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甘肃农业大学：食品科学与工程学院（文献讲义）The potential of cottonseed hull as biorefinery substrate after biopretreatment by Pleurotus ostreatus and the mechanism analysis based on comparative proteomics

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Industrial Crops Products 130(2019)151-161 Contents lists available at ScienceDirect ROP Industrial Crops Products ELSEVIER journal homepage:www.elsevier.com/locate/indcrop The potential of cottonseed hull as biorefinery substrate after biopretreatment by Pleurotus ostreatus and the mechanism analysis based on comparative proteomics Qiuyun Xiao2.b.1,Hongbo Yu,Jialong Zhang",Fei Li,Chengyun Lib,Xiaoyu Zhang", Fuying Ma.* o,Key Laboratory of Molecuar Biophysics of MOE,College of Life Science and Technology Huazhong University of Science and Technology Sr mm ARTICLE INFO ABSTRACT Keywords ntaprotcome ular m in sawdust, nseed hul cod the Agricultural and forestry residues highest saccharification rate.297,333,and 312 soluble proteins were identified in hardwood sawdust,cot- tonseed hull and corncob,respectively.P.ostreatus mobilized the corresponding antioxidant and carbon meta- bolism pathways and produced more abundant ligninolytic enzymes,especially class II peroxidases,to accom modate lgni enzymes and oyster mushroom cultivation. 1.Introduction t inclear how P.os Lignocellulosic biomass mainly composed of hemicellulose,cellu various feedstocks ith st ructure and co tion dif atus adapts to and li in sid ed to he tial feedstock for The s 、of lig bio-based p and enzy s.Glycoside hvdrolase family 1 refinery of ost agricultural and forestry bi nd GH3 R-glve sidase of p.o ere m ng as a ce th pro mi le xidase 2 (VP2) cellulose.forms an are produced on str embedded and。 otected against chemical or enzymatic degradation et al.2016).The sec ted pro oteins of other fung are also affected (Himmel et al..2007:Kuhad et al..1997). hut the nedium The additio n of stalks to various media enha e Some white-rot fungi have the abilities of de n by co showing at no and Gurdal.2002).The idase P)and Dle a oxidase (MnP)in Phlebia significantly tus as vpical white s.is nd ed d as e(Makela et al.2013).The exr edible mushro of fungi to diffe variety of lignocellulosic biomass.such as hard/soft w a s of tes Ho further studies the mol echanisms un traw cottonseed hull and cor mcob,P.ostreatus is also sed to derlying substrate adaptability of p tus,are still lacking. .Corresponding author at:College of Life Science and Technology,Huazhong University of Science and Technology,Wuhan,430074,China. du.cn(F.Ma 2018 vevised form 16 December2018;Accepted 17 December201 A2eagcv

Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop The potential of cottonseed hull as biorefinery substrate after biopretreatment by Pleurotus ostreatus and the mechanism analysis based on comparative proteomics Qiuyun Xiaoa,b,1 , Hongbo Yua,1 , Jialong Zhanga , Fei Lia , Chengyun Lib , Xiaoyu Zhanga , Fuying Maa,⁎ a Department of Biotechnology, Key Laboratory of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China b Institute for Advanced Study, Shenzhen University, Shenzhen, 518060, China ARTICLE INFO Keywords: Secretome Intracellular proteome Pleurotus ostreatus Cottonseed hull Agricultural and forestry residues ABSTRACT Oyster mushrooms use different lignocellulosic substrates with different biological efficiency, whereas deep understanding of the molecular mechanism is lacking. The extracellular/intracellular proteomes, lignocellulosic composition were analyzed after 21-day cultivation of P. ostreatus in sawdust, cottonseed hull and corncob. Lignin and hemicellulose content of three substrates significantly decreased, and cottonseed hull showed the highest saccharification rate. 297, 333, and 312 soluble proteins were identified in hardwood sawdust, cottonseed hull and corncob, respectively. P. ostreatus mobilized the corresponding antioxidant and carbon metabolism pathways and produced more abundant ligninolytic enzymes, especially class II peroxidases, to accommodate lignin-rich substrate hardwood. Ligninolytic enzymes and carbohydrate oxidases showed higher expression levels in sawdust, while carbohydrate active enzymes were highly expressed in polysaccharide-rich cottonseed hull and corncob. These results suggested P. ostreatus adapts to different substrates through regulating extra/intracellular proteins expression, and cottonseed hull is a potential source for biorefinery and oyster mushroom cultivation. 