Analysis of the Biotechnological Potential of a Lentinus crinitus Isolate

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Analysis of the biotechnological potential of a Lentinus crinitus isolate in the light of its secretome Geison Cambri, Mirta Mittelstedt Leal de Sousa, Davi de Miranda Fonseca, Fabricio Marchini, Joana Lea Meira da Silveira, and Jaime Paba J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00636 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analysis of the biotechnological potential of a Lentinus crinitus isolate in the light of its secretome

Geison Cambria, Mirta Mittelstedt Leal de Sousab, Davi de Miranda Fonsecab, c, Fabricio Marchinid ,Joana Lea Meira da Silveiraaand Jaime Pabaa*

a

Departamento de Bioquímica, Setor de Ciências Biológicas, Centro Politécnico,

Universidade Federal do Paraná. Curitiba, PR. Brazil. 81531-990 b

Department of Cancer Research and Molecular Medicine, Norwegian University of

Science and Technology, NTNU, N-7491 Trondheim, Norway.

c

Proteomics and Metabolomics Core Facility (PROMEC), Norwegian University of

Science and Technology, NTNU, N-7491 Trondheim, Norway.

d

Laboratório de Genômica Funcional, Instituto Carlos Chagas, Fundação Oswaldo

Cruz, Curitiba, PR. Brazil. Geison Cambri ([email protected]) Mirta Mittelsted Leal de Sousa ([email protected]) Davi de Miranda Fonseca ([email protected]) Fabricio Marchini ([email protected]) Joana Lea Meira da Silveira ([email protected]) *

Jaime Paba ([email protected])

Corresponding author Jaime Paba ([email protected]) Phone/fax: +55 41 33611536 / 32662042 1 ACS Paragon Plus Environment

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Abstract Analysis of fungal secretomes is a prospection tool for the discovery of new catalysts with biotechnological application. Since enzyme secretion is strongly modulated by environmental factors, evaluation of growth conditions is of utmost importance to achieve optimal enzyme production. Here, a non-sequenced wood-rotting fungus, L. crinitus, was used for secretome analysis by enzymatic assays and a proteomics approach. Enzyme production was assessed after culturing the fungus in five different carbon sources and three nitrogen containing compounds. The biomass yield and secreted protein arrays differed drastically among growing conditions. A mixture of secreted extracts derived from solid and liquid cultures was inspected by shotgun mass spectrometry and 2-DE prior to analysis via LC-MS/MS. Proteins were identified using mass spectrometry (MS)-driven BLAST. The spectrum of secreted proteins comprised CAZymes, oxidase/reductases, proteases and lipase/esterases. Although pre-separation by 2-DE improved the number of identifications (162) compared to the shotgun approach (98 identifications) both strategies revealed a similar protein pattern. Culture media with reduced water content stimulated the expression of oxidases/reductases while hydrolases were induced during submerged fermentation. The diversity of proteins observed within both the CAZyme and oxidoreductase groups revealed in this fungus a powerful arsenal of enzymes dedicated to the breakdown and consumption of lignocellulose.

Keywords lentinus, 2D-PAGE, secretome, LCMS, CAZyme

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1. Introduction Lignocellulose is the major reservoir of organic carbon in our planet. It is highly recalcitrant to physical and biological turnover due to the crystalline structure of cellulose and the protective action of lignin. Few organisms can degrade lignocellulose, most of them are wood rotting basidiomycetes. They release into the environment a complex set of enzymes that break down the lignin polymer gaining access to polysaccharides that are then used as a source of carbon and energy. Enzymatic consortia derived from wood rotting fungi include carbohydrate active enzymes (CAZymes), oxidases and peroxidases (lignin degradation) and also proteases and lipases1. Lignin-degrading enzymes and CAZymes display a wide range of industrial applications such as the modification of the final properties of fabric in textile industry2; biodegradation of recalcitrant pollutants3; saccharification of agroindustrial wastes in the production of second generation biofuels4 and generation of building blocks to supply chemical synthesis5. Within the family of wood rotting basidiomycetes the Lentinus genus comprises a group of edible fungi with around 40 different species and worldwide distribution mainly in subtropical regions. Several species and strains have been tested as an alternative source of nutrients for humans and animals6-9, bioactive compounds with antiparasitic10, antioxidant11-13, anti-nociceptive and anti-inflammatory activity14 and also for different biotechnological applications such as the production of hydrolytic and lignolytic enymes15-18, biodegradation of pollutants19 and bioconversion of biomass20,21. In the present work we approached the characterization of the secretome of a local lignolytic Lentinus crinitus isolate. In order to favor the expression of the repertory of potential secreted enzymes the fungus was grown in media with variable content of

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carbon, nitrogen and water, and the resulting soluble extracts analyzed through twodimensional gel electrophoresis (2-DE), LC-MS and conventional enzymatic assays.

2 - Materials and methods 2.1 - Organism and culture conditions L. crinitus IOC 4579 was originally collected in Campina Grande do Sul (Paraná, Brazil) and maintained by serial cultivation on potato dextrose agar at 28 °C in the dark. The fungal isolate is deposited in the Fungal Collection of the Oswaldo Cruz Institute (Rio de Janeiro, Brazil). For each assay, it was grown on plates of solid minimal medium (SMM), containing per liter: NaNO3 6.0 g; KH2PO4 1.5 g; KCl 0.5 g; MgSO4 0.5 g; FeSO4·7H2O 0.01 g; ZnSO4·7H2O 0.02 g; glucose 10.0 g; bacteriological agar 10.0 g; pH 6.8. Liquid minimal medium (LMM) had an identical composition as SMM excluding agar. After 7–10 days of growth on SMM, three mycelial plugs collected using a 4-mm diameter biopsy punch were used to inoculate 5 mL replicates of different sterilized culture media. Cultures were incubated at 28 ±0.2 °C for subsequent assays. Seven

different

carbon

substrates

(glucose,

maltose,

starch,

sucrose,

carboxymethylcellulose (CMC), glycerol and fructose) and three nitrogen containing compounds (urea, sodium nitrate and ammonium chloride) were used as substitutes of the original carbon and nitrogen sources in culture media. The assessed concentrations were 1, 5 and 20 g L-1 (for carbon sources) and 4, 20 and 100 mM (for nitrogen sources). Low water content cultures were obtained by adding vermiculite (0.12 g mL-1) to liquid media.

