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Geobacillus thermoglucosidasius is a metabolically versatile thermophilic facultative anaerobe, able to grow on a wide range of monomeric, dimeric and oligomeric carbohydrates derived from lignocellulose. It naturally carries out a mixed acid fermentation producing lactate, formate, acetate and ethanol as fermentation end products. In recent years this fermentation pathway has been redirected by metabolic engineering to produce ethanol almost exclusively, which has enabled TMO Renewables to scale-up and commercialise their cellulosic bioethanol process. Nevertheless, there are a number of potential areas for further improvement and the metabolic engineering has generated some unanswered physiological questions. Through a previous CASE studentship the group at Imperial College have developed a small scale (45ml) continuous culture system providing pH and temperature control as well as redox measurement for the economic study of metabolic flux using 13C labelling. Maintenance of a fixed redox potential and metabolic profiling of cultures at different redox potentials in continuous culture have proved to be valuable tools for reproducible physiological studies of G. thermoglucosidasius under fermentative conditions. In this project we propose to extend physiological studies of mutant and wild type strains through combined metabolic flux and transcript analysis. Selective transcriptome analysis will be done either using microarrays based on genome sequence information which is currently being assembled, or through transcriptome sequence analysis on high throughput platforms available at Imperial College or the BBSRC funded advanced genome centre. Current metabolic flux analysis uses the programme Fiatflux to generate information on flux ratios, but with the availability of genome sequence information (the TMO production strain has been sequenced and a similar strain,SB2, which is being worked on at Imperial College, is being sequenced) it is envisaged that the CASE student would build full metabolic models necessary for determining absolute fluxes. Using these approaches the initial focus would be to explore a range of issues (eg additional nutrient requirements) associated with approaching true anaerobic growth in wild type and engineered strains. In particular, we find that the knockout of pyruvate formate lyase, with or without upregulation of pyruvate dehydrogenase produces some undefined nutritional requirements. Additionally the student will investigate the regulation of the utilisation of multiple carbohydrates in G. thermoglucosidasius, which may require developing new interpretation methods based on the Fiatflux platform. Information arising from these analyses will then guide metabolic engineering strategies for strain improvement as part of an iterative programme.

The UK government recognises that 'our economy is driven by high levels of skills and creativity' and has prioritised investment in skills as a means to recovery rapidly from the current economic downturn (HM Government: New Industry, New Jobs, 2009). Bioprocessing skills underpin the controlled culture of cells and microorganisms and the design of safe, environmentally friendly and cost-effective bio-manufacturing processes. Such skills are generic and are increasingly being applied in the chemical, pharmaceutical and regenerative medicine sectors. Recent reports, however, highlight specific skills shortages that constrain the UK's capacity to capitalise on opportunities for wealth and job creation in these areas. They emphasise the need for bioprocessing skills related to the application of 'mathematical skills... to biological sciences', in core bioprocess operations such as 'fermentation' and 'downstream processing' and, for many engineering graduates 'inadequate practical experience'. UK companies have reported specific problems in 'finding creative people to work in fermentation and downstream processing' (ABPI: Sustaining the Skills Pipeline, 2005 & 2008) and in finding individuals capable of addressing 'challenges that arise with scaling-up production using biological materials' (Industrial Biotechnology Innovation and Growth Team report: Maximising UK Opportunities from Industrial Biotechnology, 2009). Bioprocessing skills are also scarce internationally. Many UK companies have noted 'the difficulties experienced in recruiting post-graduates and graduates conversant with bioprocessing skills is widespread and is further exaggerated by the pull from overseas (Bioscience Innovation and Growth Team report: Bioscience 2015, 2003 & 2009 update). The EPSRC Industrial Doctorate Centre (IDC) in Bioprocess Engineering Leadership has a successful track record of equipping graduate scientists and engineers with the bioprocessing skills needed by UK industry. It will deliver a 'whole bioprocess' training theme based around fermentation and downstream processing skills which will benefit from access to a superbly equipped £25M bioprocess pilot plant. The programme is designed to accelerate graduates into doctoral research and to build a multidisciplinary research cohort. Many of the advanced bioprocessing modules will be delivered via our MBI Training Programme which benefits from input by some 70 industry experts annually (www.ucl.ac.uk/biochemeng/industry/mbi). Research projects will be carried out in collaboration with many of the leading UK chemical and pharmaceutical companies. The IDC will also play an important role supporting research activities within biotechnology-based small to medium size enterprises (SMEs). The need for the IDC is evidenced by the fact that the vast majority of EngD graduates progress to relevant bioindustry careers upon graduation. This proposal will enable the IDC to train the next generation of bioindustry leaders capable of exploiting rapid progress in the underpinning biological sciences. Advances in Synthetic Biology in particular now enable the rational design of biological systems to utilise sustainable sources of raw materials and for improved manufacturing efficiency. These will lead to benefits in the production of chemicals and biofuels, in the synthesis of chemical and biological pharmaceuticals and in the culture of cells for therapy. The next generation of IDC graduates will also possess a better understand of the global context in which UK companies must now operate. This will be achieved by providing each EngD researcher with international placement opportunities and new training pathways either in bioprocess enterprise and innovation or in manufacturing excellence. In this way we will provide the best UK science and engineering graduates with internationally leading research and training opportunities and so contribute to the future success of the UK bioprocess industries.