1. Introduction Lignocellulosic biomass mainly composed of hemicellulose, cellulose, and lignin is considered to be a potential feedstock for producing bio-based products. Due to their recalcitrant structure, high efficient refinery of most agricultural and forestry biomasses are often problematic. Lignin acting as a cementing material, together with hemicellulose, forms an amorphous matrix in which the cellulose fibrils are embedded and protected against chemical or enzymatic degradation (Himmel et al., 2007; Kuhad et al., 1997). Some white-rot fungi have the abilities of degrading lignin component from lignocellulose selectively, showing great potential applications in lignocellulosic biorefinery. The oyster mushroom, Pleurotus ostreatus as a typical white rot fungus, is the second most cultivated edible mushroom worldwide (Sánchez, 2010). Due to growing on a variety of lignocellulosic biomass, such as hard/soft wood, all types of straw, cottonseed hull and corncob, P. ostreatus is also used to pretreat lignocellulose for biorefinery purpose (Taniguchi et al., 2005; Mustafa et al., 2016). However, it is still unclear how P. ostreatus adapts to various feedstocks with structure and composition differences. The structure and composition of lignocellulose can affect the secreted proteins and enzymes of P. ostreatus. Glycoside hydrolase family 1 (GH1) and GH3 β-glycosidase of P. ostreatus were more abundant on poplar and straw, respectively, and versatile peroxidase 2 (VP2) was overproduced on straw, while VP3 was only found on poplar (FernándezFueyo et al., 2016). The secreted proteins of other fungi are also affected by the medium. The addition of cotton stalks to various media enhanced the laccase production by Coriolus versicolor and Funalia trogii (Kahraman and Gurdal, 2002). The production of both lignin peroxidase (LiP) and manganese peroxidase (MnP) in Phlebia radiata was significantly promoted with wood as a carbon source (Mäkelä et al., 2013). The expression of these enzymes enhances the adaptability of fungi to different substrates. However, further studies on the molecular mechanisms underlying substrate adaptability of P. ostreatus, are still lacking. https://doi.org/10.1016/j.indcrop.2018.12.057 Received 18 September 2018; Received in revised form 16 December 2018; Accepted 17 December 2018 ⁎ Corresponding author at: College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China. E-mail address: mafuying@hust.edu.cn (F. Ma). 1 These authors contributed equally to this work. Industrial Crops & Products 130 (2019) 151–161 Available online 27 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved. T

In China, cottonseed hull and corncob, as well as other agricultural and forestry residues, are widely used to cultivate P. ostreatus. Some of the substrates provide the highest biological efficiency, while the molecular mechanism is unclear. In our previous study, lignin, xylan or CMC were individually supplemented to Kirk medium for evaluating adaptation of P. ostreatus to lignin, and results showed that intracellular antioxidant and anti-stress proteins are upregulated when P. ostreatus responding to lignin, and carbohydrate metabolism-related proteins are upregulated in xylan and CMC media (Xiao et al., 2017). Based on this work, cottonseed hull, corncob and sawdust, representing potential biorefinery feedstocks and cultivation materials of oyster mushrooms, were chosen to study the molecular mechanism on adaptation and preference of P. ostreatus in response to different lignocellulosic substrates. Weight loss-based composition analysis was applied to evaluate the changes in the chemical composition of three substrates after P. ostreatus cultivation. In addition, a two-dimensional gel electrophoresis (2-DE) and iBAQ label-free quantification method, followed by Mass Spectrometry (MS) were used to investigate intracellular proteomes and secretomes of P. ostreatus on three different substrates. The comparison of proteomes, together with lignocellulosic composition analysis will bring new insights into explaining the molecular mechanism on substrate preference and pretreatment effect of P. ostreatus. 