Second Generation Sustainable Bacterial Biofuels

Currently the fuels we use to provide electricity or to run motor vehicles is derived from coal, oil and gas. The availability of these 'fossil fuels' is limited and will be exhausted by the middle of this century. Furthermore, fossil fuels are a major contributor to global warming. Thus, there is considerable interest in using environmentally friendly and renewable systems for producing 'biofuels'. To date, there has been widespread adoption of ethanol production from plant derived starch using yeast in a fermentation process akin to that used in brewing. Two fundamental improvements would be of benefit. On the one hand, more effective fuels to ethanol could be produced. On the other, starch is an important component of the human diet. If we are to feed the expanding world population, alternative feedstocks to starch are required. These challenges would be met by the production of the superior biofuel, butanol, and by using microbes able to convert plant 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 without engine modification, offers better fuel economy and has, unlike ethanol, potential 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. Butanol producing bacteria are called 'solventogenic' and belong to a group called Clostridium. Although, the solventogenic species that produce butanol are unable to degrade lignocellulose, Clostridium species do exist that can due to the production of a complex of enzymes called the 'cellulosome'. The cellulosome is one of the most efficient plant cell wall degrading systems known. Using proprietary technology, we will take the genes which code for the cellulosome from one bacterium and introduce them into the chromosome of a butanol producer in a process termed 'synthetic biology'. The ability of the engineered bacterium to degrade plant cell walls and ferment the sugars generated into butanol will be evaluated. Further improvements to the process will be made by alteration of the cell's genetic makeup to improve butanol yields. The most effective strains will be tested on an industrial demonstration scale. The net result will be the creation of more environmentally friendly, sustainable processes for second generation biofuel production. Partner Roles: The programme is led by Prof Nigel P Minton (Nottingham), a world expert in the modification of Clostridium bacteria. The work is underpinned by a portfolio of patented technologies developed within his group and through the skills and expertise of Dr Dave Bolam & Prof Harry Gilbert (Newcastle) who are expert in the functional analysis of the cellulosome. The objectives are supported by key Nottingham strengths in bioinformatics (Prof Charlie Hodgman), mechanistic modeling (Prof John King) and biological circuitry engineering (Dr John Crowe). Crucial, is the participation of TMO Renewables Ltd, a world leader in the development of second generation sustainable biofuels, who will both part fund the work and undertake the small and large scale analysis of the strains generated. These skills are supplemented by input and advice from Prof Hubert Bahl (Rostock) and Dr Wilf Mitchell (Heriot Watt), expert in Clostridium physiology. Collaborative Links between P5 and other programmes: Subject to suitable agreement with the relevant industrial partners involved in other BSBEC Progammes (P1-6), we will undertake the evaluation of; - optimised feedstocks (Willow and Miscanthus) from P1 for biobutanol production, - the potential of enzymes identified by P2 & P6 for incorporation into designer cellulosomes, - a sub-set of barley genotypes showing improved saccharification from P3 will be evaluated for biobutanol production, - optimised wheat feedstocks from P4 for biobutanol production.

The generation of the butanol from lignocellulose (plant cell walls) has considerable BioEnergy potential. The major limitation to exploiting lignocellulose, however, is the rate at which these composite structures can be degraded by enzyme consortia. The most economic method of delivering these enzymes into the biomass conversion process is to engineer the fermenting organism to synthesise the plant cell wall degrading apparatus. In this project we will develop a consolidated bioprocessing system by introducing the plant cell wall degrading multienzyme complex from Clostridium cellulolyticum (cellulosome) into the butanol producing bacterium Clostridium acetobutylicum. Genes will be stably introduced into the genome using a newly developed, and patented, technological innovation which allows the construction of complex operons encoding the large number of catalytic components involved. Initially in vitro experiments will be used to develop an enzyme cocktail that is optimized for plant cell wall degradation. To assemble the enzymes into a cellulosome, to maximise the essential synergy between the catalytic components, the scaffolding protein will be inserted from Clostridium thermocellum, which will be tethered to the bacterial cell wall though a type II cohesin-dockerin interaction. By engineering promoter strengths the stoichiometries of the enzymes will be optimized for cell wall degradation. The genetic approach will also be used to identify genes that significantly enhance the degradation process by selecting for the activation of 'pro-genes'. The strains developed will be evaluated in butanol fermentation trials using plant biomass as the carbon and energy source. The readout from these initial experiments will inform modelling of cellulosome composition and action, which will inform further modification of the cellulosome through an iterative process. Finally, the influence of scale up will be evaluated through our industrial partner.