Understanding substrate translocation in polyketide synthase (PKS) assembly lines
Authors:   , Buyachuihan Lynn 
Published: February 2025

Megasynthases like polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are huge enzymatic factories found in bacteria, fungi, and plants. They produce complex natural products by condensing simple molecular building blocks. Most polyketides and nonribosomal peptides exhibit great pharmaceutical potential, such as the antibiotics erythromycin and penicillin, making megasynthases a popular target for protein engineering efforts.
In megasynthases, biosynthesis happens through the coordinated interaction of the synthase-building domains. In type I systems, these domains are covalently connected to form multidomain proteins. Important representatives of the megasynthase family are the modular PKSs due to their potential for rational protein engineering. In modular PKSs, the structure of the product can be inferred from the architecture of the synthase. This collinear biosynthetic logic paves the way toward the retrobiosynthetic production of designer drugs by altering the PKS domain composition.
Modular PKSs consist of multiple modules that interact sequentially. Each module harbors a set of domains catalyzing one round of chain elongation and potential processing. The modules are often divided across several large polypeptide subunits. The ordered arrangement of these subunits into the final PKS is mediated by docking domains located at the ends of the respective polypeptides. Docking domains are a unique feature of molecular assembly lines, aiding in the accurate assembly of the PKS-forming subunits and contributing to the fidelity of the polyketide biosynthesis.
A minimal PKS module consists of three essential domains, namely the β-ketoacyl synthase (KS), the acyltransferase (AT), and the acyl carrier protein (ACP). The AT domain is responsible for selecting the extender units and loading them onto the ACP. AT domains act as gatekeepers in polyketide synthesis, exercising tight control over the selection of the substrate. The KS can be regarded as the heart of a PKS module, enabling the growth of the polyketide chain. It accepts the freshly processed intermediate from the upstream module in a chain translocation reaction. Subsequently, it catalyzes a decarboxylative Claisen condensation between the KS-tethered intermediate and the ACP-bound extender substrate, resulting in an ACP-bound intermediate elongated by two carbon atoms. The ACP domain, a small ~9 kDa carrier protein is embedded into the module via flexible linkers. It adopts a key role in polyketide biosynthesis as it shuttles the growing polyketide chain and the substrates between the catalytically active domains within the multienzyme complex. Additional processing domains can be present within the module, setting the reductive state and stereochemistry of the intermediate. The arrangement of modules within the PKS defines the order of functional groups in the produced macrolide and the different domain compositions are reflected in the complexity of the product scaffold.
The development of general engineering principles could enable a “lego-ization” of modular PKSs towards the production of new-to-nature drugs. However, the lack of universal rules for PKS engineering is evident in the limited success of current engineered assembly lines. Previous studies have demonstrated a discrepancy between the recombinatorial potential of modular PKSs in theory and the actual performance of engineered assembly lines in the laboratory. For the successful engineering of PKSs, a comprehensive understanding of the mechanisms underlying polyketide synthesis mechanisms is essential. The availability of accurate structural data is crucial for successful protein engineering, yet structural data on these large enzyme complexes is limited, and current methods for structure elucidation are not sufficient to capture the dynamic nature of the catalytic cycle, especially the interactions of the highly dynamic ACP. User-friendly computational methods and artificial intelligence show promise in supporting the megasynthase engineering community by enhancing our understanding of the architectures and dynamics of megasynthases.
This thesis aims to improve the understanding of modular PKSs by investigating their fundamental characteristics. In four projects, existing challenges in PKS engineering were addressed to help unlock the potential of modular PKSs for the sustainable production of custom compounds.