Novel Methods to Investigate Solvent Toxicity in Bacteria

Toxicity of organic solvents to microbial hosts is a major consideration in the industrial production of biofuels such as ethanol and especially butanol, with low product concentrations leading to high recovery costs. Understanding the mechanisms involved in solvent tolerance is crucial for rational engineering of robust microbes. We have developed a bioluminescence assay to determine the effects of different genes on survival in four model inhibitors-ethanol, n-butanol, acetone and furfural. Adopting a synthetic biology approach, a library of potential solvent tolerance BioBricks (genes) was generated and tested as proof-of-concept. Using this method, we have generated a set of tolerance modules suited for these inhibitory compounds, which can be combined with genetic modules encoding substrate breakdown and product formation pathways. Ultimately, we hope to generate improved biofuel-producing systems which can generate higher product concentrations, greatly improving process economics. Further tests carried out using enzyme and fluorescence-based assays to characterise the effect of n-butanol on the cell envelope showed that the alcohol released lipopolysssacharides from the outer membrane of E. coli and resulted in ‘leakiness’ in both outer and inner membranes.