Secretome Profile of Cellulomonas Sp. B6 Growing on Lignocellulosic

Accepted Article This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/jam.14176 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has CAZymes B6 sp. Cellulomonas HEAD RUNNING Mailing address:[email protected] [email protected]/ Campos Eleonora Dr. AUTHOR CORRESPONDING Universitaria (C1428EGA), Ciudad Autónoma de Buenos Aires, Argentina. CienciasExactas yNaturales, Universidad Buenos de Aires.IntendenteGüiraldes 2160 -Ciudad por y Biológicos Químicos Estudios de c) Centro Repetto (1686), Aires, Argentina. Buenos Hurlingham, Agronómicas(CICVyA),Nacional Instituto deTecnología Agropecuaria(INTA). yNicolas Los Reseros b) Institutode Microbiología yZoología Agrícola, Centro deInvestigación enCiencias Veterinarias y Reseros y Nicolas Repetto (1686), Hurlingham, Buenos Aires, Argentina. investig de Nacional Consejo (INTA), Agropecuaria deTecnología Nacional Instituto (IABIMO), Molecular yBiología deAgrobiotecnología a) Instituto Piccinni Elizabeth Florencia of profile Secretome TITLE Article -Original : type Article DR. ELEONORA 0000-0002-0887-6627) (OrcidCAMPOS : ID Mónica Talia a , Máximo Lisandro Rivarola Lisandro , Máximo Cellulomonas a

, Ornella Mailén Ontañon Mailén Ornella , sp. B6 growing on lignocellulosic substrates substrates lignocellulosic on growing B6 sp. a , María Pia Valacco Espectrometría de Masa (CEQUIBIEM). Facultad de (CEQUIBIEM). Facultad Masa de Espectrometría aciones Científicas y Tecn Científicas aciones Version of Record. Please Version as this cite of Record. article a , SilvinaGhio c , Eleonora Campos , Eleonora a , Diego Herman Sauka Herman , Diego ológicas (CONICET). ológicas (CONICET). Los a a,b , Paola , Paola Accepted Article Growth of of Growth Conclusions and an AA10.Surprisingly,among thexylanases, sevenGH10. were This by is protectedarticle reserved. Allrights copyright. industries. biotechnological and bioprocessing Cellulomonas Study the of Impact and Significance CAZymes. of repertoire anarabinofuranosidase/ a xyloglucanase, xylanases, exo-glucanases, endo-and including cultures, SCR and/or EWS from supernatants in identified were 22 secreted, to be GHs predicted 32 the From CAZymes. of diversity ofa identification the in resulted EWS or SCR on growth while xylanases, GH10 two and GH48) and (GH6 exo-glucanases two of secretion whichproteins playedrole in a lignocellulose deconstruction.Growth resulted on CMC in the or (SCR) straw cane sugar grinded (EWS), strawwheat of analysis secretome By (GH). hydrolases glycoside of genome the in CAZymes potential 205 identified We Results and Methods when grown on residual biomass for the formulationcost-efficient of enzymaticcocktails. products. value-added and biofuels obtaining for bottleneck a is deconstruction biomass Lignocellulosic Aims ABSTRACT grants PICT2014-0791 andPICT2016-4695. by of means of Argentina Technology and of Science by the Secretary supported work was This FUNDING

Our mainOur goal was characterize to secretome the of anovel isolate, Cellulomonas Cellulomonas sp. B6 could serve as a source of lignocellulose degrading enzymes applicable to to enzymes applicable degrading as serve could a B6 sp. oflignocellulose source sp. B6 on lignocellulosic biomass induced the secretion of a diverse diverse of a secretion the induced biomass on lignocellulosic sp. B6 supernatants from cultures in either extruded extruded from ineither cultures supernatants carboximethylcellulose (CMC), we identified Cellulomonas β -xylosidase, a -xylosidase, sp. B6, 91 of which were were ofwhich 91 B6, sp. Cellulomonas β -glucosidase sp. B6, Accepted Article hemicellulose can also occur in an oxidative way and cellulose of breakdown the that noting worth is It 2016). al., et (Biely xylan in found typically (3.2.1.131/139) and feruloyl3.2.1.73) (EC are esterases also requiredthe dueto many substituents enzymes, such as arabinofuranosidases 3.2.1.55), (EC arabinases (3.2.1.99), glucuronidases and backbone enzymes,endo- primarily The deconstruction of xylan occurs in a similar fashion and it involves the concerted action of several glucosidase (EC3.2.1.21) activity is required for theconversion final glucoseto (Lynd andnon-reducing end-acting cellobiohydrolases 3.2.1.176 (EC andEC 3.2.1.91, respectively). Also, and cellobiose to converted (Pauly polymer substituted heavily and short a is cellulose, This by is protectedarticle reserved. Allrights copyright. products. value-added and biofuels obtaining for resource an is renewable and trees, abundant part pastures ofcrops, non-edible the biomass, Lignocellulosic INTRODUCTION LIGNOCELLULOSE. CELLULOMONAS; GLYCOSYL GLUCANASES; CAZYMES; HYDROLASES; XYLANASES; GH10; SECRETOME; KEYWORDS Xylan, the most abundant hemicellulose, consists of of consists hemicellulose, abundant most the Xylan, carbohydrates. are hemicelluloses alcohols, organic of made is lignin While material. recalcitrant among bonds Chemical hemicellulose. and lignin regions (Lynd amorphous some only with structure, non-soluble, crystalline, a forming therefore chains, among polymer of to and 50% to 35% 20 of plantdry weight, respectively), welllignin. as as aislinear Cellulose large (35 walls cell plant of components major the are which hemicelluloses, and cellulose polysaccharides β -1,4-linked glucose units. It is organized in fibers that are stabilized by hydrogen bonds et al. β -1,4-xylosidases (3.2.1.37)further to hydrolyze oligomers intoxylose. Debranching 2002; Mischnick and Momcilovic2010). Cellulose is coated in a complex matrix of β -1,4-xylanases 3.2.1.8) (EC that release xylooligomers (XOS) from the xylan cello-oligomers by endo- by cello-oligomers by lytic polysaccharide mono-oxygenases (LPMOs), (LPMOs), mono-oxygenases polysaccharide lytic by all components make plant cell wall a highly a highly wall cell plant make components all β -1,4-glucanases (EC3.2.1.8) and both reducing β -1,4- linked xylose units and, contrary to to contrary and, units xylose linked -1,4- et al. 2013;Zhao

It is formed by the structural structural bythe is formed It et al. 2015). Cellulose 2015). is et al. 2002). 2002). β - Accepted Article Wakarchuk 2014; Lisov activity (Din their confirming characterized, also been have of species both hydrolases glycoside individual (Ponce-Noyol utilization polysaccharides suchxylanascellulose, or revealingcomplex aarray enzymatic of involved activities in Cellulomonas flavigena are genus ofthe members Themost characterized 2015). Schumann have the ability to degrade cellulose and other polysaccharides such as xylan (Stackebrandt and The facult cellulolytic and reported Clostridiales Actinomycetales and Shoham 2003). Many aerobic cellulolytic bacteria belong to the phylum (Shallom sugars ofsmall by internalization followed chains, polysaccharide from oligosaccharides large mainly generate that enzymes soluble of secretion the in consists and microorganisms aerobic (Lamed in described first cellulosome, the called enzymes, of complex bound 2015; Lynd Bhattacharya 2000; (Bhat production bioethanol generation second and detergents laundry feed, animal paper-pulp, food, in applications potential for studied are systems lignocellulolytic This by is protectedarticle reserved. Allrights copyright. important in recyclingthe ofcarbon frommatter dead in soil (Bomble are which bacteria and fungi hydrolyzed by aremostly hemicellulose and cellulose In nature, (Lombard (http://www.cazy.org) CAZy database inthe structure their to according classified are and bonds glycosidic create or modify, degrade, al., Loose2017; etal., 2016). All these enzymes, known as CAZymes (carbohydrate active enzymes), hydrol ofglycoside activity overall the addto which Cellulomonas etal. . Among the the . Among et al. et al. 1983; Bayer whereas most anaerobic bacteria belong to the phylum phylum the to belong bacteria anaerobic most whereas 2017; Toushik 2017; genus comprises Gram positive, non sporulating, coryneform actinobacteria that that actinobacteria coryneform sporulating, non Grampositive, comprises genus 1990; Mayorga-Reyes 1990; . Their (hemi)cellulolytic system was studiedbygrowth in defined substrates, et al. Actinobacteria et al. 2017; Yang 2017; ative anaerobes (Christopherson (Christopherson anaerobes ative 1998). The strategy The 1998). for (hemi)celluloseconversion isdifferent in et al. 2017). Anaerobiccellulolytic bacteria produce amembrane , members of the genus genus the of members , et al. a and De La Torre 2001; Amaya-Delgado Amaya-Delgado 2001; La Torre De a and et al. 2015). 2015). et al. 2002; Santiago-Hernández Santiago-Hernández 2002; ases (Hemsworthases etal., 2016; Kruer-Zerhusen et 2014). et al. al. et Cellulomonas 2013). Clostridium thermocellum Cellulomonas fimi fimi Cellulomonas et al. Actinobacteria are the onlyknown the are et al. et al. Firmicutes 2007; Gao and 2017). Their 2006). 2006). Some , order , order , order , order et and

al.

