Table 1.
Oligonucleotides used in this study.
Figure 1.
Strategy for producing a mnp4 over-expressing P. ostreatus
strain. (A) Map of the VP4 (encoded by mnp4) over-expression and carboxin-resistance-conferring (CbxR) cassette, TMS12. Small arrows indicate the location of primers (Table 1) used for construction and detection of the construct. (B) PCR screening of P. ostreatus genomic DNA targeting TMS12, using primers R4 and btubTR (Figure 1A, Table 1). The arrow indicates the expected 6866 bp amplicon. M – DNA size marker (GeneRuler DNA Ladder Mix, Fermentas); PC9– wild-type; 1, 13, 51, 61, 68, 69, 70, 76– carboxin-resistant transformant strains.
Figure 2.
In-vivo decolorization of Orange II, Reactive Black 5 and Amaranth, and MnO2 precipitation.
P. ostreatus wild-type strain (PC9) and mnp4 over-expressing strain (OEmnp4) were grown for 10 days on solid GP medium containing 27 µM Mn2+ supplemented with 100 mg/l of the corresponding dye. For Mn2+ oxidation assay, the fungi were grown on solid GP medium containing 108 µM Mn2+.
Figure 3.
Expression of the P. ostreatus manganese peroxidase genes in PC9 and OEmnp4 strains.
Total RNA was extracted from P. ostreatus wild-type strain (PC9) and mnp4 over-expressing strain (OEmnp4) cultures at (A) 4 days, (B) 7 days and (C) 10 days of incubation. The fungi were grown in GP medium containing 27 µM Mn2+ supplemented with 100 mg/l Orange II. Data represent the average of three biological replicates. Bars denote SD.
Figure 4.
Time-course assay of peroxidase activity.
Mn2+-dependent (+Mn2+) and Mn2+-independent (−Mn2+) peroxidase activities of P. ostreatus wild-type strain (PC9) and mnp4 over-expressing strain (OEmnp4). The fungi were grown in GP medium containing 27 µM Mn2+ supplemented with 100 mg/l Orange II for 10 days. Data represent the average of three biological replicates. Bars denote SD.
Figure 5.
Time-course assay of in-vivo azo dyes decolorization.
P. ostreatus wild-type strain (PC9) and mnp4 over-expressing strain (OEmnp4) were grown for 10 days in GP medium containing 27 µM Mn2+ supplemented with 100 mg/l of (A) Orange II, (B) Reactive Black 5 and (C) Amaranth. Data represent the average of three biological replicates. Bars denote SD. The chemical structure of each dye is shown.
Figure 6.
Gene-expression, lignin degradation and lignocellulose digestibility under solid-state fermentation.
(A) mnp4 expression level, (B) [14C]-lignin mineralization (percentage of 14CO2 emitted from the total initial radiolabelled [14C]-lignin is presented), (C) in-vitro dry matter digestibility (IVDMD) and (D) neutral detergent fiber digestibility (NDFD) by P. ostreatus. The wild-type strain (PC9) and mnp4 over-expressing strain (OEmnp4) were incubated for 14 days under solid-state fermentation conditions, using a lignocellulosic substrate of cotton stalks containing either 8.0 (basal Mn2+ concentration) or 23.3 (concentration in Mn2+ supplemented stalks) mg/kg dry matter (DM) of Mn2+. Non-inoculated substrate was used as a control. Data represent the average of three biological replicates. Bars denote SD. Statistical analysis was performed by analysis of variance with Tukey-Kramer HSD test (significance accepted at P<0.05).