Engineering of plant polyketide biosynthesis.

A growing number of functionally divergent the chalcone synthase (CHS) superfamily type III polyketide synthases (PKSs) have been cloned and characterized, which include recently obtained pentaketide chromone synthase (PCS) and octaketide synthase (OKS) from aloe (Aloe arborescens). Recombinant PCS expressed in Escherichia coli catalyzes iterative condensations of five molecules of malonyl-CoA to produce a pentaketide, 5,7-dihydroxy-2-methylchromone, while OKS carries out sequential condensations of eight molecules of malonyl-CoA to yield aromatic octaketides, SEK4 and SEK4b, the longest polyketides generated by the structurally simple type III PKS. The two enzymes share 91% amino acid sequence identity, maintaining most of the active-site residues of CHS including the Cys-His-Asn catalytic triad. One of the most characteristic features is that the conserved Thr197 of CHS (numbering in Medicago sativa CHS) is uniquely replaced with Met207 in PCS and with Gly207 in OKS, respectively. Site-directed mutagenesis and X-ray crystallographic studies clearly demonstrated that the chemically inert single residue lining the active-site cavity controls the polyketide chain length and the product specificity depending on the steric bulk of the side chain. Finally, on the basis of the crystal structures of both wild-type and M207G-mutant PCS, a triple mutant PCS F80A/Y82A/M207G was constructed and shown to catalyze condensations of nine molecules of malonyl-CoA to produce a novel nonaketide naphthopyrone with a fused tricyclic ring system. Structure-based engineering of the type III PKS superfamily enzymes would thus lead to further production of chemically and structurally divergent unnatural novel polyketides.