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TheKetosynthaseDomainConstrainstheDesignofPolyketideSynthases

MajaKlausa+,LynnBuyachuihana+andMartinGriningera*

aInstituteofOrganicChemistryandChemicalBiology,BuchmannInstitute forMolecularLifeSciences,GoetheUniversityFrankfurt+Theseauthorscontributedequally*Correspondingauthor:grininger@chemie.uni-frankfurt.deAbstractModularpolyketidesynthases(PKSs)producecomplex,bioactivesecondarymetabolitesinassemblyline-likemultistepreactions.Thesequenceofreactionstepsdeterminestheidentityofthecompoundandisdefinedbytheorderofmoduleswithintheassemblyline.Thistypeofchemicalprogrammingmakes modular PKSs ideal templates for engineering multistep C-C bond forming reactions.However,engineeringofmodularPKSsbyrecombiningintactmodulestoproducenovel,biologicallyactive compounds has often resulted in chimeras exhibiting poor turnover rates and decreasedproductyields.RecentfindingsdemonstratethatthelowefficienciesofmodularchimericPKSsalsoresultfromratelimitationsinthetranslocationstep;i.e.,thetransferofthegrowingpolyketidefromonemoduletotheotherandfurtherprocessingofthepolyketidesubstratebytheketosynthase(KS)domain.In this study, we systematically vary protein:protein interfaces, substrate identity and substratespecificityoftheKSinvolvedreactionsofbimodularchimericPKSs.First,webroadenedthesubstratespecificity of the C-C bond forming KS domains with the aim to increase the turnover rates ofkinetically impaired chimeric PKSs. Diverse multipoint mutants were calculated with a Rosettaprogramsuitetoresultinstableactivesiteenvironments.Somemutationsefficientlyreleasedthekinetic penalties imposed by the non-native substrate and especially one mutant exhibitedunexpectedhigherturnovernumbersthanthereferencesystem.Second,wechangedtheidentityofthedonormoduleinthechimericconstructstoalsovaryprotein-proteininteractionsandsubstrateidentity.WeobservedthattheKSdomain,specificallythetranslocationreaction,issensitivetowardsboth substrate identity and protein-protein interactions in a complex and often non-intuitiveinterplayof theseconstraints.Overall,oursystematicstudycanexplainwhyearlyoversimplifiedengineeringstrategiesbasedontheplainshufflingofmodulesmostlyfailedandwhymorerecentapproachesshowimprovedsuccessrates.

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INTRODUCTIONMultimodularpolyketidesynthases(PKSs)arelargemegasynthases,whichareresponsiblefortheproductionofavarietyofpharmaceuticallyimportantcompoundssuchasantibiotics,anti-fungalandanti-cholesterol agents (Figure 1A).1 The modularity of PKSs has put them at the forefront ofengineering efforts to achieve programmable access to novel compoundswith new bioactivities.However, to datemost of the engineered systems do not reach biotechnological relevant stages,becausetheturnovernumbersandthusproductyieldsareoftentoo low.2–4Despiteconsiderableeffortsinthelast30years,nogeneralizablerulesforthegenerationofefficientchimericPKSsexisttoday.Forexample,althoughthe importanceofprotein-protein interactionse.g.betweendockingdomains5–7orbetweentheacylcarrierprotein(ACP)andtheketosynthase(KS)atdifferentstagesinthecatalyticcyclehasbeenrecognized(Figure1B),4,8–10moststrategiestomodulateandadaptthoseinteractionsinchimericmodularPKSshavefailedsofar.11–14The6-deoxyerythronolideBsynthase(DEBS)istheprototypicalexampleofthisenzymeclassandhas served as a platform to study the structure, mechanism, and engineering potential of theseenzymes (Figure 1A).15 In the past, numerous studies on DEBS have shed light on substratetolerance,6,16protein-protein interactionspecificities,5,9,17,18andstructuralcharacteristics19–21withbroad relevance for the family of modular PKSs. Recently, we analyzed a library of bimodularchimericPKSs,withmodulesmainlyderivingfromDEBS,tocomparedifferentchimericinterfaces(Figure 1C).22 From our analysis, it became apparent that some systems with native domaininterfaces across module boundaries (from the ACP of the upstream module to the KS of thedownstreammodule)sufferadecreaseinturnoverrates,likelyasaresultofatoonarrowsubstratetoleranceofthedownstreammodule(inthiscaseobservedontheexampleofM6-TE).22InorderforaPKSmoduletosuccessfullyprocessnon-naturalpolyketidesubstrates,mutagenesisapproachesarerequiredthatbroadenthesubstratetoleranceofagivenmodule.Therearemultipleoptions foradownstreammodule tobe rate-limitingwhenprocessinganon-cognatesubstrate. IncaseofourmodelPKS(Figure1C,bottom), threereactionscan inprincipleaccountforsubstratespecificityofM6-TEandcausetheabove-mentionedphenomenon:(i)eithertheKS6-catalyzedcondensationreaction,(ii) the followingNADPH-dependentreductionof theb-ketoester by KR6, or (iii) the TE-catalyzed lactonization to the triketide product. While noinformationisavailableonthesteady-statekineticsoftheindividualreactionstepswithinM6-TE,previous analysis of the hydrolysis rate of two enantiomeric diketides by the TE domain,23comparisonofthereductionratesoftwoKRdomains24andkineticanalysisoftheKSdomainofDEBSM216,25haveledtotheconclusionthattheKS-catalyzeddecarboxylativecondensationlikelylimitstherateofanativemodule.15,25IdentificationoftheKSdomainasabottleneckinPKSturnoverisfurther supported by a recentmutagenesis study on DEBS KS3 inwhich single point active sitemutagenesisresultedinsignificantlybroadenedsubstratespecificitiesandhigherturnoverrates.26BasedontheobservationthattheKSdomainsofthemycolactonesynthase(MYCL)sharea>97%sequenceidentity,despiteacceptingsubstratesofdifferentlengthandchemicalmodification,DEBSKS3residueswerereplacedwith theircounterparts fromMYCL, resulting inoneparticularpointmutant(A154WKS3)withsignificantlyincreasedpromiscuitycomparedtowild-typeKS3.26