Accepted Article 1 This by is protectedarticle reserved. Allrights copyright. a we isolated al. (Elberson polysaccharides of structural degradation novel revealed habitats cellulolytic of diverse prospection culture and metagenomic isolation, first their Since preservation, individual clones were grown in LB supplemented with 5 g l 5g with LBsupplemented in grown were clones individual preservation, at stored and 30°C at overnight Cellulomonas Cellulomonas Organism METHODS AND MATERIAL genus. aerobic degradation of structural polysaccharides (cellulose and hemicellulose) of this important of mechanism core the understand to contribute also and cocktail enzymatic cost-efficient a develop Cellulomonas The main goal of this study was to identify the (hemi)cellulose degrading enzymes secreted by sources. carbon sole as orbiomass cellulose with media minimal grow on to capacity B6, the had sp. isolation was achieved by culture in minimal saline medium (MM) (K (MM) medium saline inminimal culture by achieved was isolation native subtropical forest soil sample(NCBI BioSample: SAMN04287673) (Campos cultures in solid minimal medium MM- g 15 l medium MM- minimal solid in cultures 0.5% CMC(low viscosity Sigma C5678)withfilter paper sole as carbon sourceand successive sub- activity monitoring, 0.1g l bioting l 0.01 , 0.05 NaCl gl 2013; Zhang sp. B6 when grown on residual inexpensive lignocellulosic substrates in order to to order in substrates lignocellulosic inexpensive residual on grown when B6 sp. -1 Cellulomonas sp. strain B6 was isolated from a bacterial consortium obtained from a preserved preserved from a obtained consortium bacterial from a isolated was B6 strain sp. , nicotinic 0.02gacid l -1 et al. et Cellulomonas , MgSO 2013;Zhuang 4 ∙ 7H -1 sp 2 Trypan Blue was added to the MM-CMC agar plates. For long term long For agar plates. the to MM-CMC added Blue was Trypan O 0.1 gO 0.1 l . isolates, which suggested theimportant role ofthisgenus in the − novel strain (Campos strain novel 80°C with 20% glycerol. et al. et -1 , pantothenic acid 0.01 l g 0.01 acid , pantothenic -1 , CaCl 2015). From a subtropical forest soil cellulolytic consortium, consortium, cellulolytic soil forest subtropical a From 2015). 2 0.04 g l -1 agar (MM-CMC) (Ghio (Ghio (MM-CMC) agar et al. et al. -1 , FeCl , FeCl 0.004 gl 2000; Rusznyák2000; 2014). This isolate, This2014). named -1 , NH 2 4 HPO Cl 1.0 gl 1.0 Cl -1 , NaMoO4 4 et al. et al. 1.67 g l -1 CMC, allowed to grow -1 2012). For cellulase 2012). For ) supplemented with with supplemented ) 2011; Hatayama 2011; -1 ∙ et al. 2H , KH 2 O g0.005 l Cellulomonas 2 PO 2014). The 2014). 4 0.87 g l g 0.87 et -1 , - Accepted Article For 2000 bootstrap repetitions. with 1993) Nei and (Tamura Likelihood Maximum using obtained were results Similar outgroup. (Cole (https://rdp.cme.msu.edu/) This by is protectedarticle reserved. Allrights copyright. flavigena ofall sequences (https://www.ncbi.nlm.nih.gov/genome/13845?genome_assembly_id=259466) rRNA and 16S the genome of (Kumar MEGA7 Statistical significances assessedwere using 2000 bootstrapreplications (Felsensteinwith 1985) A phylogenetic was tree constructedusing Neighbour Joining method (Saitou andNei 1987). characterization strain and analysis Phylogenetic Microbial Genomics Laboratory (Georgia Institute of Technology) (Goris (Goris ofTechnology) Institute (Georgia Laboratory Genomics Microbial Environmental the by developed (http://enve-omics.ce.gatech.edu/) tools online the with calculated Microorganisms and Cell Cultures. Average Nucleotide Identity and Average AminoacidIdentity were of –GermanCollection DSMZ Institute Leibniz the from (http://ggdc.dsmz.de/home.php) (GCA_001462455.1) was performed using online the Genome to Genome Distance Calculator tool (bioMérieux), accordingthe manufacturer’s to indications. of characteristics Biochemical hybridization (Meier-Kolthoff (https://www.ncbi.nlm.nih.gov/assembly) (Agarwala (GCA_001315245.1) wereobtained fromAssembly NCBI the database in-silico ATCC 482 (GCA_000092865.1) ATCC482 and estimation of Hybridization, DNA-DNA et al. Cellulomonas 2016). For this analysis, we used with the full 16S rRNA coding sequence from from sequence coding rRNA 16S full the with weused this analysis, For 2016). Cellulomonas Cellulomonas et al. sp et al. al. et . sp. B6(ATM99_00120) (Piccinni etal., 2016) type strains available at the Ribosomal Database Project Project Database Ribosomal the at available strains type 2013) with 2013) 2014).The was rooted using tree sp. B6were determined sp. using API galleries 50CH

Cellulomonas the genomic sequences for sequences genomic the et al. Cellulomonas persica 2017). Estimation of DNA-DNA Estimation DNA-DNA of 2017). sp. B6 genomic B6 sp. sequence et al. et al. 2007). 2007). Micrococcus luteus ATCC 700642 ATCC Cellulomonas as as T

Accepted Article This by is protectedarticle reserved. Allrights copyright. gl 5 with supplemented 1951), (Bertani (LB) Broth Lysogeny on or source Cellulomonas assays activity for cultures Bacterial preservationpurposes. Bacterialpellet was stored -20°Cat and then suspended0.1 in mol l C5678), 10 g l (200 rpm) and harvested by centrifugation at 5000 g for 20 minutes at 4°C, at 72 h, which h,which 72 4°C,at at minutes gfor 20 5000 at centrifugation by harvested rpm) and (200 orbital an in 30°C at grown was culture The (EWS). remove traces of biomass. Sodium (NaN azide Sodium ofbiomass. remove traces on or SCR EWS were filtered through #32glass fiber (1.2 µm) filter paper (Schleicher and Schnell) to grown from cultures the free supernatants in Cell phase cases. all exponential late to corresponded temperature on CMCase and xylanase activity profiles wasstudied using0.3 mol l and of pH The effect conditions. assay the min, under per equivalents or xylose of glucose µmol 1 (IU) per ml of enzymatic extract. One IU was defi Units International as was expressed Activity calculations. prior further to subtracted were detected “substrate without enzyme” and “enzyme without subs of controls cases, all In nm. 540 at absorbance the on based equivalents sugars reducing the andBisaria 1987; Ghio (Ghose protocols described already to according mixture reaction the to (DNS) acid dinitrosalicylic agitation (400 rpm), after which resulting the re substrates, respectively. The enzymatic extracts were incubated with the substrates for 30 min with citrate/phosphate buffer,pH from50°C,and 4.5to 8 atwith at pH 7, temperaturesfrom to 35°C enzymatictested wereusing extracts) g l 10 activities xylanase and (CMCase) Endoglucanase Measurement of enzymatic activity phosphate pH7 buffer toanalyze intracellular activity. sp. B6 was grown on minimal medium (MM) with 0.1% yeast extract (YE) as a nitrogen a as nitrogen (YE) extract yeast 0.1% with medium (MM) onminimal grown B6 was sp. -1 grinded sugar cane straw (SCR) or (SCR) gl 10 canestraw sugar grinded et al. et 2016). Standard curves using glucose or xylose were used to determine determine to used were xylose or glucose using curves Standard 2016). -1 ned the ofenzymeas amount necessary to release CMC and 5 g l 5 CMC and shaker (Innova 42, NewBrunswick) with agitation agitation with NewBrunswick) 42, (Innova shaker 3 in cell the supernatants free (extracellular ) at 0.04% was added to supernatants for for supernatants to added was 0.04% at ) ducing sugars were detected by addition of of byaddition weredetected sugars ducing trate” were included and the reducing sugars reducing sugars andthe wereincluded trate” -1 wheat straw pre-treated by extrusion by extrusion pre-treated straw wheat -1 beechwood xylan (SIGMA) as (SIGMA) xylan beechwood -1 CMC(lowviscosity, Sigma -1 -1

