Microorganism growth conditions and RNA extraction
The brown rot basidiomycete Serpula lacrymans S7 strain maintained within the culture collection of Warwick HRI (School of Life Sciences) was grown in the dark on 2% malt extract agar (MEA) plate at 20 °C for 3–4 weeks. This was used to inoculate 10 g of autoclaved wheat straw and cultured under solid-state fermentation (SSF) for 41 days. Samples were taken every 3 days and RNA extracted from 100 mg using a fast RNA Pro-Soil Direct Kit (MP Biomedicals). The purified RNA was quantified using spectrophotometer NanoDropTM ND-1000 and evaluated using the RNA 6000 Nano assay Kit (Agilent 2100 Bioanalyser). First strand cDNA was synthesized using the ThermoScript™ RT-PCR system (Invitrogen) following the manufacturers guidelines.
The quantification of genes encoding iron reductase from Serpula lacrymans
The pattern of expression of the iron-reductase genes (IR1 and IR2) in wheat straw SSF cultures was determined through extraction of RNA and the use of QRT-PCR. RT-PCR amplification was performed in a 20 µl total reaction volume, using 1 µl of cDNA solution as template, 10 µl of Lightcycler 480 SYBR Green master mix (Roche Diagnostic Ltd), and 0.5 µM of each primer (Table 1). The amplification program consisted of an initial cycle (95 °C for 1 min), followed by 45 cycles of denaturation at 95 °C for 30 s, 60–62 °C for 1 min (temperature specific for each primer pairs) then extension at 72 °C for 30 s. A melting curve was obtained by performing 45 cycles at 95 °C for 1 min, 40 °C for 1 min, and 60 °C for 30 s and followed by 72 °C for 5 min. All reactions were done in triplicate in 384-well microtiter plates and a no-template control was included for each primer pair. Quantification of gene expression was determined relative to a standard curve for each target gene. Transcription of the iron-reductase genes (IR1 and IR2) was normalized and quantified by extrapolation to standard curves generated by plotting the logarithm of fluorescence versus cycle number for a serial dilution of cDNA template and to the housekeeping gene actin. The normalization of target gene and internal standard was carried out by correction with the endogenous control results .
Cloning of iron reductase from the brown rot fungus Serpula lacrymans
To clone both genes (IR1 and IR2), primers were designed for the full length coding sequence (CDS), and the genes amplified from cDNA prepared from total RNA extracted from 41 days culture of Serpula lacrymans grown on wheat straw (Table 2). These were initially cloned using the TA cloning kit (Invitrogen) following the manufacture’s protocol. Sequence verification against the S. lacrymans genome was performed following Sanger sequencing using the ABI BigDye terminator V.1.1/3.1 seq Kit and alignment to the relevant accession number carried out (IR1 18816815; IR2 18813585). To optimize protein recovery in E. coli, the signal peptide was removed using an oligonucleotide primer designed to commence 60 base pairs from the start methionine. The amplified product included the appropriate adaptors for cloning into the GatewayTM system. Cloning into this system was then carried out using the standard protocol. The plasmid clone was then expressed in BL21 cells (www.invitrogen.com).
Production of recombinant protein
A transformed E. coli colony was inoculated into 10 ml LB medium containing the selective antibiotics (50 µg/ml carbenicellin and 34 µg/ml chloramphenicol) and grown overnight at 37 °C with shaking 220 rpm. 2.5 ml overnight culture was inoculated into 50 ml of prewarmed LB media (with antibiotics) on a shaking incubator (220 rpm for approximately 1.5 h), until the OD600 was 0.5–0.7 achieved. The transformants were induced using 0.4 mM of isopropyl-β-d-thiogalactopiranoside (IPTG) and incubated at 30 °C for an additional 5–6 h. The cells were harvested by centrifugation at 5000 rpm (20 min) for the SDS-PAGE analysis, resuspended in 1 ml lysis buffer containing 50 mM Tris–HCl pH 8; 1 mM EDTA pH 8,0; 1 mM tris2 carboxyethyl-phosphine (TCEP); 1 mM phenyl methylsulfonyl-fluoride (PMSF); 200 mM NaCl; deionized water (dH2O) and the cell pellet was frozen under liquid nitrogen and thawed in cold water. The cells were then sonicated for 6 × 10 s with 10 s pauses at 200–300 W and the lysate was centrifuged at 5000×g at 4 °C for 20 min. The soluble and insoluble fractions were tested for the presence of recombinant protein using a 12% SDS-PAGE gel.
