HsPKS1 from Huperzia serrata is a type III polyketide synthase (PKS) with remarkable substrate tolerance and catalytic potential. Here we present the synthesis of unnatural novel polyketide-alkaloid hybrid molecules by exploiting the enzyme reaction using precursor-directed and structure-based approaches. HsPKS1 produced novel pyridoisoindole (or benzopyridoisoindole) with the 6.5.6-fused (or 6.6.5.6-fused) ring system by the condensation of 2-carbamoylbenzoyl-CoA (or 3-carbamoyl-2-naphthoyl-CoA), a synthetic nitrogen-containing non-physiological starter substrate, with two molecules of malonyl-CoA. The structure-based S348G mutant not only extended the product chain length, but also altered the cyclization mechanism to produce a biologically active, ring-expanded 6.7.6-fused dibenzoazepine, by the condensation of 2-carbamoylbenzoyl-CoA with three malonyl-CoAs. Thus, the basic nitrogen atom and the structure-based mutagenesis enabled additional C-C and C-N bond formation to generate the novel polyketide-alkaloid scaffold. Benzalacetone synthase (BAS) from Rheum palmatum is a structurally simple, plant-specific type III PKS, which catalyzes the one-step decarboxylative condensation of malonyl-CoA with 4-coumaroyl-CoA. The type III PKS exhibits unusually broad substrate specificity and notable catalytic versatility. Here we report that R. palmatum BAS efficiently produces a series of unnatural, novel tetramic acid derivatives by the condensation of malonyl-CoA with aminoacyl-CoA thioesters, chemically synthesized from L- and D-amino acids. Remarkably, the novel tetramic acid dimer from D-phenylalanoyl-CoA, showed moderate antiproliferative activity against murine leukemia P388 cells.
