Background: 6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase fromPenicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes—a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed inEscherichia coliandSaccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system.Results: A three--step purification procedure was developed to obtain >95% pure MSAS from extracts ofE. coli. As reported earlier for the enzyme isolated fromP. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted theN-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate.Conclusions: NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in bothE. coliand yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.