Glucose supplementation greatly enhances the transformation efficiency of A. pullulans. (A and B) Number of Hyg^R transformants and (B) number of correct ura3::HYG^R integrants from three independent transformations with or without amino acids (AA) or glucose (Glu). (C) Cell viability was calculated from the number of colonies that grow on YPD (no selection) normalized to the “no additives” condition. (D and E) Number of Hyg^R transformants and (E) % of correct ura3::HYG^R integrants from three independent transformations with 2% glucose added at different steps (see Fig. 1 D: note that when glucose is added in step (ii) it is also present for step (iii). (F) Cell viability normalized to the condition with 2% glucose added in step (i) only. In all graphs, the mean and standard deviation are shown. Statistical significance is calculated by one-way ANOVA and P values are indicated (not shown, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001).