Systems biology of the butanol-producing Clostridium acetobutylicum: new source of biofuel and chemicals/COSMIC2

By 2030, worldwide energy consumption is projected to grow by 57%. Fossil fuels cannot meet this demand, as their continued use is causing global warming and in any case are a finite reserve that will be exhausted before the end of this century. Liquid fuels derived from natural plant material (biofuels), are the most promising alternative energy source for use in the transportation sector. In this regard, an alcohol called butanol is currently receiving considerable interest due to its superior properties compared to ethanol and biodiesel. 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 without engine modification, offers better fuel economy and has, unlike ethanol, potential as aviation fuel. Traditionally, butanol is produced by a bacterium called Clostridium acetobutylicum when growing on growth media containing sugars. The fermentation process is known as the Acetone-Butanol-Ethanol (ABE) process. In the first hald of the last century, the ABE process was second in importance/ scale only to ethanol production from yeast. However, with the advent of the petrochemical industry, the process became uneconomic and was abandoned in the western world from the 1960s onwards. The process has recently been re-established in China and Brazil, where lower operating costs make a process based on inefficient 20th century technology economic. For adoption in the western world, a more efficient process is required. Its derivation will be reliant on improvements to the bacterial strains employed. However, the rational creation of such strains will require a more detailed understanding of the complex processes within the cell that catalyse and control the formation of fermentation products. Deriving a better understanding of the process is the overall objective of this proposal. For this, a new holistic approach termed systems biology will be used, which involves iterative cycles of mathematical modelling and experimental testing. It is made possible by developments made in an earlier funding round in which crucial gene tools and methodologies were developed that will allow precise changes to be made to selected genes and the consequences assessed.

The enzymes catalyzing and controlling conversion of glucose to solvents and acids are encoded by up to 40 genes. Starting with model-driven hypotheses, specific mutants will be generated by knock-in and knock-out strategies and analyzed. Selected mutants will be grown in continuous culture, allowing the imposition of reproducible, controlled perturbations. Fermentation analysis will include substrate/product concentrations, determination of key intracellular metabolites, and transcriptome time series. These data will lead to further iterative experimentation and ultimately to a fine-tuned quantitative description of the process of solventogenesis. Workpackages:- 1] Construction of artificially controlled genes required for solvent production/regulation: ACE technology will place specific chromosomal genes under inducible control. These changes, in combination with gene knock-outs, will allow rational perturbation of the system. 2] Analysis of mutants in continuous culture under standardized conditions: Strains will be grown and analyzed in continuous culture. Quantitative analysis will include substrate and product determination, identification and quantification of key intracellular metabolites, and transcriptome time series. 3] Modelling, in silico generation of hypotheses and experimental design: An iterative process of model-based hypothesis generation and experimental testing by variations of the transcriptome and the environome will be adopted for refining the model. Controlled stimuli from the environome and rapid sampling experiments will be included. 4] Data management: DaMaSys will serve as an access-controlled repository for multi-'omics' datasets as well as platform for communication and joint model development. Data pre-treatment, data consistency checks, data curation for modelling purposes, and, in part, the cyclic interaction between model-based hypothesis generation and experimental testing will be organized into automatable workflows.

Beneficaries: The ultimate goal of this project is to generate strains of Clostridium acetobutylicum that produce biobutanol with greater productivity and which can form the basis of a commercial process for biobutanol production. The major direct beneficary is, therefore, the industrial private sector concerned with chemical commodity production. A boost for the agricultural sector is also expected, as farmers will be able to profit from the demand for cellulosic waste products which form the substrates for biobutanol production. The results will also benefit national and international government green policies in helping to replace fossil fuels with biofuel. Achieving government targets in terms of green issues will also indirectly benefit the wider general public. How they benefit from this research: At the basic level, industrial producers will benefit through the availability of strains which may be employed as the basis of an economic process for biobutanol production. These beneficaries may be expected to gain a commercial advantage over competitors. The ultimate development of a biobutanol process will reduce national, and international, reliance on fossil fuels in the transportation sector, providing a cleaner environment and therefore indirectly impacting on human health. The technological developments will also provide an opportunity for export to third countries providing revenue for UK Plc. The project will also provide the opportunity for staff working directly on the project, together with postgraduate students indirectly affiliated to the project, to become trained in the arena of the strategically important areas of 'Systems Biology' and 'Bioenergy'. These skills should prove applicable to many different projects outside of butanol metabolism. To ensure that they benefit: The project PI (NPM) already has strong links with a major UK Biofuel company, who part fund other BBSRC projects at the University, and with whom a commercial agreement is already in place. A high degree of collaboration will be maintained with this company, while at the same time other collaborative ventures will be explored. To date, previous BBSRC funding to NPM has resulted in the filing of 4 patents to cover the enabling gene technologies that make this project possible. In addition to a number of academic awards, the existence of these technologies have led to 2 X BBSRC IPA awards, 4 X industrial CASE awards, 1 X industrial studentship and an industrial research contract with a major US Pharmaceutical company. Our principle measure of success will, therefore, be the filing and subsequent licensing of patents concerned with butanol production to private companies. Through liaison with Nottingham's Research & Innovations service, NPM will continue to monitor the IP and commercial potential of the research to be undertaken here, and will additionally chair a consortium wide committee concerned with IP and commercialisation of the consortia outputs. Over an above these activities, the project members will endeavour to communicate their work widely, both through the scientific and non-scientific press and through various media outlets, including websites.