In fungi, the iterative, non-reducing polyketide synthases (NR-PKSs) are responsible for the biosynthesis of aromatic polyketide products. While the modular type I PKSs have been extensively studied for 20 years, the biochemistry of NR-PKS is only now beginning to be elucidated. The NR-PKSs share a common domain architecture that is intrinsically linked to their function. The mode of biosynthesis is analogous to that of fatty acids by animal fatty acid synthases (FAS), but simplified. The three N-terminal domains, the starter unit:acyl-carrier protein transacylase (SAT), ketosynthase (KS) and malonyl acyl transferase (MAT) domains are responsible for the initiation and polyketide elongation phases. The SAT domain selects a precursor or starter unit substrate as an acyl thioester while the MAT domain introduces ketide extender units from malonyl-CoA. The KS works in collaboration with the acyl-carrier protein (ACP) to catalyze the decarboxylative Claisen condensation of these substrates generating a linear, ACP-bound β-ketone intermediate. The C-terminal domains of NR-PKSs control the final stage of biosynthesis, which includes regiospecific aldol cyclizations/aromatizations by the product template (PT) domain and product release by the thioesterase (TE) domain. In this way, the four factors governing chemical diversity in aromatic polyketides are entirely controlled by the enzyme, with the N-terminal half determining starter unit selection and chain length, and the C-terminal half controlling the cyclization pattern and mode of product evolution. Our lab has innovated the use of enzyme deconstruction in NR-PKSs to determine the catalytic program of these enzymes. The enzyme deconstruction approach requires the dissection of an individual NR-PKS into its constituent domains or multidomain fragments. Mono- and multidomain fragments are expressed and purified individually and then recombined in vitro, reconstituting wild-type activity.
Using the enzyme deconstruction approach, the catalytic activity of CTB1—the NR-PKS of cercosporin biosynthesis in the fungal plant pathogen Cercospora nicotianae—was determined. The CTB1 TE domain was demonstrated to catalyze an unprecedented enol-lactonization to form the naphthopyrone nor-toralactone—representing an expansion of known TE chemistry. The formation of nor-toralactone was unexpected and in conflict with the accepted cercosporin biosynthetic pathway. Using a combination of gene knockout strains and in vitro enzymology, a new cercosporin biosynthetic pathway was identified. Of particular interest was the activity of an unusual didomain enzyme CTB3. The flavin-dependent monooxygenase domain of CTB3 was identified to catalyze a unique oxidative aromatic ring cleavage—expanding the chemical repertoire of these ubiquitous proteins. Enzyme deconstruction served as the basis of an NR-PKS engineering project in which homologous domains from non-cognate parent NR-PKSs were swapped in vitro to produce non-native polyketide products. Using a systematic approach to domain-swapping, several rules governing rational engineering of NR-PKS were codified. Domain-swapping of deconstructed NR-PKSs facilitated rational engineering of intact, chimeric NR-PKSs. A library of chimeric NR-PKSs was prepared and selected members were assayed for activity. Combining the rules of engineering gleaned from deconstructed NR-PKS domain-swapping and the construction of the chimeric NR-PKS library, an attempt to rationally design a topopyrone synthase was unsuccessfully made. By examining the activities of individual domains and perturbing the composition of NR-PKSs, we have made invaluable insights into the NR-PKS catalytic program—understanding that will underpin future efforts towards rational NR-PKS engineering
