genomic library screens for genes involved in n-butanol tolerance in escherichia coli基因组文库的屏幕基因在大肠杆菌的正丁醇耐受性.pdfVIP

Genomic Library Screens for Genes Involved in n-Butanol Tolerance in Escherichia coli Luis H. Reyes, Maria P. Almario, Katy C. Kao* Department of Chemical Engineering, Texas AM University, College Station, Texas, United States of America Abstract Background: 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. Methodology/Principal Findings: 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 32.864.0% and 49.1 63.3%, 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 66.3%. Conclusions/Significance: We identified and experimenta