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Methods in Molecular Biology 2555 Wolfgang R. Streit Rolf Daniel Editors Metagenomics Methods and Protocols Third Edition M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Metagenomics Methods and Protocols Third Edition Edited by Wolfgang R. Streit Department of Microbiology and Biotechnology, University of Hamburg, Hamburg, Germany Rolf Daniel € Institut für Mikrobiologie und Genetik, Georg-August-Universitat Göttingen, Göttingen, Germany Editors WolfgangR.Streit RolfDaniel DepartmentofMicrobiology Institutfu¨rMikrobiologieundGenetik andBiotechnology Georg-August-Universit€atGo¨ttingen UniversityofHamburg Go¨ttingen,Germany Hamburg,Germany ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2794-5 ISBN978-1-0716-2795-2 (eBook) https://doi.org/10.1007/978-1-0716-2795-2 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. 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Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Almost 25 years after the first publications on metagenomics, it is still a key technology to the DNA- and RNA-based exploration of the genomic potential from not-yet-cultivated microbes for ecology and biotechnology. Since the term “metagenome” was coined over twodecadesago,metagenomicshasdramaticallychangedour viewonmanyresearchareas suchasmicrobialecology,communitybiology,andmicrobiomeresearch,andithasresulted in the rapid identification of many novel biomolecules with potential value to bio-based industrial processes. Function-driven metagenomics has been in the focus of many labora- toriesaroundtheworldtoquicklyencounternovelfunctionalgenesencodingenzymeswith new and/or improved traits. In this way, the diversity of biocatalysts and other valuable biomoleculesusefulfordownstreamapplicationsincreasedsignificantly. Industries demand enzymes that can be directly applied in biotechnological processes and that catalyze a wide variety of different reactions. Ideally, these biocatalysts/bioactive molecules should be highly active with a broad range of substrates under harsh reaction conditions and, at the same time, should possess a predictable substrate specificity and enantioselectivity. Today, only a limited number of truly well-suited enzymes fulfill these requirements. To identify novel biomolecules, different strategies are employed: While the sequence-baseddetectionofnovelenzymesandotherbiomoleculescertainlyprovidesrapid accesstonovelgenesandenzymes,itsuffersfromthefactthatonlysequenceswithputative function and similarities to already known genes are recovered. Function-based screens overcome this bottleneck but are limited by low hit rate due to the often poor capabilities oftheemployedhosttoexpressforeigngenesandtoproduceactiverecombinantproteins. Thus,function-drivendetectionofnovelbiocatalystsorothervaluablebiomoleculesisstilla very time-consuming process that slows down development times for novel products. However, it has the huge advantage that functional biocatalysts and bioactive compounds arerecovered. Inrecentyears,variousnoveltechnologieshavebeendevelopedtoaccessthemetagen- omes ofmicrobialcommunities usinghigh-throughputtechnologies often incombination with next-generation sequencing approaches. Within the third edition of this book, we provideup-to-dateandnoveltechnologiesonvariousfunction-basedtechnologiescurrently used in metagenomics and related areas. Our goal is that this book serves as a manual for researcherswhoareinterestedinestablishingmetagenomicsintheirlaboratories.Allwork- ing steps involved are presented in the chapters: Starting from the DNA/RNA isolation fromsoilsandmarinesamplesfollowedbytheconstructionandscreeningofthelibrariesfor