Accepted Article This by is protectedarticle reserved. Allrights copyright. p-nitrophenyl using assayed were extracts β above. described as detected was activity remaining the h and 48 and 24 5, 3, 1, 0, at collected stability of thecellsupernatants free was by tested incubation 45 andat 50°C. wereSamples xylanase and endoglucanase The respectively). activity, orxylanase CMCase (for 75°C or 60°C visualization. product for used was orcinol 1% with solution water ethanol: acid: sulphuric A3:70:20 (https://www.megazyme.com/). and Megazyme Sigma from purchased were monosaccharides and Oligo activity. cellooligosaccharides (G2 to G4) and glucose mg/ml0.25 were used as standards for CMCase and xylose 0.25mg/ml eachwereused as standards for xylanase activity. Similarly, watermix andsilica as mobile and stationaryphases, respectively. Xylooligosaccharides (X2to X6) acid: acetic 2:1:1 butanol: a TLCusing by detected were products reaction andCMCase Xylanase Layer Chromatography (TLC) Detection reaction byThin enzymatic of products hypothesis. null the as supernatant” sp. B6 on different substrates of “growth with USA), CA, Diego, San (GraphPad, v6.0 software Prism GraphPad using does not affect xylanase byof weredetermined analysis assays activity or CMCase activity (ThermoF Kit Assay Protein BCA Micro commercial ofthe cell free cultureexperiments were conducted in triplicates. Protein concentration was determined using the 1 released that enzyme of amount the cellobioside 5mmol (pNPC) l p-nitrophenol (pNP) standard curve (Ghose and Bisaria, 1987). Bisaria, and (Ghose curve standard (pNP) p-nitrophenol basedon was andactivity determined nm measured 410 was at Absorbance as Aldrich) substrates. -glucosidase, cellobiohydrolase and -1 andp-nitrophenyl- β -xylosidaseactivities in intracellular the andextracellular β μ - mol of pNP per min under the assay conditions. All All conditions. the assay under min per mol of pNP D -glucopyranoside (pNPG) 5 mmol mmol 5 l (pNPG) -glucopyranoside variance (ANOVA)followed by Tukey’s(P<0.05) test β - ischer). Significant differences enzymatic of D -xylopyranoside (pNPX) 4 mmol l mmol 4 (pNPX) -xylopyranoside

One international unit was defined as as defined was unit international One -1 , p-nitrophenyl , p-nitrophenyl Cellulomonas -1 (Sigma- β - D - Accepted Article This by is protectedarticle reserved. Allrights copyright. (Altschul (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) BLASTp using homology of sequence confirmation and dbCAN with annotation CAZY andspecific pipelines and NCBI RAST performed with Cellulomonas CAZYmes (Yin al.,et 2012) into order identify potential CAZymesgenomicthe in sequence of of models and HMM signatures unique account into takes which (http://csbl.bmb.uga.edu/dbCAN/), The predicted CAZome of CAZymes secreted of Prediction soluble lignin, %ashes7.7 and 15.9% extractives. 0.1% acetic acid. SCR:32.6% cellulose,% 23.9hemicellulose, acid 14.9% insoluble lignin,5.1% acid cellulose, 30.0 % hemicellulose, 19.8% acid insoluble lignin, 1.3% acid soluble lignin, 2.1% ashes and biomasscomposition determinedwas at CIEMAT following specifications. NREL EWS: 46.8% Pre-treated Argentina). (Tucumán, EEA INTA from Famaillá Valeiro by Alejandro provided Dr. Secades Manzanares Paloma Dr. and Ballesteros EWSstraw (pre-treated by extrusion70ºC,at NaOH pretreatment and analysis compositional Biomasses bacteria was used for prediction of signal peptides (Käll (Käll peptides signal of forprediction used was bacteria categorized as high. as categorized of secreted CAZymes of (Dyrløv Bendtsen pathways by secreted non-classical proteins extracellular SecretomeP 2.0 (http://www.cbs.dtu.dk/services/SecretomeP/)was for prediction used of wascarried out bysequ Blastp et al. sp. B6(NCBI accession number LNTD00000000.1). Full genome annotationwas 1990). SignalP v4.1(http://www.cbs.dtu.dk/services/SignalP/) for Gram positive

Cellulomonas Cellulomonas ence alignment. An identity ence identity alignment. An sp. B6 and B6 sp. sp. B6 was constructed using dbCAN algorithm algorithm dbCAN using constructed was B6 sp. Cellulomonas 6%, 2mm) was gently provided by Dr. Mercedes Mercedes by Dr. provided gently was 6%,2mm) from CIEMAT (Madrid, Spain). SCR was gently was SCR Spain). (Madrid, from CIEMAT et al. ≥ 90%, cover with query a

fimi 2004; Petersen ATCC 484 (Spertino et al., 2018)al., (Spertino et 484 ATCC et al et . 2005). Comparisons. 2005). et al . 2011) and ≥ 70%, was was 70%, Accepted Article This by is protectedarticle reserved. Allrights copyright. then precipitated with 10%trichloroacetic andacid resuspended (TCA) in water(18 supernat free cell from Proteins before. described Bacteria were grown supplementedonMM with 0.5% sucrose, 1% or CMC, 1%SCR 1% as EWS spectrometry mass by analysis Secretome resolution. The flow rate used fo used rate flow The resolution. PepMap RSLC (P/N ES801) suitable forseparating complex peptide mixtures witha high degree of phasefor min column using mm)reverse Easy-Spray 120 a x150 2 µm, µm (C18, 100A, Column 50 For the LC-MS/MS analysis, approximately 1 Scientific Q-Exactive Mass Spectrometer coupled to (Promega V5111) overnight at 37°C. digestsThe were analyzed by nano LC-MS/MS in a Thermo 56 min at mM for45 10 dithiotreitol werereducedwith Proteins (http://cequibiem.qb.fcen.uba.ar/). CEQUIBIEM at performed were analysis Spectrometry andMass digestion Protein assay. CAZYmes secretion. Sucrose was used instead of gl for control negative a as used were sucrose on grown cultures from Supernatants protein. total unassigned charge state were excluded from fragmentation for MS2. MS2. for fragmentation from excluded were state charge unassigned with or of +1 charge a with Peptides analyser. Orbitrap the in measured byHCD and fragmented of at70000 400 m/z and the 12 most intense ions in eachcycle, were sequentially isolated, were acquired in the Orbitrap analyser. The scanned mass range was 400-2000 m/z, at a resolution peptideidentification at the same time of their chromatographic separation. Full-scan mass spectra allow to configuration andequipment acquisition fordata used was software Scientific) (Thermo (HCD) for fragmentation and an Orbitrap analyzer (Thermo Scientific, Q-Exactive). XCalibur 3.0.63 Ionization(Thermo Scientific, EASY-SPRAY). The MS equipmenthas ahighcollision dissociation cell Spray Electro for used was kV 3,5 of voltage A 2µL. was volume injection The acetonitrile. in B (5 min) to 35% (120min). Solvent A was 0.1% formic acid in water whereas B was 0.1% formic acid ° C, alkylated with iodoacetamide (55 mmol l r the nano column was 300 nl min nl 300 was column nano r the μ g of peptides was loaded onto the column and eluted eluted and column the onto loaded was peptides of g -1 ) for 45 min in the dark and digested with trypsin trypsin with digested darkand the in min 45 ) for ants were quantified by Bradford assay (Promega), (Promega), by assay Bradford ants werequantified a nano HPLC EASY-nLC 1000 (Thermo Scientific). Scientific). (Thermo 1000 EASY-nLC HPLC nano a ucose to avoid interference with DNS activity -1 from and the solvent range 7% Ω ) to 1 mg/ml Accepted Article This by is protectedarticle reserved. Allrights copyright. strains the of amember as (Piccinni genome its sequenced subsequently and Argentina in sample soil forest sub-tropical native a from B6 strain the isolated had we study, previous In a (Ishihama %(emPAI%) mol in content protein the emPAI/ equation The data. identification protein using Sequest used we abundance, ofrelative the For estimation strategy. database reverse false discovery employing calculated of a by 1% rate confidence forhigh filtered were hits Protein acetylation. and N-terminal carbamidomethylation toset were modificationsof Cys,and dynamic oxidation set to were ofMet modifications Da. Static 0.05 to tolerance ion ppmandproduct of10 tolerance mass a precursor Proteomewith Discovererper peptide. performed searches were missed maximum cleavage one of depositedSRA databaseat NCBI under accession the LNTD00000000) withtrypsin specificity anda Scientific) and searched against Q Exactive raw data were processed usingProteome Discoverer software(version 2.1.1.21 Thermo Genomic and phenotypic characterization of of characterization phenotypic Genomic and RESULTS Identity (ANI). It has been reported that to consider genomes of be from the same species DDH be fromthe species of same genomes consider to that been reported Ithas (ANI). Identity resulted in an for reported been had and what 97.73% 16S rRNA sequenceidentity with 2000) (Table S1). Genome comparisons between API CHtestwhich 50 resulteddifferences in between results the obtainedfor a was novel it whether to establish made difficult C. flavigena in-silico ATCC 482 and Cellulomonas 25.40% DNA-DNA 25.40% hybridization (DDH) and 83.71% Average Nucleotide Cellulomonas C. flavigena genus by 16S rRNA sequence analysis, closely related to type type to related closely analysis, sequence rRNA by 16S genus C. persica C. flavigena ATCC700642 Cellulomonas Cellulomonas peptide matches with a maximum protein and peptide and peptide protein maximum a with matches peptide sp. B6 protein sequences database (from the genome genome the (from database sequences protein B6 sp. (Ahmed (Ahmed et al et the protein abundance index emPAI calculated by by emPAI calculated index abundance protein the species. Regarding biochemical assays, we run an an run we assays, biochemical Regarding species. Cellulomonas 2005, Shinoda ATCC 482 ATCC and 98.54% with et al T sp. B6 B6 sp. (Figure 1). . 2014) and et al. et Σ (emPAI) x 100was used to calculate sp. B6 and B6 sp. 2016). This isolate was classified et al Cellulomonas . 2010). 2010). . C. persica C. flavigena Cellulomonas (Elberson (Elberson C. persica sp. B6shared ATCC482 sp.B6 which et al . Accepted Article AAI values between AAI valuesbetween the most studiedstrain of the with the analysis conducted We also new the name and species. describe Enzymatic activities of Enzymaticof activities respectively (Table S2). assigned to either (9.14 ± mU0.28 ml This by is protectedarticle reserved. Allrights copyright. late log phase and cell density were 9.66 ± 0.19; 9.77 ± 0.23 and 10.27 ± 0.08 log xylanase activities at 72 h in the supernatant from MM-SCR cultures (figure AllS1). cultures were in and CMCase highest achieving (SCR), straw sugarcane and grinded (CMC) carboxymethylcelulose Cellulomonas strain this from sequences protein persica Cellulomonas Thompson values should be above70% and ANI values above 95% (Goris and 277.65 ± 0.18 mU ml pH 7for CMCase (0.16 ± 0.01 U ml and respectively. EWS Optimal enzymatic activities, MM-SRC culturesobtained from were50°C, at cellobioside)was also observed in the extracellular extract (2.09 ± 0.12 mU ml tolerant to higher temperatures than enzymes active on CMC. Some cellobiohydrolase activity (pNP- xylan-active enzymes secreted by that suggest could This 2). (Figure h 24 at 80% than more decreased activity CMCase hwhile 48 to forup preserved was activity of over 60% 50°C, xylanase at incubated were when the supernatants β -xylosidase activities were detected mainly in the intracellular fraction (28.58 ± 6.05 and was alsolow (29.9%),butandcouldAAI ANI not calculated, be as thecomplete genome and et al sp. B6, was able to grow on LB and minimal media (MM) supplemented with with supplemented (MM) media minimal LB and on grow to able was B6, sp. sp. . 2013) (Table S2). Average Aminoacid Identity (AAI) between between Aminoacid Average (Table (AAI) S2). Identity . 2013)

