Megazyme/AZCL-Rhamnogalacturonan I/I-AZRHI/2 grams

Lactococcuslactissubsp.lactisstrainA12wasisolatedfromsourdough.Combinedgenomic,transcriptomic,andphenotypicanalyseswereperformedtounderstanditssurvivalcapacityinthecomplexsourdoughecosystemanditsroleinthemicrobialcommunity.ThegenomesequencecomparisonofstrainA12withstrainIL1403(aderivativeofanindustrialdairystrain)revealed78strain-specificregionsrepresenting23%ofthetotalgenomesize.Mostofthestrain-specificgeneswereinvolvedincarbohydratemetabolismandarepotentiallyrequiredforitspersistenceinsourdough.Phenotypemicroarray,growthtests,andanalysisofglycosidehydrolasecontentshowedthatstrainA12fermentedplant-derivedcarbohydrates,suchasarABInoseandα-galactosides.StrainA12exhibitedspecificgrowthratesonraffinosethatwereashighastheywereonglucoseandwasabletoreleasesucroseandgalactoseoutsidethecell,providingsolublecarbohydratesforsourdoughmicroflora.Transcriptomicanalysisidentifiedgenesspecificallyinducedduringgrowthonraffinoseandarabinoseandrevealsanalternativepathwayforraffinoseassimilationtothatusedbyotherlactococci.

ThegenomeofDictyoglomusturgidumwassequencedandanalyzedforcarbohydrases.Thebroadrangeofcarbohydratesubstrateutilizationisreflectedinthehighnumberofglycosylhydrolases,54,andthehighpercentageofCAZymespresentinthegenome,3.09%ofitstotalgenes.ScreeningarandomclonelibrarygeneratedfromD.turgidumresultedinthediscoveryoffivenovelbiomass-degrADIngenzymeswithlowhomologytoknownmolecules.Wholegenomesequencingoftheorganismfollowedbybioinformatics-directedamplificationofselectedgenesresultedintherecoveryofsevenadditionalnovelenzymemolecules.Basedontheanalysisofthegenome,D.turgidumdoesnotappeartodegradecelluloseusingeitherconventionalsolubleenzymesoracellulosomaldegradationsystem.Thetypesandquantitiesofglycosylhydrolasesandcarbohydrate-bindingmodulespresentinthegenomesuggestthatD.turgidumdegradescelluloseviaamechanismsimilartothatusedbyCytophagahutchinsoniiandFibrobactersuccinogenes.

Fungus-growing(attine)antsandtheirfungalsymbiontspassedthroughseveralevolutionarytransitionsduringtheir50millionyearoldevolutionaryhistory.Thebasalattinelineagesoftenshiftedbetweentwomaincultivarclades,whereasthederivedhigher-attinelineagesmaintainedanassociationwithamonophyleticcladeofspecializedsymbionts.Inconjunctionwiththetransitiontospecializedsymbionts,theantsadvancedincolonysizeandsocialcomplexity.Hereweprovideacomparativestudyofthefunctionalspecializationinextracellularenzymeactivitiesinfungusgardensacrosstheattinephylogeny.Weshowthat,relativetosisterclades,gardensofhigher-attineantshaveenhancedactivityofprotein-digestingenzymes,whereasgardensofleaf-cuttingantsalsohaveincreasedactivityofstarch-digestingenzymes.However,theenzymeactivitiesoflower-attinefungusgardensaretargetedprimarilytowardpartialdegradationofplantcellwalls,reflectingaplesiomorphicstateofnondomesticatedfungi.Theenzymeprofilesofthehigher-attineandleaf-cuttinggardensappearparticularlysuitedtodigestfreshplantmaterialsandtoaccessnutrientsfromlivecellswithoutmajorbreakdownofcellwalls.Theadaptivesignificanceofthelower-attinesymbiontshiftsremainsunclear.Oneoftheseshiftswasobligate,butdigestiveadvantagesremainedambiguous,whereastheotherremainedfacultativedespiteprovidinggreaterdigestiveefficiency.

