TMO Renewables Ltd, the CASE collaborating company, has developed an ethanol fermentation process capable of using sugars derived from lignocellulosic substrates, based on engineering the fermentation pathways of Geobacillus thermoglucosidasius. As part of the work they have contracted to obtain the genome sequence of this organism, which is almost complete. In the initial stages, the annotation of a genome sequence is usually done automatically, based on the search for open reading frames (orfs) and bio-informatic homology searches. This means that initial annotations often contain errors and unclassified orfs. In this project the student will start to build a genomic scale metabolic model of this organism, using the initial genome sequence as a starting point. The ultimate goal is to build an in silico model of the metabolic capabilities of this organism based on Palsson's flux balance analysis approach (Palsson BO (2006) Systems Biology. Properties of Reconstructed Networks. Cambridge University Press, New York, USA). However, because Geobacillus spp are not extensively described at a biochemical and physiological level, this will require a considerable amount of experimental validation to eg confirm the gene assignments and fill in missing metabolic links. Once constructed, an in silico metabolic model can have predictive capabilities. Initially, these can be used to authenticate the quality of the model, which invariably includes an number of assumptions/estimates. However, in the long term, these may be used to predict the optimal route for metabolic engineering for a defined objective, which is one of the future aims of the company. In the interim, however, the logical iterative experimental and in silico construction of the model should generate new insights into the physiology and biochemistry of this increasingly important group of thermophiles. The combined experimental and modelling aspects of this programme will form an excellent training programme for a postgraduate student with a biological background, exposing them to one of the more accessible avenues of systems biology.

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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.

TMO Renewables Ltd, the CASE collaborating company, has developed an ethanol fermentation process capable of using sugars derived from lignocellulosic substrates, based on engineering the fermentation pathways of Geobacillus thermoglucosidasius. As part of the work they have contracted to obtain the genome sequence of this organism, which is almost complete. In the initial stages, the annotation of a genome sequence is usually done automatically, based on the search for open reading frames (orfs) and bio-informatic homology searches. This means that initial annotations often contain errors and unclassified orfs. In this project the student will start to build a genomic scale metabolic model of this organism, using the initial genome sequence as a starting point. The ultimate goal is to build an in silico model of the metabolic capabilities of this organism based on Palsson's flux balance analysis approach (Palsson BO (2006) Systems Biology. Properties of Reconstructed Networks. Cambridge University Press, New York, USA). However, because Geobacillus spp are not extensively described at a biochemical and physiological level, this will require a considerable amount of experimental validation to eg confirm the gene assignments and fill in missing metabolic links. Once constructed, an in silico metabolic model can have predictive capabilities. Initially, these can be used to authenticate the quality of the model, which invariably includes an number of assumptions/estimates. However, in the long term, these may be used to predict the optimal route for metabolic engineering for a defined objective, which is one of the future aims of the company. In the interim, however, the logical iterative experimental and in silico construction of the model should generate new insights into the physiology and biochemistry of this increasingly important group of thermophiles. The combined experimental and modelling aspects of this programme will form an excellent training programme for a postgraduate student with a biological background, exposing them to one of the more accessible avenues of systems biology.

Under the terms of the Renewable Transport Fuel Obligation (RTFO), the UK is committed to substituting 5.75% of its gasoline consumption with bio-derived fuels by December 2010. This demand is predicted to increase in the future, particularly in response to concerns about climate change and fuel security. Current, biofuel generation in the UK is negligible and demands are met by bioethanol imports from countries such as Brazil. Bioethanol is mainly produced from 'first generation' crops (e.g. maize, wheat, sugar beet and sugar cane) which are characterised by a high non-structural carbohydrate content. The technology involved is straightforward and production has become more price competitive. The feasibility of producing biofuel from such crops in the UK is limited because of the requirement for arable land which is primarily used for food production and the high energy input involved. Production of biofuel from 'second generation' lignocellulosic crops such as grasses offers a potential alternative. Grasslands comprise up to 70% of UK agricultural land greatly exceeding the area used for food crops. Perennial ryegrass achieves similar biomass yields to other lignocellulosic crops used for biofuel production. This crop has a number of traits which are desirable in a fermentable feedstock including a readily available high water-soluble sugar content, high fibre digestibility and a low lignin content in comparison with other candidate lignocellulosic crops. Perennial grasses have low annual input requirements and contribute to the rural landscape, maintaining biodiversity and environmentally sensitive landscapes which have major attractants for the tourist industry. UK farmers have the necessary expertise involved in management of these grasses which can be harvested over a long season and stored over winter. We propose that perennial ryegrass can provide an environmentally and economically viable feedstock for the production of bioethanol and that existing biological material and technologies can be readily adapted to achieve this. The main challenges for development of a sustainable, low input process, for conversion of grasses to bioethanol will be addressed in this programme. This will include reducing the major operating costs, maximising yield and carbon cost efficiency. IGER's large selection of ryegrass germplasm will be exploited to select for appropriate varieties, in particular, high sugar perennial grasses with high digestibility (low lignin). The legume, white clover, will be included in grass swards to provide nitrogen and minimise green house gas emissions associated with artificial fertilizer. This programme will test the feasiblility of juicing on-farm to generate two separate feedstocks; a water soluble carbohdyrate (fructan) rich liquid fraction and a high dry-matter stable lignocellulosic fraction. Procedures for handling, preserving/stabilising and transporting these feedstocks will be assessed. A major aim is to maximise utilisation of the full range of sugars in perennial ryegrass for fermentation to ethanol. This will be achieved by using an appropriate combination of pre-treatments, enzymes, yeast and an ethanol producing thermophilic micro-organsim, taking advantage of TMO Renewables groundbreaking method for producing ethanol from almost any type of biomass. Fermentation conditions will be optimised to maximise ethanol production from ryegrass feedstock both at laboratory and pilot scale. The carbon and energy balance as well as the economic viability of these processes will be evaluated. Data generated by this programme will provide valuable information for accurate comparisons with other crops used in bioethanol production.