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Figure1.SchematicarchitectureofDEBS,itscatalyticcycleandaccessibleinterfacesforengineeringapproaches.(A)ArchitectureofDEBS.Thethreepolypeptides(DEBS1-3),theencodedmodules(M1-M6),aswell as the loadingdidomain (LDD), the thioesterasedomain (TE), and the final product (1) aredepicted.Polyketideintermediatesareshownasattachedtotherespectiveacylcarrierprotein(ACP).Blacktabsdepictdocking domains. Domain annotations are as follows: LDD- loading didomain, AT- acyltransferase, KS-ketosynthase, ACP- acyl carrier protein, KR- ketoreductase, DH- dehydratase, ER- enoylreductase, TE-thioesterase.Module3hasaketoreductase-likedomain(denotedinlowercase)thatlacksNADPH-dependentoxidoreductaseactivitybutharborsC2epimeraseactivity.27,28(B)CatalyticcycleontheexampleofM1andM2.(I) AT-catalyzed transacylation using (2S)-methylmalonyl-CoA, followed by KS-mediated decarboxylativeClaisencondensation(chainelongation,II)togivethe(2R)-diketide.29InM1theKRcatalyzestheepimerizationof the C2 methyl group, followed by diastereospecific reduction (III) to give the (2S,3R)-2-methyl-3-hydroxydiketide condensation product. The diketide is translocated to the KS domain of the downstreammodule(chaintranslocation,IV)fromwhichthenextroundofreactionscanstart.InDEBSonlyKR1andKR3catalyzeepimerizationof theC2methylgroup.30 (C)Subsetofbimodular chimericPKSsusedpreviously.22SYNZIP domains (SZ3 and SZ4) assisted in construct design. Black tabs represent DEBS-derived dockingdomains(modulenumbersinbrackets)installedattheproteinterminitomediateweakinteractions.HavingidentifiedtheKSascandidateenzymaticdomainforadditionalengineeringapproaches,wesoughttobroadenitssubstratespecificitybymultipointmutations.Directedevolutionexperimentsrevealedthatonaverage10mutationsarerequiredtoimprovetheactivityofanenzymebyfactor1000,31indicatingthatseveralmutationswilllikelybenecessarytoincreaseturnoverofnon-nativesubstrates by PKSs. Due to the lack of practical selection methods, the application of directedevolution approaches is hampered in our experimental set-up. We therefore aimed at a moretargeted approach to guide multipoint mutagenesis of KS domains, using the FuncLib server, aprogramthatgeneratesavarietyofdiversemultipointmutants,bycalculatingstablenetworksofinteractingactivesiteresidues.32FuncLibusesphylogeneticinformationtorevealpossible,coupledmutationswithin a protein, byworking on two levels: First, it uses phylogenetic information tosuggestaminoacidexchangesatselectedpositions,andsecond, it ranks themutatedproteinsbycalculatingtheirstabilityviatheRosettaprogramsuite.Inthisprocess,mutationsthatarepredictedto destabilize the protein fold are discarded. The FuncLib approach does not target individual

