Transcriptional analysis of selected cellulose-acting enzymes encoding genes of the white-rot fungus Dichomitus squalens on spruce wood and microcrystalline cellulose Johanna Rytioja, Kristiina Hildén, Annele Hatakka, Miia R. Mäkelä ⇑ Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Viikki Biocenter 1, Finland article info Article history: Available online xxxx Keywords: White-rot fungus Dichomitus squalens Cellobiohydrolase Cellobiose dehydrogenase Lytic polysaccharide monooxygenase abstract The recent discovery of oxidative cellulose degradation enhancing enzymes has considerably changed the traditional concept of hydrolytic cellulose degradation. The relative expression levels of ten cellulose-act- ing enzyme encoding genes of the white-rot fungus Dichomitus squalens were studied on solid-state spruce wood and in microcrystalline Avicel cellulose cultures. From the cellobiohydrolase encoding genes, cel7c was detected at the highest level and showed constitutive expression whereas variable tran- script levels were detected for cel7a, cel7b and cel6 in the course of four-week spruce cultivation. The cel- lulolytic enzyme activities detected in the liquid cultures were consistent with the transcript levels. Interestingly, the selected lytic polysaccharide monooxygenase (LPMO) encoding genes were expressed in both cultures, but showed different transcription patterns on wood compared to those in submerged microcrystalline cellulose cultures. On spruce wood, higher transcript levels were detected for the lpmos carrying cellulose binding module (CBM) than for the lpmos without CBMs. In both cultures, the expres- sion levels of the lpmo genes were generally higher than the levels of cellobiose dehydrogenase (CDH) encoding genes. Based on the results of this work, the oxidative cellulose cleaving enzymes of D. squalens have essential role in cellulose degrading machinery of the fungus. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Plant biomass is an abundant renewable carbon source with a huge biotechnological potential as a raw material in several indus- tries. For the efficient and feasible use of lignocellulosic plant bio- mass, depolymerisation of the plant cell wall polymeric components, including the polysaccharides cellulose and hemicel- luloses, and the aromatic lignin, is needed. Fungi are the most effective plant cell wall degrading organisms producing diverse set of synergistically acting extracellular en- zymes (Eriksson et al., 1990; Lundell et al., 2010; Hatakka and Hammel, 2011). The main catalytic activities of lignocellulose degrading fungi for cellulose decomposition are various hydrolytic enzymes such as endoglucanases (EG, EC 3.2.1.4) and exoglucanas- es (cellobiohydrolase I and II; CBHI and CBHII; EC 3.2.1.176 and EC 3.2.1.91, respectively), and b-glucosidases (BGL, EC 3.2.1.21,). They belong to different glycoside hydrolase families (GH) classified in the Carbohydrate-Active enZyme database (CAZy, www.cazy.org) (Cantarel et al., 2009). In addition, oxidative enzymes of Auxiliary Activities (AA) group (Levasseur et al., 2013) such as cellobiose dehydrogenase (CDH, AA3, AA8; EC 1.1.99.18) and lytic polysac- charide monooxygenases (LPMOs, AA9) have been shown to en- hance the degradation of cellulose in combination with cellulases (Harris et al., 2010; Quinlan et al., 2011). Furthermore, basidiomy- cetous white-rot fungi produce lignin-modifying oxidoreductases, such as class II haem-peroxidases and laccases, endowing them the unique ability to depolymerize lignin, which is the most recal- citrant part of plant cell wall (Lundell et al., 2010). So far, most of the studies on cellulose degrading fungal enzymes have been concentrated on ascomycetous fungi. The accumulating genome data, however, reveals that also basidiomy- cetous white-rot fungi have a complete set of plant polysaccharide decomposing enzymes (Floudas et al., 2012). Since the focus of white-rot fungal research has mostly been on the oxidoreductive lignin-modifying enzymes, basidiomycetous carbohydrate active enzymes (CAZymes) have been studied to a lesser extent and thus analyses both on transcriptional and biochemical level are still scarce (Baldrian and Valášková, 2008). Recently, ascomycetous LPMOs have been reported to enhance cellulose degradation by opening of the cellulose chain oxidatively thus suggesting an important role of these enzymes together with classical cellulases (Quinlan et al., 2011). Moreover, the action of LPMO is strongly potentiated when combined with CDH (Langston et al., 2011; 1087-1845/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2013.12.008 ⇑ Corresponding author. Address: Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, Viikki Biocenter 1, P.O.B. 56, FIN-00014 Helsinki, Finland. Fax: +358 019159322. E-mail address: miia.r.makela@helsinki.fi (M.R. Mäkelä). Fungal Genetics and Biology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi Please cite this article in press as: Rytioja, J., et al. Transcriptional analysis of selected cellulose-acting enzymes encoding genes of the white-rot fungus Dichomitus squalens on spruce wood and microcrystalline cellulose. Fungal Genet. Biol. (2014), http://dx.doi.org/10.1016/j.fgb.2013.12.008