Biochemical characterization of Serpula lacrymans iron-reductase enzymes in lignocellulose breakdown
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Putative iron-reductase (IR) genes from Serpula lacrymans with similarity to the conserved iron-binding domains of cellobiose dehydrogenase (CDH) enzymes have been identified. These genes were cloned and expressed to functionally characterize their activity and role in the decomposition of lignocellulose. The results show that IR1 and IR2 recombinant enzymes have the ability to depolymerize both lignin and cellulose, are capable of the reduction of ferric iron to the ferrous form, and are capable of the degradation of nitrated lignin. Expression of these genes during wheat straw solid-state fermentation was shown to correlate with the release of compounds associated with lignin decomposition. The results suggest that both IR enzymes mediate a non-enzymatic depolymerisation of lignocellulose and highlight the potential of chelator-mediated Fenton systems in the industrial pre-treatment of biomass.
KeywordsSerpula lacrymans Lignocellulose Recombinant enzymes Iron reductase Fenton chemistry
Least significant difference
Malt extract agar
Green fluorescent protein
Biorefineries capable of the production of renewable chemicals from a range of suitable biomass feedstocks have been proposed as the basis for a biobased economy [1, 2]. Lignocellulosic biomass contains three major components: cellulose, hemicellulose, and lignin. Lignocellulosic biomass pre-treatment steps are required to make subsequent enzymatic digestion of each component efficient. Currently, such pre-treatments are expensive due to the cost of the energy or chemicals used [3, 4]. Developing a cost-effective method remains a significant challenge in the development of economic biorefineries.
In nature, fungi are the principle agents of lignocellulose degradation utilizing both enzymatic and non-enzymatic processes to convert biomass. White rot fungi produce ligninolytic enzymes such as class II peroxidases and laccases, which drive lignin depolymerisation and metabolism . Brown rot fungi, derived from a white rot ancestry, have reduced enzymatic capacity to mineralize lignin and typically target the depolymerisation of hemicellulose and cellulose [6, 7, 8]. Studies into the mechanism used by different species of brown rot fungi and the subsequent effect on woody substrates have increased our understanding of the decay process [9, 10, 11]. Depolymerisation of lignocellulose is thought to be largely driven by a non-enzymatic chelator-mediated Fenton (CMF) system  that in the presence of Fe2+ generates reactive hydroxyl radicals [12, 13, 14]. Fe3+ reduction to Fe2+ in white rot fungi has been linked to cellobiose dehydrogenase (CDH) activity, extracellular hemoflavoenzymes produced by various lignocellulose-degrading fungi [15, 16, 17]. Fungus-derived oxalic acid, quinones, and others phenolic compounds produced through the degradation of lignin have also been implicated in the reduction of iron . For example, 2,3 dihydroxybenzoic acid (DHBA) was reported as the chelating agent  released by the brown rot fungi Gloeophyllum trabeum  which is able to promote sustained iron reduction and generate reactive oxygen species (ROS). This suggests that the CMF reaction could become a self-sustaining process; however, it is unclear how the process is initiated.
Cellobiohydrolases (Glycoside hydrolases 6 and 7) associated with white rot endocelluloytic activity through the cleavage of β-1, 4 glycosidic bonds are absent or reduced in brown rots . The Boletales brown rots Serpula lacrymans and Coniophora puteana retained one and two copies of GH6 cellobiohydrolase, respectively . However, transcriptomic and proteomic analysis failed to detect the expression of this gene in S. lacrymans wood cultures [7, 20]. While the role of the S. lacrymans GH6 remains uncertain, a role for a chelator-mediated Fenton system in the depolymerisation of cellulose has been proposed for brown rot species . Cellulose-targeted iron reduction, combined with substrate induction of iron-reducing phenolate biosynthesis, might explain the particular ability of brown rot fungi in the Boletales, such as the dry rot fungus S. lacrymans, to degrade crystalline cellulose without the presence of lignin . Unusually, for the brown rots, two cellobiose dehydrogenase (CDH) genes were identified in S. lacrymans, but neither was expressed when the fungus grew on a wood substrate . Two putative genes (number IR1 452187; IR2 417465) with similarity to the CDH iron-reductase domain were identified in the S. lacrymans genome . These iron-reductase genes (IR1 and IR2) were postulated to play a role in lignocellulose decomposition, since IR1 contained cellulose-binding module-1 (CBM1) domain and was up regulated 122-fold when grown on wood compared to glucose medium .
The uncertainty in how S. lacrymans converts lignocellulose led us to investigate the iron-reductase genes to determine if they could be involved in the breakdown of lignocellulose and microcrystalline cellulose and, if so, their function. Recombinant S. lacrymans IR1 and IR2 proteins were produced using gateway plasmids cloned in E. coli. The function of these proteins was then tested for their iron reduction capabilities and the ability to release sugars and phenolic compounds through a putative chelator-mediated Fenton attack on lignocellulose. The results help to explain the particular ability of this brown rot fungus to degrade lignocellulose, and hence their potential as tools for the pre-treatment of biomass.
Materials and methods
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
Primer sequences for IR1 and IR2 genes used for QRT-PCR
Primer sequence 5′–3′
Primer sequence 5′–3′
Iron reductase (IR1)
Iron reductase (IR2)
Cloning of iron reductase from the brown rot fungus Serpula lacrymans
Primer sequences for IR1 and IR2 genes as used for cloning into the gateway system
Primer sequence 5′–3′
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).
