the TAQing2.0 system application. Colony forming units (CFU) were calculated for each experimental group shown in (b), and cell viabilities were determined by setting CFU without protein (i.e., TaqI- BSA-) to 100%. Bars and error bars, respectively, represent the mean and the standard deviation from three independent experiments (n = 3) and one-tailed Welch’s t-test was applied (*p < 0.05, **p < 0.01). Experiments were performed in triplicates. d Morphological images of wild type (WT) Cu×5 (Control after five passages), AG4×5 (TAQed mutant AG4 cells after five passages), and AG9×5 (TAQed mutant AG9 cells after five passages). Bright-field images were obtained by microscopy BZ-X700. The scale bars are 5 µm. e Chromosome sizes of WT Cu (Control) and TAQed Cu (AG4, AG4×5, AG9, and AG9×5) cells. Chromosomal DNA prepared from Cu cells along with size marker DNA fragments (Bio-Rad Catalog #170-3667) were analyzed by pulsed-field gel electrophoresis. Red triangles in the short exposure panel indicate appearance of bands in the TAQed mutants. We noticed that the longest chromosome in wild type (chromosome I) exhibited smear bands (the vertical bar in the long exposure panel) possibly due to heterogeneity of rDNA repeat number. Instead, we detected shortened chromosomes in AG4 and AG4x5 (the three red triangles). The AG9 and AG9x5 mutants had elongated chromosome I (the red triangle). Since a duplication at a position proximal to rDNA region was observed in AG9x5 (Fig. 4a), it is possible that this duplication may affect the rDNA stability.