Development of probing strategies to investigate metabolic flux of biofuel production pathways in Clostridia

Currently, fossil fuels contribute a large number of high value chemicals that are used on a daily basis. Crude oil is cracked to give a number of high value chemicals, including vehicle fuels as well as chemicals and solvents that are used daily both commercially and industrially. However, fossil fuel reserves are in decline, with research going into alternatives to obtain these useful chemicals, one of which is biofuels. Biofuels can be generated in a number of ways, one of which is the fermentation of acetogenic bacteria, microorganisms that generate acetate as a product of anaerobic metabolism. Clostridium autoethanogenum is an acetogen that can grow on one carbon gases as its feedstock, and can be used to generate valuable chemicals, with scope to develop the range of metabolic products further. One aim of this project was to investigate the metabolic flux through pathways of the bacterium using isotopically labelled compounds, which would be assessed by mass spectrometry and NMR. Following on from this, design of inhibitors for the enzymes of the pathways with a view to drive the metabolic processes towards higher value chemical compounds by ‘switching off’ other branches of the pathway. Putative small molecule mimics of acetyl-CoA, SNAC thioesters, were synthesised and tested for uptake and activity in whole cell growth experiments with C. autoethanogenum, and determined to be unsuccessful. Further to this, compounds were designed and synthesised to replace pantothenic acid in the growth media, which were not tested in growth experiments. A library of inhibitor compounds was synthesised and tested against recombinantly purified acetate kinase. A number of compounds were shown to inhibit the enzyme, and the mode of inhibition was determined, as well as IC50 and Ki values for each. This project operated as part of a larger GASCHEM project in the Synthetic Biology Research Centre at the University of Nottingham