Thedigestionofdietaryfibersisamajorfunctionofthehumanintestinalmicrobiota.SofarthisfunctionhasbeenattributedtothemicroorganismsinhABItingthecolon,andmanystudieshavefocusedonthisdistalpartofthegastrointestinaltractusingeasilyaccessIBLefecalmaterial.However,microbialfermentations,supportedbythepresenceofshort-chainfattyacids,aresUSPectedtooccurintheuppersmallintestine,particularlyintheileum.Usingafosmidlibraryfromthehumanilealmucosa,wescreened20,000clonesfortheiractivitiesagainstcarboxymethylcelluloseandxylanschosenasmodelsofthemajorplantcellwall(PCW)polysaccharidesfromdietaryfibres.ElevenpositiveclonesrevealedabroadrangeofCAZymeencodinggenesfromBacteroidesandClostridialesspecies,aswellasPolysaccharideUtilizationLoci(PULs).Thefunctionalglycosidehydrolasegeneswereidentified,andoligosaccharidebreak-downproductsexaminedfromdifferentpolysaccharidesincludingmixed-linkageβ-glucans.CAZymesandPULswerealsoexaminedfortheirprevalenceinhumangutmicrobiome.Severalclustersofgenesoflowprevalenceinfecalmicrobiomesuggestedtheybelongtounidentifiedstrainsratherspecificallyestablishedupstreamthecolon,intheileum.Thus,theilealmucosa-associatedmicrobiotaencompassestheenzymaticpotentialforPCWpolysaccharidedegradationinthesmallintestine.

Background:Thediscoveryanddevelopmentofnovelplantcellwalldegradingenzymesisakeysteptowardsmoreefficientdepolymerizationofpolysaccharidestofermentablesugarsfortheproductionofliquidtransportationbiofuelsandotherbioproducts.TheindustrialfungusTrichodermareeseiisknowntobehighlycellulolyticandisamajorindustrialmicrobialsourceforcommercialcellulases,xylanasesandothercellwalldegradingenzymes.However,enzyme-ProspectingresearchcontinuestoidentifyopportunitiestoenhancetheactivityofT.reeseienzymepreparationsbysupplementingwithenzymaticdiversityfromothermicrobes.Thegoalofthisstudywastoevaluatetheenzymaticpotentialofabroadrangeofplantpathogenicandnon-pathogenicfungifortheirabilitytodegradeplantbiomassandisolatedpolysaccharides.Results:Large-scalescreeningidentifiedarangeofhydrolyticactivitiesamong348uniqueisolatesrepresenting156speciesofplantpathogenicandnon-pathogenicfungi.Hierarchicalclusteringwasusedtoidentifygroupsofspecieswithsimilarhydrolyticprofiles.Amongmoderatelyandhighlyactivespecies,plantpathogenicspecieswerefoundtobemoreactivethannon-pathogensonsixofeightsubstratestested,withnosignificantdifferenceseenontheothertwosubstrates.Amongthepathogenicfungi,greaterhydrolysiswasseenwhentheyweretestedonbiomassandhemicellulosederivedfromtheirhostplants(commelinoidmonocotordicot).AlthoughT.reeseihasahydrolyticprofilethatishighlyactiveoncelluloseandpretreatedbiomass,itwaslessactivethansomenaturalisolatesoffungiwhentestedonxylansanduntreatedbiomass.Conclusions:Severalhighlyactiveisolatesofplantpathogenicfungiwereidentified,particularlywhentestedonxylansanduntreatedbiomass.Therewerestatisticallysignificantpreferencesforbiomasstypereflectingthemonocotordicothostpreferenceofthepathogentested.Thesehighlyactivefungiarepromisingtargetsforidentificationandcharacterizationofnovelcellwalldegradingenzymesforindustrialapplications.

Therecentlyisolatedbacterialstrain80/3representsoneofthemostabundant16SrRNAphylotypesdetectedinthehealthyhumanlargeintestineandbelongstotheRuminococcaceaefamilyofFirmicutes.Thecompletedgenomesequencereportedhereisthefirstforamemberofthisimportantfamilyofbacteriafromthehumancolon.Thegenomecomprisestwolargechromosomesof2.24and0.73Mbp,leadingustoproposethenameRuminococcusbicirculansforthisnewspecies.Analysisofthecarbohydrateactiveenzymecomplementsuggestsanabilitytoutilizecertainhemicelluloses,especiallyβ-glucansandxyloglucan,forgrowththatwasconfirmedexperimentally.Theenzymaticmachineryenablingthedegradationofcelluloseandxylanbyrelatedcellulolyticruminococciishoweverlackinginthisspecies.Whilethegenomeindicatedthecapacitytosynthesizepurines,pyrimidinesandall20aminoacids,onlygenesforthesynthesisofnicotinate,NAD⁺,NADP⁺andcoenzymeAweredetectedamongtheessentialvitaminsandco-factors,resultinginmultiplegrowthrequirements.Invivo,thesegrowthfactorsmustbesuppliedfromthediet,hostorothergutmicroorganisms.OtherfeaturesofecologicalinterestincludetwotypeIVpilins,multipleextracytoplasmicfunction-sigmafactors,aureaseandabilesalthydrolase.

