Abstract
BackgroundUnderstanding ethanol tolerance in microorganisms is important for the improvement of bioethanol production. Hence, we performed parallel-evolution experiments using Escherichia coli cells under ethanol stress to determine the phenotypic changes necessary for ethanol tolerance.ResultsAfter cultivation of 1,000 generations under 5% ethanol stress, we obtained 6 ethanol-tolerant strains that showed an approximately 2-fold increase in their specific growth rate in comparison with their ancestor. Expression analysis using microarrays revealed that common expression changes occurred during the adaptive evolution to the ethanol stress environment. Biosynthetic pathways of amino acids, including tryptophan, histidine, and branched-chain amino acids, were commonly up-regulated in the tolerant strains, suggesting that activating these pathways is involved in the development of ethanol tolerance. In support of this hypothesis, supplementation of isoleucine, tryptophan, and histidine to the culture medium increased the specific growth rate under ethanol stress. Furthermore, genes related to iron ion metabolism were commonly up-regulated in the tolerant strains, which suggests the change in intracellular redox state during adaptive evolution.ConclusionsThe common phenotypic changes in the ethanol-tolerant strains we identified could provide a fundamental basis for designing ethanol-tolerant strains for industrial purposes.
Highlights
Understanding ethanol tolerance in microorganisms is important for the improvement of bioethanol production
We found that genes involved in the iron ion transport and biosynthesis pathways of some amino acids, including tryptophan, histidine, valine, leucine, and isoleucine, were commonly up-regulated in tolerant strains, which suggests that these gene functions are involved in ethanol tolerance
Parallel laboratory evolution experiments of E. coli under ethanol stress Before starting the evolution experiments under ethanol stress, we performed a laboratory evolution experiment in M9 synthetic medium without the addition of ethanol to distinguish the phenotypic and genetic changes that occurred in the adaptive evolution to M9 synthetic medium from those that occurred due to ethanol stress
Summary
After cultivation of 1,000 generations under 5% ethanol stress, we obtained 6 ethanol-tolerant strains that showed an approximately 2-fold increase in their specific growth rate in comparison with their ancestor. Biosynthetic pathways of amino acids, including tryptophan, histidine, and branched-chain amino acids, were commonly up-regulated in the tolerant strains, suggesting that activating these pathways is involved in the development of ethanol tolerance. In support of this hypothesis, supplementation of isoleucine, tryptophan, and histidine to the culture medium increased the specific growth rate under ethanol stress. Genes related to iron ion metabolism were commonly up-regulated in the tolerant strains, which suggests the change in intracellular redox state during adaptive evolution
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