Dissecting the Chain Length Specificity in Bacterial Aromatic Polyketide Synthases using Chimeric Genes

Many aromatic polyketides are synthesized by bacterial enzyme complexes called polyketide synthases (PKSs). Previous work has indicated that two subunits in the PKS, called the ketosynthase (KS) and chain length factor (CLF), play a central role in determining the chain length of the polyketide product. The purpose of this study was to construct and analyze chimeric KS and CLF proteins in order to identify the structural determinants of chain length. The genes encoding KS and CLF subunits from different aromatic PKSs share significant (50–70%) sequence identity. This similarity provides a means to genetically engineer the PKSs by directed gene shuffling methods. A set of hybrid KS and CLF genes were designed based on the sequences of the actinorhodin (a 16 carbon polyketide) and tetracenomycin (a 20 carbon polyketide) PKS genes. Analysis of these chimeric genes ruled out a role for most of the KS and reinforced the importance of the CLF in controlling polyketide chain length. Homology modeling of the quaternary structures of the functional hybrid KS–CLF dimers based on recent crystal structures of the ketosynthase homodimer from E. coli suggested that the regions of the KS and CLF that are most important for activity and specificity are located at the interface of this dimer.