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2.2 - Sample preparation Culture supernatants were separated from the mycelium with filter paper and then sieved through 0.45 µm nitrocellulose membranes. For cultures on solid media, two extractions with 2 mL of minimum media (carbon and nitrogen free) were performed in an orbital shaker (150 rpm) for 30 min. Samples were stored at -20 °C until utilization. When required, protein precipitation was carried out by the addition of trichloroacetic acid (20% final concentration) followed by overnight incubation at -20 °C. Samples were then centrifuged (15,000 g) for 10 min and washed twice with cold ethanol. Proteins were solubilized in water prior to measurement of protein concentration via Bradford assay22.

2.3 Protein electrophoresis 2.3.1 SDS-PAGE Culture supernatants (80 µL) were diluted with the same volume of 2X gel loading buffer (100 mM Tris-Cl pH 6.8; 200 mM DTT; 4% SDS; 0.2% SDS and 20% glycerol), boiled for 5 min and then subjected to electrophoresis on 10% SDS-PAGE gels23 in a Hoeffer SE 600 Ruby vertical cube (GE Healthcare, São Paulo, Brazil), at 30 mA constant current. Protein profiles were revealed by silver staining24.

2.3.2 2D-SDS-PAGE For two-dimensional gel electrophoresis, 13 cm IPG strips pH 4-7 (GE Healthcare, São Paulo, Brazil) were loaded with 80 µg protein and then submitted to isoeletric focusing (IEF) in an Ettan IPGphor II system (GE Healthcare, São Paulo, Brazil) for a total of 20 kVh at 20 °C, according to manufacturer instructions. Reduction and alkylation of IPG gels were performed in 3 mL of equilibrium buffer (50 mM Tris pH 8.0, 6 M Urea, 30%

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glycerol and 2% SDS) containing 125 mM DTT for 30 min followed by incubation in the same buffer containing 300 mM acrylamide instead of DTT for 30 min25. The second dimension was performed in 10% SDS-PAGE gels and proteins visualized by silver staining. Silver-stained gels were digitized with a SHARP JX-330 scanner (Tokyo, Japan) at 300 dpi resolution and the resulting images were analyzed with Image Master 2D Elite software (GE Healthcare, São Paulo, Brazil).

2.4 LC-MS analysis 2.4.1 Sample preparation Lyophilized fungal secretion was resuspended in 100 µL of lysis buffer consisting of 8 M urea, 0.5% Triton, 100 mM DTT, 2X phosphatase inhibitor cocktails II and III (Sigma Aldrich, St Louis, USA ), 2X Complete-EDTA free protease inhibitor cocktail (Roche, Oslo, Norway) and protein concentration was determined using the Bio-Rad Protein assay. The lysate (50 µg protein) was submitted to methanol-chloroform precipitation as described elsewhere26. Briefly, 400 µL of methanol were added to the lysate, followed by the addition of 100 µL chloroform and 300 µL of MilliQ water. Samples were briefly mixed using a vortex and centrifuged at 16,000 g for 2 min. The upper layer was then discarded without disturbing the protein layer and 300 µL of methanol were added to the sample, followed by another round of vortex and centrifugation. After removing the supernatant, the protein pellet was resuspended in 50 mM NH4HCO3 containing 5 mM tris (2-carboxyethyl phosphine (TCEP) and incubated for 30 min prior to alkylation with 1 µmol/mg protein of iodoacetamide for 30 min in the dark. Overnight protein digestion was performed using LysC-Trypsin mix (Promega, Madison, WI, US) at 1:50 ratio (w/w, enzyme:protein) at 37 °C. Digests

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were diluted to final concentration 0.5 µg/µL in 0.1% formic acid prior to mass spectrometry analysis.

2.4.2 In-gel tryptic digestion Protein bands excised from a 10% SDS-PAGE gel were reduced and alkylated with iodoacetamide as described above while protein spots excised from 2-DE gels were alkylated with acrylamide prior to in-gel tryptic digestion as described with minor modifications27. Briefly, in-gel tryptic digestion was performed by washing the gel pieces twice in 50 mM NH4HCO3:CH3CN (1:1) for 15 minutes followed by incubation in CH3CN for 10 minutes. For gel bands, 10 mM DTT was added for rehydration followed by incubation at 56 °C for 1 hour and subsequent incubation with 55 mM iodoacetamide in the dark for 45 minutes. Gel bands were washed again twice in 50 mM NH4HCO3:CH3CN (1:1) for 15 minutes followed by incubation in CH3CN for 10 minutes. Both gel bands and protein spots were then incubated with 12.5 ng/mL trypsin in 50 mM NH4HCO3 for 30 minutes on ice prior to overnight incubation in 50 mM NH4HCO3 at 37 °C. Tryptic digests were dried out followed by resuspension in 0.1% formic acid for mass spectrometry analysis

2.4.3 Mass spectrometry analysis Protein digests were analyzed on a Thermo Scientific Q Exactive mass spectrometer operating in FullMS-ddMS2 mode coupled to an EASY-nLC 1000 UHPLC system (Thermo Scientific, Oslo, Norway). Peptides (2 µg) were injected onto a Acclaim PepMap100 C-18 column (75 µm i.d. × 2 cm, C18, 3 µm, 100 Å) (Thermo Scientific, Oslo, Norway) and further separated on a Acclaim PepMap100 C-18 analytical column (75 µm i.d. × 50 cm, C18, 2 µm, 100 Å) (Thermo Scientific, Oslo, Norway). For the 7 ACS Paragon Plus Environment

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fungal secretion peptides, a 240 min method was employed consisting of a 250 nL/min flow rate, starting with 100% buffer A (0.1% formic acid) with an increase to 5% buffer B (100% acetonitrile, 0.1% formic acid) in 5 min, followed by an increase to 40% buffer B over 220 min and an increase to 100% buffer B in 8 min, where it was held for 10 min. Peptides extracted from gel bands and protein spots were processed using a 60 min method starting with 100% buffer A with an increase to 5% buffer B in 2 min, followed by an increase to 35% buffer B over 46 min and a rapid increase to 100% buffer B in 3 min, where it was held for 9 min. The peptides eluting from the column were ionized by a Nanospray Flex Ion Source (Thermo Scientific, Oslo, Norway) and analyzed on the Q Exactive operating in positive-ion mode using electrospray voltage 1.9 kV and HCD fragmentation. For the fungal secretion each MS scan (m/z 300–1600) was acquired at a resolution of 70 000 FWHM, automatic gain control (AGC) target value of 3 × 106, maximum injection time of 200 ms. TopN 10 MS/MS scans were acquired at a resolution of 17 500 FWHM, maximum injection time of 100 ms, normalized collision energy (NCE) 30, AGC target value of 1 × 105, and isolation window 2 m/z, dynamic exclusion 30 s. MS and MS/MS scans were acquired with similar parameters for gel bands and protein spots, however, AGC of 1 × 106 and maximum injection time of 250 ms were applied for MS scans and AGC target value of 1 × 105 with maximum injection time of 100 ms were employed for MS/MS scans. For all analysis, charge exclusion was set to unassigned, 1.