The use of fossil fuels in the energy and chemical industries is no longer tenable; they represent a finite resource and their use results in carbon dioxide emissions, which is a major cause of global warming. There is, therefore, an urgent need to find alternative sources of liquid fuels that are renewable and do not have an adverse effect on the environment. Lignocellulosic biomass is a promising substrate for biofuel production as it is not a food source, is more abundant than starch, and its use is carbon dioxide neutral. A significant limitation in the use of lignocellulosic biomass in the biofuel industry is its recalcitrance to enzyme attack. Thus, cellulose, the major polysaccharide in lignocellulosic biomass, is chemically simple but its highly crystalline structure makes it inaccessible to enzymes that act as hydrolases. Recent studies, however, have identified novel enzymes that could improve the efficiency of plant cell wall deconstruction. Thus, several reports have shown that oxidases cleave bonds in crystalline regions of cellulose, leading to increased access to hydrolase attack. Significant advances have also been made in the degradation of xylan, the major matrix polysaccharide in lignocellulosic biomass. It was widely believed that degradation of the main chain of xylan required the removal of side chains prior to attack by xylanases. It is now apparent that a cohort of xylanases not only accommodate side chains, but actually display an absolute requirement for these decorations. We have also shown that it is possible to introduce novel functionalities into the active site of biotechnologically significant arabinofuranosidases that assist in removing the side chains from xylan. The generation of such multifunctional enzymes has the potential to simplify the biocatalysts required to deconstruct plant cell walls, and thus increase the economic potential of lignocellulosic biomass as a substrate for the biofuel industry. In this project we will explore the mechanism by which cellulose oxidases, arabinoxylanases and multifunctional arabinofuranosidase/xylanases recognize their target substrates. The data will provide a blueprint for further enhancing the efficiency of the plant cell wall degrading catalytic toolbox.

Currently the fuels we use to provide electricity or to run cars and other vehicles is derived from coal, oil and gas. The availability of these 'fossil fuels,' however, is limited and it is projected that current sources will be exhausted by the middle of the 21st century. Furthermore, it is now apparent that the use of fossil fuels is a major contributor to global warming through the production of carbon dioxide. Thus, there is considerable interest into using more environmentally friendly and renewable systems for producing liquid fuels, now widely referred to as 'biofuels,' for cars and other vehicles. As a consequence there has been widespread adoption of the production of ethanol from plant derived starch using yeast in a fermentation process akin to that used in brewing. Two fundamental improvements to the process would be of benefit. On the one hand, more effective fuels to ethanol could be produced. On the other hand, starch is an important component of the human diet, and as the world population expands and agricultural land diminishes through global warming, it will be impossible to sustain the competition between the use of this polysaccharide for human consumption and biofuel production. The above two improvements would be met by developing a process for the large scale production of the superior biofuel, butanol, and by developing microbes able to convert plant cell derived lignocellulose into biofuel. Butanol has a higher energy content than ethanol, can make use of existing petrol supply and distribution channels, can be blended with petrol at higher concentrations than ethanol without engine modification, offers better fuel economy than petrol-ethanol blends and has, unlike ethanol, potential to be used as aviation fuel. Lignocellulose, the most abundant source of organic carbon on the planet, is both renewable and does not represent a human food source. The bacteria that produce butanol are called 'solventogenic' and belong to a group called Clostridium, typified by Clostridium acetobutylicum. Although those solventogenic species that can produce butanol are unable to efficiently degrade lignocellulose, there are examples of clostridial species, such as Clostridium thermocellum, that can. This is a consequence of the production of a specialised complex of enzymes called the 'cellulosome', one of the most efficient plant cell wall degrading systems known. Cellulosome-producing bacteria do not, however, produce butanol, only ethanol. Using proprietary, patented technology developed at Nottingham, and drawing on knowledge gained from a current BBSRC project concerned with metabolic engineering of the butanol pathway in C. acetobutylicum, we will take the genes which encode the butanol pathway, and introduce them into C. thermocellum using synthetic biology principles. The ability of the engineered bacterium to degrade plant cell walls and ferment the sugars generated into butanol will be evaluated. The net result will be the creation of more environmentally friendly, sustainable processes for second generation biofuel production.