Energy insecurity, global warming and fluctuations in oil prices and has resulted a rapid increase in the demand for the biofuel, bioethanol. However, the alcohol biobutanol is widely acknowledged as a 'superior biofuel'. It is traditionally produced via a sugar-based fermentation process using a bacterium Clostridium acetobutylicum. The process, however, remains uneconomic due to feedstock prices, which represents 60-80% of production costs.

Another member of the same family of bacteria, Clostridium pasteurianum is a robust, fast growing bacterium which is also able to grow on glycerol as well as sugars and produce valuable 3-carbon (1,3 propanediol) and 4-carbon chemicals (butyrate & butanol). The microbe has tremendous potential as a fermentation host but has not been exploited due to its limited substrate range and mixed array of chemical products that result in low butanol yield compared to current commercial. In this project we aim to use synthetic biology to introduce new metabolic pathways for starch utilisation and butanol production into C. pasteurianum with the aim to produce butanol in high yield from starch. Genetic manipulation based on gene knockout will also be used to inactivate competing pathways.

The partners have extensive experience with solventogenic Clostridia. Nottingham's CRG has developed an impressive range of gene tools and recently completed the determination of the complete genetic blueprint (genome sequence) of C. pasteuranium. GBL has identified novel pathways for both starch degradation and butanol production from its unique collection of commercial strains and leads recommercialisation efforts for the butanol fermentation. This collaboration between GBL and GRG enables, for the first time, a targeted and rational approach for strain improvement in a novel host using synthetic biology and metabolic engineering. This project clearly demonstrates the potential application of synthetic biology for industrial biotechnology. Fermentation performance from the engineered C. pasteurianum strains will be benchmarked against GBL's best performing commercial strains and if promising taken forward for further development. The project output will also support additional projects with this microbe focused on extending the substrate range to include cellulosic feedstocks and developing glycerol metabolism for the production of 3-carbon chemicals.

Technical Abstract:
This project is a collaboration between the Clostridia Research Group (CRG) at Nottingham and Green Biologics Ltd (GBL).

GENOME SEQUENCING & ANNOTATION: CRG has just completed sequencing of the C. pasteurianum ATCC 6013 genome. Data will be used to finalise the assembly and complete annotation of the genome.

DEVELOPMENT OF A METABOLIC MODEL: GBL will use the annotated genome sequence to construct a metabolic model. Engineered strains will be used to validate the model. The model will be used as a predictive tool for future manipulations.

PLASMID TRANSFORMATION: Existing CRG modular shuttle vectors will be used for gene transfer experiments. Optimisation of transformation will be by both the empirical alteration of electroporation parameters, and by instigating strategies designed to circumvent any barrier posed by restriction-modification systems identified in the genome.

GENE KNOCK-OUT/KNOCK-IN: CRG will use the genome sequence data and transformation system to identify any knockout genes encoding enzymes involved in by-product formation. The genes will initially be knocked out using ClosTron technology. Thereafter, using novel in-frame deletions methods.

BUTANOL PRODUCTION: In parallel, CRG will modify expression of enzymes involved in butanol formation, replacing C. pasteurianum 'butanol genes' with synthetic equivalents (identified from hyper-butanol producing clostridia). In addition, endogenous promoters will be replaced with stronger promoters.

STARCH UTLISATION: GBL have identified a novel operon containing four hydrolytic enzymes involved in starch metabolism. Synthetic genes and appropriate orthogonal promoters and terminators will be designed and assembled in BioBrick format using GBlock assembly. These will be integrated into the 6013 genome using ACE.

FERMENTATION PERFORMANCE TESTING: GBL will test fermentation performance with both wildtype & engineered strains at lab-scale with glucose, glycerol and starch.

Potential Impact:
WHO WILL BENEFIT?

The overall aim of this project is to enhance and extend the capabilities of solventogenic bacteria in terms of fuel and chemical production from cost effective feedstocks. As this is an Industrial Partnership, the primary beneficiary is GBL. They will directly commercialise all useful strains that emerge from the project and will have first refusal on any foreground intellectual property that arises.

Both parties have extensive global networks of existing commercial contacts and strategic partners. For example, GBL have partnerships with Guangxi Jinyuan Biochemical and Lianyungang Union of Chemicals in China. Nottingham have partnerships/ collaborations with EBI, Lanxess and Genencor (N America), Evonik, Universities of Munich, Ulm and Berlin (Germany), TMO Renewables Ltd, Invista and Unilever (UK), Metabolic Explorer Ltd, INRA and CNRS (France), Chinese Academy of Sciences (Shanghai and Tsinghau, China), the Mumbai Institute of Chemical Technology (India) and LanzaTech (New Zealand). Working together, the partnership will seek to maximise these links for the benefit of both parties.

The successful commercialization of anticipated outputs will have a rapid and global impact for both humanity and the environment. It will reduce greenhouse gas emissions and environmental pollution, provide an alternative to the use of food or farm resources for the production of low cost low carbon fuels and chemicals. It is therefore of benefit to society, ultimately impacting on health and well-being.

HOW WILL THEY BENEFIT?

Project outcomes will allow improved fermentation process economics and product diversity, thus encouraging more rapid and wide spread adoption of clostridia-based butanol fermentation as a process to produce high volumes of low cost butanol as a chemical commodity and potentially as a biofuel. The partnership are anticipated to directly benefit from the outputs of the project through their commercial adoption via the pipeline established by GBL to scale-up and commercially produce fuel and chemical products by clostridia fermentation. Additionally the partnership intends to explore strategic licensing deals with third party organisations. These will take the form of up front and milestone payments as well as ongoing royalty streams.

The successful scale-up and commercialization of processes will assist the UK and Europe in meeting challenging 'greenhouse' gas reduction targets, and contribute to indirectly to food security. The generation of butanol from cellulosic feedstocks will additionally impact on reducing reliance on fossil reserves, and therefore increase national fuel security.

The use of low carbon fuels to displace petrol reduces localized pollution from transport, and has a positive influence on public health and thus national productivity, ie, EtOH petrol blends reduce smog formation (the American Lung Association credits ethanol-blended petrol with reducing smog-forming emissions by 25% since 1990), and toxic exhaust emissions (CO emissions by as much as 30%, toxic content by 13% (mass) and 21% (potency), and tailpipe fine particulate matter emissions by 50%).

Our programme is tailored to allow definitive benefits to be realized within the project's timeframe. Thus, our target is to improve the productivity of the existing GBL process, allowing its immediate transfer to commercial operations in China. The project will provide the opportunity for staff working directly on the project, together with indirectly affiliated postgraduate students, to become trained in the strategically important areas of 'Synthetic Biology' and 'Industrial Biotechnology and Bioenergy'. These skills will be translatable to many different areas outside of butanol-producing clostridia, enhancing future job prospects.