Application of novel methods using synthetic biology tools to investigate solvent toxicity in bacteria
Fletcher, Eugene Kobina Arhin
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Toxicity of organic solvents to microbial hosts is a major consideration in the economical production of biofuels such as ethanol and especially butanol, with low product concentrations leading to high recovery costs. The key to rational engineering of solvent tolerant microorganisms for such processes lies in obtaining appropriate tolerance genes (modules) suited for different compounds. In this project, a synthetic biology approach was adopted to generate a library of standardised BioBrick parts involved in different stress responses. Using a multiple-assay approach, including a bioluminescence assay, these stress response genes were tested individually and in combination to determine their effects on survival in ethanol, nbutanol, acetone and fermentation inhibitors produced by biomass pre-treatment. A set of tolerance modules was obtained for ethanol and n-butanol. Proof-of-concept tests suggested that ethanol and n-butanol toxicity was mainly due to damage to membrane, cellular proteins and DNA possibly by oxidative stress. No synergistic interactions were observed from a combination of different tolerance genes. Further tests carried out using enzyme and fluorescence-based assays to elucidate the effect of n-butanol on the cell envelope showed that the solvent released lipopolysaccharides from the outer membrane of E. coli and also caused both outer and inner membranes to be leaky. Very high n-butanol concentrations resulted in an altered cell shape and bleb formation suggesting an impairment in cell division and peptidoglycan biosynthesis. The cell membrane was modified by cis-trans isomerisation of unsaturated fatty acids in the phospholipids resulting in a reduction of membrane leakage which in effect, increased n-butanol tolerance in E. coli. In conclusion, results from this research suggest that strategies to protect the membrane and cellular proteins should be included in rational engineering of n-butanol tolerant bacteria.