Introduction:Inwood-dwellingfungus-farmingweevils,theso-calledambrosiabeetles(Curculionidae:ScolytinaeandPlatypodinae),woodintheexcavatedtunnelsisusedasamediumforcultivatingfungibythecombinedactionofdigginglarvae(whichcreatemorespaceforthefungitogrow)andofadultssowingandpruningthefungus.Thebeetlesareobligatelydependentonthefungusthatprovidesessentialvitamins,aminoacidsandsterols.However,towhatextentmicrobialenzymessupportfungusfarminginambrosiabeetlesisunknown.Herewemeasure(i)13plantcell-walldegradingenzymesinthefungusgardenmicrobialconsortiumoftheambrosiabeetleXyleborinussaxesenii,includingitsprimaryfungalsymbionts,inthreecompartmentsoflaboratorymaintainednests,atdifferenttimepointsaftergalleryfoundationand(ii)fourspecificenzymesthatmaybeeitherinsectormicrobiallyderivedinX.saxeseniiadultandlarvalindividuals.Results:Wediscoveredthattheactivityofcellulasesinambrosiafungusgardensisrelativelysmallcomparedtotheactivitiesofothercellulolyticenzymes.Enzymeactivityinallcompartmentsofthegardenwasmainlydirectedtowardshemicellulosecarbohydratessuchasxylan,glucomannanandcallose.Hemicellulolyticenzymeactivitywithinthebroodchamberincreasedwithgalleryage,whereasirrespectiveoftheageofthegallery,thehighestoverallenzymeactivityweredetectedinthegallerydumpmaterialexpelledbythebeetles.Interestinglyendo-β-1,3(4)-glucanaseactivitycapableofcallosedegradationwasidentifiedinwhole-bodyextractsofbothlarvaeandadultX.saxesenii,whereasendo-β-1,4-xylanaseactivitywasexclusivelydetectedinlarvae.Conclusion:Similartocloselyrelatedfungiassociatedwithbarkbeetlesinphloem,themicrobialsymbiontsofambrosiabeetleshardlydegradecellulose.Instead,theirenzymeactivityisdirectedmainlytowardscomparativelymoreeasilyaccessIBLehemicellulosecomponentsoftheray-parenchymacellsinthewoodxylem.FurThermore,thedetectionofxylanolyticenzymesexclusivelyinlarvae(whichfeedonfunguscolonizedwood)andnotinadults(whichfeedonlyinfungi)indicatesthatonlylarvae(pre-)digestplantcellwallstructures.ThisimpliesthatinX.saxeseniiandlikelyalsoinmanyotherambrosiabeetles,adultsandlarvaedonotcompeteforthesamefoodwithintheirnests-incontrast,larvaeincreasecolonyfitnessbyfacilitatingenzymaticwooddegradationandfunguscultivation.

Aspergillushancockiisp.nov.,classifiedinAspergillussubgenusCircumdatisectionFlavi,wasoriginallyisolatedfromsoilinpeanutfieldsnearKumbia,intheSouthBurnettregionofsoutheastQueensland,Australia,andhassincebeenfoundoccasionallyfromothersubstratesandlocationsinsoutheastAustralia.ItisphylogeneticallyandphenotypicallyrelatedmostcloselytoA. leporisStatesandM.Chr.,butdiffersinconidialcolour,otherminorfeaturesandparticularlyinmetaboliteprofile.Whencultivatedonriceasanoptimalsubstrate,A. hancockiiproducedanextensivearrayof69secondarymetabolites.Elevenofthe15mostabundantsecondarymetabolites,constituting90%ofthetotalareaunderthecurveoftheHPLCtraceofthecrudeextract,werenovel.ThegenomeofA. hancockii,approximately40Mbp,wassequencedandminedforgenesencodingcarbohydratedegradingenzymesidentifiedthepresenceofmorethan370genesin114geneclusters,demonstratingthatA. hancockiihasthecapacitytodegradecellulose,hemicellulose,lignin,pectin,starch,chitin,cutinandfructanasnutrientsources.LikemostAspergillusspecies,A. hancockiiexhibitedadiversesecondarymetabolitegeneprofile,encoding26polyketidesynthase,16nonribosomalpeptidesynthaseand15nonribosomalpeptidesynthase-likeenzymes.

Chineseliquorisoneoftheworld"sbest-knowndistilledspiritsandisthelargestspiritcategorybysales.Theuniqueandtraditionalsolid-statefermentationtechnologyusedtoproduceChineseliquorhasbeenincontinuoususeforseveralthousandyears.Thediverseanddynamicmicrobialcommunityinaliquorstarteristhemaincontributortoliquorbrewing.However,littleisknownabouttheecologicaldistributionandfunctionalimportanceofthesecommunitymembers.Inthisstudy,metatranscriptomicswasusedtocomprehensivelyexploretheactivemicrobialcommunitymembersandkeytranscriptswithsignificantfunctionsintheliquorstarterproductionprocess.Fungiwerefoundtobethemostabundantandactivecommunitymembers.Atotalof932carbohydrate-activeenzymes,includinghighlyexpressedauxiliaryactivityfamily9and10proteins,wereidentifiedat62°Cunderaerobicconditions.Somepotentialthermostableenzymeswereidentifiedat50,62,and25°C(maturestage).Increasedcontentandoverexpressedkeyenzymesinvolvedinglycolysisandstarch,pyruvateandethanolmetabolismweredetectedat50and62°C.Thekeyenzymesofthecitratecyclewereup-regulatedat62°C,andtheirabundantderivativesarecrucialforflavorgeneration.Here,themetabolismandfunctionalenzymesoftheactivemicrobialcommunitiesinNFliquorstarterwerestudied,whichcouldpavethewaytoinitiateimprovementsinliquorqualityandtodiscovermicrobesthatproducenovelenzymesorhigh-valueaddedproducts.