(10c) Heterologous Expression Of Fungal Polyketide Synthases

Protein engineering with the bacterial polyketide synthases (PKSs) has enabled the biosynthesis of novel bioactive compounds. In contrast, the mechanistically distinct fungal PKS machinery has not been well studied and the general programming rules are lacking. This is largely due to difficulties associated with manipulating the genetics of the producing organisms. We present various strategies to study and engineer fungal PKSs using the genetically well characterized organism Escherichia coli. Using the genetically amendable host, we have expressed and investigated LovB (330 kDa), a megasynthase involved in the biosynthesis of the cholesterol-lowering therapy lovastatin from Aspergillus terreus, as well as PKS4 (270 kDa), a nonreducing PKS involved in the biosynthesis of the anticancer compound bikaverin in Gibberella fujikuroi. Expression of both PKSs in E. coli were facilitated by the coexpression of the surfactin phosphopantetheinyl transferase (sfp) gene from Bacillus subtilis, which posttranslationally modified the active site serines of the acyl carrier domains of the fungal PKSs. We verified 1) the activities of the ketosynthase and acyltransferase domains in both PKSs; 2) channeling of the substrates between these domains; and 3) coordinated catalysis of the domains to afford an elongated polyketide product from malonyl-CoA. We were also able to obtain and characterize the expected polyketide products from the reconstituted PKSs in vitro. Lastly, using different acyl-CoAs as starter units, we were able to synthesize compounds that are not observed previously. Our work demonstrates that heterologous expression and successful posttranslational modification of complex fungal PKSs in a bacterial host are powerful approaches towards understanding and engineering the biosynthesis of fungal polyketides.