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

Luis H Reyes,Maria P Almario,Katy C Kao,Arnold Driessen

doi:10.1371/journal.pone.0017678

Abstract

Backgroundn-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 FindingsUsing 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%.Conclusions/SignificanceWe 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.

Highlights

There has been renewed interest in the four-carbon alcohol, nbutanol, within the scientific and industrial fields due to its potential as an alternative liquid fuel. n-Butanol has physiochemical properties comparable to gasoline, allowing its use as a fuel replacement in internal combustion engines without any modification [1]
Conclusions/Significance: We identified and experimentally verified 14 genes that decreased the inhibitory effect of nbutanol tolerance on E. coli
Using a genomic library enrichment strategy, we identified genes involved in n-butanol tolerance in E. coli

Summary

Introduction

There has been renewed interest in the four-carbon alcohol, nbutanol, within the scientific and industrial fields due to its potential as an alternative liquid fuel. n-Butanol has physiochemical properties comparable to gasoline, allowing its use as a fuel replacement in internal combustion engines without any modification [1]. The solvent production in Clostridia is coupled to its complex growth phases, which creates difficulties in the engineering of the organism for improved nbutanol production. The complex growth and production phases and the strict anaerobic nature of the native producers have prompted researchers to pursue heterologous hosts for biobutanol production. In the last few years, with the advances in metabolic engineering, non-native producers of n-butanol such as Escherichia coli [4,5,6], Saccharomyces cerevisiae [7], Lactobacillus brevis [8], Pseudomonas putida [9] and Bacillus subtilis [9], have been demonstrated as potential hosts for use in n-butanol production. Understanding the mechanisms involved in n-butanol response can help to facilitate the engineering of production hosts for improved tolerance

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