Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation

Luo, Lian Hua; Seo, Pil-Soo; Seo, Jeong-Woo; Heo, Sun-Yeon; Kim, Dae-Hyuk; Kim, Chul Ho

doi:10.1007/s10529-009-0092-4

Cited by 49 publications

(31 citation statements)

References 11 publications

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“…Ethanol induces broad transcriptional changes in E. coli that extend beyond membrane-stress responses (12,13), however, suggesting that membrane effects explain only a part of the toxicity of ethanol. Consistent with this idea, widely varied approaches have successfully been used to achieve modest ethanol tolerance in E. coli, including random transposon insertion (14,15), overexpression of native gene libraries (16)(17)(18), overexpression of protein chaperones (19), engineered oxidation of ethanol (20), transcriptional rewiring through the catabolite activator protein (21) or the transcription factor σ 70 (22), and modulation of cellular fatty-acid composition (23). The mechanisms by which these genetic changes confer tolerance remain unclear.…”

mentioning

confidence: 99%

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Haft

Keating

Schwaegler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

116

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The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

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mentioning

confidence: 99%

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Haft

Keating

Schwaegler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

116

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“…Although previous reports show that ethanol damages the permeability barrier of bacterial cell membranes and therefore inhibits cell growth [8,11,18], it is believed that ethanol tolerance is a multiple-gene-controlled trait. Changes in membrane composition such as fatty-acid chain length, and/or trans-fatty acid, membrane protein, or phospholipid composition, were all reported to contribute to ethanol tolerance [2,9,10,[14][15][16][17][18]. Therefore, to improve the Fig.…”

Section: Discussionmentioning

confidence: 99%

Adaptive evolution of nontransgenic Escherichia coli KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

Wang

Manow

Finan

et al. 2010

J Ind Microbiol Biotechnol

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Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l(-1) ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l(-1)) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l(-1) and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.

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“…Isobutanol tolerance of E. coli was studied in detail [4]. Only few reports have addressed the potential of alternative hosts for alcohol production and focused on the selection of microorganisms tolerant to these compounds [15,17]. Considering the signiWcant recent progress in the development of membrane technologies for higher alcohol separation and the promising results of screening for and characterizing alcohol-tolerant species, further engineering of microorganisms for eVective conversion of biomass-derived sugars into fusel alcohols should create a basis for implementing this approach to the production of biotechnological fuels.…”

Section: Discussionmentioning

confidence: 99%

Use of the valine biosynthetic pathway to convert glucose into isobutanol

Savrasova

Kivero

Шакулов

et al. 2010

J Ind Microbiol Biotechnol

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Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259-2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also--as has been demonstrated previously (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009)--used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used.

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Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation

Cited by 49 publications

References 11 publications

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Adaptive evolution of nontransgenic Escherichia coli KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

Use of the valine biosynthetic pathway to convert glucose into isobutanol

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