Figure 5

Vector construction for the simultaneous overexpression of PfXyn5 and PfEgl16 in PfMig1⁸⁸. (a) Schematic diagram showing the construction cassette for the dual overexpression of the Xyn5 and Egl16 genes from P. funiculosum NCIM1228. Abbreviations: Pro, promoter; SP, signal peptide; Ter, terminator. (b) Transformants of pOAO10 after AMTM transformation in PfMig1⁸⁸. Transformants were selected on 100 µg/ml hygromycin. (c) Determination of integration of the dual cassette (Xyn5/Egl16) in the genome of PfMig1⁸⁸ transformants by PCR using the primers PgpdA-F and TrpC-R. The expected size is 7 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 pOAO10 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 PfOAO8. (d) The transcriptional expression of endoglucanase in NCIM1228, PfMig1⁸⁸, and all the five positive strains of PfOAO8 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, lichenase, laminarase and FPase activities in the fermentation broth of PfMig1⁸⁸ and the five transformants of PfOAO8. (f) Saccharification performance of PfOAO8 secretomes on SCB. Total sugar released from hydrolysis of SCB by secretomes of PfMig1⁸⁸ and three transformants of PfOAO8. (g) Percentage holocellulose conversion measured at 72 h saccharification period. ** shows the significant difference at α = 0.05 using Tukey’s multiple comparison test.