2.4.4 Data analysis Prior to protein identification, MS spectra were analyzed using Preview software28 revealing the parameters to be used in the database searches. Due to differences in sample preparation and protein separation methodologies employed prior to mass

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spectrometry analysis, the Preview software revealed distinct static and dynamic modifications for each type of sample. Protein identification was performed via analysis of acquired MS spectra using Thermo Proteome Discoverer version 1.4.0.288 software running Mascot 2.2.6 and the Sequest HT search algorithms (search parameters are listed in Supporting Information Table S-1); peptides were identified with high degree of confidence as having False Discovery Rate (FDR) ≤ 0.01 using Percolator 2.04. Furthermore, to exclude false positive hits, minimum score of 5.0 was set for Sequest HT identifications from gel bands and LC-MS/MS approaches. Moreover, to maximize high-confidence protein identification, protein spots spectra were searched in Sequest HT against a database from a closely related organism, namely Lentinus tigrinus, instead of the Polyporales database. The Lentinus tigrinus genome was completely

sequenced29

and

it

is

available

at

JGI

Genome

Portal

(http://genome.jgi.doe.gov/Lenti6_1/Lenti6_1.home.html). The corresponding proteome was also generated, and for the present work we downloaded the filtered protein list from JGI and named it Lenti6 database. However, the proteome is not yet annotated, hence, to determine protein identities and their functions, peptide sequences identified for each spot using the Lenti6 database were further submitted to MS-BLAST˗based protein identification. MS-BLAST searches were performed as described30. Briefly, peptides generated from each protein spot were spaced with minus symbols (-), merged into a single string and edited according to the rules described. Searching was carried out at http://genetics.bwh.harvard.edu/msblast/, using default settings and the PAM30MS matrix against nrdb95 database. Although a minimum score was not set for Sequest HT identifications prior to MS-BLAST, matches were considered significant only if matched fungal proteins and displayed a high-scoring segment pairs value of 85 or above.

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The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE31 partner repository with the dataset identifier PXD003901.

2.5 Hydrolases Glycoside hydrolase activity was determined by measuring the amount of reducing sugar released after incubation of secretion extracts with each particular substrate using the 2,4 dinitrosalicylic acid (DNS) method32. Spectrophotometric assays were performed in Epoch Microplate Spectrophotometer (Biotek, Winooski, VT, US). The amount of reducing sugar released in the reactions was subtracted from the control replicates of each sample. An enzyme unit was defined as the amount of sample which releases 1 µmol of reducing sugar per minute.

2.5.1 Cellulase Cellulase activity was measured using the filter paper assay, FPase33. Briefly the reaction mixture consisted of 1 mL of culture secretion extract and 1 mL of 200 mM acetate buffer pH 5.0, added to 50 mg of filter paper (4 pieces of 0.5x1.5 cm). After incubation for 50 min at 45 °C, 3 mL of DNS reagent were added and the mixture incubated in a boiling water bath for 20 min. Absorbance was then measured at 570 nm and reducing sugar concentration was determined by a standard curve using 0-3 µmol glucose.

2.5.2 Xylanase Xylanase activity was measured according to Kim et al34. Sample volumes of 20 µL were added to 20 µL of 1% xylan solution and 10 µL of 100 mM phosphate buffer pH 5 with subsequent incubation for 1 hour at 40 °C. Next, 200 µL of DNS solution were

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added followed by incubation in boiling water for 20 min and the absorbance determined at 570 nm. Controls were carried out by replacing substrate by water. The standard graph was prepared using 0-1 µmol xylose.

2.5.3 Pectinase Pectinase assays were carried according to Biz et al35. A 0.25 mL pectin solution (0.5% pectin in 200 mM acetate buffer pH 4.5) was added to 0.25 mL fungal secretion extracts and incubated for 20 min at 30 °C. Next, 0.5 mL of DNS was added and the test tube transferred to a boiling water bath for 20 min. Absorbance was measured at 545 nm and the amount of released reducing sugar was defined using a galacturonic acid standard graph (0-10 µmol).

2.6 Oxidases/reductases 2.6.1 Laccase Total oxidase/peroxidase activity was followed by the oxidation of 0.3 mM 2,2′-azinobis (3-ethylthiazoline-6-sulfonate) (ABTS) to its cation radical (ε420: 36,000 cm-1 M-1) in 50 mM acetate buffer pH 5.0, 20 mM MnSO4 and 0.05 mM H2O2 at 5 min intervals during 20 min at 28 °C36. Laccase activity was determined in the same conditions without MnSO4 and peroxide. One enzyme unit was defined as the amount of enzyme which oxidizes 1 µmol of ABTS per minute.

2.6.2 Dye Peroxidase (DyP) DyP activity was monitored by the decolorization of the anthraquinone dye Reactive Blue 19 (RB19) (ε595: 10,000 cm-1 M-1). Briefly, fungal soluble extracts were incubated with 50 µM RB19 solution in the presence and absence of 0.1 mM H2O2 in 100 mM

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acetate buffer pH 4.0. The absorbance was monitored during 90 min at 28 °C. One enzyme unit was defined as the amount of enzyme which destained 1 µmol of RB19 per minute37.

2.6.3 Azoreductase Enzyme activity was measured by the decrease in absorbance of methyl red (ε430: 23,360 M-1 cm-1) after reduction38. The reaction mixture contained 150 µL of sample supernatants in 4.45 µM methyl red, 20 µM NADH and 50 mM phosphate buffer pH 5.5. Absorbance was monitored during 90 min at 28 °C. One enzyme unit was defined as the amount of enzyme which reduced 1 µmol of methyl red per minute.

2.6.4 Alcohol veratryl oxidase (VO) The enzyme activity was tested in a reaction mixture containing 1 mM of veratryl alcohol and 100 mM tartarate buffer pH 3.0. Absorbance was measured at 310 nm after 0 and 90 min incubation at 28 °C39.

2.6.5 Lignin peroxidase (LiP) LiP activity was determined in a reaction mixture containing 100 mM n-propanol, 250 mM tartaric acid and 10 mM H2O2. Absorbance was measured at 300 nm after 0 and 90 min incubation at 28 °C39.