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substrate specificities, but delivers a set of stable variants that can then be screened for thespecificitiesandactivitiesofinterest.Here we identify KS domains as a bottleneck in assembly line PKSs and show that multipointmutagenesiswithin theKS active site can increase the turnover rate of chimeric PKSs. Our datademonstrates that the translocation reaction inmodular PKSs is constrained by protein-proteininteractionsandsubstratespecificityoftheKSdomain,andcanexplainwhyengineeringapproachesbasedonthesimpleshufflingofmoduleshaveoftenfailedinthepast.RESULTSANDDISCUSSIONDesignofKSMultipointMutants-TotestthefeasibilityofKSengineeringtobroadenthesubstratespecificity of PKS modules, we decided to apply the FuncLib approach on KS domains of thedownstreammodules(alsotermedacceptormodulesinthefollowing)oftwobimodularchimericPKSs (Figure 2A). Specifically, we introduced the KS mutations in the context of the full-lengthmodulesM3-TEandM6-TE(DEBSTEinbothsystems)andcomparedtheturnoverratesofwild-type(nonKS-mutated)vs.mutantmodules(Figure2B).Inthesebimodularsystems,e.g.LDD(4)+(5)M1-SZ3+SZ4-M3-TE, docking domains (indicated as (4)+(5))33 and SYNZIP domains (indicated asSZ3+SZ4)34,35wereemployedattheproteininterfacestomediatenon-covalentinteractionsbetweenseparatelypurifiedmodules(Figure2B).

Figure2.KSmutagenesis-StructuralorganizationofKSactivesitesandsetupofbimodularchimericPKSs.(A)StructuralorganizationoftheactivesiteofKS3andKS6.Thecatalytictriadisdepictedasgraysticks.Residueschosenformutagenesiswithina12Å-rangeoftheactivecysteineC202KS3orC172KS6areshownasorangesticks.(B)BimodularchimericPKSsusingLDD(4),(5)M1-SZ3,andSZ4-M3-TEorSZ4-M6-TEasthefinalmodule. Matching DEBS-derived docking domains (black tabs; (4)+(5)) or matching SYNZIP domains(SZ3+SZ4)areinstalledattheproteinterminitomediatenon-covalentinteractions.ThechimericACP1harborsthe(2S,3R)-2-methyl-3-hydroxydiketide(naturaldiketide,NDK)astheincomingsubstrateforwild-typeandmutant KS domains. Depending on the acceptor module either unreduced triketide lactone (TKLUR) 2 orreducedTKL(TKLR)3arethepredictedproducts.

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ThechoiceofM3andM6wasguidedbytheirdifferingturnoverrateswithM122andthepreviouslyobservedsubstraterestrictions.16 X-raystructuraldataexists forKS3-AT3,36,37whichgaveustheopportunitytoruntheFuncLibmethodforthecalculationofstableactivesitesonauthenticdata.ForKS6-AT6,ahomologymodelwasgeneratedon thebasisofasequencesimilarityof~55%to thestructurallysolvedKS-ATdidomainsKS3-AT336andKS5-AT519andthemodelwasalsosubmittedtoFuncLib. To determine suitable residues for KS active sitemutagenesis, we considered residueswithinafirstshellaroundtheactivecysteinesthatdirectlydecoratethebindingsiteforsubstratesandmapped themonto amultiple sequence alignment (Figure S1&S2). From the subset of non-conservedresidues,allthosepointingtowardstheactivecysteineswerechosenformutagenesis,astheymightbe involved insubstratebindingduringtheenzymaticreaction, leadingtoa totalof7residuesforKS3and9residuesforKS6(Figure2A).Submission of the KS3-AT3 and KS6-AT6 didomains and selection of the target residues withinFuncLib(includingthepossibilityofatryptophanatA154KS3andA124KS6)26resultedinasequencespaceof152,826designsforKS3-AT3and285,822designsforKS6-AT6withalargevariabilityinmutatedresidues(TableS1).Inanextstep,Rosettacalculationrevealedthatfromtheinitialdesigns1,011designsforKS3-AT3and3,683designsforKS6-AT6resultinmorestableproteinsthantherespectivewild-typeproteins.Fromthe50highestrankeddesigns,wechose12forKS3-AT3and10forKS6-AT6 forsubsequentanalysis,basedontheir largevariability inmutatedresidues(TablesS2&S3).Alldesignsharboredaminimumofthreeandamaximumoffivemutations.Inaddition,thepreviously reported single point mutants A154WKS3 and A124WKS6 were also generated as areference.26 The mutations are predicted to change the geometry of the respective active sites,potentiallyleadingtodifferentpropertiesinsubstratebindingorcatalysis(FiguresS3&S4).GenerationofMultipointKSMutantsandTurnoverAnalysis-To evaluate the performance of the different KS designs under PKS turnover conditions, all KSmutationswereintroducedinthebackgroundofthefull-lengthmodulesSZ4-M3-TEandSZ4-M6-TE.Allmutantproteinsexhibitedsimilarpurificationbehaviorandpurity(FigureS5A&B),withyieldscomparableorhigherthanthewild-typemodules(between5-17mgperliterE.coliculture,TableS4).Proteinoligomerization,asmeasuredbySEC,showedthatbesidesMut07andMut10allmutantproteinseluted inasinglepeak indicativeofahomodimericprotein fold(FigureS6&S7). Ingoodagreementwiththeseresults,thermalshiftassaysofeachmutantindicatednosignificantdifferencein thermal stability betweenwild-type andmutantmodules (Table S5).By analyzing theproteinqualityof all generatedmutants,we conclude that inour caseFuncLib serves as auseful tool tocalculatestablemutantswithalargevarietyinactivesiteresidueswithoutdecreasingproteinyieldaswellasoligomericandthermalstability. TurnoverratesofbimodularchimericPKSsharboringKS3mutationsTheturnoverratesofallM3mutantswasfirstassessedinthebimodularchimericPKSconsistingofLDD(4), (5)M1-SZ3 and SZ4-M3-TE; a system harboring a non-cognate ACP1:KS3 interface andchallengingKS3withtheDEBS-derivednaturaldiketide(NDK,(2S,3R)-2-methyl-3-hydroxydiketide,Figure3A).Asmeasuredbefore,thechimericsystemwithwild-typeSZ4-M3-TEexhibiteda>30-foldloweractivitythanthereferencesystemwithSZ4-M2-TE(Figure3A,graybar).Comparedtowild-typeM3,thesinglepointmutantMut01(A154WKS3),followingthedesignofMurphyetal.,26hadaroughly2-foldincreasedactivity.AmongthemultipointmutantsconstructedbytheFuncLibmethod,Mut06,Mut07,Mut10,Mut14,andMut15showednoimprovementorevenlossofactivity(Mut07,Figure3A).SimilartoMut01,Mut03andMut05showeda2-foldincrease(Figure3A).ThemultipointmutantsMut04,Mut08,Mut09,Mut11andMut13exhibitedthelargestincreaseinturnoverrate(2.5-fold),narrowingthegaptowardsthereferencesystemtoa15-folddifference.Ingoodagreement

