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

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.

Future bioethanol production will need to use cellulose and hemicellulose from biomass (eg short rotation crops and plant waste) in a multi-step 'biorefinery'. Ideally, this will involve continuous production (and ethanol removal eg at high temperatures) with organisms which grow on pentoses, hexoses and complex polysaccarides and tolerate processed biomass derived toxins. We will explore two strategies to convert any genetically amenable glycolytic thermophile into an ethanol producer. The first is to create a thermophilic pyruvate decarboxylase. The enzyme from Zymomonas mobilis, although moderately thermostable once folded, does not fold correctly in Geobacillus spp above 50oC, probably because the affinity between the unfolded enzyme and TDP-Mg2+ cofactors is too low. Using selection based on growth, we will use directed evolution methods including error prone PCR and oligonucleotide shuffling (based on sequences encoding TDP-Mg2+ binding sites from thermophiles), to incrementally improve the thermophilicity of PDC. The resulting PDC gene may then be combined with that for a thermophilic alcohol dehydrogenase (ADH) to create a metabolic engineering 'cassette' that could be applied in other thermophiles. The second strategy exploits the observation that pyruvate dehydrogenase, together with elements of the pyruvate formate lyase pathway have a redox balance and metabolic outcome identical to that of PDC-ADH. As all of these functions are present in thermophiles it should be possible to create a novel homoethanol fermentation pathway based on PDH, CoA dependent acetaldehyde dehydrogenase and ADH. Approaches for anaerobic expression of PDH will be explored, together with the possibility of converting non/poorly-fermenting strains into ethanol producers. In the latter context we will test G. denitrificans K1041 under conditions of low nitrate availability. Finally, we intend to apply these tools to a newly isolated 'process optimised' thermophile.