| Iterative fungal polyketide synthases (PKSs) use a unique set of biochemical rules in the synthesis of complex polyketides. These rules dictate polyketide starter unit selection, chain length control, and post polyketide synthase processing. In contrast to bacterial polyketide synthases, the biosynthetic mechanism of fungal polyketide synthases are not well understood, and their potential for combinatorial biosynthesis has not yet been realized. Engineered biosynthesis of fungal polyketides in heterologous hosts paves the road towards facile synthesis of important fungal polyketide megasynthases such as lovastatin, an important therapeutic for hypercholesterolemia, and the anticancer fungal polyketide bikaverin. We have shown soluble expression of the fungal genes: pks4 from Gibberella fujikuroi, lovB from Aspergillus terreus, and lovC from Aspergillus terreus in E. coli. In addition, we have shown that recombinant PKS4 from E. coli is functional, and its entire repertoire of catalytic activates can be reconstituted. We were able to generate the unoxidized and unmethylated precursor of bikaverin, as well as engineer and characterize the in vitro synthesis of new compounds with an artificial starter unit and the recombinant PKS4 protein. Heterologous expression of lovB in Saccharomyces cerevisiae yielded the functional megasynthase. In vitro assays containing this recombinant LovB enzyme is able to synthesize tri-, tetra-, hexa-, and heptaketide intermediates solely from the starter and extender unit malonyl-CoA. We biochemically characterized the minimal polyketide synthase from the LovB megasynthase with in vitro experiments containing only the dissected ketosynthase (KS), malonyl:acyl transferase (MAT), and acyl carrier domain (ACP) from LovB expressed as standalone proteins from Escherichia coli. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with kcat/Km values of 0.62 min-1·mum-1 and 0.032 min -1·mum-1, respectively. Finally, we were able to completely reconstitute the early biosynthetic pathway in lovastatin biosynthesis. With LovB heterologously expressed from S. cerevisiae and LovC (the dissociated enoyl reductase (ER)) heterologously expressed from E. coli in the presence of pertinent cofactors in vitro experiments showed the production of dihydromonacolin L. Experiments replacing LovC with analogous MlcG from the compactin biosynthetic pathway, also heterologously expressed in E. coli, we demonstrated the gate-keeping function for dihydromonacolin L lies in the partner enzyme LovC. |