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withtheturnoverrates,productanalysisafterovernightincubationconfirmedthepresenceoftheexpectedunreducedtriketidelactone2(TKLUR)inallsystemswithmeasurableturnover(FigureS8).

Figure3.TurnoverratesofbimodularchimericPKSsusingwild-typeormutantM3-TE(A)andM6-TE(B), respectively.BimodularPKSsconsistedofLDD(4), (5)M1-SZ3,andSZ4-M3-TE/SZ4-M6-TE(wild-type(WT)oroneoftheirmutants).ForbothbimodularPKSs,thereferencesystemconsistedofLDD(4),(5)M1-SZ3,andSZ4-M2-TE(graybar).Allinitialratedatawasobtainedat4µMenzymeconcentrationandnon-limitingconcentrations of propionyl-CoA, methylmalonyl-CoA, and NADPH. Measurements were performed intechnicaltriplicate.Notethatforfurtherconfidence,theturnoverrateofMut26isdisplayedasanaverageofthreeindependentlypurifiedproteinsamples(biologicaltriplicate)measuredintriplicate.TurnoverratesofbimodularchimericPKSsharboringKS6mutationsAsanextstep,weevaluatedtheturnoverratesofbimodularchimericPKSsusingM6-TEoroneofitsmutants(Figure3B).Thesebimodularsystemsharboranon-cognateACP1:KS6interface.Analysisofwild-typeM6-TE in thepresenceof (5)M1-SZ3showedsimilar resultsas reportedearlier.22 Incontrast toKS3mutants, the single pointmutantMut02 (A124WKS6)26 and 8 out of 9multipointmutants revealeddiminished turnover rates of thebimodular systemcompared towild-typeM6(Figure3B).Tooursurprise,onemutant,Mut26,didnotonlyshowsignificantlyincreasedturnovercomparedtowild-typeM6(~2.5-foldimprovement),butperformedevenbetterthanthereferencesystemM2-TE(1.5-foldimprovement,Figure3B).Mut26containsonlythreeKSmutationsofwhichS174AandV207Larepredictedtocauseonlyminimalstericchanges,whileF235Misanticipatedtoenlarge the binding site of KS6 (Figure S4) and to increase the similarity betweenKS2 andKS6(FigureS9).22NotethatforallacceptormodulesshowninFigure3B,amixtureoftheexpectedreducedTKLR3andunreducedTKLUR2wasobtained from the reactionmixtureaftera reaction timeofonly10min(TableS6,FigureS10).SimilardatahasbeenreportedbeforeforDEBSM6-TE.38AstheturnoverratesweremeasuredbycorrelatingthereductionratesoftheKRdomains(NADPHconsumption)withproduct formation and assuming a 100% conversion to reduced TKLR,33 the absolute turnovernumbers presented in Figure 3B would all be higher if corrected by the amount of producedunreducedTKLUR.Yet,astheratiosofreducedtounreducedTKLaresimilarinallM6samples(TableS6),therelativeratiosofturnoverratesarestillcapturedinFigure3B.UsingdifferentdonorACP-substratecombinationstoanalyzetheturnoverratesofKSmutantsAs the same donor module (5)M1-SZ3 was used to evaluate the performance of all generatedbimodularPKSs,theanalysissofaronlygaveinsightintotheeffectofeachmutantwhenpresentedwith the chimeric ACP1 bearing NDK (Figure 2B). With the aim of additionally analyzing thecontributionofACP:KSrecognitionandsubstrate:KSinteractionontheturnoverofKSmutants,we

