Discovery and Exploitation of Novel Lytic Polysaccharide Monooxygenase Redox Partners.

Plants contain a vast amount of sugar. Sugar is important because it can be fermented by microorganisms into ethanol which can be used as a fuel - a commodity that we are running out of. The advantage of using fuels derived from plants is that this creates a carbon neutral cycle, where carbon dioxide released from fuel combustion is reabsorbed by photosynthesis during growth of new crops. Biofuels are already being produced, but they are largely derived from crops that could be better utilized as food sources. A significant challenge is, therefore, ensuring that we move towards a more sustainable solution by making use of the large amount of sugar that is found in the inedible parts of plants which currently goes to waste. These sugars are locked away in structures known as polysaccharides, which effectively act as the plants skeleton giving them rigidity. The most abundant polysaccharide is called cellulose, which is already utilized extensively in the paper and cotton industries, and is the main component of wood. The sugars in cellulose are all joined together in a highly ordered structure that is incredibly difficult to break down. The challenge then is to find an efficient means of deconstructing cellulose to release the sugars so that they can be used to generate bioethanol.

Fungi and bacteria have evolved over millions of years to live in all sorts of environments. Some of these organisms are able to degrade and live off wood. In order to do this, they produce protein-based molecular machines known as enzymes. As a structural biologist I am able to study these enzymes and gain a fuller understanding of how they break down cellulose. Recently a new family of enzymes has been identified in this process known as LPMOs (lytic polysaccharide monooxygenases). LPMOs show great promise in enhancing the efficiency of biofuels production but our knowledge of how they function is limited.

LPMOs require two things - oxygen and electrons. Oxygen is readily available from the air but electrons need to be supplied from some other source. In bacteria, this source of electrons has not yet been identified. The aim of this fellowship is to characterize a range of enzymes that are potential electron sources for LPMOs. By determining their three-dimensional structures and biochemical properties I aim to build up a thorough understanding of the interplay between these various enzymes at the molecular level. Modern enzyme engineering approaches will then be applied to make more efficient and novel enzymes for deployment in industry for biofuels production. Electron transport, and the so-called redox reactions which these processes support, are also of importance to other industries beyond the biofuels sector and so this fellowship also offers ample opportunities to explore the application of these proteins to solve other scientific problems. Having worked extensively on LPMOs this fellowship offers me the perfect opportunity to branch out into my own research area answering a fundamental question in the field and expanding my knowledge of new scientific approaches to tackle real world problems.

The breakdown of waste plant material, in the form of lignocellulose, for subsequent fermentation into biofuels holds tremendous promise for securing humankind's future energy needs. Lytic Polysaccharide Monooxygenases (LPMOs) have emerged as key players in the natural and applied breakdown of these difficult substrates. These copper dependent enzymes utilize free oxygen and an electron donor to oxidatively deconstruct biomass. However, there remain some key questions regarding what the natural electron source is in this process. Answering these questions will be key to optimizing both the efficiency of enzymatic lignocellulose deconstruction and maximizing the lifetime of the reaction mixture.

We propose that some carbohydrate binding proteins contain "X-domains" capable of electron transport to LPMOs. The aim of this proposal is to isolate and characterize these extracellular redox proteins. Using bioinformatics I will supplement the current set of X-domains that have been identified with additional targets to be characterized. These proteins will be produced and purified to homogeneity using modern molecular biology approaches to facilitate a full structural and functional analysis of their roles. Structures of individual domains will be determined by X-ray crystallography, whilst the full-length proteins and complexes with binding partners offer an excellent opportunity to branch out into cryo-electron microscopy. Underpinning these structural studies will be protein-protein interaction work, enzymology, spectroscopic characterization, and protein-film voltammetry to further investigate and derive a holistic understanding of the electron transport processes involved in LPMO activation. Ultimately by exploiting the structural and biochemical knowledge gained using protein engineering it will be possible to produce even more efficient systems for lignocellulose deconstruction whilst also investigating other potential applications for these proteins.

One of the biggest obstacles to the successful implementation of industrial scale bioethanol production from lignocellulose is the highly recalcitrant nature of biomass to enzymatic breakdown. This has spurred a surge in interest in the natural enzymes produced by microorganisms for the degradation of biomass and has resulted in the discovery of the lytic polysaccharide monooxygenases (LPMOs). These enzymes are now viewed as key players in biomass breakdown, and use a novel oxidative mechanism to induce chain breaks in polysaccharide chains. Their rapid inclusion in commercially available enzyme cocktails for use in the bio-refinery attests to their importance in the field.

Following the discovery of these enzymes there is now a worldwide drive to ensure that these enzymes are used effectively for industrial application. One of the key aspects of how these enzymes function, that is incompletely understood, is how electrons are transported to the enzymes to support their activity. This proposal seeks to tackle this problem by first providing a platform for the investigation of possible redox partners to LPMOs, but then to also allow the exploitation of the knowledge gained using enzyme engineering approaches to ensure maximal enzyme turnover for cellulose degradation while also investigating other potential applications for these electron transporting proteins. The main route for dissemination of research results will be via publication in peer-reviewed journals with interested parties including researchers working on biomass deconstruction, bio-inorganic chemistry, enzyme engineering, and protein mediated electron transport mechanisms. There is also interest in important oxidations for the production of both high-value chemical precursors as well as for the production of diverse compounds as potential drug candidates in the pharmaceutical industry, and applications for these electron transport proteins in these areas will also be investigated. One of the key applications of electron transport proteins to date is in the development of biofuel cells in which the proteins produced from this research may also find utility. By presenting the research outcomes at national and international conferences I will ensure the widespread dissemination of this research to as wide an audience as possible across both academia and industry.

This research also offers impact to the British general public by allowing a transition towards a greener, more sustainable economy. The British biofuels industry lags behind the rest of the world at present with the majority of biofuels produced in the US, Brazil and Scandinavia. The research outcomes from the current proposal will add to the knowledgebase of UK researchers in this area, opening up new opportunities for investment and advances in UK biofuels production. By engaging with the public I also hope to raise awareness of the "green" credentials of this research and communicate how the research results can positively impact the British quality of life.

I will stand to benefit greatly from the proposed research, which will provide ample personal development opportunities for me to set out as an independent researcher, building up new expertise across new fields of research, whilst also providing opportunities to perform more outreach activities and to disseminate research results more widely by engaging with the press. The research also offers ample training opportunities for a postdoctoral research associate to work on the project with the chance to learn general laboratory skills across molecular biology and structural biology through to enzyme engineering. These opportunities will leave both the PDRA and myself in excellent positions to further pursue careers in either academia or industry, with many of the skills gained equally applicable across sectors.