Results and discussion
The quantification of genes encoding iron reductase from Serpula lacrymans and the relationship to phenols released
The expression and purification of the recombinant protein in E. coli (BL21)
Determination of the function of recombinant iron reductases (IR1 and IR2)
The ability of iron reductases to degrade nitrated lignin
In the absence of iron (Fe3+) and 2,3-DHBA recombinant, IR1 and IR2 had no significant impact on the absorbance after 20 min (data not shown), indicating no decomposition of nitrated lignin. However, in the presence of Fe3+, a significant increase (P < 0.05, ANOVA using LSD) in the release of phenols was found using recombinant IR1 following 20 min incubation. IR2 on the other hand, only showed a significant difference when Fe3+ was present together with 2,3-DHBA. Without the 2,3-DHBA, there was no significant difference even after 20 min (Fig. 4). When 2,3-DHBA was added, it appeared to act synergistically with the IR genes, but showed no effect by itself (Fig. 4). In the absence of hydrogen peroxide (H2O2), IR1 and IR2 did not release phenolic products. The fact that H2O2 was required to support the idea that lignin degradation might occur via the Fenton reaction. This is supported by other research that implies the generation of hydroxyl radicals and/or superoxide radicals are required to break down lignocellulose and that the presence of iron, H2O2, and chelating agent also plays a role [14, 35, 36, 37, 38, 39]. Figures showing the production of quinone (2,5 dimethoxy-1,4-benzoquinone) and oxalic acid during the culture of the fungus on straw support the evidence that these genes act as strong chelators with a function in non-enzymatic lignocellulose degradation (Supplemental materials).
Release of reducing sugars from Avicel and wheat straw powder
While the data demonstrate that the potential of both IR proteins facilitates reactions that ultimately depolymerize the polymers found in lignocellulose, full elucidation of their role requires further study. The gene expression data clearly show high and differential expression of both IR genes during S. lacrymans growth on biomass, where the CMB1-containing IR1 has greater expression, as decay proceeds and cellulose becomes exposed. This does not mean that the enzyme is directly involved in cellulose depolymerization and instead might provide a greater localization of chelator-mediated cycling close to the substrate as cellulose binding becomes available (NB there is no known lignin-binding module). S. lacrymans has retained a GH6 cellobiohydrolase gene and exocellulases from the GH5 family are expanded and expressed during growth on plant biomass , suggesting that enzymatic cellulose depolymerization is present in the fungus. It is important to note that core iron-reductase activity of the IR proteins emerged from an existing cellobiose dehydrogenase enzyme, and this process involved further gene duplication event to create IR1 and IR2, whereby IR1 gained a CBM1, even though S. lacrymans showed lower CBM1 complement (23 total) compared with white rot species, e.g., 48 in Phanaerochaete chrysosporium . Such events are unlikely to have occurred for a functionally redundant gene. Taken collectively, the data indicate that the IR proteins are utilized by S. lacrymans during lignocellulose decomposition to drive iron cycling most likely linked to a chelator-mediated Fenton mechanism targeted towards the disruption of lignin. Moreover, there is potential for enzymes such as IR1 and IR2 to drive cell-free chemically mediated biomass processing in a biorefinery setting.
We would like to thank to Directorate General of Higher Education (DGHE)—Ministry of National Education and the Universitas Brawijaya Indonesia for providing a studentship and EPSRC for funding the research (IMRC69). Volkan Cevik provided advise on the use of the Gateway cloning system.
Compliance with ethical standards
Conflict of interest
The author(s) declare that they have no conflict of interest.
- 5.Hatakka A (2001) Biodegradation of Lignin. In: Hofrichter M, Steinbüchel A (eds) Biopolymers. Biology, chemistry, biotechnology, applications. Vol 1. Lignin, Humic Substances and Coal. Wiley-VCH, Weinheim, pp 129–145Google Scholar
- 6.Curling S, Clausen CA, Winandy JE (2001) The effect of hemicellulose degradation on the mechanical properties of wood during brown rot decay. In: Int Res Gr Wood Prot 32nd Annu Meet IRG/WP 01:1–11Google Scholar
- 12.Tamaru Y, Yoshida M, Eltis LD, Goodell B (2019) Multiple iron reduction by methoxylated phenolic lignin structures and the generation of reactive oxygen species by lignocellulose surfaces. Int J Biol Macromol 128:340–346. https://doi.org/10.1016/j.ijbiomac.2019.01.149 CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Goodell B, Jellison J, Liu J et al (1997) Low molecular weight chelators and phenolic compounds isolated from wood decay fungi and their role in the fungal biodegradation of wood. This is paper 2084 of the Maine Agricultural and Forest Experiment Station. 1. J Biotechnol 53:133–162. https://doi.org/10.1016/s0168-1656(97)01681-7 CrossRefGoogle Scholar
- 22.Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16:144–158Google Scholar
- 23.Sambrook J, Russell DW (2001) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- 24.Arantes V, Qian Y, Kelley SS et al (2009) Biomimetic oxidative treatment of spruce wood studied by pyrolysis-molecular beam mass spectrometry coupled with multivariate analysis and 13C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood. J Biol Inorg Chem 14:1253–1263. https://doi.org/10.1007/s00775-009-0569-6 CrossRefPubMedGoogle Scholar
- 32.Henriksson G, Pettersson G, Johansson G et al (1991) Cellobiose oxidase from Phanerochaete chrysosporium can be cleaved by papain into two domains. Eur J Biochem 196:101–106. https://doi.org/10.1111/j.1432-1033.1991.tb15791.x CrossRefPubMedGoogle Scholar
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