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generatedalternativeM1donormodulesthatwouldallowustoprobetheremainingACP-substratecombinations(Figure4&5).BasedontheobservationsthatKRdomainscanefficientlyturnoverwithavarietyofchimericACPs,39andthatchainelongationatachimericACP:KSinterfaceinafusionproteinisnotrate-limiting,22wegenerated6alternativeM1donorsbypermutatingKR1andACP1.TheexchangeofACP1withACP2and ACP5, the native upstreamACPs ofM3/M6, generated donormodules carrying NDK on therespectiveACP(Figure4A,donor2&Figure5A,donor5).SinceKRdomainsareresponsible forinstallingthestereogeniccentersatthea-andb-positionofthepolyketide,anexchangeofKR1byKR2 allowed us to generate the DEBS-derived enantiomeric diketide (EDK, (2R,3S)-2-methyl-3-hydroxydiketide)onACP1(Figure4A&5A,donor3).4Asalastengineeringapproach,bothKR1andACP1wereexchangedbythenativeupstreamdomainsofM3/M6,resultingintheformationofEDKon either ACP2 (Figure 4A, donor 4) or ACP5 (Figure 5A, donor 6). All donor proteins could bepurified in similar quantities and were pure as judged by SEC and SDS-PAGE (Table S4, FigureS11&S5C).Withthesedonormodulesinhand,weexaminedtheactivityofbimodularPKSsinthepresenceofeitherwild-typeM3orasubsetofmutantsthatshowedhighturnoverrateswithdonor1(Mut01,Mut08, Mut11, Mut13) (Figure 4A). Among all acceptors, donor 2, that reconstitutes the nativeACP2:KS3interactionandpresentsKS3withtheNDKsubstrate,emergedasthebestdonormodule.Inmanycases, thesecond-bestdonormoduleappeared tobedonor4,alsoemployingACP2,butdisplayingtheenantiomericsubstrate.Overall,thecomparisonofdifferentdonorsforwild-typeM3andMut01highlightstheimportanceofprotein-proteininteractionsattheACP:KSinterfaceinthischimericPKS.4Inaddition,whereaswild-typeM3andthesinglepointmutantMut01showedastrongsubstratepreferenceforNDKoverEDK,someofthemultipointmutantsefficientlybroadenedthesubstratetoleranceofKS3andexhibitedareleasedACPpreference.

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Figure4.TurnoverratesofbimodularchimericPKSsusingM3-TEanddifferentdonormodules.(A)Thedonor modules used to generate bimodular PKSs. Substrates and the employed ACPs are indicated. (B)TurnoverratesofbimodularchimericPKSusingM3-TEasacceptormodule.AllbimodularPKSsconsistedofLDD(4),followedbyadonormoduleasindicatedin(A),andSZ4-M3-TEoroneofitsmutants.ThereferencesystemconsistedofLDD(4),(5)M1-SZ3,andSZ4-M2-TE(graybar).Allinitialratedatawasobtainedat4µMenzyme concentration andnon-limiting concentrations of propionyl-CoA,methylmalonyl-CoA, andNADPH.Measurementswereperformedintriplicate.TheexpectedunreducedTKLURwasdetectedinallsamplesafterovernightincubation(FigureS12).ThealternativedonormoduleswerefurtheranalyzedinbimodularchimericPKSswithwild-typeM6andMut26astheacceptormodules(Figure5A).Again,wenotethatallbimodularPKSsusingwild-typeM6orMut26producedamixtureofTKLRandTKLUR(Figure5B),whichhamperskineticanalysisofthebimodularPKSsand,specifically,underestimatestheactualnumberofturnovers(Figure5C).Intriguingly,whilesimilarratiosofTKLR:TKLURwereobservedforbothacceptors(Figure5C,TableS7),therelativeratiosdifferedbetweentheuseddonors.Thishighlightsthatbothwild-typeM6andMut26processthepolyketidesubstrateinasimilarmanner.However,asthelactonizationofTKLURcanonlyoccurafterasuccessfulreductionreactioninthedonormodule,thedifferencesinproductratiosindicatethattheidentityofthedonormoduleaffectsthereductionreactionofKR6withintheacceptormodule.Forwild-typeM6andMut26,theexpectedTKLRismoreefficientlygeneratedwitheitherACP1orACP5carryingEDKasthesubstrate(Figure5B,donor3&6),whereasahigheramountofTKLURaccumulatesinthepresenceofNDKboundtoeitherACP1orACP5(Figure5B,donor1&5).Analysis of the product ratios highlights that the identity of the substrate impacts the reductionreaction by KR6. A pronounced substrate specificity of KR6 was observed before.40 Overall, no

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unambiguouscorrelationofturnovernumberswitheithertheinvolvedprotein-proteininteractionsnorsubstrate:KSinteractionscanbedrawnbasedontheturnoverdataforbimodularchimericPKSsusingdifferentdonormodulesforloadingofM6.Whileitseemsthatforwild-typeM6,usageofdonor1and3correlateswiththehighestactivities,donor1appearstobethepreferredforMut26(Figure5C). Inotherwords,whilewild-typeM6onlyshowshigh turnoverrates in thepresenceofACP1carryingeitherNDKorEDK,Mut26exhibitshighturnovernumberswith(almost)alldonors,butespeciallyinthepresenceofACP1-NDKasthedonor.

