Synthetic approaches towards the production of biofuels from lignocellulosic feedstocks in yeast

The construction and integration of new pathways and systems into organisms for the production of useful metabolites is a key area of synthetic biology. The brewers' yeast (Saccharomyces cerevisiae) is a biotechnological workhorse; it has been used for the production of foods and beverages for centuries. More recently, this organism has been central to the production of the biofuel, bioethanol. Saccharomyces cerevisiae also serves as a model organism in molecular and cell biology, biochemistry and genetics. Studies on this yeast have pioneered the transition into the genomic and post-genomic era. For instance, S. cerevisiae was the first organism to have its genome sequenced and it has the most comprehensive compilation of gene expression data and systematic mutant collections. Probably as a result, much of the data, tools and technologies of systems biology have been developed in this yeast. This combination of a long, successful history of biotechnological application and unprecedented tools and resources make S. cerevisiae an ideal host for synthetic biology.
We have introduced a pathway that allows the production of biobutanol in S. cerevisiae. Biobutanol is viewed as a superior biofuel to bioethanol; perhaps most fundamentally, as it can be used directly in vehicles without engine modification and it is non-corrosive allowing transportation via existing pipelines. The design strategies that we have employed in the integration of this pathway, have improved the yield about 100-fold relative to other published studies. However, there is further optimisation and development required before the strains would be applicable in an industrial context.
Therefore, the first aim of this proposal is to optimise the production of biobutanol from this strain. We will assess the efficiency of the added enzymes, we will alter endogenous metabolism to channel metabolites towards the butanol production pathway and we will minimise the production of contaminants such as ethanol.
A second goal is to extend the feedstock range such that our butanol producing strain can make maximal use of the resources available in lignocellulosic-based feedstocks. For example, wheat fed bioethanol refineries, like those in the UK, use only the starch rich endosperm (within the seed). Our proposed strain could potentially use a much greater proportion of the plant as a feedstock. A final overarching goal running through the proposal is to develop mathematical models that accurately recapitulate levels of butanol production in various mutants under a variety of conditions. The model can then be used to predict genetic alterations that would lead to further enhancements of butanol yield. The impact of these predictions on butanol yield would ultimately be assessed.
Overall, the project aims to generate yeast strains with the capacity to produce an environmentally clean biofuel from renewable energy sources.

The yeast, Saccharomyces cerevisiae, has been used for centuries in ethanol production from plant crops. Yeast preferentially metabolise monosaccharides such as glucose or fructose to ethanol, and more recently this property has proved crucial in bioethanol production from yeast anaerobic fermentations. However, bioethanol is widely acknowledged to be sub-optimal as a biofuel; principally due to its lower energy content and hygroscopic properties. Furthermore, yeast is limited by its inability to ferment sugars such as xylose that are released from the lignocellulose portion of plants.

Biobutanol does not suffer from the disadvantages described for bioethanol; being higher in energy content, less influenced by water contamination and less corrosive. It has also been claimed that 100% biobutanol can be used directly in existing motor engines. The traditional biobutanol production route is ABE (acetone, butanol, ethanol) fermentation in Clostridial species of bacteria from high-energy plant feedstocks. In the background work to this project, we have used synthetic biology strategies to insert four Clostridial genes and one optimised yeast gene, which encode butanol production enzymes, into the yeast genome. These five genetic manipulations have generated a strain that has the capacity to produce 100-fold higher levels of butanol than previously observed in yeast; highlighting the potential S. cerevisiae has as a vehicle for biobutanol production.

In this project, we propose to combine the tools of metabolic engineering and modelling to produce a strain with highly optimised biobutanol production that is thus relevant to industrial application. We will also generate strains where the biobutanol production pathway is integrated with a similarly honed pathway for xylose metabolism. Overall, a yeast strain will be created with the capacity to produce a superior biofuel, biobutanol, from renewable lignocellulosic carbon sources.

Who will benefit from this research?
The most important impact of this project will be on society as a whole in the area of energy. An important component in the diversification of energy sources is to increase the use of biofuels, which are renewable sources of energy. Butanol and ethanol are readily usable biofuels that have been produced from agricultural food sources (mostly wheat in the UK). A consequence of increased use of these crops for biofuel production is that production of food is decreased and food prices increased. This is obviously insupportable in our society and thus other sources of biofuels must be sought out. The most obvious sources that would not be competing with food production (in fact it could be synergistic with it) are lignocellulosic sources, such as straw, bagasse, or waste paper. The project will develop experiments and simulations that will lead to a better understanding of the regulation of nutrient utilisation by Saccharomyces cerevisiae. This is important as a biotechnological output since S. cerevisiae is a widely used microorganism for this purpose.

How will they benefit from this research?
The result of the research proposed here will be new strains of yeast and methods of fermentation with increased yield and flux of biobutanol production. Our results will also impact on the general understanding of the interplay of energy metabolism, redox regulation and genetic regulation in all eukaryotes (since S. cerevisiae is also one of the best model organisms for studies of eukaryotic biochemistry). Our strategy combining experiments and modelling will be pioneering in the development of biotechnological solutions. From the biotechnology point of view, we will develop strains that have improved production of biofuels (butanol and ethanol) from mixtures of hexoses and pentoses, exactly what is needed for fermentation of lignocellulosic sources.

What will be done to ensure that they benefit from this research?
Results will be disseminated through research seminars, presentations at conferences and publications in scientific journals. Funding is requested to attend national and international research conferences to allow the researchers to publicize this research. Resources generated from this project are likely to include modelling data, yeast strains and plasmids and will be made available to the scientific community upon request. Detailed protocols and primary data will be made freely available to academic collaborators. Manchester University has a good track record of encouraging public engagement. This includes regular open days to inform school children and the public about University research and tours of the research facilities at Manchester. This will allow the researchers to share their research findings with the wider public and to raise awareness of the importance of basic research. One important opportunity that is available to us for doing this will be to interact with colleagues at Manchester University's Sustainable Consumption Institute. This Research Institute houses a multidisciplinary programme of research examining issues, and disseminating findings, linked to sustainable consumption and sustainable development. Manchester University maintains excellent links with the business sector, which will allow us to exploit any potential for collaboration with industry. This is managed by the faculty Business Development Team, who provide support and information for staff wishing to develop relationships with business.