B6 was 79.14%, which further supported these differences. differences. these supported further which 79.14%, was B6 C. flavigena C. flavigena -1 -1 ) and Cellulomonas on pNPG and pNPX, respectively). Very low extracellular extracellular Very low respectively). pNPX, and pNPG on Cellulomonas β -glucosidase activity 0.14 (0.58± mU ml Cellulomonas or -1 were not available. Therefore available. were Therefore not sp. B6 vs Cellulomonas C. persica ) and sp. B6 B6 sp.

at 60°C,6 for pH xylanase 0.01ml U(0.41 ± genus. Percentage of genus. 16S rRNAPercentage similarity, DDH, ANI and . Additional assays are being conducted in order to to in order conducted being are assays Additional . C. fimi sp. B6 when grown on MM-SCR could be more more be could on MM-SCR grown when B6 sp. were 95.07%,21.70%, 80.95% and 70.36% Cellulomonas Cellulomonas et al -1 ) were observed.) Consistent . 2007; Auch C. fimi -1 In-silico ) while β sp 10 typestrain, it is as -xylosidase activity activity -xylosidase ml . C. flavigena B6 could not be be not could B6 -1 -1 β etal ) DDHwith for CMC,for SCR . -glucosidase In addition, addition, In . 2010; and C. Accepted Article a GH18 chitinase (EC 3.2.1.14) and aGH5 one could be secreted by non-classical pathways. Additionally, a GH74 xyloglucanase (EC 3.2.1.151), xylooligosaccharides (Shallom and Shoham 2003). Twoother GH43 had signalpeptide and another hypo could that intracellular, be to predicted were GH43 and two GH39 two identified We hydrolysis. xylan from role adifferent indicate may which analysis), (byboth beintracellular to predicted was (KSW28941.1) GH10 was bySecretomeP server. Anotherwas asextracellular but predicted identified, peptide nosignal so portion aminoterminal the in incomplete was sequence (KSW14744.1) GH10 (EC 3.2.1.8), of which only one was classified andGH11 as restwere GH10. the From these, one in more detail (Figure 3, Table 1). Regarding hemicelluloses deconstruction,we identified 9xylanases analyzed were LPMOs, as well as polysaccharides, structural on activity GH predicted Enzymes with S3).SecretomeP server(Table secretion by for werethreshold considered below the Bendtsen (Dyrløv sequence) This by is protectedarticle reserved. Allrights copyright. se by non-classical beextracellular to predicted bysecreted classical pathwaysignal peptide (Petersen polysaccharide lyase (PL) was identified (Table S3). From the 205CAZYmes, were predicted48 to be catalytic and domain) (CBM) 10 (5%) were proteins with auxiliary activities (AA), while only one known not (and modules only carbohydrate-binding with proteins were (6%) (CE), 12 esterases glycoside hydrolases (GH), (28%) were 57 glycosyl transferases, 34(17%) carbohydratewere the CAZy database (Yin of in genome) the encoded CAZymes potential Inthe interest ofassigning observed the activities to specific enzymes,studied we the CAZome(all In silico cello-oligosaccharides,while no glucose andonly trac with these results, the main reaction products and of CMCase xylanase activities were xylo- and prediction of extracellular CAZome CAZome extracellular of prediction et al . Weidentified2012). a total of 205CAZymes, fromwhich (48%)91 were et al . Interestingly,2005). proteins12 with predicted signalpeptide β -mannosidase (EC3.2.1.25) werealso predicted to be Cellulomonas cretion pathways (independe pathways cretion theticallybe involved infinal the degradation of es of xylose (X1) were detected (Figure 2). 2). (Figure detected were (X1) xylose of es et al. 2011) and additional 28 proteins were proteins 28 andadditional 2011) sp. B6, using the dbCAN algorithm and and algorithm dbCAN the using B6, sp. β -xylosidases (EC 3.2.1.37) which nt of signal peptide Accepted Article grown on 10 g l g on 10 grown mg ml mg This by is protectedarticle reserved. Allrights copyright. analyzedshotgun by proteomics the secretomeof To clarify further which predicted CAZYmes were proteomics shotgun by supernatants culture EWS and SCR CMC, of analysis Secretome modules. these of function biological the clarify would proteins purified the with reports are available in the literature regarding simi domain (cd00161), involved in carbohydrate binding beta-trefoil aricin-type as annotated but algorithm dbCAN by domain GH5 a as identified module anadditional with associated were (KSW21678.1) GH43 the of and one (KSW17752.1) GH62 The S2). (Figure strains both between of CAZome the found in bynon-cla be secreted to predicted were which families GH1 (3) and GH3 which (8) were mostly predicted to beintracellular, exceptfor two GH3, cellulose deconstruction by oxidation. Finally we identified 11 on havearole could which AA10 offamily monooxygenases secreted potentially two identified and sixextracellular endo-glucanases(EC 3.2.1.4) (twoGH6 and GH9). four Furthermore,we glucanases,GH6 and GH48(non-reducing EC 3.2.1.91 and reducing3.2.1.176, end EC respectively), Regarding breakdown the of cellulose oligosaccharides, to weidentified only two extracellularexo- study. this in identified domains catalytic multiple with protein only the was and modules, catalytic four presented xyloglucanase GH74 the Noteworthy, secreted. activities were higher in theextracts from and SCR than EWS from CMC(0.79 ± 0.10, 0.58 ± 0.07 and activity in thewere tested culture supernatants.expected, As and protein enzymatic the content Prior secretion). to CAZymes for control (negative 0.11 0.05 ± mgml -1 ) as CMC,butwithnegative) as results regarding enzymatic activities 4). (Figure -1 CMC, 10 g l -1 , respectively). resulted sucrose The control Cellulomonas -1 SCR and 10 g l g 10 and SCR sp. B6 and B6 sp. -1 extrudedwheat straw (EWS), using5 g l sucrose ssical pathways (Table S3). Also, the differences differences the Also, S3). (Table pathways ssical proteomic analysis, protein secretion and overall overall and secretion protein analysis, proteomic lar proteins. Therefore, only experimental assays assays only experimental Therefore, proteins. lar effectively being expressed andsecreted, we C. flavigena , by NCBI. To thebest of our knowledge, no Cellulomonas in similar protein content (0.13 ± 0.05 (0.13 content protein similar in further supported the differences differences the supported further β -glucosidases (EC 3.2.1.21) from B6 supernatants from cultures cultures from B6 supernatants -1 as Accepted Article This by is protectedarticle reserved. Allrights copyright. Bendtsen (Dyrløv understood yet not is secreted be could they bywhich pathways) ornon-classical independent peptide (signal mechanism The server. SecretomeP by extracellular be to werepredicted but sequence peptide signal a have not did that supernatants culture in identified proteins those closely analyzed also in CMCase and xylanse activitycorrelated levels withthe total proteins and identified. CAZYmes We observed differences the activity, its to related directly not is enzyme particular a of abundance the 22 and18 were CAZYmes, respectively, corresponding proteins and65 and 78 EWS, SCR In were CAZYmes. of which four identified, were peptide signal with proteins seven supernatants, InCMC analysis. pr experimental during cell lysis dueto presumably proteins, intracellular to correspond could SecretomeP, by extracellular be to predicted and not taken intoconsideration Proteins S4). (Table identifiedsupernatants in the without signalpeptide were replicates one of the in peptide unique one matching least by at detected sequence peptide compared to the potentially secreted enzymes.All CAZy domain-containingproteins with signal and bymass spectrometry analyzed were supernatants cell-free the in proteins Extracellular (KSW14744.1 and (KSW14744.1 KSW20567.1 and KSW18330.1) and theonly GH11(KSW19115.1), whileadditional two GH10s InSCR and EWS culture supernatantswe alsoidentified other three GH10 xylanases (KSW16349.1, Cellulomonas et al. secretomethe of in identified recentlybeen also had GH6 GH48 and offamilies Cellobiohydrolases utilization. forpolysaccharides importance andKSW29966.1) were secreted bygrowth withall substrates (CMC, SCR and EWS),indicating their familiesGH6 (KSW15663.1) and GH48 (KSW15916.1),GH10asxylanasesas well three (KSW23552.1 2, (Table identified were 29 dependent), peptide (signal pathway byaclassical secreted potentially proteins domain-containing CAZy the 48 From 2018), supporting the supporting 2018), relevance of theseenzymes the genus. genus. KSW21671.1 et al ) were also present in SCR (Table 2). Therefore, all extracellular extracellular all Therefore, 2). (Table SCR in present also ) were . 