Biofuels and biochemicals are a promising alternative to traditional chemical sources that are fraught with negative environmental impacts and rising costs. However, many useful chemicals are toxic to production hosts and are thus produced at low levels [1]. A solution is to engineer or evolve production organisms that are tolerant to industrially relevent solvent concentrations.Toward the goal of highly tolerant production hosts, we have developed alcohol-tolerant strains of Escherichia coli and Saccharomyces cerevisiae using multiple strategies. For E. coli, we used directed evolution to alter the substrate specificity of the solvent pump AcrB to act on short-chain alcohols. The strain exhibits growth and stationary phase cell density increases, but expression of the pump itself is toxic. Therefore, we developed a regulatory system to permit optimal expression of the pumps in the absence of external inducer, and further enhanced the performance of the strain.We took a different approach with S. cerevisiae and directly evolved strains with log-phase growth rates more than double that of the wild-type strain in the presence of toxic levels of n-hexanol. The phenotype is stable and dominant, and extends to other short-chain alcohols from n-butanol to n-heptanol. Using polysome analysis, we have demonstrated that the wild-type strain is translationally inhibited by these alcohols whereas the solvent-tolerant strains are not inhibited. We have sequenced the genomes of the tolerant strains and identified causative mutations in several translation initiation factors. Together, we provide two powerful examples in which microbes are engineered for solvent tolerance using evolution-based strategies.1) Dellamonoco et al., Nature, 2011.

Engineering microbial hosts with increased tolerance to short-chain alcohols

247th National Meeting (2014)

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