Methods and options for the heterologous productio

MethodsandoptionsfortheheterologousproductionofcomplexnaturalproductsHaoranZhang,BrettA.Boghigian,JohnArmandoandBlaineA.Pfeifer*Received1stSeptember2010DOI:10.1039/c0np00037jCovering:1990to2010Heterologousbiosynthesishasemergedasaviableroutetoaccessthebeneficialpropertiesofnaturalproducts.Thisdevelopmentprimarilyowestothedifficultiesencounteredwhenusingtraditionalmethodsofnaturalproductdiscoveryandproduction.However,theprocessofheterologousbiosynthesisalsopresentsanumberofchallengesthathaveproducedanarrayofcreativesolutionsandnoteworthysuccessstories.Inthisreview,ahistoricalperspectivewillbepresented,togetherwithananalysisoftheexperimentalapproachesthusfarusedtoaddresstheuniqueissuesassociatedwithheterologousnaturalproductbiosynthesis.1Introduction2Heterologousbiosynthesis2.1Nativehosts2.2Choosingaheterologoushost2.3Earlyheterologoushostsystems2.4Emerginghostsandtheaccompanyingmolecularbiologytools3Heterologousproductionofpolyketides3.1Streptomycescoelicolor3.2Streptomyceslividans3.3Escherichiacoli3.4Fungalhosts3.5Casestudy:Erythromycin4Heterologousproductionofnonribosomalpeptideandhybridnonribosomalpeptide-polyketidecompounds4.1Streptomycesspp.4.2E.coli4.3Bacillussubtilis4.4Pseudomonasputida4.5Casestudy:Epothilone5Heterologousproductionofisoprenoids5.1Artemisinin5.2Taxol6Summary7Emergingthemesandfuturedirections8Acknowledgements9References1IntroductionNaturalproductscontinuetofascinatebothbasicandappliedscientistsduetothecomplexbiochemicalpathwaysthatgiverisetothemandtheirincredibletherapeuticrange.Naturalproductsmaybegroupedintoseveralclasses;thisreviewwillfocusonthepolyketide,nonribosomalpeptide,andisoprenoidgroups.Fig.1highlightsexamplesfromthesefeaturedgroups.Theenzymology,biochemistry,andproteinstructuraldetailbehindnaturalproductbiosynthesishavebeenthefocusofnumerousresearchprograms.Thisintereststemsfromthearrayofuniquebiosyntheticstepsusedtoproduceequallydiversechemicalarchitectures.Briefly,thoughtherearedifferentsubdivisionsofbiosyntheticmechanisms,polyketideandnonribosomalpeptidebiosynthesisoftenfeatureslargeenzymes(referredtoasmegasynthasesormegasynthetases,dependingonthebiosyntheticcontext)characterizedbyamodularormulti-domainnature.PolyketidebiosynthesiscanbefurthersubdividedintotypeI,typeII,andtypeIIIsystems,withthetypeIandIIsystemssharingsimilaritiesandnomenclaturewithfattyacidsynthases.Isoprenoidbiosynthesisfeaturesterpenesynthasescapableofcyclizingpoly-isopreneunits.Foreachnaturalproductclasstobefeaturedinthisreview,biosynthesismayalsoincludeseveraltailoringtransformationssuchasglycosylation,hydroxylation,methylation,halogenation,orreductionbeforethefinalnaturalcompoundisproduced.Readersaredirectedtoseveralrecentreviewsthatfurtherdetailthebiosyntheticprocessesunderlyingthesenaturalproductclasses.1–9Besidesprovidingarichenvironmentforgenetic-andbiochemical-basedresearch,thekeyattractionofthestudyofnaturalproductbiosynthesisistheimpressiverangeofDepartmentofChemical&BiologicalEngineering,Science&TechnologyCenter,TuftsUniversity,4ColbyStreet,Medford,MA,02155,USA.E-mail:[e-mail]ªTheRoyalSocietyofChemistry2011Nat.Prod.Rep.,2011,28,125–151|125DynamicArticleLinksCNPRCitethis:Nat.Prod.Rep.,2011,28,125://pubs.rsc.org|doi:10.1039/C0NP00037JViewOnline/JournalHomepage/TableofContentsforthisissuetherapeuticvalueassociatedwiththefinalcompoundsproduced.ThisispartiallycapturedinFig.1,butthecompoundsfeaturedareonlyafractionofthosethathavebeeninstrumentalintransformingmodernmedicine.Forexample,analysisofsmallmoleculecompoundsintroducedintotheclinicfrom1981–2006indicatesthat76%ofantibacterialsand78%ofanticanceragentswerenaturalproductsorderivedfromnaturalproducts.10Beyondtheimpactnaturalproductshavehadinantibacterialandanticancertreatment,theyhavealsofoundapplicationsasanti-cholesterol,antimalarial,antiparasitic,andantifungalagents.Thetherapeuticpotentialofnaturalproductshasalsoattractedtheinterestofappliedscientistsandengineerseagertoaccesstheassociatedmedicinalvalue.Thissameinterestwasthebasisforsignificantindustrialinvestmentinnaturalproductprograms.Asaresult,sincethe1940s,acommunityofscientistsandengineershaspropelledeffortstounderstandandover-produceclinically-relevantnaturalproducts.Thisreviewwilllookattheemergingfrontiersfornaturalproductbiosynthesiswithaparticularfocusonheterologousbiosynthesis.Inpartic-ular,thereviewwilldetailthestepsrequiredtotransfertheresponsiblegeneticmaterialtoagivenhostandestablishnaturalproductformation(Fig.2).Specialemphasiswillbeplacedonguidingmethodologyandcurrentexperimentaltoolsavailabletocompletethistask.Severalexcellentreviewsprovidesimilarsummariesofheterologousbiosynthesis.11–14Here,weintendtoupdateandexpandtheanalysisacrossadditionalnaturalproductclassesandhostsystems.However,tobesomewhatsuccinct,thereviewhasbeenconstrainedtoaselectnumberofhostsandnaturalproductsystemsandexcludescertainnaturalproducttypes(suchasflavonoids,stillbenes