2005). . 2005). C. fimi Table S3). Among them, the fromexo-glucanases when grown on CMCand wheat straw (Spertino ocedure, and were therefore not included in the the in included not therefore were and ocedure, with signal peptide were identified, from which which from identified, were peptide signal with to approximately 28% bothin cases. Although in the lignocellulose degrading activity of the the of activity degrading lignocellulose the in Accepted Article containing protein in the extract from the EWS culture, both predicted to be secreted by signal bysignal secreted be to predicted both culture, EWS the from extract the in protein containing This by is protectedarticle reserved. Allrights copyright. a GH3 detected we Interestingly, substrates. cellulosic by induction secretion confirming and secretion for CAZymes described endogalactosaminidase GH114 domain), thus validating this culturecondition as control doma CBM32 a with function ofundefined a protein need to befurther investigated. Inthesucrose cont ina transcriptional unit, although proteins roleand the ofthese the regulation of expression their down components forABCtransporter coding genes so with co-regulated possibly be could expression may of beinvolved in uptake the proteins These S5). (Table abundant highly were and extracts, all in present were transporters ABC carbohydrate from proteins binding substrate- that mentioning worth is It in EWS extracts. identified and theonlypredicted PL3(KSW13730.1), while a GH13 of two the predictedin the genome), GH27 a Other proteins were only present in SCR culture supernatants, including an AA10 (KSW19119.1) (one supporting relevance its in further CAZymes, abundant most the one also was secretomes inall identified (KSW23552.1) GH10 The (Table S5). estimated was in sample each proteins all abundance of relative The activity. KSW29693.1, KSW30160.1 and KSW29929.1) with no assignedcatalytic module or enzymatic putative esterases Pfam domains (CE1), and four CBM-containing proteins (KSW23491.1, as hypothetical three proteins (KSW15560.1, KSW21470.1 and KSW21507.1), which presented weidenti andEWS secretomes, SCR In conditions. assay under our supernatants, culture CMC in present were noendoglucanases Surprisingly, GH6). GH9 and a four (the cultures biomass from supernatants in endoglucanases predicted seven the account for the xylanolyticactivity observed in and SCR EWS supernatants. We also identified five of presen were genome the in encoded endoxylanases β -glucosidase in supernatants from the SCR and EWS cultures and a CBM32 domain- andaCBM32 cultures EWS and SCR the from supernatants in -glucosidase Cellulomonas the small sugars resulting from polysaccharides hydrolysis and their their and hydrolysis frompolysaccharides resulting sugars small the sp. B6 polysaccharides utilization. utilization. polysaccharides B6 sp. α -galactosidase (KSW30136.1), a GH43 (KSW21678.1) fied enzymes from GH62, GH18 and GH74, as well well as GH74, and GH18 GH62, from enzymes fied rol we only identified three proteins (an esterase, (anesterase, proteins three identified only we rol me of the enzymes. For example, we identified stream of the GH6 exo-glucanase gene, possibly possibly gene, the GH6 exo-glucanase of stream t in biomass culture supernatants, which could could which supernatants, culture biomass in t in and a hypothetical protein with a recently a recently with protein hypothetical a in and α -1,6-glucosidase (KSW29886.1)was Accepted Article This by is protectedarticle reserved. Allrights copyright. activities lignocellulose-based industrial to feature anadvantageous is which offungi, those than shorter is cycle life biological Its production. 2G-ethanol as such applications, forseveral enzymes fungal or complement replace could Cellulomonas of proteins when grown on lowcost residual lignocellulosic biomass,sole as carbon source. In this work,we studied a novel isolate from the DISCUSSION identified in culture supernatants of for core the lignocellulose degradingmechanism the of enzymes with high sequence identity identifiedwere in both studies,corroborating their importance Eight results. of comparison the by drawn be can conclusions interesting used, was methodology Although straw. wheat study, In arecent Spertino solublecellulosic substrate, such (Figure 3). CMC as a pure on growth to compared secreted, was enzymes of diversity and number higher a substrate, culture as used was biomass lignocellulosic when summary, In pathways. peptide-independent Cellulomonas glucosidase (KSW29886.1) and the only GH18 (KSW18655.1). Other CAZymes secreted by GH13 a (KSW29966.1), xylanase aGH10 KSW29539.1), and (KSW29417.1 endoglucanases GH9 identityto CAZymes by encoded Cellulomonas by secreted CAZymes other However, S6). (Table lyase pectate PL3 a and arabinofuranosidase GH48 exoglucanases, GH6 and GH9 endoglucanases,a GH10 xylanase,GH74 a xyloglucanase, a GH62 were eight enzymes identifi is a very interesting genus as it is a bacterial source of extracellular CAZYmes, which which CAZYmes, extracellular of source bacterial a is it as genus interesting a very is sp. B6 had low sequence identity to proteins encoded by encoded proteins to identity lowsequence had B6 sp. sp. B6 when sp.grownon B6 cellulosic biomassthatalsohave homologues of high sequence Cellulomonas et al ed ed in thesecretome of (2018) analysed the secretome of C. fimi C. Cellulomonas sp. B6 and B6 sp. (Gomez Del Pulgar and Saadeddin 2014). 2014). Saadeddin Del and (GomezPulgar werenot identified in itsextracellularextract, suchtwo as sp. B6 (Table S6)(Spertino sp. etal., 2018). Cellulomonas C. fimi C. fimi Cellulomonas that are different species and a different adifferent and species different are

did not have homologues or were not not were or homologues have not did genus that presented high secretion secretion high presented that genus C. fimi genus. These were GH6 and GH6 and were These genus. when cultured on CMC and C. fimi . Conversely, there α -1,6- Accepted Article sugarml mgcaneto straw,between 1 0.5 varied culture by inlab yieldprotein secretion achieved and two GH10 were present in all culture conditions. Remarkably, the GH48 was the only one the GH48 was the Remarkably, conditions. culture inall werepresent GH10 and two GH6, GH48 a them, a Among pathways). by and3 non-classical peptide signal with (29 besecreted to By mass spectrometryof biomass-culture supernat more distant phylogenetically to isolate S2). B6(Figure al. Reyes reported levelsofprotein secretion(Din contrast, This by is protectedarticle reserved. Allrights copyright. described previously other of range Enzymatic activities and thermal stability of enzymatic extracts from to species a assign unequivocally to undertaken be will studies Further species. novel a be considered novelspecies,rather Cellulomonas and the presence of more than one GH11one we than more of the presence and and 113predicted GHs (CAZy Database) These results differentiated further genome of an organism) indicated the presence of 205CAZY proteins, from which were 91 GHs. The standardization. evidences reported here (including biochemical API results) indicated that that indicated results) API biochemical (including here reported evidences we (DDH), hybridization genome DNA AverageNu Aminoacid and Identity(AAI) 2007; Rusznyák Cellulomonas Cellulomonas et al.

