Optimization of A. pullulans transformation. (A and B) Number of Hyg^R transformants and (B) cell viability calculated from three independent transformations with and without carrier ssDNA. (C–E) Number of Hyg^R transformants, (D) % of correct ura3::HYG^R integrants, and (E) cell viability calculated from three independent transformations with different heat shock conditions. “None” indicates cells were incubated at room temperature for 15 min without heat shock. (F–H) Number of Hyg^R transformants, (G) % of correct ura3::HYG^R integrants, and (H) cell viability calculated from three independent transformations with cells harvested at different densities. In all transformations, a total of 10⁸ cells were used per condition. In all graphs, the mean and standard deviation are shown. Statistical significance was calculated by two-tailed Student’s t test in A and B and by one-way ANOVA in C–H with P values indicated (not shown, P > 0.05; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001).