Figure 5. Turnover rates of bimodular chimeric PKSs using M6-TE or Mut26 and different donormodules.(A)ThedonormodulesusedtogeneratebimodularPKSs.SubstratesandtheemployedACPsareindicated. (B) Products of bimodular PKSs usingM6-TE orMut26. Different ratios of TKLR-to TKLURwereproducedwithdifferentdonors,yetwild-typeM6andMut26showthesametrends.CalculationbasedonpeakareasshowninTableS7.N.A.,notapplicable.(C)TurnoverratesofbimodularchimericPKSusingM6-TEastheacceptormodule.AllbimodularPKSsconsistedofLDD(4),followedbyadonormoduleasindicatedin(A),andSZ4-M6-TEorMut26.ThereferencesystemconsistedofLDD(4),(5)M1-SZ3,andSZ4-M2-TE(graybar).Allinitialratedatawasobtainedat4µMenzymeconcentrationandnon-limitingconcentrationsofpropionyl-CoA,methylmalonyl-CoA,andNADPH.Measurementswereperformedintriplicate.ConclusionInthiswork,12multipointmutantsofKS3and10mutantsofKS6weregeneratedandtestedfortheir ability to productively process two different substrates presented by either the naturalupstreamACPorachimericACP.Despitestrongdifferenceinactivesiteresidues,allmutantscould

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bepurified as stable proteins andwith high yields, highlighting the successful use of FuncLib tocalculatestableactivesitemutantsonthebasisofaproteinstructureorahomologymodel.ThisisespeciallyremarkableaspurificationofPKSmodulescanoftenbedifficult.Analysisoftheturnoverrates of bimodular PKSs using the M3 and M6 acceptors in the presence of M1 revealed rateimprovementorimpairmentfordifferentmutants(Figure3).FuncLibdoesnotdesignmutationsforturnoverwithspecificsubstrates,butratheryieldsalargevarietyofmutationsthatcanbescreenedfor specificities and activities of interest.32 In that light, the increase as well as the decrease inturnoverrateswasexpected,providedthatitisthesubstratespecificityoftheacceptorKSthatlimitstherateofachimericPKS.26,41In addition, we systematically varied the protein-protein interactions within the chimeric KS-mutatedPKSs; i.e., theprotein:protein interfaceemerging from theupstreamACPdocking to thedownstreamKSfortranslocationandthestructureofthesubstratepresentedtothedownstreamKSforuptake andprocessing.Overall, our approachyielded a largebodyof data giving insight intomechanisticdetailsofPKS-mediatedproductsynthesisoutlinedinthefollowingonthreeexamples:

(i) Theinitialobservationthatwild-typeM3prefersNDKoverEDK16hasbeenconfirmedinthis study. While the low turnover rates with ACP1-NDK as the donor have beenpreviouslyobservedtostemfromarate-limitingeffectbasedonthenon-nativeACP1:KS3interface,4 theusageofACP2-NDKovercomes theserate limitationsandresults in thehighestmeasuredturnoverratesofabimodularPKSusingwild-typeM3-TE(Figure4B).Inthisregard, thereconstitutionof thenative interface improvedtheefficiencyof thesystem.

(ii) BytakingacloserlookatkeyresiduesmutatedinthedifferentM3mutants,itisstrikingthat from the 12 chosen designs, Mut08, Mut11, and Mut13 all include the S306Pmutation (Table S2, Figure S3). Overall, a picture emerges in which the chimericbimodularPKSsusingM3-TEoftenbenefitsfromretainingthenativeACP2:KS3interface(see (i)) and can be engineered towards higher turnover rates aswell as broadenedsubstratespecificitiesviaKSmultipointmutagenesis.

(iii) DataonmutagenesisofKS6providesfurtherindicationastowhyDEBSM6-TEhasbeenrecognized as one of the best suited modules for engineering approaches.2,4,22,38,42Previously, the high sequence similarity between KS2 and KS6 has served as anexplanationforwild-typeM6beingalmostasgoodasanacceptorasM2withM1asthedonormodule.22Assuch,itwasnotpredictedthatmultipointmutagenesisofKS6wouldresultinmutantswithimprovedactivitywithM1,butratherleadtoimpairedmutants.Indeed,mostofthescreenedmutantsexhibitedlowerturnovernumbersthanwild-typeM6,exceptforasinglemutant(Mut26,Figure3B).AcloserlookattheactivesiteofthismutantrevealedthattheF235MmutationlikelyhasthestrongestimpactonbindingsitegeometrybyenlargingtheactivesitewhilealsoincreasingthesimilaritybetweenKS2andKS6evenfurther(FigureS9).ThisdatasupportstherelevanceofthespecificityofthedownstreamKSasabottleneckinchimericPKSsandthecapabilityofKSmutagenesistoimproveefficiencies(see(ii)).