2. Experimental 2.1. Estimation of fungal growth Pleurotus ostreatus BP2 was maintained on potato dextrose agar (PDA) slant at 4 °C and transferred onto fresh PDA plate before use. Three mycelial discs cut from the margins of 7-day fungal colony were transferred into 100 ml potato dextrose broth (PDB) and cultivated at 28 °C for 7 days at 150 r/min as inoculum. Ten grams (dry weight) of oak sawdust, cottonseed hull or corncob with less than 5 mm of fine particles was respectively added into 250 ml Erlenmeyer flask, then 20 ml distilled water was added and mixed. The flasks were sterilized for 30 min at 121 °C. After cooling down to room temperature, each flask was inoculated with 10 ml mycelial suspension, the non-inoculated as control, statically cultured at 28 °C. Mycelial extension rates on solid substrates were determined, according to the methods described (Philippoussis et al., 2001; Zervakis et al., 2001). Ten grams (dry weight) of each substrate mixed with 20 ml distilled water was uniformly filled in the graduated test tube (150 mm X 20 mm) with a total volume of 100 mL, and sterilized by autoclaving for 1 h at 121 °C. After cooling, a mycelial plug (6 mm diameter) from the margin of 4-day P. ostreatus grown on PDA was transferred onto the top of the substrate of each tube. All cultures were incubated at 28 °C. Mycelial extension rates were determined by measuring the mycelial growth front distal from the inoculum every 2 days and by averaging the growth measurements at four equidistant points around the circumference of each tube. Three biological replicates were carried out for each substrate. 2.2. Isolation and analysis of intracellular proteins The total mycelial proteins were extracted from 1 g freeze-drying mycelia of P. ostreatus by the TCA-acetone precipitation method as mentioned before (Xiao et al., 2017), then followed by 2-DE. Ready strip™ IPG strips (18 cm, 4–7 linear pH gradient, Bio-Rad) were rehydrated in 800 μg protein sample for 12 h and subjected to the first electrophoretic dimension. IPG were carried out in a Protean IEF-Cell (Bio-Rad), performing with a limiting current of 50 μA/strip following the program setting: (i) 250 v, rapid, 0.5 h. (ii) 1000 v, rapid, 0.5 h. (iii) 9000 v, liner, 4.5 h. (iv) 9000 v, rapid, 75,000 vh(v) 500 v, rapid, 1 h. The IPG strips were treated twice for at least 30 min with SDS equilibration buffer (6 mmol/L urea, 1.5 mmol/L Tris-Cl with pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS, 0.001% bromophenol blue). DTT (10 mg/ml) was added to the equilibration buffer in the first step, and iodoacetamide (25 mg/ml) was added in the second step. The second dimensional SDS-polyacrylamide electrophoresis (SDS-PAGE) was performed on acrylamide gel (12.5%, w/v) with SDS (2%, w/v) using a Protean II xi Cell system (Bio-Rad). Coomassie PAGE Blue (Bio-Rad) was used to stain the gels. The finished gels were scanned with GE Gel Scan system (GE) and analysed with PDQuest™ software 7.0.1 version (Bio-Rad). and focusing on those spots which were present in all three biological replicates. The spots apparent in all gels and with more than two-fold intensity difference when compared with each other were selected and identified by MALDI-TOF/TOF. A combined search (PMF and MS/MS) was piloted by 4000 Series Explorer Software over JGI database using MASCOT search engine (http://matrixscience.com). 2.3. Gene expression verification by real-time quantitative PCR (RT- qPCR) Total RNAs from mycelia cultured in SD, CH, CC solid substrate were isolated using Eastep Super Total RNA Extraction Kit (Promega, Cat. LS1030), following the manufacturer's protocol. RNase-free DNase I was used to completely digest DNA. After RNA purity and integrity being evaluated by electrophoresis and no DNA bands being visible, total RNAs were reverse transcribed using the Prime Script RT reagent kit (Takara, Japan) to generate cDNA. RT-qPCR was conducted according to the manufacturer’s instructions (Takara, Japan). The reaction condition was optimized. The resultant melting curves were visually inspected to ensure the specificity of product detection. The amplification efficiency of each primer pair was derived from a standard curve generated after the optimization, to ensure more than 100% qPCR efficiency. After optimization, the reaction condition was as follows: 20 μL reaction mixture containing 1.5 μL of cDNA, 0.8 μL of