2.6.6 Cellobiose Dehydrogenase (CDH) CDH activity was determined by following the decrease in absorbance of 2,6diclorophenol-indophenol (DCIP) at 520 nm. The reaction mixture contained sample supernatants diluted in 30 mM lactose, 0.3 mM DCIP, 30 mM sodium azide and 50 mM

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phosphate buffer pH 4.0. Absorbance was measured after0 and 90 min incubation at 28 °C40.

2.6.7 NADH-DCIP reductase Enzyme activity was measured in a 5.0 mL reaction mixture containing 25 mM DCIP (2,6-dichlorophenol indophenol) and 0.1 mL enzyme solution in potassium phosphate buffer (50 mM, pH 7.4). 2.0 mL of reaction mixture was assayed at 590 nm after adding 250 mM NADH. The DCIP reduction was calculated using the extinction coefficient of 0.019 mM−1 cm−1 41.

3. Results and discussion L. crinitus IOC 4581 was grown in seven carbon sources (maltose, glycerol, glucose, fructose, starch, sucrose and CMC) in concentrations ranging from 1 to 20 g L-1. In general, increases in carbon concentration stimulated both fungus growth and yield of secreted proteins (Figure 1). The highest biomass production was observed in maltose, fructose and glycerol (20 g L-1) based cultures. However, the amount of secreted proteins was markedly higher in maltose cultures (20 g L-1) compared to other growth conditions. The protein profile also displayed qualitative changes according to the carbon source (Figure 2). Maltose, sucrose and fructose based media induced the expression of polypeptides in a wide range of molecular masses while growth on glycerol, glucose and starch based media resulted in rather poor protein profiles. Analysis of CMC derived samples was unfeasible due to excessive production of exopolysaccharide, hindering protein separation. In order to check the effect of nitrogen culture content on the protein secretion profile, three previously used carbon sources (maltose, starch and glycerol 20 g L-1) were combined with one of three different

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nitrogen compounds (urea, sodium nitrate and ammonium chloride) in concentrations ranging between 4 and 100 mM. Maltose-based media was chosen due to the complex pattern of secreted proteins, while glycerol and starch based media resulted in low protein yield. The rationale behind this is the synergistic effect among C and N sources previously observed42 and the use of low cost carbon sources (glycerol and starch) that can be obtained as by-products of the synthesis of biodiesel and agroindustry43,44. Notably, changes in nitrogen content resulted in distinct protein secretion profiles according to the carbon source (Figures 3 and 4). In glycerol or maltose basedmedium, different N sources and changes of N concentration had little effect on fungal growth. However, drastic changes were observed in starch based media in each condition, with the highest amount of biomass obtained in medium containing urea (4 and 20 mM) (Figure 3). Protein secretion was also affected by the addition of N compounds. Here, the highest protein levels were induced by the mixtures maltose (20 g L-1)/urea (100 mM) and maltose (20 g L-1)/nitrate (20 mM). The most complex protein profiles were observed when the fungus was grown in media containing maltose or glycerol (20 g L-1) with 20 mM urea and 20 mM ammonium chloride or sodium nitrate respectively (Figure 4). The main protein groups produced by wood rotting fungi are lignin-modifyingenzymes (LMEs) (such as Lac, MnP, LiP), CAZymes (carbohydrate active enzymes) and peptidases. These have been shown to be differentially regulated in response to a wide variety of environmental signals such as the availability of carbon and nitrogen in culture media45,46, among several others. Ligninolytic enzymes are mainly produced during secondary metabolism in response to nitrogen and/or carbon depletion47-52. Apparently, the carbon-regulated expression of LMEs occurs via a cAMP mechanism, since cAMP-responsive elements (creA) have been described in Lac, LiP and MnP gene 14 ACS Paragon Plus Environment

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promoters53,54. The nitrogen repression response element (NIT2), which activates the expression of many structural genes in nitrogen-limited conditions as well as other regulatory elements such as Mig (carbon response element) have been described in fungi55-59, although only in P. sajor-caju among the white rot fungi60. Thus, the changes here observed in protein secretion suggest that C and N response elements are also present in this particular L. crinitus isolate. Indeed, as further described, our data show that LMEs and CAZymes are major constituents of L. crinitus secretion extracts.

Due to the high protein yield of maltose-based cultures combined with the increased protein profile complexity induced by urea, maltose-urea cultures were chosen for the subsequent experiments. Thus, to assess the effect of water content on protein secretion, L. crinitus was cultured in liquid (submerged fermentation, SmF) and solid media (solid state fermentation, SSF) containing maltose (20 g L-1) and urea (100 mM) and the protein profiles were analyzed by 1D-SDS PAGE. Interestingly, changes in water content led to drastic differences both in secreted protein pattern (Figure 5) and total protein yield (Figure 6). While a large number of proteins with molecular weight varying between 20 and 100 kDa was detected in the secretion from fungi cultivated in liquid medium, growth on solid media resulted in quite simple protein profiles exhibiting intense protein doublets around 42 kDa and 58 kDa. Notably, most of the polypeptides particularly expressed in liquid media were strongly suppressed in solid media based cultures. Tryptic digestion and MS analysis of the 42 kDa doublet resulted in 11 matches to known proteins with ten corresponding exclusively to enzymes involved in lignin degradation such as laccase and MnP, and only one match to a CAZyme (Table 1 and Supporting Information Table S-2). Hereinafter the activity of some CAZymes and oxidases was assessed in secretion extracts derived from

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cultures in media with variable content of water. The evaluated CAZymes were xylanase, pectinase and cellulase each one displaying a distinct expression profile (Figure 7). Pectinase and xylanase were induced by high water content since better titers were obtained when the fungus was grown in liquid media. Cellulase activity, evaluated via filter paper activity (FPase) assay, remained unchanged in the assessed conditions.

