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Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

Significance Benzenediol lactone (BDL) polyketides are privileged structures whose various members bind to distinct receptors or modulate the heat shock response and the immune system. BDLs are biosynthesized by collaborating polyketide synthase enzyme pairs in fungi. Coexpressing random heterocombinations of these enzymes from different BDL biosynthetic pathways in yeast cells is shown here to lead to the one-pot, one-step combinatorial biosynthesis of structurally diverse polyketides in practical amounts. Combinatorial biosynthesis promises to generate a novel unexplored source of bioactive molecules in an environmentally sustainable, economical, and inherently scalable manner. Broadening the medicinally relevant chemical space of polyketides by such methods will provide unnatural products as valuable entry points for drug discovery and development. Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into “build–couple–pair” combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.