Purification of recombinant iron reductases
500 ml of LB culture was prepared for the purification of recombinant protein (IR1 and IR2) as described above. All protein purification was undertaken at 4 °C. The culture was centrifuged at 5000×g for 20 min at 4 °C and the pellet resuspended in lysis buffer containing 50 mM Tris–HCl pH 8; 1 mM EDTA pH 8.0; 1 mM tris2 carboxyethyl-phosphine (TCEP); 1 mM phenyl methylsulfonyl-fluoride (PMSF); and 200 mM NaCl. The cells were lysed using a combination of freeze–thaw and sonication method and then centrifuged at 13,000×g for 10 min at 4 °C and the supernatant was collected for the purification. The soluble fractions of recombinant protein (IR1 and IR2) were purified using Glutathione Sepharose 4B beads (GE Healthcare, UK) according to the manufacturer’s instructions. The crude cell extract was passed through a column pre-equilibrated with binding buffer PBS pH 7.5 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4). The columns were prepared according to the manual (Glutathione Sepharose 4B, 52-2303-00 AK). After extensive washing using binding buffer, the GST fusion proteins were eluted with elution buffer (50 mM Tris–HCl, 20 mM reduced glutathione, pH 8.0). Concentration of the recombinant protein was determined using the Bradford RC–DC protein assay (from Bio-Rad) using 1 mgml-1 BSA as the standard and absorption measured at 750 nm.
Western blotting was carried out using the standard protocols . The IR1 and IR2 recombinant proteins were transferred onto nitrocellulose membrane for 1.5 h and treated for 2–3 h at room temperature using 5% skimmed milk as the blocking agent. The membrane was then incubated overnight at 4 °C with primary antibody (monoclonal anti-GST antibody (SIGMA G-1160) at a dilution of 1:2000. The membrane was washed using three washes of PBST (Phosphate Buffer Saline with Tween 20) for 5–10 min each, then incubated with secondary antibody (Sigma-A4416 anti-GST antibody-peroxidase conjugate produced in mouse diluted in 1:10,000) for 2 h at room temperature. The blot was washed three-to-five times for 15 min using buffer PBST and then incubated with ECL (Enhance chemiluminesence) Western-blotting detection reagents (according to manufacture’s instructions from Amersham) for 5 min at room temperature before imaging.
Determination of total soluble phenols released following SSF culture with the recombinant enzymes
Phenols were measured colourimetrically from wheat straw samples following inoculation with iron-reductase recombinant enzymes. This was carried out using the Folin–Ciocalteau method . The concentration of phenols (per gram of substrate dry weight) was determined by reference to a standard curve prepared using gallic acid as the standard.
A ferrozine-based colorimetric assay was used to detect release of Fe2+ by reduction of Fe3+. A modified method combining the approaches of  and  was developed to determine the reduction of iron using Ferrozine reagent [3-(2-pyridyl)-5,6-bis-(4-phenylsulfonic acid)-1,2,triazine] (Sigma). Using partially purified recombinant proteins (IR1 and IR2), the experiment was conducted in 96-well micro titer plates. A glutathione S-transferase green florescent protein (GFP) plasmid was also used expressed to provide a control protein for the experiment. 50 µl of crude extract/supernatant from soluble fusion protein of IR1 and IR2 cultures were combined with 0.1 mM FeCl3, 1 M acetate buffer pH 4.6, in the presence and absence of 50 µM 2,3 dihydroxybenzoic acid (DHBA). After 10 min incubation, 10 µM ferrozine reagent was added to the reaction. The absorbance was then measured at 550 nm using a spectrophotometer TECAN-Genious plate reader. Two readings were taken at 1 and 30 min.