The production of hydrolytic enzymes is widely described in white rot fungi since they allow the microorganisms to obtain energy from lignocellulosic substrates. In the Lentinus genus, cellulase, endoglucanase, beta glucosidase, endoxylanase, and xylosidase activities have been reported in L. tigrinus61, L. sajor-caju62 and L. polychrous63 but most of the experiments were performed in SSF and yield comparisons with SmF were not determined. In wood rotting fungi the expression of hydrolytic enzymes is usually improved by submerged fermentation. Elisashvili et al64. using three Lentinula strains and six Pleurotus isolates observed that submerged fermentation resulted in strong induction of CMCase, xylanase and FPase activities. In SSF low levels of hydrolytic enzymes were similarly secreted by all Lentinula strains. Conversely, the amount of hydrolytic enzymes produced by Pleurotus species varied greatly. Lentinula strains showed a 15-45 fold increase in the production of xylanase and cellulase respectively when cultured by SmF in comparison with SSF64. Additionally, culture medium with a rich content of easily metabolizable sugars led to a similar effect. The same authors working with several genera of wood rotting fungi (Pseudotremella, Fomes, Trametes, Trichaptum, Ganoderma and Pleurotus) reported significant differences in the production of hydrolytic enzymes using several substrates in liquid and solid fermentation65 showing that the enzyme yield and ratio in enzyme

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preparations significantly depends on the fungus species, substrate, and cultivation method. In general, submerged fermentation and a high content of soluble sugars favored hydrolases production.

To investigate the contribution of oxidases and accessory proteins namely Lac, DyP, azoR, MnP, VO, LiP, NADH-DCIP-R (2,6 dichlorophenol indophenol reductase) and CDH (cellobiose dehydrogenase) in vitro activity assays were performed in secretions collected from L. crinitus cultured in liquid or solid media. Only Lac, MnP and DyP activities were detected in one or more soluble extracts. Moreover, Lac, DyP and azoR were mostly detected in solid medium (Figures 8 and 9). Thus, the overall higher yield of total oxidase activity was observed in solid cultures with high carbon content. No oxidase activity was detected in liquid cultures with low carbon content and supplemented with 1% lignin (data not shown). In Lentinus as well as in most wood rotting fungi, the expression of lignin degrading enzymes can be modulated by water availability and the presence of lignocellulosic material, among several other variables such as carbon or nitrogen content, supplementation with metal ions66, aromatic compounds67 and alcohol68. L. polychrous grown in different solid substrates produced high levels of Lac and MnP. Although hydrolytic enzymes were also detected the production ratio among lignolytic and carbohydrate degrading enzymes was markedly high63. The same behavior was here observed with the L. crinitus isolate. When growth media is supplemented with lignin different outcomes have been described. By studying several white rot fungi Kenkebashvili et al.45 observed a fivefold improvement in Lac production due to lignin supplementation. However, huge variations were observed in response to different types of lignocellulosic substrate used. Notably, no substrate resulted in the same response for

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every strain/isolate. This great variability was also described by Elisashvili et al.64 using several Lentinula and Pleurotus isolates where SSF and supplementation with raw plant material favored the secretion of oxidative enzymes. The degradation of RR19 (anthraquinone dye) and methyl red, the current substrates of DyP and azoR respectively, was already described in Lentinus sp.69, L. tigrinus70 and L. polychrous71-73, although these activities were assigned to the presence of Lac, MnP and LiP. Our data show different activity patterns for each substrate suggesting that either DyP or azoR are present, or alternatively, the pool of oxidase/reductase enzymes in the secretion extract displays wider substrate specificity.

DNA response elements

necessary for controlling expression through metal ions, xenobiotics54 and carbohydrates53,74 have been described in Lac and MnP genes but none of them were associated to water content effects on culture medium.

A major goal of this work was to induce the expression of the maximum number of potential secreted proteins encoded on the L. crinitus genome through the modulation of growth conditions. According to our data, highest levels of secreted protein were induced by the combination urea-maltose in culture medium. Therefore, secretion extracts derived from solid and liquid cultures containing maltose (20 g L-1) and urea (100 mM) were pooled and submitted to two strategies: (1) shotgun LC-MS/MS proteome analysis and (2) protein separation by two-dimensional gel electrophoresis prior to protein identification by mass spectrometry analysis. LC-MS/MS analysis of the whole secretion extract resulted in 98 protein identifications using the non-redundant NCBI database (nrdb95) (Supporting Information Table S-3 and S-4). These corresponded to 25 CAZymes, 20 oxidase/reductases, 3 proteases, 5 lipase/esterases and 9 proteins with non-related functions, which we named miscellany proteins. Several

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matches (36) corresponded to hypothetical or unknown predicted proteins. Within the CAZymes group, glycoside hydrolases from different families, glucanases, alpha/beta glycosidases, amylases and galacturonases were detected revealing a powerful arsenal of enzymes dedicated to the consumption of polyssacharides. Also, several oxidases associated to lignin depolymerization were identified such as laccases, peroxidases, glucose-methanol-choline (GMC) oxidoreductases, MnP and glyoxal oxidases. By using the 2D gel approach, a protein map containing 89 spots was resolved (Figure 10). Most of the detected polypeptides displayed acidic pI and relative molecular mass among 20 and 100 kDa. MS analysis of the resolved spots resulted in 162 protein identifications corresponding to CAZymes (49), oxido/reductases (25), proteases (36), lipase/esterases (6), miscellany (34), hypothetical/uncharacterized proteins (4) and nonidentified spots (8) (Supporting Information Table S-5). The group of identified enzymes classified as CAZymes in this study is quite heterogeneous. It comprises alpha/beta hydrolases, cellulases, cellobiohydrolases, xylanases, polygalacturonases and glycoside hydrolases from at least 11 CAZyme families suggesting that L. crinitus can grow in a wide variety of saccharide substrates. Furthermore, several oxidase/reductases were identified including 2 alcohol oxidases, 10 multicopper oxidases, 4 glyoxal oxidases, 3 MnPs and 2 LiPs. The overall distribution of proteins identified in each experimental strategy was quite similar except for the protease family (3 proteases by shotgun proteomics vs. 32 proteases by 2D-LC-MS/MS). A slightly larger number of proteins was identified via 2D-SDS PAGE combined with downstream MS analysis compared to the shotgun proteomics approach (Figure 11). The good performance obtained with the 2D-SDS PAGE approach was quite surprising since several limitations of this technique have been related when compared to high throughput LCMS strategies, such as low load ability and poor separation of hydrophobic, acidic and

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alkaline proteins as well as loss of proteins with molecular mass above 100 kDa or below 20 kDa75. Possibly, challenges in discerning highly similar proteins from a pool characterized by a limited number of functional classes and high content of isoforms and multiple post translational variants contributed to lower number of IDs obtained by shotgun mass spectrometry. Pre-fractionation, using 2D PAGE, drastically diminishes the complexity of the analysis due to the generation of a smaller amount of peptides belonging to a restricted number of proteins (1-5)76. In addition, the database used for SEQUEST-based searches likely increased the number of relevant protein identifications for protein spots. Here, a database of a closely related species was chosen instead of a broad database consisting of sequences from a variety of organisms belonging to the Polyporales order employed in the shotgun analysis. As the Lenti6 genome was not annotated, peptide sequences identified for each spot were manually submitted to MS-BLAST homology searches to determine the identity and therefore functionality of characterized proteins. Although this strategy is suitable to analyze protein spots, where a limited number of peptide sequences belonging to one or a few proteins match a particular spot, it would be impractical for the analysis of the whole fungal secretion by LC-MS/MS. In the latter, thousands of peptides would be identified based on the Lenti6 database. However, since protein IDs are not annotated, manual inspection of peptide sequences by MS-BLAST to assign protein identity would become extremely laborious and challenging. To overcome this limitation, data from LCMS/MS analysis of fungal secretion was searched against a broader database composed by 9 proteomes of organisms belonging to the Polyporales order for protein identification.