Severalrecombinantfungalenzymes(endoglucanaseandpectinase)werestudiedfortheirinteractionswithα-expansinincellwallextensionandpolysaccharidedegradation.BothCel12AandCel5AwereabletohydrolyzecelluloseCMC-Naandmixed-linkageβ-glucan.IncontrasttoCel5A,Cel12Acouldalsohydrolyzexyloglucanandinducewallextensionofcucumberhypocotylsinaninvitroassay.Combiningα-expansin,evenathighconcentrations,withCel12Adidnotenhancethemaximum/finalwallextensionrateinducedbyCel12Aalone.Theseresultsstronglysuggestthatmodification/degradationofthexyloglucanmolecule/networkisthekeyforcellwallextension,andα-expansinandCel12Amaysharethesameactingsiteinthesubstrate.Pectinase(Pel1,apectinlyase)enhancedα-expansin-inducedwallextensioninaconcentration-dependentmanner,suggestingthatthepectinnetworkmaynormallyregulateaccessibilityofexpansintothexyloglucan–cellulosecomplex.α-ExpansinenhancedCel12A"shydrolyticactivityoncelluloseCMC-Nabutnotonxyloglucanandβ-glucan.ExpansindidnotaffectCel5A"shydrolyticactivity.Interestingly,expansinalsoenhancedPel1"sactivityondegradinghighesterifiedpectin.Apotentialexplanationforwhyexpansincouldsynergisticallyinteractwithonlycertainenzymesonspecificpolysaccharidesisdiscussed.Additionalresultsalsosuggestedthatcellwallswellingmaynotbeasignificanteventduringtheactionofexpansinandhydrolases.

Xyloglucaniswidelybelievedtofunctionasatetherbetweencellulosemicrofibrilsintheprimarycellwall,limitingcellenlargementbyrestrictingtheabilityofmicrofibrilstoseparatelaterally.Totestthebiomechanicalpredictionsofthis“tetherednetwork”model,weassessedtheabilityofcucumber(Cucumissativus)hypocotylwallstoundergocreep(long-term,irreversibleextension)inresponsetothreefamily-12endo-β-1,4-glucanasesthatcanspecificallyhydrolyzexyloglucan,cellulose,orboth.Xyloglucan-specificendoglucanase(XEGfromAspergillusaculeatus)failedtoinducecellwallcreep,whereasanendoglucanasethathydrolyzesbothxyloglucanandcellulose(Cel12AfromHypocreajecorina)inducedahighcreeprate.Acellulose-specificendoglucanase(CEGfromAspergillusniger)didnotcausecellwallcreep,eitherbyitselforincombinationwithXEG.Testswithadditionalenzymes,includingafamily-5endoglucanase,confirmedtheconclusionthattocausecreep,endoglucanasesmustcutbothxyloglucanandcellulose.Similarresultswereobtainedwithmeasurementsofelasticandplasticcompliance.BothXEGandCel12Ahydrolyzedxyloglucaninintactwalls,butCel12AcouldhydrolyzeaminorxyloglucancompartmentrecalcitranttoXEGdigestion.XyloglucaninvolvementintheseenzymeresponseswasconfirmedbyexperimentswithArabidopsis(Arabidopsisthaliana)hypocotyls,whereCel12Ainducedcreepinwild-typebutnotinxyloglucan-deficient(xxt1/xxt2)walls.Ourresultsareincompatiblewiththecommondepictionofxyloglucanasaload-bearingtetherspanningthe20-to40-nmspacingbetweencellulosemicrofibrils,buttheydoimplicateaminorxyloglucancomponentinwallmechanics.Thestructurallyimportantxyloglucanmaybelocatedinlimitedregionsoftightcontactbetweenmicrofibrils.

Componentsofmodularcellulases,type-Acellulose-bindingmodules(CBMs)bindtocrystallinecelluloseandenhanceenzymeeffectiveness,butstructuraldetailsoftheinteractionareuncertain.WeanalyzedcellulosebindingbyEXLX1,abacterialexpansinwithabilitytoloosenplantcellwallsandwhosedomainD2hastype-ACBMcharacteristics.EXLX1stronglybindstocrystallinecelluloseviaD2,whereasitsaffinityforsolublecellooligosaccharidesisweak.Calorimetryindicatedcellulosebindingwaslargelyentropicallydriven.WesolvedthecrystalstructuresofEXLX1complexedwithcellulose-likeoligosaccharidestofindthatEXLX1bindstheligandsthroughhydrophobicinteractionsofthreelinearlyarrangedaromaticresiduesinD2.Thecrystalstructuresrevealedauniqueformofligand-mediateddimerization,withtheoligosaccharidesandwichedbetweentwoD2domainsinoppositepolarity.Thisreportclarifiesthemoleculartargetofexpansinandthespecificmolecularinteractionsofatype-ACBMwithcellulose.

