Mining microbial genomes for new natural products微

SGMSpecialLectureFlemingPrizeLecture2007Deliveredatthe160thmeetingoftheSGM,[e-mail]fornewnaturalproductsandbiosyntheticpathwaysGregoryL.ChallisDepartmentofChemistry,UniversityofWarwick,CoventryCV47AL,UKAnalysesofmicrobialgenomesequenceshaverevealednumerousexamplesof‘cryptic’or‘orphan’biosyntheticgeneclusters,withthepotentialtodirecttheproductionofnovel,structurallycomplexnaturalproducts.Thisarticlesummarizesthevariousmethodsthathavebeendevelopedfordiscoveringtheproductsofcrypticbiosyntheticgeneclustersinmicrobesandgivesanaccountofmygroup’sdiscoveryoftheproductsoftwosuchgeneclustersinthemodelactinomyceteStreptomycescoelicolorM145.Thesediscoverieshintatnewmechanisms,rolesandspecificitiesfornaturalproductbiosyntheticenzymes.Oureffortstoelucidatethesearedescribed.TheidentificationofnewsecondarymetabolitesofS.coelicolorraisesthequestion:whatistheirbiologicalfunction?Progresstowardsansweringthisquestionisalsosummarized.IntroductionAlexanderFleming’sdiscoveryofpenicillinin1928anditssubsequentdevelopmentintoamedicinebyFloreyandChaininthe1940sprovidedthefoundationfordevel-opmentofmicrobialnaturalproductsasacornerstoneofnewdrugdiscoveryinthe20thcentury(Fleming,1929;Chainetal.,1940).Bytheendofthecenturymanymicrobialnaturalproductshadfoundtheirwayintotheclinicasantibacterial,antifungal,antiparasitic,anticancerandimmunosuppressiveagents.Yettheturnofthecenturyalsowitnessedamasswithdrawaloflargepharmaceuticalcompaniesfromnewmicrobialnaturalproductdiscoveryandmicrobialnaturalproductsresearch(Koehn&Carter,2005).Severalfactorsspurredthisretreat,includingrediscoveryofknownnaturalproductswithhighfre-quency,thetechnicalchallengesassociatedwithpurifica-tionandstructureelucidationofnaturalproductsfrommicrobialfermentationsandtheadventofcombinatorialchemistry,whichpromisedtoprovideawealthofnewcompoundstoscreenforbiologicalactivity.Attheverytimenaturalproductdiscoveryprogrammeswererampingdowninthelate1990s,large-scalemicrobialgenomesequencingbegantorampup,fuelledbythepioneeringapplicationofwhole-genomeshotgunsequencingtoHaemophilusinfluenzae,publishedin1995,whichdemon-stratedthatmicrobialgenomesequencescouldbeobtainedwithhithertounimaginedrapidity(Fleischmannetal.,1995).Atpresenttherearemorethan580completemicrobialgenomesequences.Dramaticandsustainedincreasesinourunderstandingofthegeneticsandenzymologyofmicrobialnaturalproductbiosynthesisthroughoutthe1990shavealsofacilitatedtheidentificationandanalysisofgeneclusterslikelytoencodenaturalproductbiosyntheticpathwaysinsequencedmicrobialgenomes(Fischbach&Walsh,2006).ItwasextremelyfortunatethattheWellcomeTrustfundedmetocarryoutresearchasapostdoctoralfellowintheDepartmentofGeneticsattheJohnInnesCentrein2000–2001,justastheStreptomycescoelicolorA3(2)genomesequencingprojectwasnearingcompletion.S.coelicolorwasoneofthefirstsequencedmicrobesinwhichitwasrecognizedthattherearemanymoregeneclustersencodingnaturalproduct-likebiosyntheticpathwaysthanthereareknownnaturalproductsoftheorganism(Bentleyetal.,2002).Similarobservationshavenowbeenreportedforseveraldiversesequencedmicro-organisms(Boketal.,2006;Ikedaetal.,2003;Kelleretal.,2005;Oliynyketal.,2007;Omuraetal.,2001;Paulsenetal.,2005;Udwaryetal.,2007).Thisdiscoveryprovidedastrongcounter-argumenttotheideathatfewnovelcompoundsremaintobediscoveredfromnaturalsourcesandsuggestedthatthewithdrawalofbigpharmaceuticalcompaniesfromnaturalproductdrugdiscoverywaspremature.Attheinceptionofmyindependentacademiccareer,wesetouttoinvestigatetheso-called‘cryptic’or‘orphan’naturalproductbiosyn-theticgeneclustersfoundwithinthegenomesofS.coelicolorandothersequencedmicrobesthatencodenaturalproductbiosynthesis-likeproteinsnotassociatedwiththeproductionofknownmetabolites.Overthepast6yearsnumerousgroupsaroundtheworldhavefocusedonsimilarobjectivesandtoday‘genomemining’fornewnaturalproductsandbiosyntheticpathwayshasbecomeadynamicandrapidlyadvancingfield(Corre&Challis,2007;Challis,2008;Gross,2007;Wilkinson&Micklefield,2007).Abbreviations:A,adenylation;ACP,acylcarrierprotein;CDA,calcium-dependentantibiotic;FAS,fattyacidsynthase;fhOrn,N5-formyl-N5-hydroxyornithine;hOrn,N5-hydroxyornithine;NRPS,nonribosomalpep-tidesynthetase;PCP,peptidylcarrierprotein;PKS,polyketidesynthase;TE,thioesterase.Microbiology(2008),154,1555–1569DOI10.1099/mic.0.2008/018523-02008/018523G2008SGMPrintedinGreatBritain1555InthefirstpartofthisarticleIwillbrieflyreviewthedifferentapproachesvariousgroupshavetakenfordiscoveringthemetabolicproductsofcrypticnaturalproductbiosyntheticgeneclusters.AnaccountofourdiscoveryofnewmetabolitesofS.coelicolorfollows,alongwithadiscussionoftheinvestigationsbyusandothersofthebiosynthesisandbiologicalfunctionsofthesemetabolites.StrategiesforidentifyingthemetabolicproductsofcrypticgeneclustersManymicrobialnaturalproducts,inparticularcomplexpolyketidesandnonribosomalpeptides,areassembledbybiosyntheticassemblylinesinvolvingmodularmega-synthasesandsynthetases(Fischbach&Walsh,2006).Inmanycasesthenumberofm