Developing strategies and a toolbox for metabolic engineering of thermophiles for ethanol production

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The UK is committed to replacing an increasing fraction of current liquid fuel consumption with biologically derived fuels. While this is attractive given the current price of oil, the primary driver for this was a commitment made under the Kyoto protocol, to reduce greenhouse gas emissions. Unlike fossil fuels, those derived from green plants are virtually carbon dioxide neutral. Ethanol, produced by the fermentation of sugars, is an established biofuel which is already extensively used in Brazil and in the USA, and the technology to run cars on either pure ethanol or ethanol-petroleum mixtures exists. The classic method for ethanol production uses yeast to ferment either sucrose (from sugar cane or beet) or glucose (from starch). Yeast is one of the few organisms that can ferment sugars exclusively to ethanol and carbon dioxide, which it does by employing the enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). PDC is rarely found in bacteria, which is the main reason why the brewing industry, and more recently, fuel ethanol production have used yeast. However, the energy balance of ethanol production from sucrose and starch is marginal and it is clear that this, and the overall economics would be much improved if it was possible to use all of the sugars present in biomass, particularly those available in hemicellulose and cellulose, which together comprise the most abundant global sources of carbohydrates. Unfortunately, bakers/brewers yeast does not naturally ferment the pentose (C5) sugars found in hemicellulose, and those yeast strains that do, grow very slowly. Furthermore, it would be more efficient to run continuous fermentation processes than the sequential batch processes typical of industrial yeast fermentations, which incur significant 'dead time' between runs. A continuous process implies continuous removal of ethanol, which is most efficiently achieved by operating at elevated temperature (the boiling point of ethanol is 78oC). Thus, an ideal organism for ethanol production would rapidly ferment a wide range of sugars, including pentoses, and possibly more complex substrates such as cellulose, at temperatures around 70oC (ethanol can be removed at this temperature using gas stripping). However, such an organism has not been isolated yet. Yeasts do not grow at temperatures above 50oC, while thermophilic bacteria that can often metabolise a range of sugars, including complex polymers, tend to produce multiple fermentation products, the composition of which may depend on growth conditions. This proposal presents two strategies for constructing thermophilic bacteria which produce ethanol exclusively from a range of biomass-derived sugars. It starts from the premise that, given the range of different biomass substrates and pretreatments that are likely to be used, it would be more feasible to isolate bacteria able to grow on the various substrates and engineer their downstream metabolism to ethanol production, than to find and engineer a good thermophilic ethanol producer to use a wide range of substrates. The first strategy is to use a 'directed evolution' approach to produce a modified PDC which works in a thermophile at 65-70oC. This essentially involves mutating the relevant gene at high frequency, and/or recombining elements from known similar genes present in thermophiles, combined with a powerful selection method for improved variants. The second strategy involves creating a novel fermentation pathway based on combinations of enzymes known to be expressed in thermophiles, but which are not normally expressed together. In particular it involves expressing pyruvate dehydrogenase, an enzyme usually associated with aerobic growth, under anaerobic conditions, together with two normally anaerobic enzymes. Together, these would have the same outcome as the PDC pathway. We have a precedent that this pathway already operates in mutants of the thermophile Geobacillus thermoglucosidasius.