Mapping Protein-Protein Interactions in Modular Polyketide Synthases by Carbene Footprinting

Bioactive natural products from plants and microorganisms have indispensable applications in both medicine and agriculture. They are often employed to treat life-threating conditions, including bacterial, fungal and viral infections and cancer. Moreover, these valuable molecules are also used as herbicides, insecticides and fungicides that are essential for the protection of food crops. Many of these compounds are assembled by giant enzymatic 'assembly lines', much like a car production line, with the compound being constructed in a logical stepwise manner. During production, each component of the assembly line must engage in productive communication with other enzymes in the assembly line. These interactions are critical to ensuring that the overall process is efficient and maintains product fidelity.

In this project, I aim to use a combination of established and recently-developed techniques to develop a better understanding of the interactions driving these biosynthetic processes. My focus will concern the enzymes involved in production of a potent antiviral compound called quartomicin, which targets the viral reverse transcriptase and is part of a larger family of compounds called tetronates. This particular system provides the opportunity to study an array of interactions important for producing tetronate-containing natural products. This will broaden our understanding of the role played by protein-protein interactions in natural product assembly and allow us to establish the common principles underlying the way in which they recognise their interaction partners. The results of this research will reveal common principles that can be harnessed to produce more efficient enzymes for biocatalysis, and by extension novel derivatives of bioactive polyketides.

Overall, this project will significantly deepen our understanding of the roles played by protein-protein interactions during natural product assembly and will establish a rational basis for exploiting such interactions to construct engineered assembly lines capable of producing novel natural product analogues.

Modular polyketide synthases (PKSs) consist of enzymatic domains that must interact with each other in a programmed manner to ensure a high degree of fidelity in the overall biosynthetic process. Interactions between domains can occur in both an intra- and inter-modular manner, and are relatively difficult to characterise due to their transient nature. This project builds on recent study conducted by the applicant, showing that a recently-developed carbene footprinting methodology can be successfully deployed to characterise transient interactions in modular PKSs, affording ground-breaking insights into domain communication. The proposed research aims to build on this initial success by applying the technique to an entire PKS module, to obtain the first detailed insights into the interactions governing the biosynthesis of important bioactive compounds.

The work focuses on the PKS responsible for production of the antiviral compound quartromicin, which belongs to the spirotetronate family. The quartromicin PKS affords the opportunity to examine intra-modular interactions, by virtue of a subunit (QmnA3) consisting of ketosynthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains. The interactions between each of these domains will be mapped in molecular detail providing insights into PKS intra-modular dynamics. Additionally, QmnA3 interacts with a cassette of stand-alone enzymes responsible for releasing the polyketide chain via tetronate formation. This is a ubiquitous mechanism for tetronate biosynthesis, and results from this can applied to other systems that employ analogous enzymology. Preliminary work has shown that all of the proteins required for the study can be overproduced and purified, providing confidence that the project's objectives are achievable. The insights gained from this work will find utility for guiding rational pathway engineering to generate novel natural product derivatives.

Natural products are the basis for many the commercially important compounds used in both medicine and agriculture. However, resistance against many of these bioactive molecules has resulted in a requirement to produce new bioactive natural product analogues with lower toxicity and greater efficacy. This imperative has led to the discovery of increasingly complex biosynthetic pathways. However, we currently lack the armoury of analytical tools required to fully understand these pathways at the molecular level, and by extension modify them to produce novel bioactive derivatives. This research project aims to obtain unprecedented molecular insights into the enzymes producing such high value chemicals using a combination of established and cutting-edge structural techniques, thus informing future engineering efforts. Several sectors stand to benefit from the results of this work including: UK-based pharmaceutical and agrochemical companies, researchers in the fields of bioengineering and synthetic biology, and in the long run, healthcare practitioners, farmers and the wider public in the UK.
The development of new biosynthesis strategies and processes for industrial production of new bioactive natural products is a challenging task. This project will address this by generating insights and tools for the manipulation of polyketide biosynthetic pathways, which will have a useful impact on biotechnology companies. An important outcome of the work will be communication to a wide audience of both the basic science entailed in the project and the motivation for pursuing the research. This will be achieved via publication of results in high-impact journals and presentations at conferences, and by effective engagement with the general public via outreach activities. The programme of research will also greatly benefit the applicant. The opportunity to design and execute a cutting edge research project will facilitate his transition to an independent academic career.