Additional file 1 of Genetic modifications of critical regulators provide new insights into regulation modes of raw-starch-digesting enzyme expression in Penicillium

Additional file 1: Fig. S1. Southern blot analysis of the genomic DNA of OamyR, OhepA, ΔCreA, and Mpga3 mutants. The location of the probes and restriction enzyme sites for Southern blot analysis are shown. The primers are listed in Table S2. (A) The overexpression strains OamyR and OhepA generated 5.6 and 4.7 kb DNA bands, respectively, while the parental strain 114–2 did not produce a detectable band, indicating that the overexpression cassettes were integrated into the genome. (B) Southern blot analysis of the genomic DNA of 114-2 and Mpga3. The 114-2 strain generated a 1.3 kb DNA band, while a 3.1 kb band was obtained in Mpga3 indicating that the targeted gene was correctly replaced. Sequencing of the mutated pga3 (Mpga3) gene in strain Mpga3. The mutated codon is indicated. (C) Southern blot analysis of the genomic DNA of 114-2 and ΔCreA. The 114-2 strain generated a 3.3 kb DNA band, while a 6.3 kb band was obtained in ΔCreA indicating that the targeted gene was correctly replaced. Fig. S2. RSDE activity assay of WT and various mutants. The strains were cultivated in liquid VMM supplemented with 1% starch and cultivated at 30 °C for 5 days. Soluble starch was used as a reaction substrate for RSDE activity assays. Fig. S3. RSDE activity assay of WT, ΔHepA and ΔPGA3 strain. The strains were cultivated in liquid VMM supplemented with 1% starch and cultivated at 30 °C for 5 days. Raw rice starch was used as a reaction substrate for RSDE activity assays. Fig. S4. Extracellular proteins of 114-2 on starch analysed by SDS-PAGE. 32 μL culture supernatant of 114-2 strain was loaded after cultivating in 1% starch medium for 72 h, respectively. Lane M was the molecular weight marker, lanes 1 and 2 represent two independent cultivations for 114-2 strain. Fig. S5. SDS-PAGE analysis of the purified raw starch digesting enzyme Amy15A. Lane M indicates protein molecular weight marker; lane 1 indicates the purified Amy15A. Fig. S6. Effects of pH and temperature on enzymatic activity and the stability of the RSDE Amy15A. (A) The effect of pH on enzyme activity. The enzyme activity was assayed in a citrate–phosphate buffer (pH 3.0–7.0) at 37 °C. (B) The pH stability of Amy15A was measured by pre-incubating the enzyme in various buffers for 24 h at 4 °C, and the residual enzyme activity was determined using the standard method. (C) The influence of temperature on enzyme activity. The enzyme activity was determined between 35 °C and 80 °C under optimum pH condition. (D) The influence of temperature on enzyme stability. Temperature stability was determined by the standard method after pre-incubating the enzyme at pH 4.5 between 30 °C and 75 °C for 1 h. Data given are mean ± standard deviation from three replicates. The results are from a representative experiment, and similar results were obtained in two other independent experiments. Table S1. Quality control statistics. Table S2. Primers used in this study.