Intrinsic And Extrinsic Programming Of Product Chain Length And Release Mode In Fungal Iterative Polyketide Synthases

Bezenediol lactones(BDLs)are a class of structurally and functionally diverse fungal polyketide natural products,which display great application potential in the pharmaceutical,agricultural,and food industries.The biosynthesis of BDLs requires two single-module,multi-domain fungal polyketide synthases(BDL synthase,BDLS)acting in sequence.In this study,BDLS subunit shuffling,domain swapping,and active-site remodeling approaches were employed to combinatorially biosynthesize"unnatural" BDLs.The results further expand the structural diversity of BDLs,and reveal novel intrinsic(preference and catalytic competence towards a given intermediate)and extrinsic programing rules(domain interactions and the kinetic competition for a given intermediate)of starter unit acyltransferase(SAT)and thioester(TE)domains,and their influences on BDL biosynthesis.A pair of novel BDLS(RrDa1S1-RrDa1S2)encoding genes were discovered from the plant pathogenic fungus Rhytidhysteron rufulum through genome mining efforts.Their catalytic functions were evaluated by co-expressing RrDa1S1 or RrDa1S2 with non-reducing polyketide synthases(nrPKS)or highly reducing PKS(hrPKS)from non-cognate BDLSs in heterologous host Saccharomyces cerevisiae.The results showed that RrDa1S1 synthesizes a tetraketide 4Pc,which features a α,β,γ,δ-unsaturated keto functionality,as on-program product and passes it to downstream non-cognate nrPKSs.Like the nrPKS from curvularin synthase(AtCurS2),RrDa1S2 catalyzes four more rounds of chain extension and forms an S-type aromatic ring after downloading the priming unit from non-cognate hrPKSs.However,these two S-type nrPKSs have clear differences in priming unit selecting and product releasing modes:AtCurS2 is a promiscuous enzyme that accepts the on-program products of non-cognate hrPKSs to prime its biosynthesis,and releases dihydroxyphenylacetic acid lactones(DALs)as products via intro-molecular esterification;while RrDalS2 favors shorter off-program priming units and off-loads acyl dihydroxyphenylacetic acid esters(ADAEs)through inter-molecular transesterification.Domain swap strategy was employed to unveil the intrinsic and extrinsic programing rules of SAT and TE domains,and how they regulate the priming unit selecting and product releasing processes during BDL biosynthesis.First,properties like the volume,shape,and charge distribution of the active-site determines whether TE releases the product via intro-or inter-molecular esterification.Such selectivity is strongly influenced by the identities of amino acids residing on the Lid area of a given TE.Mutation of either of the two amino acids locating on the Lid of TEAtCurS2(F1929V or T1948Q)leads to significant changes in product type without altering the overall productivity(DAL→ADAE).However,modifying the selectivity of TERrDalS2 requires much more extensive exchanges of polypeptide segments.This implies that the amino acids on the Lid as well as those from other parts of TE should be taken into consideration when re-modeling TERrDaiS2 active site to direct the polyketide intermediate into the configuration that favors macrolactonization.Next,the chain length of the hrPKS product is strongly influenced in trans by the off-loading preferences of the SAT domain from the downstream nrPKS.SATAtCurS2,Which displays good catalytic promiscuity,is able to accept the on-program products of different hrPKSs and hand the priming unit over to downstream domains.In contrast,SATRrDaiS2 exhibits stronger preference and favors shorter,if possible triketide,priming units by proactively intercepting the immature off-program products from hrPKS.Last,TE is capable of facilitating or obstructing product formation from certain priming units based on its in-built preference for certain type of polyketide intermediates.Such characteristic of TE,together with the priming unit preference of SAT,may contribute to significant increase or decrease of productivity for specific polyketide.Phylogenetic analysis suggested that the SAT and TE domains of RrDalS1-RxDalS2 may travel different evolutionary trajectories:SAT RrDa1S2 developed a preference for triketide priming units,while TE evolved a priority of releasing tetraketide-primed products.Such divergence of the intrinsic and extrinsic programs creates potential conflicts in the metaprogram,and eventually incapacitates the BDLS.The current study afforded a panel of "unnatural" S-type BDLs and their derivatives through combinatorial biosynthesis,which greatly expands the chemical spaces of this type of fungal polyketide.Meanwhile,the discovery of intrinsic and extrinsic programs of SAT and TE domains and their influences on BDLS metaprogram(the overall observable program of the whole system)may guide future fungal PKS rational design and engineering studies.