each gene-specific primer (10 μmol/L) (Table 1), 10 μL SYBR Premix Ex Taq II (Takara, Japan), 0.4 μL ROX Reference Dye II (50×), 6.5 μL ultrapure water; the thermal cycling profile with 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 30 s. β-actin gene was chosen as a reference gene (Pezzella et al., 2013). Each amplification run contained positive and negative controls. Mean quantification cycle (Cq) values of each tenfold dilution were plotted against the logarithm of the cDNA dilution factor. Comparative Ct method (ΔΔCT method) was used to quantify mRNA (Schmittgen and Livak, 2008). The ΔΔCT was calculated as the difference between the normalized CT values (ΔCT = CT of target gene - CT of endogenous control gene) of the treatment and the control samples: ΔΔCT=ΔCT treatment-ΔCT control. All of the genes were amplified in triplicate, and the results are presented as the mean ± standard deviations. 2.4. Analyses of lignocellulosic composition and enzymatic saccharification Acid soluble lignin (ASL), acid-insoluble lignin (AIL), cellulose, hemicellulose and ash contents in the treated and raw materials at different time were determined according to the standard biomass analytical method from the National Renewable Energy Laboratory (Sluiter et al., 2012). The enzymatic hydrolysis was performed at a substrate concentration of 2% (w/v) in 50 mM sodium acetate buffer (pH 4.8) with cellulase originating from Aspergillus niger (10 FPU/g substrate, 5 mg/mL; Sigma − Aldrich, St. Louis, MO, USA) at 45 °C. After enzymatic hydrolysis for 72 h at 100 rpm, the hydrolyzed materials were filtered and the filtrates were collected for determining the amount of reducing sugars according to the classical dinitrosalicyclic (DNS) method (Miller, 1959). All experiments were carried out in triplicate. The amount of reducing sugar was calculated as follows (Ma et al., 2010): Reducing sugar yield from enzymatic hydrolysis(mg/g dry substrate) amount of reducing sugar produced after enzymatic hydrolysis amount of dry substrate = Q. Xiao et al. Industrial Crops & Products 130 (2019) 151–161 152

2.5. Isolation and analysis of secreted proteins After 21-day cultivation at 28 °C, 100 ml 0.05 mol/L citrate-phosphate buffer (pH 7.0) was added into each flask and incubated for 30 min at 200 r/min, then filtered through several layers of Miracloth (Merck). The process was repeated for three times so that extracellular proteins were totally brought into solution. The supernatant was concentrated by ultrafilter. The proteins were confirmed using BCA assay and separated by SDS-PAGE. 10 mmol/L Dithiothreitol (DTT), 200 μL UA buffer (8 mol/L Urea, 150 mmol/L pH 8.0 Tris-HCl) were added into 200 μg supernatant proteins and concentrated with Millipore (10 kDa), then adding 50 mmol/L iodoacetamide. The supernatant was subjected to overnight digestion with sequencing grade modified Trypsin buffer (5 μg Trypsin in 40 μL dissolution buffer) at 37 °C A Q-exactive mass spectrometer (LTQFT Ultra mass spectrometer, Thermo) coupling a capillary HPLC (easy Nlc1000, Thermo) was applied to analyze the resulting tryptic peptides of the supernatant. The peptide separation from an auto-sampler was packaged with a Trap column (EASY column SC001 traps 150μm*20 mm (RP-C18)) in solvent A (0.1% formic acid in 2% acetonitrile solution). The peptide mixtures were dissolved with a linear gradient (0 to 45%) of solvent B (0.1% formic acid in 84% acetonitrile) for 120 min that comprised of 100 min (0 to 45%), followed by 8 min (45 to 100%) and maintained 12 min at 100%. HPLC was maintained at constant flow rate of 0.3 μl/min. The samples were injected into Q-Exactive mass spectrometer (Thermo Finnigan). The QExactive mass spectrometer was set to perform data acquisition in the positive ion mode and full MS scan (300–1800 m/z, resolution 70,000). The collection of peptide ions and measurement of peptide ion fragments generated by collision-induced dissociation was achieved through the linear ion trap. 20 fragment to graphy (MS2 scan, HCD) were collected after every full scan (Michalski et al., 2011). All LC-MS/MS raw data files were imported into Maxquant software (version 1.3.0.5) and quantified with iBAQ label-free quantification analysis. JGI database (PleosPC15_1_GeneModels_FrozenGeneCatalog20100405_ aa, PleosPC15_2_GeneModels_AllModels_20100427_aa, PleosPC15_2_ GeneModels_FilteredModels1_aa) was used to identify the proteins with default parameters. The search was performed versus full tryptic peptides with mass tolerance of 20 ppm for the precursor masses and 20 ppm for the fragment ions. Minimal peptide length was set to six amino acids and limited to a maximum of 2 missed trypsin cleavages. Carbamidomethyl on cysteine was accepted as a static modification and methionine oxidation, and N terminal acetylation was considered to be variable modifications. Targetdecoy strategy was used to filter peptide- and protein-level false discovery rates (FDRs) to 1%. The proteins with PFP score> 0.002 and single peptides were filtered out. All the proteins were identified with at least two peptides and quantified based on extracted ion currents of peptides from each LC/MS. The sum of intensities of all tryptic peptides for each protein was divided by the number of theoretically observable peptides and presented as iBAQ (intensity based absolute quantification). The iBAQ intensities provided an accurate determination of the relative abundance of all proteins identified in a sample. All intensities were log2-transformed. Perseus software (1.3.0.4) was used to evaluate the level of correlation between biological repeats and treatments. 3. Results 3.1. Decomposition characteristics of lignocellulosic substrates by P. ostreatus The growth rate of P. ostreatus in CH was the highest, followed by SD and CC (Fig. 1). The lignocellulosic compositions of three substrates were evaluated. SD contained the highest lignin content (35.7%), followed by CH (20.4%) and CC, (12.2%). The hemicellulose content of CC and SD was 39.1% and 29.1%, respectivly, whereas CH showed the lowest content of hemicellulose (23.4%). CH contained the highest cellulose content (41.2%), followed by CC (31.6%) and SD (20.6%) (Fig. 2). Over thirty-five days of cultivation, the weight loss for each substrate increased with time (Table 2). On day thirty-five, P. ostreatus showed the greatest extent of lignin decomposition on CH, resulting in a 64.9% absolute weight loss, which was principally due to high hemicellulose losses (77.9% absolute loss), followed by SD (62.0%), and CC Fig. 1. Time course of mycelial extension rates of P. ostreatus grown in the three types of lignocellulosic substrates (SD-sawdust, CH-cottonseed hull, CCcorncob). Table 1 Primer sequences and optimized reaction conditions of the seven candidate reference genes. Gene JGI accession No. Prime sequence Amlicon size(bp) Ta(℃) PCR efficiency(%) Regression Coefficient(R2 ) xr jgi|1102061 F:AGATGCCATTGGTCGGGTTT R:GGCCTCGTATACGGTGTCAG 71 60 93 0.996 xk jgi|1019376 F:AAGAGTGGAGGCCATCTTGC R:CGTACGCTTTCATGCCGAAG 125 60 98.2 0.995 gpdh1 jgi|1096444 F:GAAAACGCCGGGTCTCTACA R:CTCGGGTATCTTCGTGTCCG 88 60 99 0.994 gpdh2 jgi|1108563 F:AAGGAAAGCTCGAGGAACGG R:AGCTCGACTTTTCCGCGTTA 114 60 104 0.995 gadph jgi|1075210 F:GGGGTCTGGCAGAAATGACA R:GCTGCACGTAAGGAAGAGGT 101 60 97 0.997 eno jgi|1054502 F:CATGCCGGAAATAAGCTGGC R:TTCATTGCCTCCGTGAACGA 77 60 97.5 0.99 pyk jgi|1070334 F:TCCCCAAGACTGAGGTCGAT R:AAGATCATGTCGACGCCGTT 91 60 101.7 0.992 F, Forward; R, Reverse; Ta, Annealing temperature. Q. Xiao et al. Industrial Crops & Products 130 (2019) 151–161 153

(41.6%), weight losses of cellulose were low in the three substrates (50.6%, 54.1% and 34.2% in SD, CH and CC, respectively). The initial carbohydrate/lignin ratio in SD, CH and CC were 1.39, 2.02 and 2.59, respectively. After thirty-five days of fungal pretreatment, the carbohydrate/lignin ratio changed to 1.45, 3.82 and 6.54 for SD, CH and CC, respectively. 3.2. Effect of P. ostreatus pretreatment on lignocellulosic substrate saccharification After 35 days pretreatment by P. ostreatus, saccharification rates of SD, CH and CC increased 3.3, 5.0 and 3.0 folds, with the amount of reducing sugars released 202.57, 330.17 and 264.4 mg/g, respectively (Fig. 3). The effect of P. ostreatus pretreatment on the cellulose accessibility towards commercial cellulolytic enzyme preparation has been evaluated. The release of reducing sugars (i.e. substrate saccharification) increased significantly (P < 0.01), indicating the improved accessibility of cellulose after the fungal pretreatment towards commercial cellulase. 