Polyketides are one of the largest families of small molecule natural products.  They serve as lead compounds and provided structural inspiration for multibillion-dollar drugs that save lives.  Biosynthesis of polyketides in fungi involves iterative multi-domain polyketide synthase enzymes (iPKS) of >300 kDa that catalyze the recursive condensation of malonyl-CoA in a manner conceptually similar to fatty acid biosynthesis.  However, these iPKS enzymes follow a complex and currently not well understood biosynthetic program to create structural diversity. 

Generation of novel natural product analogues by combinatorial biosynthesis is a longstanding goal of chemical biology, and promises “unnatural products” that can serve as valuable entry points for drug discovery.  However, combinatorial biosynthesis with fungal iPKSs is an especially demanding area of study, considering the unknown programming rules of these iterative enzymes and the complexity of the molecular engineering work required.  We have recently reported a working example of combinatorial biosynthesis within the benzenediol lactone (BDL) polyketide family, using subunit heterocombinations of ortholgous iPKSs (“subunit shuffling”, PNAS 111, 12354, 2014).  With the current work, we expand this methodology by combining iPKSs from the BDL family with those from an unrelated fungal polyketide family, the azaphilones.  We show that heterocombinations of the iPKS subunits of these two non-orthologous polyketide families create unprecedented branched acyl benzaldehydes by “merging” structural elements of BDLs and azaphilones. Thus, this work significantly extends the remit of iPKS subunit shuffling to non-orthologous enzymes producing bio-orthogonal biosynthons.  Such morphing of one family of metabolites into another is a longstanding, but hitherto rarely achieved goal of chemical biology.

Pairwise heterocombinations of BDL and azaphilone iPKS subunits also helped us to illuminate the innate, idiosyncratic programming of iPKS enzymes.  Importantly, we show that exchange of the starter acyltransferase (SAT) domain is necessary, but not sufficient for a productive communication between non-orthologous iPKS subunits, emphasizing that other gating functions are also operational within these enzymes.  We also show that some heterocombinations provoke the relaxation of chain length control by the iPKSs to assemble larger, or conversely, smaller homologous products, phenomena referred to as “stuttering”, and “sputtering”, respectively. 

In conclusion, our research describes a concise but high quality study, with immediate import to chemical biologists working towards combinatorial biosynthesis.