P122: Classification, Prediction and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

The fungal iterative nonreducing polyketide synthases (NRPKSs) synthesize aromatic polyketides, many of which have important biological activities.  In this work, we verified the function of PKS4-PT (product template domain) through excising the PT domain from the PKS4 domain-domain interaction.  As PT mediate the regioselective cyclization of the highly reactive polyketide backbones and dictate the final structures of the products, understanding the sequence-activity relationships of different PT domains is therefore an important step towards the prediction of polyketide structures from NRPKS sequences, which can also enable the genome mining of hundreds of cryptic NRPKSs uncovered via genome sequencing.  Therefore, we performed phylogenetic analysis of PT domains from NRPKSs of known functions and showed that the PT domains can be classified into five groups – each group corresponding to a unique product size or cyclization regioselectivity, among which group V contains the formerly unverified PT domains that were identified as C6-C11 aldol cyclases. The regioselectivity of PTs from this group were verified by product-based assays using the PT domain excised from the asperthecin AptA NRPKS. When combined with dissociated PKS4 minimal PKS, or replaced the endogenous PKS4 C2-C7 PT domain in a hybrid NRPKS, AptA-PT directed the C6-C11 cyclization of the nonaketide backbone to yield a tetracyclic pyranoanthraquinone 1.  The PT phylogenetic analysis was then expanded to include 100 PT sequences from unverified NRPKSs.  Using the assays developed for AptA-PT, the regioselectivities of additional PT domains were investigated and matched to those predicted by the phylogenetic classifications.