Journal Article

Genomic Library Screens for Genes Involved in n-Butanol Tolerance in Escherichia coli

Kao, Katy C., Almario, Maria P., Reyes, Luis H.

2011

Overview

n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance. Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%. We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: 1-Butanol - pharmacology / Acid resistance / Adaptation, Physiological - drug effects / Adaptation, Physiological - genetics / Bacillus subtilis / Biofuels / Biology / Biomass / Biomass energy / Biosynthesis / Butanol / Cell Membrane - drug effects / Cell Membrane - genetics / Chemical engineering / Cloning / Clostridia / Comparative Genomic Hybridization / E coli / Engineering / Enrichment / Escherichia coli / Escherichia coli - drug effects / Escherichia coli - genetics / Escherichia coli - growth & development / Fuels / Gene expression / Gene Expression Profiling / Gene Knockout Techniques / Genes / Genes, Bacterial - genetics / Genetic screening / Genetic Testing / Genomes / Genomic libraries / Genomic Library / Iron / Iron transport / Kinetics / Laboratories / Lactobacillus brevis / Libraries / Library collections / Metabolic engineering / Metabolism / Metabolites / Microorganisms / Molecular Sequence Annotation / Physiological aspects / Proteins / Robustness / Screens / Solvents / Stress response / Studies / Transport / Viability