Biofuels can be produced by microbes, but biofuel toxicity is a major obstacle to efficient production. Here, the authors identify efflux pumps that can effectively export biofuels, improving cell viability and increasing biofuel yields.

n-Butanol has been proposed as an alternative biofuel to ethanol, and several industrially used microbes, including Escherichia coli, have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell-wide studies were conducted at the transcript, protein, and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicates that n-butanol stress has components common to other stress responses, including perturbation of respiratory functions (nuo and cyo operons), oxidative stress (sodA, sodC, and yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, cpxR, and cpxP), and metabolite transport and biosynthesis (malE and opp operon). Assays using fluorescent dyes indicated a large increase in reactive oxygen species during n-butanol stress, confirming observations from the microarray and proteomics measurements. Mutant strains with mutations in several genes whose products changed most dramatically during n-butanol stress were examined for increased sensitivity to n-butanol. Results from these analyses allowed identification of key genes that were recruited to alleviate oxidative stress, protein misfolding, and other causes of growth defects. Cellular engineering based on these cues may assist in developing a high-titer, n-butanol-producing host.

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