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The spectrum of detected proteins in the secretion extract of L.crinitus IOC 4579 resembles the expected profile of a good candidate for the delignification and saccharification of lignocellulose1. Currently, there is genomic (genome.jgi.doe.gov; kwanlab.bio.cuhk.edu.hk)

and

transcriptomic

information

(genome.jgi.doe.gov/Lentignscriptome) available for only one related Lentinus species, L. tigrinus.

However, these data are not readily available and lack throughout

annotation, which is paramount for the prediction or comparison of the number of potential secreted proteins by the fungus through bioinformatic resources. When contrasting the data from the L. crinitus secretome with other basidiomycetes according to their lifestyle (white rot, brown rot, symbionts or parasites), the abundance of lignin oxidases and glycoside hydrolases fits well with the profile observed in the majority of analyzed white rot fungi1. Additionally, the total number of identified proteins (162) is quite comparable with the observed in other wood rotting fungi secretomes such as Phlebia brevispora (178 proteins)77, Bjerkandera adusta (157)78, Phanerochaete chrysosporium (190)79 and Ganoderma lucidum (71)80 among others1. Several of the enzymes here described have been reported in other Lentinus species but none of them using a high-throughput approach. In L. tigrinus, it has been demonstrated that enzyme secretion is regulated by the fungus growth phase. It was shown that production of lignocellulosic enzymes was high during colonization and ligninases declined drastically during fruit body formation61. Oxidative enzymes were preferentially produced in SSF by L. strigellus81 and L. polychrous63. Moreover, several hydrolytic enzymes such as endoglucanase, cellobiohydrolase, beta-glucosidase and xylanase have been described in different Lentinus species. Despite the fact that basidiomycetes are scarcely described as pectinase producers, polygalacturonase activity was previously detected in a Lentinus sp. isolate82.

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Importantly, there are only a few reports on L.crinitus describing the production of laccase and MnP and its general lignolytic activity42,83,84. Thus, the current lack of information on secreted enzymes and influence of different growth conditions on the L. crinitus secretome prompted us to investigate this species. Our findings shed light into the nature of secreted proteins as well as modulation of protein expression levels and enzymatic activities via alterations in growth conditions.

4. Conclusion Secretome analysis reveals the extraordinary variability among fungi as well as the wide range of potential applications they offer. By studying the secretome of a poorly characterized Lentinus species, we identified 162 proteins in a mixed soluble extract containing polypeptides produced in solid and liquid cultures. Our findings reveal that the L. crinitus secretome displays a diverse array of lignocellulolytic and proteolytic enzymes with promising applications on several branches of industry. The relatively high number of hypothetical or uncharacterized proteins matched in database search indicates a higher complexity of the L. crinitus fungal secretome and highlights the need for genome information in order to better understand the physiology of this organism.

Supporting Information Table S-1. Parameters used in the database searches. Table S-2 Proteins identified in the 42 kDa doublet gel band exclusively expressed by L. crinitus in solid cultures Table S-3. Proteins identified in the L.crinitus soluble extract through LC-MS/MS (overall). Table S-4. Proteins identified in the L.crinitus soluble extract through LC-MS/MS (detail). Table S-5 Proteins identified in the L.crinitus soluble extract using the 2-DE-LC MS/MS approach. 22 ACS Paragon Plus Environment

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Acknowledgements Cambri, G is grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing a graduate scholarship. Paba, J. acknowledges International Foundation for Science (IFS, Sweden) for financial support (grant No F/4196). We also thank Professor David Hibbett for providing access to unpublished genome data produced by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Role of the funding source The present work was supported by grant No F/4196-1 from International Foundation for Science (IFS, Sweden).The authors declare that IFS did not participate in the study design, collection, analysis, and interpretation of data, in the writing of the report, nor in the decision to submit the paper for publication. The Proteomics and Metabolomics Core Facility (PROMEC) at the Norwegian University of Science and Technology (NTNU) is funded by the Faculty of Medicine at NTNU and the Central Norway Regional Health Authority

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(52) Jeffries, T. W.; Choi, S.; Kirk, T. K. Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 1981, 42 (2), 290-296. (53) Galhaup, C.; Goller, S.; Peterbauer, C.; Strauss, J.; Haltrich, D. Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology (London, U. K.) 2002, 148, 2159-2169. (54) Xiao, Y. Z.; Hong, Y. Z.; Li, J. F.; Hang, J.; Tong, P. G.; Wang, W.; Zhou, C. Z. Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression. Appl. Microbiol. Biotechnol. 2006, 71 (4), 493-501. (55) Marzluf, G. A. Regulation of nitrogen metabolism and gene expression in fungi. Microbiol. Ver. 1981, 45 (3), 437-461.

(56) Mach, R. L.; Strauss, J.; Zeilinger, S.; Schindler, M.; Kubicek, C. P. Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei. Mol. Microbiol. 1996, 21 (6), 1273-1281. (57) Ilmén, M.; Onnela, M. L.; Klemsdal, S.; Keränen, S.; Penttilä, M. Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei. Mol. Genet. Genomics 1996, 253 (3), 303-314. (58) Strauss, J.; Horvath, H. K.; Abdallah, B. M.; Kindermann, J.; Mach, R. L.; Kubicek, C. P. The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level. Mol. Microbiol. 1999, 32 (1), 169-178. (59) Suto, M.; Tomita, F. Induction and catabolite repression mechanisms of cellulase in fungi. J. Biosci. Bioeng. 2001, 92 (4), 205-311.

