The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) are structurally simple homodimeric proteins that produce basic skeletons of flavonoids as well as a variety of plant polyphenols with remarkable biological activities [1,2]. The type III PKSs of plant origin usually share 50-75% amino acid sequence identity with each other, and maintain a common 3D overall fold with an absolutely conserved Cys-His-Asn catalytic triad. The polyketide formation reaction is thought to be initiated by starter molecule loading at the active site Cys, which is followed by sequential decarboxylative condensations of malonylCoA. The functional diversity of the type III PKSs derives from the differences of their selection of starter substrate, number of polyketide chain elongations, and mechanisms of the final cyclization ⁄ aromatization reactions. It has been demonstrated that the shape and volume of the active site cavity greatly influence the Aloesone synthase (ALS) and chalcone synthase (CHS) are plant-specific type III poyketide synthases sharing 62% amino acid sequence identity. ALS selects acetyl-CoA as a starter and carries out six successive condensations with malonyl-CoA to produce a heptaketide aloesone, whereas CHS catalyses condensations of 4-coumaroyl-CoA with three malonyl-CoAs to generate chalcone. In ALS, CHS's Thr197, Gly256, and Ser338, the active site residues lining the initiation ⁄ elongation cavity, are uniquely replaced with Ala, Leu, and Thr, respectively. A homology model predicted that the active site architecture of ALS combines a 'horizontally restricting' G256L substitution with a 'downward expanding' T197A replacement relative to CHS. Moreover, ALS has an additional buried pocket that extends into the 'floor' of the active site cavity. The steric modulation thus facilitates ALS to utilize the smaller acetyl-CoA starter while providing adequate volume for the additional polyketide chain extensions. In fact, it was demonstrated that CHS-like point mutations at these positions (A197T, L256G, and T338S) completely abolished the heptaketide producing activity. Instead, A197T mutant yielded a pentaketide, 2,7-dihydroxy-5-methylchromone, while L256G and T338S just afforded a triketide, triacetic acid lactone. In contrast, L256G accepted 4-coumaroyl-CoA as starter to efficiently produce a tetraketide, 4-coumaroyltriacetic acid lactone. These results suggested that Gly256 determines starter substrate selectivity, while Thr197 located at the entrance of the buried pocket controls polyketide chain length. Finally, Ser338 in proximity of the catalytic Cys164 guides the linear polyketide intermediate to extend into the pocket, thus leading to formation of the hepataketide in Rheum palmatum ALS.