α-Linkedxyloseisamajorcomponentofxyloglucansinthecellwallsofhigherplants.Anα-xylosidase(AxlA)waspurifiedfromacommercialenzymepreparationfromAspergillusniger,andtheencodinggenewasidentified.Theproteinisamemberofglycosylhydrolasefamily31.Itwasactiveonp-nitrophenyl-α-D-xyloside,isoprimeverose,xyloglucanheptasaccharide(XXXG),andtamarindxyloglucan.WhenexpressedinPichiapastoris,AxlAhadactivitycomparabletothenativeenzymeonpNPα-XandIPdespiteapparenthyperglycosylation.ThepHoptimumofAxlAwasbetween3.0and4.0.AxlAtogetherwithβ-glucosidasedepolymerizedxyloglucanheptasaccharide.AcombinationofAxlA,β-glucosidase,xyloglucanase,andβ-glucosidaseintheoptimalproportionsof51:5:19:25or59:5:11:25couldcompletelydepolymerizetamarindXGtofreeGlcorXyl,respectively.Tothebestofourknowledge,thisisthefirstcharacterizationofasecretedmicrobialα-xylosidase.Secretedα-xylosidasesappeartoberareinnature,beingabsentfromothertestedcommercialenzymemixturesandfromthegenomesofmostfilamentousfungi.

Matureplantcellwallslosetheirabilitytoexpandandbecomeunresponsivetoexpansin.Thisphenomenonisbelievedtobeduetocross-linkingofhemicellulose,pectin,orphenolicgroupsinthewall.Byscreeningvarioushydrolyticenzymes,wefoundthatpretreatmentofnongrowing,heat-inactivated,basalcucumber(Cucumissativus)hypocotylswithpectinlyase(Pel1)fromAspergillusjaponicuscouldrestorereconstitutedexogenousexpansin-inducedextensioninmaturecellwallsinvitro.RecombinantpectatelyaseA(PelA)andpolygalacturonase(PG)fromAspergillusspp.exhibitedsimilarcapacitytoPel1.Pel1,PelA,andPGalsoenhancedthereconstitutedexpansin-inducedextensionoftheapical(elongating)segmentsofcucumberhypocotyls.However,theeffectiveconcentrationsofPelAandPGforenhancingthereconstitutedexpansin-inducedextensionweregreaterintheapicalsegmentsthaninthebasalsegments,whereasPel1behavedintheoppositemanner.Thesedataareconsistentwithdistributionofmoremethyl-esterifiedpectinincellwallsoftheapicalsegmentsandlessesterifiedpectininthebasalsegments.Associatedwiththedegreeofesterificationofpectin,morecalciumwasfoundincellwallsofbasalsegmentscomparedtoapicalsegments.PretreatmentofthecalciumchelatorEGTAcouldalsorestorematurecellwalls"susceptibilitytoexpansinbyremovingcalciumfrommaturecellwalls.Becauserecombinantpectinasesdonothydrolyzeotherwallpolysaccharides,andendoglucanase,xylanase,andproteasecannotrestorethematurewall"sextensibility,wecanconcludethatthepectinnetwork,especiallycalcium-pectatebridges,maybetheprimaryfactorthatdeterminescucumberhypocotylmaturecellwalls"unresponsivenesstoexpansin.

Cottonfiberisanimportantnaturaltextilefiberduetoitsexceptionallengthandthickness.Thesepropertiesariselargelythroughprimaryandsecondarycellwallsynthesis.Thecottonfiberofcommerceisacellulosicsecondarywallsurroundedbyathincuticulatedprimarywall,buttherewereonlysparsedetailsavailableaboutthepolysaccharidesinthefibercellwallofanycottonspecies.Inaddition,Gossypiumhirsutum(Gh)fiberwasknowntohaveanadhesivecottonfibermiddlelamella(CFML)thatjoinsadjacentfibersintotissue-likebundles,butitwasunknownwhetheraCFMLexistedinothercommerciallyimportantcottonfibers.WecomparedthecellwallchemistryoverthetimecourseoffiberdevelopmentinGhandGossypiumbarbadense(Gb),thetwomostimportantcommercialcottonspecies,whenplantsweregrowninparallelinahighlycontrolledgreenhouse.Underthesegrowingconditions,therateofearlyfiberelongationandthetimeofonsetofsecondarywalldepositionweresimilarinfibersofthetwospecies,butasexpectedtheGbfiberhadaprolongedelongationperiodanddevelopedhigherqualitycomparedtoGhfiber.TheGbfibershadaCFML,butitwasnotdirectlyrequiredforfiberelongationbecauseGbfibercontinuedtoelongaterapidlyafterCFMLhydrolysis.Forbothspecies,fiberatsevenageswasextractedwithfourincreasinglystrongsolvents,followedbyanalysisofcellwallmatrixpolysaccharideepitopesusingantibody-basedGlycomeProfiling.Togetherwithimmunohistochemistryoffibercross-sections,thedatashowthattheCFMLofGbfibercontainedlowerlevelsofxyloglucancomparedtoGhfiber.Xyloglucanendo-hydrolaseactivitywasalsohigherinGbfiber.Ingeneral,thedataprovidearichpictureofthesimilaritiesanddifferencesinthecellwallstructureofthetwomostimportantcommercialcottonspecies.