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(60) Soden, D. M.; Dobson, A. D. The use of amplified flanking region-PCR in the isolation of laccase promoter sequences from the edible fungus Pleurotus sajor-caju. J. Appl. Microbiol. 2003, 95 (3), 553-562. (61) Lechner, B. E.; Papinutti, V. L. Production of lignocellulosic enzymes during growth and fruiting of the edible fungus Lentinus tigrinus on wheat straw. Process Biochem. 2006, 41 (3), 594-598. (62) Singh, M. P.; Pandey, A. K.; Vishwakarma, S. K.; Srivastava, A. K.; Pandey, V. K.; Singh, V. K. Production of cellulolytic enzymes by Pleurotus species on lignocellulosic wastes using novel pretreatments. Cell. Mol. Biol. 2014, 60 (5), 59-63. (63) Budda, W.; Sarnthima, R.; Khammuang, S.; Milintawisamai, N.; Naknil, S. Ligninolytic enzymes of Lentinus polychrous grown on solid substrates and its application in black liquor treatment. J. Biol. Sci. 2012, 12 (1), 25-33. (64) Elisashvili, V.; Penninckx, M.; Kachlishvili, E.; Tsiklauri, N.; Metreveli, E.; Kharziani, T.; Kvesitadze, G. Lentinus edodes and Pleurotus species lignocellulolytic enzymes activity in submerged and solid-state fermentation of lignocellulosic wastes of different composition. Bioresour. Technol. 2008, 99 (3), 457–62. (65) Elisashvili, V.; Kachlishvili, E.; Tsiklauri, N.; Metreveli, E.; Khardziani, T.; Agathos, S. N. Lignocellulose degrading enzyme production by white rot basidiomycetes isolated from the forests of Georgia. World J. Microbiol. Biotechnol. 2009, 25, 331-339. (66) Shutova, V. V.; Revin, V. V.; Myakushina, Yu. A. [The effect of copper ions on the production of laccase by the fungus Lentinus (Panus) tigrinus]. Prikl. Biokhim. Mikrobiol. 2008, 44 (6), 619-623.

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(74) Zhuo, R.; Ma, L.; Fan, F.; Gong, Y.; Wan, X.; Jiang, M.; Zhang, X.; Yang, Y. Decolorization of different dyes by a newly isolated white-rot fungi strain Ganoderma sp.En3 and cloning and functional analysis of its laccase gene. J. Hazard. Mater. 2011, 192 (2), 855-873. (75) Bunai, K.; Yamane, K. Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2005, 815 (1-2), 227-36.

(76) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (17), 9390-9395. (77) Hori, C.; Gaskell, J.; Samejima, M.; Hibbett, D. S.; Henrissat, B.; Cullen, D. Genome analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay. Mycologia 2013, 105 (6), 1412-1427. (78) Reina, R.; Kellner, H.; Jehmlich, N.; Ullrich, R.; García-Romera, I., Aranda, E.; Liers, C. Differences in the secretion pattern of oxidoreductases from Bjerkandera adusta induced by a phenolic olive mill extract. Fungal Genet. Biol. 2014, 72, 99-105.

(79) Wymelenberg, A.; Gaskell, J.; Mozuch, M.; Kersten, P.; Sabat, G.; Martinez, D.; Cullen, D. Transcriptome and secretome analyses of Phanerochaete chrysosporium reveal complex patterns of gene expression. Appl. Environ. Microbiol. 2009, 75 (12), 4058-4068. (80) Manavalan, T.; Manavalan, A.; Thangavelu, K. P.; Heese, K. J. Secretome analysis of Ganoderma lucidum cultivated in sugarcane bagasse. Proteomics 2012, 77, 298-309. 34 ACS Paragon Plus Environment

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(81) Gomes, E.; Aguiar, A. P.; Carvalho, C. C.; Bonfá, M. R. B.; Da Silva, R.; Boscolo, M. Ligninases production by basidiomycetes strains on lignocellulosic agricultural residues and their application in the decolorization of synthetic dyes. Braz. J. Microbiol. 2009, 40 (1), 31-39. (82) Xavier-Santos, S.; Carvalho, C. C.; Bonfá, M.; Silva, R.; Capelari, M.; Gomes, E. Screening for pectinolytic activity of wood-rotting basidiomycetes and characterization of the enzymes. Folia Microbiol (Praha) 2004, 49 (1), 46-52. (83) Hossain, S.; Anantharaman, N.; Das, M. Studies on lignin biodegradation of wheat straw using Trametes versicolor and Lentinus crinitus. J. Inst. Eng. (India), Chem. Eng. Div. 2007, 87, 42–50. (84) Valle, J. S.; Vandenberghe, L. P.; Santana, T. T.; Almeida, P. H.; Pereira, A. M.; Linde, G. A.; Colauto, N. B.; Soccol, C. R. Optimum conditions for inducing laccase production in Lentinus crinitus. Genet. Mol. Res. 2014, 13 (4), 8544-8551.

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Tables Table 1. Results of the MS analysis of the 42 kDa protein doublet exclusively expressed by L. crinitus in solid cultures MW [kDa]

calc. pI

STSTPTADLAVISVTK, Oxidoreductase ANPNFGTTGFADGVNSAILR, YSFVLNANQDVDNYWIR

50,1

5,00

Pycnoporus cinnabarinus

Oxidoreductase FPLGADATLINGLGR

56,0

5,12

Q01775_PH ACH

Phanerochaete chrysosporium

Oxidoreductase TACEWQSFVANQSK

39,2

4,49

Manganese peroxidase# Melanindecolorizing enzyme# Mn peroxidase MNP3#

G9I531_9AP HY

Lenzites gibbosa Oxidoreductase

38,0

4,63

B3IWB3_9A Ceriporiopsis sp. Oxidoreductase LQSDHDLAR PHY

38,3

5,10

G0Z9F2_9A PHY

Manganese peroxidase 2*

G9I531_9AP HY

Mn peroxidase MNP3*

G0Z9F2_9A PHY

Polyporus brumalis

Oxidoreductase

Mn peroxidase MNP6*

G0Z9F5_9A PHY

Polyporus brumalis

Oxidoreductase

Identifier

Organism

Laccase#

LAC1_PYC CI

Pycnoporus cinnabarinus

Laccase#

LAC1_PYC CI

Ligninase#

Protein / ID

Laccase (Fragment)* Glucoamylase*

Polyporus brumalis

Group

Sequence aligned

GTLFPGTGGNQGEVESPLHG EIR, LQSDSELAR

Oxidoreductase

LTFHDAIGISPAIASR, LQSAFAAAFR, LQSDSELAR

38,1

4,58

Lenzites gibbosa Oxidoreductase

GTLFPGTGGNQGEVESPLHG EIR, LQSDSELAR

38,0

4,63

LTFHDAIGISPAIASR, LQSAFAAAFR, LQSDSELAR

38,1

4,58

38,0

4,42

50,1

5,00

61,0

4,68

LQSAFAAAFR, LQSDSELAR, LTFHDAIGISPAIAAR ANPNFGTTGFADGVNSAILR, Q5EBY5_9A Lentinus tigrinus Oxidoreductase STSTPTADLAVISVTK, PHY YSFVLNANQDVDNYWIR Phanerochaete K5WMZ0_P SLIDEFVSAEATLQQVTNPSG carnosa (strain CAZyme HACS SVTTGGLGEPK HHB-10118-sp)

Proteins identified from the upper and lower protein bands of the 42 kDa doublet are marked by # and * symbols, respectively.

