Figure 4
Construction of the PfEgl16 expression cassette and its overexpression in PfMig188. (a) Schematic diagram showing the assembly of the PfEgl16 cassette from the P. funiculosum NCIM1228 genome. (b) Transformants of pOAO9 after AMTM transformation in PfMig188. Transformants were selected on 100 µg/ml hygromycin. (c) Determination of PfEgl16 integration in the genome of PfMig188 transformants by PCR using the primers PgpdA-F and TrpC-R. The expected size is 5 kbp, corresponding to a fragment spanning the region of gpdA promoter and TrpC on the expression cassette. Lane M is DNA molecular mass marker; lane (1) is the pOAO9 plasmid which served as a positive control while lanes 2–5 are the transformants of PfOAO7. Lane (6) is the gDNA of PfMig188 which was the negative control. (d) The transcriptional expression of endoglucanase in NCIM1228, PfMig188, and all the three positive strains of PfOAO7 was measured by quantitative real-time PCR after growing the strains for 60 h in the presence of Avicel and SCB. The expression levels were normalized to those in NCIM1228 and plotted. (e) The lichenase, laminarase and FPase activities in the fermentation broth of PfMig188 and three transformants of PfOAO7. (f) Saccharification performance of PfOAO7 secretomes on SCB. Total sugar released from hydrolysis of SCB by secretomes of PfMig188 and three transformants of PfOAO7. (g) Percentage holocellulose conversion measured at 72 h saccharification period. ** shows the significant difference at α = 0.05 using Tukey’s multiple comparison test.