3.3. Analyses of differential mycelial proteome of P. ostreatus in different lignocellulosic substrate Mycelial proteins of P. ostreatus, grown in SD, CH and CC were separated by 2-DE, three biological replicates were performed for each treatment. Total 376 ± 25, 597 ± 19, and 483 ± 35 protein spots were detected in SD, CH and CC medium, respectively (Fig. 4). Among them, 55 significantly quantitative differential proteins in three substrates were divided into six categories based on the JGI database and GO (http://geneontology.org/) classification system, according to their molecular functions and biological processes (Table 3). Family and domain databases (Inter Pro and Pfam) were utilized to annotate the proteins that lacking exact function according to their conserved domains. These identified proteins involved in (i) redox processes: play a role in determining the cellular redox environment. (ii) stress response: include anti-oxidation proteins and proteins involving in response to toxic stress, which plays a role in protecting cells from damage. (iii) carbohydrate metabolism and energy metabolism. The abundance of 11 proteins of P. ostreatus in SD medium involving in this metabolism process significantly decreased, whereas 3 proteins identified as Fig. 2. Time course of lignocellulosic composition and weight loss of three types of lignocellulosic substrates when P. ostreatus growing (SD-sawdust, CH-cottonseed hull, CC-corncob). Table 2 Degradation rate of lignocellulosic components. Sawdust Cottonseed hull Corn cob 0-1week 0-3weeks 0-5weeks 0-1week 0-3week 0-5week 0-1week 0-3week 0-5week lignin 12.9 ± 0.16 36.7 ± 0.25 52.9 ± 0.16 18.9 ± 0.25 45.1 ± 033 64.9 ± 0.31 11.6 ± 0.21 32.5 ± 0.25 40.6 ± 0.27 hemicellulose 11.0 ± 0.25 40.5 ± 0.27 62.0 ± 0.40 23.9 ± 0.24 58.1 ± 0.18 77.9 ± 0.17 10.6 ± 0.34 24.5 ± 0.32 41.6 ± 0.31 cellulose 10.3 ± 0.11 27.8 ± 0.13 43.9 ± 0.13 16.9 ± 0.14 32.6 ± 0.56 54.1 ± 0.52 7.9 ± 0.11 19.5 ± 0.19 34.2 ± 0.19 Q. Xiao et al. Industrial Crops & Products 130 (2019) 151–161 154

adenylate kinase (JGI-ID# 1087999, 120839, 120839) and one protein identified as carbon catabolite-derepressing protein (JGI-ID# 1082594) kinase exhibited higher expression. Interesting, two enzymes involving in xylose degrading (D-xylose reductase, JGI-ID# 1102061; xylulose kinase, JGI-ID# 1019376) were more abundant in CH and SD than in CC. The abundance of a catabolite-derepressing protein kinase (CCDK, JGI-ID# 1082594) in SD was 1.4 times than that in CH and 2.2 times in CC. (iv) protein and amino acid synthesis, (v) nucleotide metabolism, and (vi) others. In group (vi), the functions of these proteins were unknown or the proteins were related to other types of metabolism. The expression levels of nine proteins in hardwood sawdust were significantly higher than those in other substrates. Among them, mitogenactivated protein kinase (MAPK) involving carbohydrate metabolism and responding to stress, was 1.3 times higher than that in CH and 2.6 times higher than that in CC. Probable FAD synthase (No. 25), involving in the oxidation process, was the highest expressed protein in CH, and probable inactive dehydrogenase (EasA, No.9) and putative aryl-alcohol dehydrogenase (ADH, No. 44) were the highest expressed proteins in CC. The expression levels of some genes related to glucose and xylose metabolism in P. ostreatus BP2 were detected using RT-qPCR (Fig. 5). xr coding xylose reductase (XR) had peak expression in P. ostreatus grown in CH medium after 3 weeks of cultivation, followed by a gradual decrease, while it showed constant increase in expression in SD and CC during the whole cultivation period of 5 weeks. The trends in the expression of xk coding xylulokinase were similar within SD and CH, with Fig. 3. Time course of the reducing sugar yield from enzymatic hydrolysis of three types of lignocellulosic substrates pretreated by P. ostreatus (SD-sawdust, CH-cottonseed hull, CC-corncob). Fig. 4. 2-DE analysis of differential expressed intracellular proteins in P. ostreatus grown in the three types of lignocellulosic substrates. Arrows and numbers refer to differential expressed proteins. (A) sawdust; (B) cottonseed hull; (C) corncob. The top arrows of each gel indicated the isoelectric focusing gel ranging (pH 4–7). The left arrows of each gel indicated the molecular weight (MW) of proteins. Q. Xiao et al. Industrial Crops & Products 130 (2019) 151–161 155

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