(1,3)(1,4)-β-D-Glucan(mixed-linkageglucanorMLG),acharacteristichemicelluloseinprimarycellwallsofgrasses,wasinvestigatedtodeterminebothitsroleincellwallsanditsinteractionwithcelluloseandothercellwallpolysaccharidesinvitro.BindingisothermsshowedthatMLGadsorptionontomicrocrystallinecelluloseisslow,irreversible,andtemperature-dependent.MeasurementsusingquartzcrystalmicrobalancewithdissipationmonitoringshowedthatMLGadsorbedirreversiblyontoamorphousregeneratedcellulose,formingathickhydrogel.Oligosaccharideprofilingusingendo-(1,3)(1,4)-β-glucanaseindicatedthattherewasnodifferenceinthefrequencyanddistributionof(1,3)and(1,4)linksinboundandunboundMLG.ThebindingofMLGtocellulosewasreducedifthecellulosesampleswerefirsttreatedwithcertaincellwallpolysaccharides,suchasxyloglucanandglucuronoarabinoxylan.ThetetheringfunctionofMLGincellwallswastestedbyapplyingendo-(1,3)(1,4)-β-glucanasetowallsamplesinaconstantforceextensometer.Cellwallextensionwasnotinduced,whichindicatesthatenzyme-accessibleMLGdoesnottethercellulosefibrilsintoaload-bearingnetwork.

Background:Unlikeinabscissionordehiscence,fruitofkiwifruitActinidiaerianthadeveloptheabilityforpeeldetachmentwhentheyareripeandsoftintheabsenceofamorphologicallyidentifiableabscissionzone.Twoclosely-relatedgenotypeswithcontrastingdetachmentbehaviourhavebeenidentified.The‘good-peeling’genotypehasdetachmentwithcleandebondingofcells,andapeeltissuethatdoesnottear.The‘poor-peeling’genotypehaspoordetachability,withcellsthatruptureupondebonding,andpeeltissuethatfragmentseasily.Results:Structuralstudiesindicatedthatpeeldetachabilityinbothgenotypesoccurredintheouterpericarpbeneaththehypodermis.Immunolabellingshoweddifferencesinmethylesterificationofpectin,wheretheinterfaceoflabellingcoincidedwiththelocationofdetachmentinthegood-peelinggenotype,whereasinthepoor-peelinggenotype,nosuchinterfaceexisted.Thiszoneofdifferenceinmethylesterificationwasenhancedbydifferentialcellwallchangesbetweenthepeelandouterpericarptissue.Althoughbothgenotypesexpressedtwopolygalacturonasegenes,noenzymeactivitywasdetectedinthegood-peelinggenotype,suggestinglimitedpectinbreakdown,keepingcellwallsstrongwithouttearingorfragmentationofthepeelandfleshupondetachment.Differencesinlocationandamountsofwall-stiffeninggalactaninthepeelofthegood-peelinggenotypepossiblycontributedtothisphenotype.Hemicellulose-actingtransglycosylasesweremoreactiveinthegood-peelinggenotype,suggestinganinfluenceonpeelflexibilitybyremodellingtheirsubstratesduringdevelopmentofdetachability.Highxyloglucanaseactivityinthepeelofthegood-peelinggenotypemaycontributebyhavingastrengtheningeffectonthecellulose-xyloglucannetwork.Conclusions:InfruitofA.eriantha, peeldetachabilityisduetotheestablishmentofazoneofdiscontinuitycreatedbydifferentialcellwallchangesinpeelandouterpericarptissuesthatleadtochangesinmechanicalpropertiesofthepeel.Duringripening,thepeelbecomesflexibleandthecellscontinuetoadherestronglytoeachother,preventingbreakage,whereastheunderlyingouterpericarplosescellwallstrengthassofteningproceeds.Togethertheseresultsrevealanovelandinterestingmechanismforenablingcellseparation.