diverseenzymesandbiomolecules.Thebookprovidesacomprehensiveoverviewoncurrent methods used to isolate DNA and construct large-insert and small-insert libraries form terrestrialandmarinehabitats.Itfurthersummarizesmethodsforestablishingmetagenome libraries in non-E. coli hosts such as Streptomyces, and it highlights novel molecular tools ready to use for function-driven mining of metagenomic DNA. Lastly, several chapters providedetailedinsightsintoscreeningandanalyzingprotocolsforawidearrayofdifferent genesencodingenzymeswithrelevancetobiotechnologyandecology.Protocolsareoffered for the screening of polymer active enzymes, acting on natural and non-natural polymers. v vi Preface Among those, we offer protocols to identify novel polyethylene-degrading enzymes (PETases) using bioinformatic searches in combination with rapid in vitro expression protocols. Furthermore, the book provides detailed screening protocols for phosphatases, carbon-metabolism-related genes, and hydrogenases. In addition, approaches for viral metagenomeanalysiswereincluded. Inour view,thisbookprovidesacomprehensivecollectionofup-to-datemetagenome protocolsandtoolsfor therecoveryofmanymajor typesofbiocatalystsandallowsaneasy setupofthesescreensinanymicrobiologylaboratory. Hamburg,Germany WolfgangR.Streit Göttingen,Germany RolfDaniel Contents Preface ..................................................................... v Contributors................................................................. ix 1 ConstructionofSmall-InsertandLarge-Insert MetagenomicLibraries ..... ........ ....... ....... ........ ....... ........ 1 CarolaSimonandRolfDaniel 2 ExtractionofTotalDNAandRNAfromMarineFilterSamples andGenerationofaUniversalcDNAasUniversalTemplate forMarkerGeneStudies.... ........ ....... ....... ........ ....... ........ 13 JacquelineHollensteiner,FranziskaWemheuer,DominikSchneider, BirgitPfeiffer,andBerndWemheuer 3 FunctionalMetagenomicsasaTooltoTapintoNaturalDiversity ofValuableBiotechnologicalCompounds.... ....... ...... .. ...... ....... .. 23 NancyWeiland-Bra€uer,LivıáSaleh,andRuthA.Schmitz 4 FunctionalandSequence-SpecificScreeningProtocolsfor theDetection ofNovelAntimicrobialResistanceGenesinMetagenomicDNA...... ........ 51 SupathepTansirichaiya,WilliamHutton,andAdamP.Roberts 5 IdentificationofPKSGeneClustersfromMetagenomicLibrariesUsing aNext-GenerationSequencingApproach.... ....... ........ ....... ........ 73 AlinneL.R.Santana-Pereira 6 Activity-BasedScreeningofMetagenomicFosmidLibraries forHydrogen-UptakeEnzymes...... ....... ....... ........ ....... ........ 91 NicoleAdam-BeyerandMirjamPerner 7 FunctionalMetagenomicsApproachfor theDiscoveryofNovel GenesEncodingPhosphataseActivity .. ..... ...... ......... ....... ........ 103 GenisA.CastilloVillamizar,HeikoNacke,andRolfDaniel 8 IsolationofGenesEncodingCarbonMetabolismPathwaysfromComplex MicrobialCommunities..... ........ ....... ....... ........ ....... ........ 115 ArankshaThakor,JiujunCheng,andTrevorC.Charles 9 ScreeningMetagenomesforAlgaeCellWallCarbohydratesDegrading HydrolasesinEnrichmentCultures ......... ....... ........ ....... ...... .. 125 JaschaF.H.Macdonald,InesKrohn,andWolfgangR.Streit 10 ThePET-DegradingPotentialofGlobalMetagenomes:FromInSilico MiningtoActiveEnzymes.......... ....... ....... ........ ....... ........ 139 JenniferChow,PabloPe´rez-Garcıá,RobertF.Dierkes,HongliZhang, andWolfgangR.Streit 11 High-ThroughputScreeningforThermostable PolyesterHydrolases ....... ........ ....... ....... ........ ....... ........ 153 YannickBranson,ChristoffelP.S.Badenhorst,LaraPfaff, CarolinBuchmann,RenWei,andUweT.Bornscheuer vii viii Contents 12 MetagenomicScreeningofaNovelPETEsteraseviaInVitro ExpressionSystem ......... ........ ....... ....... ........ ....... ........ 167 YuchenHan,RobertF.Dierkes,andWolfgangR.Streit 13 AssigningFunctionsofUnknownEnzymesbyHigh-ThroughputEnzyme Characterization.... ....... ........ ....... ....... ........ ....... ........ 181 PatriciaMolina-Espeja,LauraFernandez-Lopez,PeterN.Golyshin, andManuelFerrer 14 IsolationofaHost-ConfinedPhageMetagenomeAllowstheDetection ofPhagesBothCapableandIncapableofPlaqueFormation ........ ....... .. 195 InesFriedrichandRobertHertel 15 CRISPR-Cas9ShapedViralMetagenomesAssociated withBacillussubtilis........ ........ ....... ....... ........ ....... ........ 205 KatharinaKohm,VeronikaTheresaLutz,InesFriedrich,andRobertHertel 16 CloningandExpressionofMetagenomicDNAinStreptomyceslividans andItsSubsequentFermentationforOptimizedProduction......... ........ 213 YuriyRebets,JanKormanec,AndriyLutzhetskyy,KristelBernaerts, andJozefAnne´ 17 DNA-,RNA-,andProtein-BasedStable-IsotopeProbingforHigh-Throughput BiomarkerAnalysisofActiveMicroorganisms ....... ........ ....... ........ 261 EleanorJameson,MartinTaubert,RoeyAngel,SaraCoyotzi,YinChen, O¨zgeEyice,HendrikScha€fer,J.ColinMurrell,JoshD.Neufeld, andMarcG.Dumont Index ...................................................................... 283 Contributors NICOLEADAM-BEYER • Geomicrobiology,DepartmentofMarineBiogeochemistry,GEOMAR HelmholtzCentreforOceanResearchKiel,Wischhofstraße,Kiel,Germany ROEYANGEL • Soil&WaterResearchInfrastructureandInstituteofSoilBiology,Biology CentreCAS,Cˇeske´Budeˇjovice,Czechia JOZEFANNE´ • DepartmentofMicrobiology,ImmunologyandTransplantation,lab. MolecularBacteriology,RegaInstitute,KULeuven,Leuven,Belgium CHRISTOFFELP.S.BADENHORST • DepartmentofBiotechnology&EnzymeCatalysis, InstituteofBiochemistry,UniversityofGreifswald,Greifswald,Germany KRISTELBERNAERTS • DepartmentofChemicalEngineering,ChemicalandBiochemical ReactorEngineeringandSafetyDivision,KULeuven,Leuven,Belgium UWET.BORNSCHEUER • DepartmentofBiotechnology&EnzymeCatalysis,Instituteof Biochemistry,UniversityofGreifswald,Greifswald,Germany YANNICKBRANSON • DepartmentofBiotechnology&EnzymeCatalysis,Instituteof Biochemistry,UniversityofGreifswald,Greifswald,Germany CAROLINBUCHMANN • DepartmentofBiotechnology&EnzymeCatalysis,Instituteof Biochemistry,UniversityofGreifswald,Greifswald,Germany GENIS A.CASTILLO VILLAMIZAR • Corporaci(cid:2)onparalainvestigaci(cid:2)ondelacorrosi(cid:2)on(CIC), Santander,Piedecuesta,Colombia;InstituteofMicrobiologyandGenetics,GeorgAugust UniversityGo¨ttingen,Go¨ttingen,Germany TREVOR C.CHARLES • DepartmentofBiology,UniversityofWaterloo,Waterloo,ON, Canada JIUJUN CHENG • DepartmentofBiology,UniversityofWaterloo,Waterloo,ON,Canada YINCHEN • SchoolofLifeSciences,UniversityofWarwick,Warwick,England JENNIFERCHOW • DepartmentofMicrobiologyandBiotechnology,Universita€tHamburg, Hamburg,Germany SARACOYOTZI • DepartmentofBiology,UniversityofWaterloo,Waterloo,Canada ROLFDANIEL • InstituteofMicrobiologyandGenetics,Georg-August-UniversityGo¨ttingen, Go¨ttingen,Germany;InstituteofMicrobiologyandGenetics,GeorgAugustUniversity Go¨ttingen,Go¨ttingen,Germany ROBERTF.DIERKES • DepartmentofMicrobiologyandBiotechnology,Universita€tHamburg, Hamburg,Germany;DepartmentofMicrobiologyandBiotechnology,Universityof Hamburg,Hamburg,Germany MARCG.DUMONT • SchoolofBiologicalSciences,UniversityofSouthampton,Southampton, England O¨ZGEEYICE • SchoolofBiologicalandBehaviouralSciences,QueenMaryUniversityof London,London,England LAURA FERNANDEZ-LOPEZ • ICP,CSIC,Madrid,Spain MANUELFERRER • ICP,CSIC,Madrid,Spain INESFRIEDRICH • InstituteofMicrobiologyandGenetics,GeorgAugustUniversityGo¨ttingen, Go¨ttingen,Germany PETERN.GOLYSHIN • ICP,CSIC,Madrid,Spain;CentreforEnvironmentalBiotechnology, SchoolofNaturalSciences,BangorUniversity,Bangor,UK ix