Ethanol tolerance engineering in ethanologenic bacteria.

Bioethanol is considered as a valuable, green fuel-alternative because greenhouse gas emission can be significantly reduced. Moreover, the polymer industry shows great interest in this alcohol as a precursor molecule for the production of a wide variety of plastics, including Plexiglas (PMMA), polyethylene (PE), polyvinyl chloride (PVC) and polyethylene terephthalate (PET). Because of its high demand in the industrial and transport sector, it is clear that the supply of bioethanol has to be guaranteed in the coming future.

Bioethanol is, most commonly, derived from a microbial fermentation process with sugars (from algal, lignocellulosic, sucrose-rich or starchy biomass) as substrate. However, ethanol is a toxic solvent and reduces cell viability of the producer strain which inevitably hampers production. Hence, improving the tolerance level of producers might be a suitable approach to increase the conversion efficiency from biomass into bioethanol. In this project, we focus on the adaptation mechanisms of  Escherichia coli in response to lethal ethanol concentrations. Therefore, we first composed a list of candidate point mutations from a mutational dataset which was originated from a long-term evolution experiment under increasing ethanol conditions. For further research, we prioritized a limited set of SNPs which appeared frequently in different parallel-evolved populations. One of those was located into the EnvZ/OmpR signal transduction system which monitors the osmolarity of the environment and plays a key role in cellular adaptations to extreme osmotic conditions. The others were situated in genes involved in the membrane biosynthesis pathway. To assess the relevance of these diverse SNPs for ethanol tolerance in vivo, these mutations were isolated from their corresponding evolved clones and transferred into the original, ethanol-sensitive ancestor. Once their causal relationship was confirmed, the mechanism underlying the tolerance-conferring potential of these SNPs was closely investigated as well. These results have revealed that improving cell envelope integrity by stabilizing the outer membrane and optimizing the membrane fatty acid composition is essential to survive lethal ethanol stress. Finally, the CRISPR-engineered E. coli mutants were also evaluated in terms of ethanol production to verify whether improved ethanol tolerance promotes microbial productivity.