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Figure Captions Figure 1. Biomass and protein concentration from cultures of Lentinus crinitus in media with variable carbon content. CMC: Carboxymethylcellulose. Error bars represent standard deviation of three independent samples. Statistical significance from One-way ANOVA followed by Tukey’s multiple comparisons test analysis is demonstrated in the table with adjusted P-values of: < 0.0001 (****), < 0.001 (***), < 0.01 (**), < 0.05 (*). Each letter represents a particular culture condition. Letters in parenthesis displayed no significant differences among them.

Figure 2. Effect of the culture media carbon-content on the protein secretion profile of Lentinus crinitus. Equal volumes of each culture supernatant were submitted to 10% SDS-PAGE

and

the

resulting

profile

revealed

by

silver

staining.

CMC:

carboxymethylcellulose. Carbon sources concentrations were 20 g L-1, with exception to CMC that is 1 g L-1. Each lane represents biological replicates.

Figure 3. Effect of nitrogen content on culture media in the growth and protein secretion of Lentinus crinitus. 20 g L-1 starch, 20 g L-1 glycerol and 20 g L-1 maltose. Results are mean of three biological replicates and error bars represent the standard deviation of samples. One-way ANOVA followed by Tukey’s multiple comparisons test analysis were performed and statistical significance is demonstrated in the table with adjusted P-values of: < 0.0001 (****), < 0.001 (***), < 0.01 (**), < 0.05 (*). Each letter represents a particular culture condition. Letters in parenthesis displayed no significant differences among them.

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Figure 4. Effect of the culture media nitrogen-content on the protein secretion profile of Lentinus crinitus. Equal volumes of each culture supernatant were submitted to 10% SDS-PAGE and the resulting protein profiles revealed by silver staining. Nitrogen concentrations were: 100 mM NH4Cl, 20 mM NaNO3 and 100 mM urea. Carbon sources were used at 20 g L-1. Lanes represent different biological replicates.

Figure 5. Effect of water content on the secretion of proteins by Lentinus crinitus. Supernatants derived from liquid and solid cultures containing 20 g L-1 maltose and 100 mM urea were submitted to 10% SDS-PAGE and silver staining. Each lane represents biological replicates. Low water content cultures (solid) were obtained by adding vermiculite (0.12 g mL-1) to liquid media.

Figure 6. Dry biomass and concentration of secreted proteins in solid and liquid cultures. Assessed conditions were 20 g L-1 maltose and 100 mM urea in liquid media (liquid) and 20 g L-1 maltose and 100 mM urea in solid medium (solid). Results are mean of three independent samples with error bars representing the standard deviation. Statistical analysis was performed by unpaired t test with Welch’s correction. (*) indicates P-value < 0.05.

Figure 7. Lentinus crinitus secreted CAZymes activity in liquid and solid cultures. Secreted pectinase (A), xylanase (B) and FPase (C) activities were assessed in the following conditions: 20 g L-1 maltose and 100 mM urea in liquid media (liquid) and 20 g L-1 maltose and 100 mM urea in solid medium (solid). Error bars represent the standard deviation of the biological replicates from 3 independent experiments. EU/mL: 38 ACS Paragon Plus Environment

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enzyme units per milliliter of secretion extract used in the enzyme reaction. Unpaired t test with Welch’s correction was performed and P-values indicated: p < 0.001 (***) and P < 0.05(*).

Figure 8. Secreted oxidase/peroxidase activity in liquid and solid cultures of Lentinus crinitus. Assessed conditions were 20 g L-1 maltose and 100 mM urea in liquid media (liquid) and 20 g L-1 maltose and 100 mM urea in solid medium (solid). Total oxidase/peroxidase assays (A) used ABTS as substrate in the presence of MnSO4 and H2O2. Laccase assays (B) used only ABTS. Results are mean of three independent samples. Error bars represent the standard deviation of samples. EU/mL: enzyme units per milliliter of secretion extract used in the enzyme reaction. Statistical analysis was performed by unpaired t test with Welchs’s correction. P value < 0.001 (***).

Figure 9. Secreted DyP and azoR activities in solid and liquid cultures of Lentinus crinitus. Assessed conditions were 20 g L-1 maltose and 100 mM urea in liquid media (liquid and 20 g L-1 maltose and 100 mM urea in solid medium (solid). DyP activity in the presence (black) and absence (gray) of H2O2 (A). Azoreductase activity (B). Results are mean of triplicate experiments with errors bars representing the standard deviation between samples. EU/mL: enzyme units per milliliter of secretion extract used in the enzyme reaction. Unpaired t test with Welch’s correction was performed and p-values indicated: P < 0.01 (**) and P < 0.05 (*).

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Figure 10. Two dimensional gel electrophoresis of Lentinus crinitus secreted proteins derived from a mixture of liquid and solid cultures containing 20 g L-1 maltose and 100 mM urea. Numbered spots were submitted to protein identification. Box represents protein spots from different replicates of 2D-SDS-PAGE gels with different intensity of silver staining for better visualization.

Figure 11. Functional classification of proteins identified from Lentinus crinitus. Proteins were identified from four different sources: two intense protein bands (~42 kDa) from supernatant of solid cultures submitted to 1D-SDS-PAGE (named solid B1 and solid B2), LC-MS analysis and spots from 2D-SDS-PAGE. LC-MS and 2D-SDSPAGE results are derived from a pool of proteins from solid and liquid cultures containing 20 g L-1 maltose and 100 mM urea. Percentages are given according to total numbers of proteins from each experiment. ni: non identified proteins

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Figures Figure 1

Figure 2

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Figure 3

Figure 4

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Figure 5

Figure 6

Figure 7

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Figure 8

Figure 9

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Figure 10

Figure 11

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