Elucidating and exploiting docking domain-mediated carrier protein recognition in natural product megasynthetases

Bioactive natural products from plants and microorganisms have numerous important applications in medicine and agriculture. They are used to tackle life-threating conditions, such as bacterial and fungal infections, organ transplant rejection and cancer, and as herbicides, insecticides and fungicides that play an essential role in the protection of food crops. Many natural products are assembled by enzymatic "assembly lines", akin to a car production line. Each component of the assembly line must engage in effective communication with the next to ensure the overall process is efficient. Such communication is typically mediated by dedicated "docking domains" attached to the ends of the components, which interact with each other in a specific way. We have been studying the role played by a particular type of docking domain and its interaction partner in the assembly of enacyloxin IIa. This antibiotic is produced by Burkholderia species and has potent activity against Acinetobacter baumannii, a bacterium that causes life threatening diseases in humans for which there is a critical need to find effective new treatments. Our data have shown that this type of docking domain is involved in the assembly of many more bioactive natural products than previously thought, including several anti-cancer agents and an antibiotic that are used in the clinic.
In this project we aim to use a combination of established and recently-developed techniques to develop a better understanding of the way the docking domain involved in the production of enacyloxin recognises its interaction partner. The insights we obtain will be used to modify the enacyloxin assembly line to see whether it behaves in the way we predict. We also aim to investigate a related system in which two components, both of which have a docking domain that is similar to the one involved in enacyloxin production, appear to interact with the same partner to execute sequential tasks in the assembly of the aeruginosins, an unusual group of protein degradation inhibitors produced by cyanobacteria. This will broaden our understanding of the role played by docking domains in natural product assembly and allow us to establish the common principles underlying the way in which they recognise their interaction partners. Finally, we will investigate whether our understanding of these common principles can be harnessed to substitute one of the components of the aeruginosin assembly line with the corresponding component from the enacyloxin system. Overall, this project will significantly deepen our understanding of the roles played by an important type of docking domain in natural product assembly and will establish the feasibility of exploiting such docking domains to construct engineered assembly lines capable of producing novel natural product hybrids.

Modular polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs) and hybrid PKS-NRPSs typically consist of several subunits that must interact with each other in a programmed manner to ensure a high degree of fidelity in the overall biosynthetic process. Interactions between subunits are typically mediated by various types of structurally complementary N- and C-terminal docking domains (DDs).
This project builds on recent collaborative BBSRC-funded work by the applicants showing that a type of DD found at the N-terminus of amide-forming condensation (C) domains and thiazoline forming heterocyclisation (Cy) domains, and previously thought to be associated with only a handful of systems, is in fact present in more than forty NRPS and hybrid PKS-NRPS assembly lines, including several responsible for the biosynthesis of clinically-approved anticancer agents and antibiotics. Moreover our work has revealed that this type of DD is associated with additional classes of catalytic domain, such as ester-forming C domains, oxazoline-forming Cy domains and flavin-dependent chlorinases. Such DDs interact with a largely unstructured peptide appended to the C-terminus of carrier proteins (CPs) to which the substrates for the downstream catalytic domains are covalently attached.
We have determined the structures of an ester-forming C domain with its N-terminal DD attached and its partner CP containing the C-terminal interacting peptide by X-ray crystallography and NMR spectroscopy, respectively. Here we aim to pursue an interdisciplinary approach to understanding the interaction between these two proteins. We also aim to characterise a system in which a halogenase and an amide-forming C domain, each containing an N-terminal DD, appear to interact with same CP to first chlorinate, then elongate its bound substrate. The utility of our insights for guiding rational pathway engineering will be tested by constructing modified and hybrid assembly lines.

The development of new techniques for natural product bioengineering is an important cornerstone of industrial biotechnology. For example, there is an urgent need to produce novel derivatives of natural product antibiotics that overcome antimicrobial resistance and this is difficult to achieve using chemical synthesis. Also many herbicides, insecticides and fungicides, which play an essential role in the protection of food crops, are natural products and new derivatives with lower toxicity and greater efficacy are needed to feed the burgeoning global population. Therefore, a number of beneficiaries stand to gain from this research, including the UK-based pharmaceutical and agrochemical companies, the researchers employed by the project and, in the long run, the wider public and health-care in the UK.
The researchers employed by the project will receive top-quality training in the methods and philosophy of highly collaborative and cutting-edge interdisciplinary research at the Chemistry/Biology interface. This will ensure that they are attractive potential employees for UK companies across the pharmaceutical, agrochemical and biotechnology sectors, with an ideal skill set to ensure such companies continue to contribute strongly to the UK economy. The training they receive will also equip them to become leading contributors to the development of a knowledge-based bio-economy, which is predicted to become a strong driver of economic growth in the UK, and indeed across Europe, in the coming decades.
An important part of the work to be carried by the researchers employed on this project is the development and delivery to schoolchildren across the West Midlands region of presentations on the important part played by natural products in industrial biotechnology. This will educate the children about the need to develop new methods for producing natural product analogues and will inspire them to take up the challenge of developing a knowledge-based bio-economy by pursuing a career in science.
New strategies for natural product bioengineering will be an important outcome of this project. This will be of benefit to UK-based pharmaceutical, agrochemical and biotechnology companies actively-engaged in the quest to develop novel natural product-based consumer products, which will ultimately be of benefit to wider society.