had a GH74 and only one GH11 but presented a larger number of GH1 and GH3, and was was GH3, and GH1 and of number alarger GH11 presented but one only and GH74 a had 2002; Amaya-Delgado 2002; sp. B6 was closely related phylogenetically to the well-known well-known the to phylogenetically related closely was B6 sp. sp. B6isolate.sp. sp. B6 CAZome (the collection of carbohydrate-active enzymes encoded by the encoded enzymes carbohydrate-active of collection B6 CAZome (the sp. et al. C. persica 2011; Yang . However,. observed Average owing differences to inthe the B6 from the type strains of et al Cellulomonas et al. et could not place it in one species or the other. Inone other. theor fact, species itin the could not place . 2006; Sánchez-Herrera Sánchez-Herrera . 2006; . et al et In cleotidewell Identity as as (ANI) 2015; Kane 2015; and French In our 2018). study, the total C. flavigena, . 1990; Ponce-Noyola and DeLa 2001; Torre Mayorga- re among the most interesting differences. differences. most interesting the re among -1 species, although there were differences with the differences were species, althoughthere ants,we identified 21out of 32GHsthe predicted scale flasksusing biomass,wheat either straw or opening a promissory way for improvement and and improvement way for promissory a opening theabsence of GH74 and GH18 enzymes C. flavigena et al. et Cellulomonas 2007; Santiago-Hernández Santiago-Hernández 2007; Cellulomonas and and C. flavigena C. fimi, in-silico sp . B6 in were the which had 86 86 had which genome-to- sp. sp. B6 could C. fimi and to a a andto , by et Accepted Article supernatants when the bacterium was grown on wheat straw (Spertino (Spertino straw wheat on grown was bacterium the when supernatants This by is protectedarticle reserved. Allrights copyright. (Spertino supernantants culture straw (Shen supernatants exo-glucanase CbhB of (GH48), the from when GH11 detected xylanses were (Wakarchuk supernatants culture CMC of GH11 the Similarly, CMC. and not andEWS, on SCR grown of GH11 only The (Spertino of supernatants the in proteomics byshotgun detected substrateaffinity, remains unclear. In consistency with our results, GH10 enzymes were also in biomass. Whether all GH10 xylanases are necessary composition ofxylan heterogeneity the to could related be xylanases GH10 of redundancy apparent on grown biomass, When on CMC, cellulosicpurelya substrate. culture by induced were xylanases GH10 two that noteworthy also was It genome. the encoded in had been previously described (Mayorga-Reyes (Mayorga-Reyes described had been previously 2016) and thetranscriptional upregulationGH11 ofa supernatants of all cultures (biomass and CMC), supporting their role as the main cellulases of the cellulases main the as role their supporting and CMC), (biomass cultures all of supernatants 2018). In study,this exo-glucanaseswethe identified GH48GH6 ofand formingstable a complexand that this interaction couldresult in exo-exo synergy (Spertino ofthe presence cellulosic both a Previousthe identification studies reported different cultureconditions still needs further clarification. the although clear, seems biomass lignocellulosic et al. et al. 2018; Wakarchuk2018; Cellulomonas Cellulomonas et al . 1995). CbhA (GH6) and CbhB (GH48) were also present in in present were also (GH48) CbhB and CbhA(GH6) 1995). . Cellulomonas nd hemicellulosic substrates (Meinke substrates nd hemicellulosic sp. B6 was detected exclusively in the supernatants fromwas cultures sp. B6 detected exclusively supernatants inthe et al. et C. flavigena 2016). et al sp. B6 secreted seven GH10 and a GH11. The nature of this C. fimi et al . It2018). wassuggested that couldthese enzymes be of CbhA, a secreted GH6CbhA, of exo-glucanase, from . 2016). However, itwas not detected in et al. et and and was cultured on CMC and xylan (Wakarchuk (Wakarchuk xylan and CMC on cultured was fine-tunning of which xylanses are expressed under under areexpressed xylanses ofwhich fine-tunning 2002). Therefore, the induction of xylanases by xylanases of induction the Therefore, 2002). C. flavigena, in the presenceof lignocellulosic inthe a substrate for biomass deconstruction or have different different have or deconstruction biomass for C. fimi C. fimi C. fimi in CMC (but not in xylan) culture culture xylan) in not (but CMC in and was detected in xylan but not in in not but inxylan detected was C. et al et al. et al.

flavigena . 1994) and the presence presence the and 1994) . Cellulomonas 2018). Moreover,2018). three grown on CMC CMC on grown C. fimi sp. B6in C. fimi wheat wheat C. fimi et al. et al in .

Accepted Article This by is protectedarticle reserved. Allrights copyright. No conflict declared. of interest CONFLICT OFINTEREST Sabio y García for English language editing assistance andcritical reading of manuscript. the of ScientificmembersMR, the PT PVare Rese and article.post-doctoral has a OO fellowship fromholds SG CONICET. a fellowship from INTA.EC,DS, Argentina of Research for Council National the from fellowship doctoral a held and (UBA) Aires of Buenos University the of (FCEN) Sciences Exact and Natural of School the of (QB) Chemistry Biological of Department the of student Ph.D. a is FP ACKNOWLEDGMENTS cocktails. enzymatic bacterial (hemi)cellulolytic Cellulomonas resultsThe presentedthis work incontribute to establish core enzymesbythe used the polysa of degradation the for paradigm a establish Cellulomonas genus to degrade lignocellulosic biomass in order to develop novel cost-efficient cost-efficient novel develop to order in biomass lignocellulosic degrade to genus sp. genus. Whether they act individually or forming a stable complex will help to to help will complex astable orforming individually they act Whether genus. sp. ccharides by aerobic bacteria soluble enzymes. enzymes. soluble bacteria byaerobic ccharides (CONICET) during the activities described in in the described activities the during (CONICET) arch Career of CONICET. Authors thank Dr. Julia Dr. Authors thank arch CONICET. of Career Accepted Article This by is protectedarticle reserved. Allrights copyright. Biol Struct Opin Bayer, E.A., Chanzy, H., Lamed, R. and Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Sci Genomic comparison. sequence genome-to-genome of means by delineation species microbial for hybridization DNA-DNA Digital Göker, M. (2010). and Klenk, H.-P. M., vonJan, Auch, A.F., of densities cell different at bagasse cane bysugar xylanases of Induction (2006). M.C. Montes-Horcasitas, and M.E. Hidalgo-Lara, L., Dendooven, L.B., Flores-Cotera, J., Vega-Estrada, L., Amaya-Delgado, tool. search alignment local Basic (1990). D.J. Lipman, and E.W. Myers, W., Miller, W., Gish, S.F., Altschul, 2311. pakistanensis (2014). M. T. Ohkuma, and Fujiwara, T., Iino, M., Ehsan, S., T., Abbas, Kudo, Ahmed, I., Information. Biotechnology for Center National the of Resources Database (2017). K. Zbicz, E.and Yaschenko, W.J., M., Wilbur, Ward, Y., D., Wang, Vakatov, B.W., Trawick, K., Todorov, T.A., Tatusova, G., Starchenko, V., Soussov, A., Soboleva, D., Slotta, K., Siyan, K., Sirotkin, S.T., Sherry, G.D., Schuler, C.L., Schoch, V., Schneider, E.W., Sayers, W., Rubinstein, K., Rodarmer, K.D., Pruitt, L., Phan, A.R., Panchenko, V., Palanigobu, C., O’Sullivan, J., Ostell, R., Orris, T., Murphy, I., Karsch-Mizrachi, A., T., Marchler-Bauer, Madej, T.L., Madden, Z., Lu, D.J., Lipman, J.M., Lee, D., Landsman, M.J., Landrum, A., Kuznetsov, S., Krasnov, W., Klimke, P., Kitts, M., Kimelman, A., V., Kimchi, M., Khotomlianski, B., M., Holmes, Johnson, W., Hoeppner, Hlavina, V., L.Y.,Gorelenkov, Geer, K., Funk, M., Feolo, I., Dondoshansky, M., DiCuccio, K., Clark, C., Charowhas, K., Canese, S.H., Bryant, J.R., Brister, D., Bourexis, E., Bolton, C., Bollin, D.A., Benson, J., Beck, T., Barrett, R., Agarwala, REFERENCES J Mol Biol J Mol Cellulomonas flavigena 2 sp. nov., a moderately halotolerant Actinobacteria. Actinobacteria. halotolerant moderately a nov., sp. , 117–134.

