Dissecting and Exploiting Lytic Polysaccharide Monooxygenases

The complex sugars, termed "polysaccharides", that are found in the cell-walls of plants and in insects and marine organisms impact on the everyday lives of us all. Plant cellulose, the most abundant of these sugars, is the basis of the textile and paper industries (both paper and cotton are made of cellulose). Plant cell-wall polysaccharides, especially cellulose, are also very important if we are ever able to produce sustainably so-called "second-generation" biofuels such as bioethanol. The reason that cellulose is very important in this regard is that it is highly abundant (100 billion kilogrammes are produced globally every year), is present in all plants, and is the principal component of plants, such as switchgrass, that can be grown densely on marginal land. However, there is one major problem which is the production of bioethanol from cellulose is limited by the chemical inertness of cellulose, which cannot currently be broken down into sugars in a sustainable way. This is a major problem. Indeed, reflecting the thoughts of all authorities on energy, The International Energy Agency says that bioethanol will only play a major role in meeting sustainable energy demands if the key technological barrier of cellulose obduracy can be overcome.

The aim of this BBSRC grant application is to study how natural catalysts which can be isolated from fungi and bacteria, termed "enzymes", can actually be used to digest and breakdown polysaccharides including cellulose from plants and chitin from insects/crustaceans. These catalysts are the vanguard of the industries that are springing-up which have the goal of harnessing complex as a biofuel source. As part of our work we will discover new enzymes with diverse functions, study their three-dimensional structures and crucially dissect their highly unusual dependence on metals such as copper for their catalytic ability. We will then use the emerging area of "synthetic biology" to design completely novel catalysts using components not found naturally in nature. Our work will provide the community with insight into the function and action of these catalysts and provide a foundation for their exploitation in the biofuel and other industries.

Recalcitrant polysaccharides from insect, terrestrial plant or marine biomass, are the most abundant biopolymers on Earth. These refractory substrates have enormous potential for exploitation as a renewable and secure energy source for "second generation" biofuels, and beyond. The drawback with these materials is their extraordinary stability. There is therefore a world-wide effort to discover, understand and exploit new enzymatic strategies for polysaccharide deconstruction in a way that benefits society. One of the most recent, and important, advances was the discovery of novel Cu-oxygenases, that complement and enhance the current use of classical glycoside hydrolases, improving biomass conversion from 2-6 times in un-optimised initial experiments. These "Lytic Polysaccharide Monooxygenases" LPMOs harness powerful oxidation chemistry to disrupt the crystalline lattice of polysaccharides facilitating access by classical enzymes. The work described in this grant will target the discovery, dissection and exploitation of novel LPMOs. Building on considerable preliminary data we will uncover enzymes with high utility against diverse substrates. Genome mining will reveal new families of LPMOs greatly increasing the utility of these enzymes in biotechnology. 3-D structural analysis will inform spectroscopic probing of their using EPR, XAS, EXAFS, magnetic circular dichroism, and computational (DFT) analysis allowing a description of their metal geometry, electron transfer and oxidative species. In the final part of the programme we will probe the role of the histidine N-methylation of fungal LPMOs and build upon that to replace the methyl group with a variety of non-genetically encoded amino-acids to develop hybrid catalysts. Work will define the fundamental action of one of nature's most unusual and interesting metalloenzyme superfamilies and support the widespread exploitation of these catalysts in the biotechnology industry.

This proposal aims to tackle head-on the single major limitation in bioethanol production. This limitation is the inability to degrade recalcitrant biomass into glucose effectively. Cellulose, for example, is synthesized in large quantity by all known plants (equivalent in energy to 20 times global oil usage) and its effective conversion to bioethanol, via fermentation, is of major importance. Indeed, all commentators on biofuels identify cellulose as one of the key long-term sustainable sources of biomass. This process is hindered by the recalcitrance of such polysaccharides to degradation.

Of the more promising solutions that are being explored, the enzyme-catalyzed conversion of biomass to glucose is the one that is attracting most commercial attention. For instance, whilst enzymatic degradation of polysaccharides has been the focus of research for the last fifteen years, it has been held back by a lack of understanding of how the substrate is initially attacked by oxidative enzymes. However, the field recently gained considerable momentum when the full structure of a fungal cellulose-degrading LPMO was published in late 2011. This structure, together with the Cu AA10 structures in 2013 are highly significant as they enable the the key catalytic factors behind the oxidative degradation of recalcitrant biomass to be studied. Given the importance of lignocellulosic bioethanol there is now to be a major worldwide effort to build on these discoveries.

The benefits of novel enzymes in the area of biomass conversion are enormous, to both industry and society.

In gauging the future impact of this work it has been possible to draw upon the extensive and detailed predictive models that map out the UK's future energy demands to 2050. These models balance the very many competing and complex factor (economic, societal, technological) and optimise the cost basis of energy production. Notwithstanding the many possible outcomes that arise from such a multivariate scenario, the models see as essential the increased and innovative use of biomass. For example, drawing on very recent work using the MARKAL optimisation model (the leading UK modelling package), to ensure energy security UK energy production from biomass needs to increase from ca 300 PJ pa in 2010 to over 1500 PJ pa in 2050. Of this huge increase, ~50% is from biofuels. Of the bioethanol production, the model(s) assumes significant contributions from UK cellulosic sources, with key assumptions made about the increased efficiency of enzymatic conversion and processing of cellulose to bioethanol.

Industry, therefore, has to rise to a formidable challenge; the UK already lags significantly behind the US, Scandinavia and Brazil where 90% of the world's bioethanol is produced. If it is to compete at all, then it simply must have primary access to the key technological breakthroughs, both in terms of intellectual know how and practical innovation. Through our work we aim to provide exactly this. If the work in this proposal is successful, then UK bioethanol producers stand to gain significant commercial benefit as they become able to switch their biofuel source from specially-grown starch crops to whole plants and waste plant matter, and to make use of marginal land for crop growth.

This project will provide excellent broad training for two PDRAs and experience opportunities across a range of disciplines ranging from molecular biology through structural biology to detailed bio-inorganic chemistry using international facilities and modern synthetic biology. When this is complemented with industrial and science outreach activities, the PDRAs will be extremely well-equipped for future work in the academic and industrial sectors. The generic skills are equally well applicable to UK pharmaceutical industry.