Figure 3

Construction of the PfXyn5 expression cassette and its overexpression in PfMig1⁸⁸. (a) Schematic diagram showing the assembly of the PfXyn5 cassette from the P. funiculosum NCIM1228 genome. (b) Transformants of pOAO7 after AMTM transformation in PfMig1⁸⁸. Transformants were selected on 100 µg/ml hygromycin. (c) Determination of PfXyn5 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 pOAO7 plasmid which served as a positive control while lane (2) is the gDNA of PfMig1⁸⁸ which was the negative control. Lanes 3–7 are the transformants of PfOAO6. (d) The transcriptional expression of xylanase in NCIM1228, PfMig1⁸⁸, and all the three positive strains of PfOAO6 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 xylanase, β-xylosidase and FPase activities in the fermentation broth of PfMig1⁸⁸ and three transformants of PfOAO6. (f) Total sugar released from hydrolysis of SCB by secretomes of PfMig1⁸⁸ and three transformants of PfOAO6. (g) Saccharification performance of PfOAO6 secretomes on SCB. Total xylose yield was estimated after 72 h hydrolysis and the hemicellulose conversion were determined and plotted (h) Percentage holocellulose conversion measured at 72 h saccharification period. ** shows the significant difference at α = 0.05 using Tukey’s multiple comparison test.