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45 is a beta-1,4- is a , 202–211. , 202–211. Cellulomonas Genome Genome 14 , 60. , 60. FEMS Accepted Article This by is protectedarticle reserved. Allrights copyright. Archaea and Bacteria, ManualBergey’s Trust. DOI: 10.1002/9781118960608.gbm00063. P (2015). Schumann, and E. Stackebrandt, ( Borsodi, A.K.(2011). and K. Márialigeti, P., Vladár, G., Szabó, J., Makk, C., Spröer, P., Schumann, E.M., Tóth, A., Rusznyák, Shen, H., Gilkes, N.R., Kilburn, D.G., Miller, R.C. and Warren, R.A.Cellobiohydrolase (1995). a B, Shallom,D. and Shoham, Y.(2003). Microbialhemicellulases. xylanases the from mesophilic thermophilic bagasse-absorbable sugarcane two of characterization and Purification (2007). M.E. and Hidalgo-Lara, M. CarmenMontes-Horcasitas, Del J., Vega-Estrada, A., Santiago-Hernández, sources. carbon different (2007). Differential expression of cellulases and xylanases by T. andPonce-Noyola, L.M. Salgado, M., Torre, La De A.C., Ramos-Valdivia, L.M., Sánchez-Herrera, Phylogenetic Trees. Saitou, N.and Nei, M. (1987). The Neighbor-Joining Method - a New Method for Reconstructing Biotechnol fimi Cellulomonas (2018). M. Cavaletto, and E. Marengo, C., Cattaneo, M., Manfredi, S., Icardi, L., Boatti, S., Spertino, Bioinformatics spectrometry. mass chromatography-tandem liquid by data identification from large-scale Ishihama M., Shinoda,K., Tomita, 1), 67–74. second exo-cellobiohydrolase from cellulolytic the bacterium Phragmites australis

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flavigena sp. nov., α - L 15 ,

Accepted Article This by is protectedarticle reserved. Allrights copyright. sequences. partial to corresponds genome the in *annotation Note: server. 2.0 SecretomeP the using 0.5 above score and a peptide SignalPv4.1. Non-classical secretion was assigned topredicted proteinswith no identified signal pare in indicated are domains Multiple dbCAN. using of (LPMO) monooxygenases polysaccharide Table 1 TABLES α arabinan endo-1,5- arabinan - L -arabinofuranosidase (EC 3.2.1.55) (EC -arabinofuranosidase α -glucuronidase (EC3.2.1.139)-glucuronidase β -glucosidase (EC 3.2.1.21) GH1 KSW21931.1 N - - N KSW21931.1 GH1 -glucosidase (EC 3.2.1.21) Predicted glycosyde hydrolases (GH) active on cellulose or hemicellulose and lytic and lytic hemicellulose or cellulose on active (GH) hydrolases glycosyde Predicted rdce ciiy AyDmis GenBank Domains CAZy Predicted Activity (EC 3.2.1.99)(EC α - L -arabinosidase -arabinosidase

H2 H KW75. Y Classical Y KSW17752.1 GH62, GH5 GH51 KSW29942.1 N - - GH51 KSW29942.1 N - GH51 KSW14247.1 N GH43 KSW21922.1 N - - GH43 KSW21922.1 N - GH43 KSW21776.1 N GH67 KSW29842.1 N - - GH67 KSW29842.1 N GH3 KSW21826.1 N - - GH3 KSW21826.1 N Non- GH3 KSW21825.1 N Non- GH3 KSW21816.1 N - GH3 KSW21537.1 N - GH3 KSW19794.1 N - GH1 KSW29522.1 N - GH1 KSW29322.1 N Cellulomonas

ntheses. Signal peptides were predicted using using werepredicted peptides Signal ntheses. sp. B6.CAZy domainswere identified Accession Accession Peptide Signal Signal mechanism classical classical Predicted secretion secretion Accepted Article This by is protectedarticle reserved. Allrights copyright. arabinofuranosidase (EC 3.2.1.55) 3.2.1.55) (EC arabinofuranosidase endo- β β -xylosidase (EC 3.2.1.37)/ β -mannosidase (EC 3.2.1.25) GH2 KSW13761.1 N - - N KSW13761.1 GH2 -mannosidase (EC 3.2.1.25) β -1,4-xylanase (EC 3.2.1.8) 3.2.1.8) (EC -1,4-xylanase -xylosidase (EC 3.2.1.37) GH39 KSW30127.1 N - - N KSW30127.1 GH39 -xylosidase (EC 3.2.1.37) htns E ...4 G1,CM KW85. Y Classical Y KSW18655.1 CBM2 GH18, Chitinase 3.2.1.14) (EC β -1,4-glucanase (EC 3.2.1.4) α - L - H,CM 2 KW93. Y Classical Classical Y Y KSW29539.1 GH9, (2) CBM4 KSW29108.1 GH9, (2) CBM4 GH10, CBM22 KSW14744.1* Incomplete Incomplete KSW14744.1* CBM22 GH10, GH43, CBM6, CBM6, GH43, GH9, CBM2, CBM2, GH9, H,CM KW09. Y Classical Classical Y Y KSW30095.1 KSW30082.1 GH6, CBM2 GH6, CBM2 H3 H KW17. Y Classical Y KSW21678.1 GH43, GH5 CBM13 CBM3 H3KW95. Non- GH43 KSW29954.1 N - GH43 KSW21730.1 N - GH43 KSW29213.1 N GH10 KSW29966.1* Y Classical Classical Y KSW29966.1* GH10 Classical Y GH10 KSW18330.1 - GH10 KSW28941.1 N GH39 KSW28373.1 N - - GH39 KSW28373.1 N GH9 KSW29417.1* Y Classical Classical KSW29417.1* GH9 Y - GH5 KSW14495.1 N GH5 KSW28923.1* Y Classical Classical KSW28923.1* GH5 Y - GH3 KSW29840.1 N - GH3 KSW29220.1 N - GH3 KSW28963.1 N S3171Y Classical KSW30137.1 Y S2031Y Classical KSW29083.1 Y sequence classical * classical Non- Accepted Article This by is protectedarticle reserved. Allrights copyright. Lytic Polysaccharide Monooxygenase Monooxygenase Lytic Polysaccharide exo- exo- yolcns E ...5) H4() B2 S2761 Classical Y KSW29726.1 GH74 (4), CBM2 Xyloglucanase (EC 3.2.1.151) β β -1,4-glucanase (EC 3.2.1.176) -1,4-glucanase3.2.1.91) (EC (LPMO) H0 B1 KW17. Y Classical Y KSW21671.1 GH10, CBM13 GH10, CBM9, CBM9, GH10, CBM9, GH10, H1 B2 S1151 Classical Y KSW19115.1 GH11, CBM2 Classical Y KSW20567.1 GH10, CBM2 H8 B2 S1961 Classical Y KSW15916.1 GH48, CBM2 A0 B2 S1191 Classical Y KSW19119.1 AA10, CBM2 A0 B2 S3091 Classical Y KSW30009.1 AA10, CBM2 H,CM KW56. Y Classical Y KSW15663.1 GH6, CBM2 CBM22 (2) CBM22 S1391Y Classical KSW16349.1 Y Classical KSW23552.1 Y Accepted Article This by is protectedarticle reserved. Allrights copyright. sequences. partial to corresponds genome the in *annotation Note: parentheses. in indicated are domains of supernatants Table 2 KSW21671.1 GH10, CBM13 CMC, SCR 53.2/8.70 53.2/8.70 SCR CMC, CBM13 GH10, KSW21671.1 arabinofuranosidase (EC 3.2.1.55) (EC arabinofuranosidase β -xylosidase (EC 3.2.1.37)/ endo- rdce ciiy GenBank Predicted Activity Extracellular proteins involved incarbohydrate utilization identified in culture β -1,4-xylanase -1,4-xylanase (EC 3.2.1.8) (EC 3.2.1.4) β -1,4-glucanase -1,4-glucanase Cellulomonas sp α - L -

. B6 grown on CMC, SCR or EWS by mass spectrometry. Multiple Multiple spectrometry. by mass EWS or SCR onCMC, grown . B6 KSW29966.1* GH10 CMC,KSW29966.1* GH10 SCR, S1741 H0 B2 SR 54.8/ KSW14744.1* GH10, SCR 4.75 CBM22 S2471 H SCR 65.8/6.48 KSW29417.1* GH9 S2591 H,CM 2 SR W 118.8/4.58 SCR, EWS 114.1/4.58 GH9, CBM4 (2) SCR, EWS KSW29539.1 GH9, CBM4 (2) KSW29108.1 GH9, KSW29083.1 CBM2, KSW21678.1 GH43, GH43, KSW21678.1 76.0/9.07 SCR GH5 36.5/9.36 EWS SCR, GH10, KSW23552.1 CBM2 GH11, CBM9, GH10, KSW20567.1 SCR,EWS KSW19115.1 50.6/8.76 CBM2 GH10 SCR, KSW18330.1 EWS GH10, KSW16349.1 65.4/5.27 CBM9, Accession Accession

