(27c) Understanding and Engineering Chain Translocation in Assembly-Line Polyketide Synthases

Polyketide synthases (PKSs) biosynthesize thousands of complex bioactive natural products, such as rapamycin and erythromycin. PKSs are arranged as “modules” minimally containing a ketosynthase-acyltransferase (KSAT) didomain and an acyl carrier protein (ACP) domain that catalyze the C-C bond formation and extension of a growing polyketide chain. The polyketide is shuttled from an upstream module’s ACP to a downstream module’s KSAT during chain translocation for successive polyketide chain extension.

PKSs are likened to “assembly lines” due to their vectorial biosynthesis and the collinearity of the PKS’s protein domain organization and the chemical structure of its produced polyketide. These properties have led to great interest in engineering these machines, where changes to the PKS’s gene sequence alter the polyketide’s structure. An early promising strategy known as module-exchange engineering demonstrated that swapping the gene sequence of one module in a PKS for the sequence of a module from a foreign PKS could enable control of the stereochemistry, degree of reduction, and alkyl substituent at different parts of the polyketide’s chemical structure. However, it quickly became apparent that these chimeric PKSs often did not retain catalytic efficiency. Before efficiently engineering PKSs, we must first characterize the key protein-protein interactions and structural dynamics at each step of its catalytic cycle.

Recent studies have identified the interactions between a module’s KSAT didomain and its upstream ACP partner during chain translocation as a crucial bottleneck limiting the efficacy of chimeric PKSs. Due to the inherent flexibility of the ACP, it has been challenging to structurally characterize this interaction. In this work, we characterized the crosslinking agent 1,3-dibromoacetone (DBA) as an efficient tool to study KSAT-ACP protein-protein interactions and structural dynamics. Despite being a simple, highly reactive chemical, DBA forms a specific, covalent linkage between the phosphopantetheine arm of an ACP and the active site cysteine of the KSAT didomain. Using DBA, we obtained a crosslinked structure of an upstream ACP interacting with its cognate downstream KSAT via cryo-EM, providing a model of how an ACP interacts with a KSAT during chain translocation.

Previous studies have analyzed chain translocation in multiple-module assays, where the chain translocation reaction is coupled to the rest of the module’s catalytic cycle, thus limiting our ability to discern the impact of solely chain translocation. Radiolabeling has also been utilized, although it can be experimentally challenging and limiting. Here, we describe the development of a liquid chromatography-mass spectrometry (LC-MS) based assay to probe the chain translocation reaction. We monitor chain translocation in a dissociated system consisting of a KSAT didomain and an upstream ACP domain instead of utilizing full modules. To quantify chain translocation, we feed the system a mimic of the natural polyketide substrate and monitor substrate occupancy on the ACP via mass spectrometry. This experiment requires minimal sample preparation or processing, allowing for analysis at higher throughputs than previously studied. Additionally, the experimental setup is generalizable, allowing for the interrogation of different KSAT and ACP pairs and substrates.

We use this assay in tandem with our model from our cryo-EM studies to understand the recognition epitope of an ACP and KSAT during chain translocation. We also quantify the kinetic penalty arising from the interactions between non-cognate ACP and KSAT pairs. With this assay, we can understand the impact of impaired chain translocation during module-exchange engineering. Our findings provide a structural model and a quantitative assay to inform engineering efforts on the protein-protein interactions involved in chain translocation. Improvements to these interactions can improve the catalytic efficiency of chimeric PKSs, allowing for improved titers of novel polyketides.