Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen

Schwalbach, Michael S.; Keating, David H.; Tremaine, Mary; Marner, Wesley D.; Zhang, Yaoping; Bothfeld, William; Higbee, Alan; Grass, Jeffrey A.; Cotten, Cameron; Reed, Jennifer L.; Sousa, Leonardo da Costa; Jin, Mingjie; Balan, Venkatesh; Ellinger, James J.; Dale, Bruce E.; Kiley, Patricia J.; Landick, Robert

doi:10.1128/aem.07329-11

Cited by 58 publications

(82 citation statements)

References 95 publications

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“…The medium for fermentations was synthetic corn stover hydrolysate (73) supplemented with glucose (60 g/L) and xylose (30 g/L) to mimic 9% glucan loading. End product detection was done as described previously (73).…”

Section: Methodsmentioning

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.

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130

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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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Section: Methodsmentioning

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.

Self Cite

130

121

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“…Relevant examples in this sense include (but are certainly not limited to) the production of ethanol (31,58,69), succinate (64), D-lactate (45), and polyhydroxyalkanoates (24,40), often by using redox and/or regulatory E. coli mutants as the biocatalyst. These metabolic engineering approaches underscore the need for a complete understanding of the cell physiology and metabolic network operativity under anoxic growth conditions.…”

mentioning

confidence: 99%

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Bidart

Ruiz

Almeida

et al. 2012

Appl Environ Microbiol

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Bioprocesses conducted under conditions with restricted O 2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O 2 and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ⌬arcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of D-glucose catabolic fluxes, different from that observed in the ⌬arcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, D-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD ؉ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes.A noxic fermentation of different carbon sources by Escherichia coli is increasingly gaining momentum in biotechnological setups designed to obtain reduced biochemicals. Relevant examples in this sense include (but are certainly not limited to) the production of ethanol (31, 58, 69), succinate (64), D-lactate (45), and polyhydroxyalkanoates (24, 40), often by using redox and/or regulatory E. coli mutants as the biocatalyst. These metabolic engineering approaches underscore the need for a complete understanding of the cell physiology and metabolic network operativity under anoxic growth conditions. In fact, the relative lack of knowledge on the cellular wiring of these regulatory networks under conditions relevant to both laboratory and industrial applications represents a hurdle that has to be overcome for the efficient design of industrial processes. Metabolic fluxes through the central carbon pathways constitute the backbone of cell metabolism and represent the in vivo reaction rates of cognate enzymatic steps (62). The observed fluxome is the phenotypic consequence of both gene transcription and translation, as well as of the enzymatic activity and the regulation exerted at the metabolite level (48). Fluxome analysis is thus a useful approach to...

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“…These methods will be useful in any case where researchers seek to identify the genetic basis of a selectable trait in a specific microbial population. Two particularly pertinent exam-ples are the identification of specific genetic variations giving rise to different levels of antibiotic resistance in natural or clinical populations (39) and the optimization of strains for biosynthetic applications by improving their usage of particular carbon sources or their tolerance of toxic intermediates or by-products (23,40,41).…”

Section: Discussionmentioning

confidence: 99%

Revealing the Genetic Basis of Natural Bacterial Phenotypic Divergence

2014

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b Divergent phenotypes for distantly related strains of bacteria, such as differing antibiotic resistances or organic solvent tolerances, are of keen interest both from an evolutionary perspective and for the engineering of novel microbial organisms and consortia in synthetic biology applications. A prerequisite for any practical application of this phenotypic diversity is knowledge of the genetic determinants for each trait of interest. Sequence divergence between strains is often so extensive as to make bruteforce approaches to identifying the loci contributing to a given trait impractical. Here we describe a global linkage analysis approach, GLINT, for rapid discovery of the causal genetic variants underlying phenotypic divergence between distantly related strains of Escherichia coli. This general strategy will also be usable, with minor modifications, for revealing genotype-phenotype associations between naturally occurring strains of other bacterial species.

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Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen

Abstract: ABSTRACTThe physiology of ethanologenicEscherichia coligrown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into howE. coliresponds to such hydrolysates, we studied anE. coliK-12 ethanologen fermenting a hydrolysate … Show more

Cited by 58 publications

References 95 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

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Revealing the Genetic Basis of Natural Bacterial Phenotypic Divergence

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