Unlike first generation biofuels, those produced from ligno-cellulosic waste material (second generation) have the potential to offer sustainable fuel production without competition for food products, whilst making significant savings in terms of greenhouse gas emissions. Second generation bioethanol has the potential to offer a stop-gap between current vehicle fuelling technologies and future solutions such as biohydrogen.TMO Renewables Ltd, a leading developer of the second-generation conversion of biomass to biofuel, has engineered the organism Geobacillus thermoglucosidasius to optimise its production of ethanol. This thermophilic bacterium grows optimally at 60-65°C, on a wide range of different substrates including both C5 and C6 sugars. The enzyme responsible for ethanol production has been shown to be a highly-expressed bifunctional enzyme (ADHE) that possesses both an acetylating aldehyde dehydrogenase (aldDH) and an alcohol dehydrogenase (ADH) activity. This enzyme is responsible for catalysing the reduction of acetyl-CoA to ethanol via an acetaldehyde intermediate:acetyl-CoA + NADH + H+ → acetaldehyde + CoA-SH + NAD+acetaldehyde + NADH + H+ → ethanol + NAD+Here we report the characterisation of the bifunctional ADHE in terms of catalytic activity, substrate promiscuity and multimeric assembly. The properties of this enzyme in relation to competing pathways in fermentative metabolism, including its expression pattern during fermentation, have also been determined.Investigations included the sub-cloning and separate recombinant expression of the aldDH and ADH halves of the protein, followed by the determination of a high-resolution crystal structure of the active ADH domain. The structure of the aldDH domain has been modelled, including its interaction with the ADH component of ADHE.An additional aldDH gene was identified in Geobacillus thermoglucosidasius; this has also been cloned and expressed, and the recombinant enzyme characterised and its high-resolution crystal structure determined.Through fusion of ADH and aldDH genes, a series of novel ADHE enzymes have been generated and their effect on ethanol production within the engineered G. thermoglucosidasius strain evaluated.
|Date of Award||7 Dec 2012|
|Supervisor||Michael Danson (Supervisor) & David Hough (Supervisor)|