Beyond the boundaries of theDEBS PKSs that served as amodel system in this study, our datasuggestslimitationsinengineeringmodularchimericPKSsingeneral.AsayetunmatchedsystematicinvitroanalysisofparametersinvolvedintheKS-mediateduptakeandprocessingofsubstrates(aspartof thetranslocationandelongationreactions), itcanhighlightwhyconventionalengineeringstrategieshavefailedinthepast.2–4Inmostcases,turnoverefficiencycanneitherbededucedfromtheidentityoftheACPdonor,norfromtheincomingsubstrate,asthehighestturnoverratescannotbecorrelatedwitheitherof theseparameters.Evenworsefromaperspectiveofsimplemix-and-matcheingeeringapproaches,itappearsthatacumulatedeffectandanon-intuitiveinterplayofthe

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identity of bothACPand substrate influence the turnoverofmanydownstreammodules. Futureengineeringstrategiesarelikelytobemoresuccessfulbyeitherretainingsubstratespecificities,43orby preserving the natural domain-domain interfaces,44–47 a task thatmight be achievedwith thesupportfromevolutionaryanalysis.48METHODSReagents: CloneAmp HiFi PCR Premix was from Takara. Restriction enzymes were from NewEnglandBiolabs.AllprimersweresynthesizedbySigmaAldrich.ForDNApurification,theGeneJETPlasmidMiniprepKitandtheGeneJETGelExtractionKitfromThermoScientificwereused.StellarCompetent Cells were from Takara and One Shot® BL21 (DE3) Cells were from Invitrogen. AllchemicalsforbufferpreparationswerefromSigmaAldrich.Isopropyl-β-D-1-thiogalactopyranoside(IPTG),kanamycinsulfate,andcarbenicillinwerefromSigmaAldrich/CarlRoth.LBbroth(Lennox)and2xYTmediaforcellcultureswerefromCarlRoth,Ni-NTAaffinityresinwasfromClontech.Foranion exchange chromatography the HiTrapQ column was from GE Healthcare. For proteinconcentrationAmiconUltracentrifugalfilterswerefromMillipore.CoenzymeA(CoA),reducedβ-nicotinamideadeninedinucleotide2’-phosphate(NADPH),sodiumpropionate,methylmalonicacid,andmagenesiumchloridehexahydratewerefromCarlRoth.Adenosine-5’-triphosphate(ATP)wasfrom SigmaAldrich. Reducing agent tris(2-carboxyethyl)-phosphine (TCEP) was from ThermoScientific.UV-StarhalfareamicrotiterplateswerefromGreiner.Plasmids:PlasmidsharboringgenesencodingindividualPKSmoduleswereeithergeneratedinthisstudyviaIn-FusionCloning(Takara),sitedirectedmutagenesis,orreportedpreviously.TablesS8-S11specifythecloningstrategy,primersequences,andtheresultingplasmids.PlasmidssequenceswereverifiedbyDNAsequencing(SeqLab).ProteinsequencesofdonormodulesnewlygeneratedinthisstudyarepresentedinTableS12.Bacterial Cell Culture andProteinPurification:All PKSproteinswere expressed andpurifiedusingsimilarprotocols.Forholo-proteins(wheretheACPdomainispost-translationallymodifiedwithaphosphopantetheinearm)E.coliBL21cellswereco-transformedwithaplasmidencodingforthe phosphopantetheine transferase Sfp fromB. subtilis (pAR35749). All proteins contained a C-terminalHis6-tagforpurification.Culturesweregrownona1-2Lscalein2xYTmediaat37°CtoanOD600of0.3,whereuponthetemperaturewasadjustedto18°C.AtOD600of0.6,proteinproductionwasinducedwith0.1mMIPTG,andthecellsweregrownforanother18h.Cellswereharvestedbycentrifugationat5000gfor15minandlysedbyFrenchPress(lysisbuffer:50mMsodiumphosphate,10mM imidazole, 450mM NaCl, 10% glycerol, pH 7.6). The cell debris was removed bycentrifugation at 50,000 g for 50 min. The supernatant was further purified using affinitychromatography(Ni-NTAagaroseresin,5mLcolumnvolumes).Afterapplyingthesupernatanttothe column, a first wash step was performedwith the above lysis buffer (10 column volumes),followedbyasecondwashstepusing10columnvolumesofwashbuffer(50mMphosphate,25mMimidazole,300mMNaCl,10%glycerol,pH7.6).Proteinswereelutedwith6columnvolumeselutionbuffer(50mMphosphate,500mM,10%glycerol,pH7.6).TheeluatewasfurtherpurifiedbyanionexchangechromatographyusingaHitrapQcolumnonanÄKTAFPLCsystem.BufferAconsistedof50mMphosphate, 10%glycerol, pH7.6,whereas buffer B contained 50mMphosphate, 500mMNaCl,10%glycerol,pH7.6.TheyieldsofallproteinperliterofculturearenotedinTableS4.EnzymesMatB,SCME,andPrpEwerepurifiedasdecribed.33,50ProteinconcentrationsweredeterminedwiththeBCAProteinAssayKit(ThermoScientific).Sampleswerestoredasaliquotsat−80°Cuntilfurtheruse.Size Exclusion Chromatography: To determine the purity of the proteins after ion exchangechromatography,samplesofeachproteinwereanalyzedbysizeexclusionchromatographyonan

