Engineering Bacteria for the Enhanced Production of High-value Chemicals

There is a growing interest in the use of microbial cell factories to produce butanol, an industrial solvent and platform chemical. Biobutanol can also be used as a biofuel and represents a cleaner and more sustainable alternative to the use of conventional fossil fuels. Solventogenic Clostridia are the most popular microorganisms used due to the native expression of butanol synthesis pathways. A major drawback to the wide scale implementation and development of these technologies is the product toxicity of butanol. This study aims to develop a deeper understanding of butanol toxicity at the membrane. Using liposome membrane models and in vitro assays to investigate characteristics such as permeability, fluidity, and diameter, it was found that altering the composition of membranes can convey tolerance to butanol. The effect of butanol on membrane proteins was also investigated, with it causing unfolding of bacteriorhodopsin. The changes to the lipidome of Clostridium saccharoperbutylacetonicum N 1-4 in different butanol environments were investigated with thin layer chromatography and mass spectrometry. In higher butanol concentrations, levels of phosphatidylglycerol and oleic acid had increased significantly. Several metabolic targets were selected for the genetic engineering of Clostridium saccharoperbutylacetonicum N 1-4 (HMT) in an attempt to improve tolerance in butanol. The three targets investigated consisted of two membrane proteins and one enzyme. The first membrane protein, GlpF, a putative butanol channel which appeared to grant a butanol-independent advantage to growth when overexpressed. The second membrane protein, TtgB, is a transporter and overexpression increased cellular growth rate during fermentation. The final protein PssA is involved in lipid synthesis and showed no effect when overexpressed and knockouts appeared to inhibit growth in fermentation. Ultimately, this work highlights the detrimental impact of butanol-membrane interactions, how the cell responds and presents some novels strains, some of which have produced promising results