Biochemical dissection and interface mapping in fungal highly reducing polyketide synthases

Fungal polyketide natural products exhibit significant structural diversity. The multi-domain enzymes which biosynthesise polyketides – polyketide synthases (PKSs) – do so using a common set of enzymatic domains, with each domain catalysing one specific chemical reaction. Fungal PKSs classed as highly reducing (hrPKS) incorporate the most structural diversity into their products via highly-programmed iterative biosynthetic trajectories, varying the combinations of domains to create functionality. This contrasts the well-studied iterative trajectory of the structurally comparable mammalian fatty acid synthase, which repeats a single combination of these same domains to yield saturated fatty acid chains. While the hrPKS can reliably biosynthesise a structurally specific polyketide, little is known about how its intrinsically complex programming pattern is achieved to ensure product fidelity. Establishing understanding of hrPKS function will create opportunities for bioengineering efforts towards the generation of novel, optimised polyketides. To date, much of the work investigating hrPKS programming has focussed on domains responsible for decorating the carbon scaffold. However, little attention has been paid to the ketosynthase (KS) and acyltransferase (AT) domains, which are visited by the acyl carrier protein (ACP) regularly throughout polyketide biosynthesis, catalysing acyl unit selection and elongation reactions to build the carbon scaffold. The work reported herein details the first in vitro characterisation of key biosynthetic steps catalysed by the KS and AT domains of a model hrPKS, SimG. Through biochemical assays analysed by intact protein mass spectrometry, highly-tuned gatekeeping and proofreading roles are demonstrated for the KS and AT domains, highlighting their role in ensuring biosynthetic fidelity. Using an alanine scanning mutagenesis approach, the first residue-level mapping of an AT:ACP interface within a hrPKS is reported, capturing contributions made by both domains towards a productive interface. Taken together, this work provides new insights into how the hrPKS controls biosynthetic flux and lays important foundations for future studies.