CAZy Domains Growth Growth Domains CAZy CBM22 (2) (2) CBM22 CBM22 CBM3 CMC, SCR, Substrate Substrate C,ES 130.7/ 4.42 SCR, EWS EWS EWS EWS SCR 86.4/5.68 86.4/5.68 SCR MW[kDa]/pI MW[kDa]/pI 112.3/4.82 Calculated 37.8/8.51 37.8/8.51 Accepted Article This by is protectedarticle reserved. Allrights copyright. exo- exo- Sugar ABC transporter substrate- transporter Sugar ABC α Carbohydrate binding protein KSW23491.1 CBM44 (2), (2), CBM44 KSW23491.1 protein binding Carbohydrate α yolcns E ...5) S2761 H4() B2 C,ES 95.7/5.00 SCR, EWS GH74 (4), CBM2 KSW29726.1 Xyloglucanase 3.2.1.151)(EC -1,6-glucosidase (EC 3.2.1.20)-1,6-glucosidase (EC β β -galactosidase (EC 3.2.1.22) α -glucosidase (EC 3.2.1.21) KSW21825.1 GH3 SCR, EWS 83.5/4.48 83.5/4.48 EWS SCR, GH3 KSW21825.1 3.2.1.21) (EC -glucosidase β -1,4-glucanase (EC 3.2.1.176) htns E ...4 KW85. G1,CM SR W 56.3/5.77 SCR, EWS GH18, CBM2 KSW18655.1 Chitinase (EC 3.2.1.14) -1,4-glucanase (EC 3.2.1.91) (EC3.2.1.91) -1,4-glucanase - L -arabinofuranosidase -arabinofuranosidase binding protein protein binding Pectate lyase KSW13730.1 PL3, CBM13 (2) SCR 30.2/4.4 30.2/4.4 SCR (2) CBM13 PL3, KSW13730.1 lyase Pectate (EC 3.2.1.55) Esterases KSW21470.1 KSW21470.1 Esterases CE1 (2) SCR, EWS 93.6/4.75 LPMO KSW19119.1 AA10, CBM2 SCR 37.0/5.92 37.0/5.92 SCR CBM2 AA10, KSW19119.1 LPMO

KSW21936.1 - - SCR, KSW21936.1 98.0/ 4.67 SCR, EWS - SCR, KSW21774.1 EWS - KSW19791.1 EWS 45.1/4.65 CE1 (2) EWS 59.4/4.64 KSW15560.1 48.6/4.2 CE1 SCR, KSW21507.1 EWS 30.2/4.4 SCR, CBM2 KSW29693.1 EWS 61.7/4.63 CBM32 KSW29929.1 EWS KSW30160.1 CBM32 SCR, 76.7/4.84 EWS 102.2/4.54 KSW17752.1 GH62, GH5 SCR, EWS 53.6/8.29 53.6/8.29 EWS SCR, GH5 GH62, KSW17752.1 KSW30082.1 GH6, GH6, KSW30082.1 47.3/8.41 EWS CBM2 KSW29886.1 GH13 GH13 KSW29886.1 (2), KSW30136.1 GH27, CBM13 SCR 60.6/6.79 GH27, 60.6/6.79 KSW30136.1 SCR CBM13 CMC, SCR, GH48, CBM2 KSW15916.1 GH6, KSW15663.1 CBM2 CMC, SCR, CBM20, CBM48 CBM66 C,ES 144.8/5.22 SCR, EWS C,ES 207.1/4.75 SCR, EWS EWS EWS EWS 90.6/ 5.33 63.4/ 6.47 Accepted Article This by is protectedarticle reserved. Allrights copyright. KSW19409.1 KSW19409.1 - CMC 46.7/4.33 KSW28372.1 - - SCR, KSW28372.1 EWS 61.0/4.61 KSW30268.1 - - SCR, KSW30268.1 - SCR, KSW29892.1 - SCR, KSW29516.1 EWS - SCR, KSW28971.1 EWS 48.6/4.46 EWS 39.9/4.40 EWS 60.6/4.32 48.2/4.26 Accepted Article way ANI and AAI were calculated with the online tools developed by the Environmental Microbial Microbial Environmental the by developed tools online the with calculated were AAI and ANI way Two- (http://ggdc.dsmz.de). DMSZ Institute Leibniz the of Calculator Distance Genome to Genome subspecies) and differences in %G+C ( (same or >79% species) (same >70% DDH that probability length), pairs score (DDH) (identities/high the from obtained were content G+C and length Cellulomonas flavigena hybridizationNucleotide and Average Identity (ANI)/Average Aminoacid(AAI) with Identity Table S2. determined. of Archea and Bacteria (Stackebrandt and Schumann, 2015).Key: +positive; -negative;not ND Microbiology (Ahmed et.al, 2014; Elbertson etal., 2000)and in the Bergey’s Manual of Systematics flavigena This by is protectedarticle reserved. Allrights copyright. Table S1. domain. GH43 ora GH62 a presented also which of two proteins, four in were found domains GH5 ** proteins; containing GH11 and GH10 of both one of protein identified in modulewere Cellulomonas flavigena Figure S2. et al., 2016). culture supernatants.or 0.2% 10–0.2% SDS-PAGE CMC ***P<0.001; P<0.0001).*** c) Zymogram ofxylanase CMCase and CMC and SCR activities from determined by of variance (one-way analysis supernatants). Culture media is indicated inthe X-axis. Statistically significant differences were Figure S1. Information Supporting the for Legends Genomic taxonomy analysis of of analysis taxonomy Genomic and and API50CH results of Number of proteins containing each CAZy domain in in domain CAZy each containing proteins of Number of activity xylanase b) and CMCase a) C. persica C. and and ATCC 482and ATCC published in the International Journal of Systematic and Evolutionary Cellulomonas persica Cellulomonas ≥ Cellulomonas 1 between1 distinct species) were estimated using the Cellulomonas C. flavigena sp. B6 compared toavailable B6 sp. information about ANOVA) followed by Tukey´s test (**P<0.01; (**P<0.01; test Tukey´s by followed ANOVA) Cellulomonas NCBI Database. Distance, DNA-DNA hybridization DNA-DNA hybridization Distance,NCBI Database. type strains. Genomic and proteomic sequences, sequences, proteomic and Genomic strains. type sp. B6. sp. GH10 Key: *A domain aswellGH11 a as and taken account into for the is number total sp. B6 in terms of computationalof B6interms DNA-DNA sp. XY were used as previously described (Ghio (Ghio described previously as were used XY sp. B6extracellular extracts (culture Cellulomonas fimi Cellulomonas ATCC ATCC 484, C. C. Accepted Article

This by is protectedarticle reserved. Allrights copyright. cover 90% (query identity sequence A BLASTp. NCBI using calculated was identity Sequence biomass. lignocellulosic Table S3 deviation standard SD: interval, confidence CI: (computational), hybridization DNA-DNA DDH: ofTe Institute Georgia from Laboratory Genomics protein content in mol % (emPAI%). The differences observed in the emPAI% correlate with the emPAI% observedcorrelate with in the the % differences mol (emPAI%). proteinThe contentin emPAI/ equation The used. was data identification protein using Sequest by calculated emPAI index abundance protein The shown. are Palgorithm Secretome cellulose. biologicallyOnly relevant proteins with signal peptide consideredor extracellular by carboxi-methyl- CMC: residue; cane sugar SCR: straw; wheat extruded EWS: substrates. cellulosic on Table S5 transporters. ABC carbohydrate of proteins substrate-binding and CAZymes secreted for supernatants CMC Table S4 SecretomeP was 2.0 prediction extrace used of for bacter positive Gram (for v4.1 SignalP parentheses. identified ingenomic the sequence using the dbCAN algorithm. Multiple domains are indicated in Table S6. identified. proteins of number total the in differences . CAZome of . Relative abundance of proteins identified in the secretomes of of secretomes the in identified proteins of abundance Relative . and SCR EWS, in spectrometry bymass detected peptides matching of number and Score . CAZymes identified insecretomes of CAZymes identified ≥ was70%) categorized as high. Cellulomonas sp. B6. All predicted CAZy domain-containing proteins were proteins domain-containing CAZy predicted All B6. sp. Cellulomonas chnology (http://enve-omics.ce.gatech.edu/). Key: Key: (http://enve-omics.ce.gatech.edu/). chnology llular proteins secreted by non-classical pathways. pathways. non-classical by secreted proteins llular ia) was used for prediction of signal peptides and and peptides signal of prediction for used was ia) Σ (emPAI) x 100 was used to calculate the to was x used 100 (emPAI) sp. B6 and C. fimi Cellulomonas ATCC 484 cultured 484 on ATCC sp. grown sp. B6, ≥

Accepted Article This by is protectedarticle reserved. Allrights copyright.