Nitrated lignin assay
This method was modified from  through the addition of Fe3+ as a substrate. 110 µl diluted nitrated organosolv lignin (prepared according to Ahmad et al.  was added to each well of a 96 well plate, followed by 40 µl 0.1 mM FeCl3, 10 µl of 50 µM 2,3-dihydroxybenzoic acid (2,3-DHBA), 30 µl recombinant protein of IR1 or IR2, and 10 µl 4 mM H2O2. The assay was monitored at 430 nm every minute for 20 min and carried out in quadruplicate. The whole plate was repeated as above but with 2,3-DHBA and/or H2O2 being replaced by deionized H2O. The bacterial lignin degrading enzyme Rhodococcus jostii DypB  and an E. coli GFP construct was used as positive and negative controls, respectively.
Cellobiose dehydrogenase (CDH) enzyme assay
The CDH assay was slightly modified from Baminger  and . The assay based on reduction of benzoquinone or dichlorophenolindophenol in the presence of cellobiose or lactose is recognized as a means of detecting CDH activity [30,31,32]. Recombinant enzyme activity was determined at room temperature using 0.1 M 2,6-dichlorophenol indophenol (DCPIP; Sigma-Aldrich) as an electron acceptor in two different buffers 50 mM sodium acetate buffer (pH 5) with cellobiose as the substrate. The reaction mixture (in a total volume 200 µl) containing: 100 µl of recombinant protein of IR1 or IR2; 40 µl of 0.6 mM cellobiose; 10 µl of 0.1 mM Fe3+ (Ferric chloride); 10 µl 2,3 dihydroxyl-benzoic acid 2,3 DHBA); 10 µl 4 mM H2O2; and 10 µl 0.5 mM DPCIP. The CDH activity was measured by following a decrease in the absorbance of the electron acceptor DCPIP. The decrease in absorption of DCPIP was monitored using kinetic spectrophotometry (TECAN GENios) at 540 nm every minute from the first 60 s until 30 min. The whole assay was repeated without cellobiose, 2,3-DHBA, and recombinant proteins. All readings were taken in quadruplicate.
Determination of the IR enzymes’ ability to release sugars from powdered wheat straw and cellulose (Avicel)
The total amount of reducing sugars released by S. lacrymans-derived iron reductases was quantified using the 3,5 dinitrosalicylic acid (DNS) method . 1 ml of partially purified iron-reductase (crude extract) sample (IR1 and IR2) was prepared according to the method above. This was mixed with the two substrates (30 mg/ml) of Avicel-PH 101 (Sigma–Aldrich) and wheat straw powder. Samples were incubated for 24 h at 50 °C, pH 5.5 then allowed to cool to room temperature. An aliquot of the crude extract (250 μl) was taken and centrifuged for 1 min at 13,000 rpm. The experiment was carried out in the presence and absence of iron (0.1 mM Fe3+) and 4 mM H2O2 to test if a chelator-mediated Fenton system had a role in the release of sugar. 50 μM 2,3-DHBA was used as a positive control. Cellulase (1,4 β-d-glucan-,4 glucanohydrolase; Sigma–Aldrich C1184) at the same estimated concentration of the iron-reductase enzymes (0.312 mg/ml) was used as a positive control. The amount of reducing sugar released was measured at 540 nm in a plate reader (TECAN GENios) using four replicate and buffer without recombinant protein include as negative controls. The absorbances refer to the amount of total reducing sugars released as calculated from the glucose standard curve.
The results were analyzed using analysis variance (ANOVA) in Genstat version 11 and the error bars represent LSD (P < 0.05).