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ÄKTA FPLC system using a Superose 6 Increase 10/300 GL column (buffer: 50mM phosphate,500mMNaCl,10%glycerol,pH7.6).ForproteinpuritybySDS-PAGEandSECprofilesrefertoFiguresS6,S7&S11.Thermal Shift Assay:Themelting temperatures of all proteins in a buffer consisting of 50mMphosphate,500mMNaCl,10%glycerol,pH7.6weremeasuredaccordingtopublishedproceduresusingaproteinconcentrationof1.2mg/mL.51PKSEnzymaticAssays:PKSenzymaticassayswereperformedaccordingtopublishedprocedures.4PKSenzymeswereusedat4µMfinalconcentration,forinsitusubstrategenerationenzymesMatB,PrpE,andSCMEwereusedatfinalconcentrationsof4µM,2µM,and8µM,respectively.Liquid Chromatography-Mass Spectrometry Analysis of Polyketides: Dried samples werereconstitutedin100μLmethanol,separatedonaAcquityUPLCBEHC181.7µmRP2.1x50mmcolumn (Waters) with an Acquity UPLC BEH C18 1.7µm RP 2.1 x 5 mm pre-column (Waters),connectedtoanUltimate3000RSLCoranUltimate3000LC(Dionex)HPLCovera16minlineargradientofacetonitrilefrom5%to95%inwater,andsubsequentlyinjectedintoanImpactIIqTof(Bruker) or anAmaZonX (Bruker) equippedwith an ESI Source set to positive ionizationmode.Reducedandunreducedtriketideproductswerelocatedbysearchingforthetheoreticalm/zforthe[M+H]+-ion.Unreducedtriketides:[M+H]+=171.098,reducedtriketides[M+H]+=173.118.BioinformaticalAnalysis:SequencealignmentsweregeneratedwithClustalWSasimplementedinJalview2.10.5.52HomologymodelsweregeneratedwithSWISS-MODEL.19TheFuncLibwebserverwasusedtocalculatediversemultipointmutationswithintheKSactivesite.32ThecalculationforKS3wasbasedontheX-raystructureoftheKS3-AT3didomain.36ThecalculationforKS6wasbasedonthehomologymodelgeneratedwithSWISS-MODELbasedontheX-raystructureofKS5-AT5.19Inthefirststep,forthecalculationofthesequencespace,chainsA+BwereincludedforKS3.TodecreasethecalculationeffortonlychainBwasincludedforKS6.SevenresidueswerechosenformutagenesisofKS3andninepositionsforKS6.Inaddition,residuesofthecatalytictriad(KS3:Cys202,H337andH377; KS6: Cys172, H307 and H347) were selected not to be altered during simulations. Thenomenclatureofresiduesisderivedfromstructuralmodels.C202KS3correspondstoUniProtEryA2(Q03132)position202andC172KS6toUniProtEryA3(Q03133)position1661.Forgenerationofthemultiplesequencealignmentthedefaultparameterswereusedduringallcalculations(MinID0.3,Maxtargets3000,Coverage0.6,andEvalue0.0001).Inthesecondcalculationstep,theparameterswerechosentoyieldbetween500-500,000designs.ForKS3152,826andforKS6285,822designsharboring3-5mutationswereselectedforRosettacalculation.Acknowledgments:WethankHelgeBodeforuseoftheirLC-MSinstruments.WefurtherwanttothankAleksandraNivinaand Chaitan Khosla for helpful discussions. In using the FuncLib method, we received fantasticsupportfromSarelFleishman,RosalieLipshandJaimePrilusky.FundingSources:This work was supported by a Lichtenberg grant of the Volkswagen Foundation to M.G. (grantnumber 85701). Further supportwas received from the LOEWE program (Landes-Offensive zurEntwicklungwissenschaftlich-ökonomischerExzellenz)ofthestateofHesseconductedwithintheframeworkoftheMegaSynResearchCluster,aswellastheDFGgrantGR3854/6-1.Authorscontribution:

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