Evolved Cobalamin-Independent Methionine Synthase (MetE) Improves the Acetate and Thermal Tolerance of Escherichia coli

Mordukhova, Elena A.; Pan, Jae-Gu

doi:10.1128/aem.01952-13

Cited by 30 publications

(27 citation statements)

References 38 publications

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“…Improvement of acetate tolerance in E. coli via modification of the methionine biosynthesis enzymes has been performed through the isolation of improved versions of MetA and MetE enzymes which catalyze, respectively, the synthesis of methionine production from homoserine and the conversion of homocysteine to methionine. Strains expressing an improved metA showed increased tolerance of 30 mM acetic acid and also showed improved thermotolerance (Mordukhova et al 2008), while strains expressing the improved metE showed improved tolerance of acetate and propionate, but not benzoate (Mordukhova and Pan 2013).…”

Section: Inhibition Of the Methionine Biosynthetic Pathwaymentioning

confidence: 98%

Adaptation and tolerance of bacteria against acetic acid

Trček

Mira

Jarboe

2015

Appl Microbiol Biotechnol

195

100

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Acetic acid is a weak organic acid exerting a toxic effect to most microorganisms at concentrations as low as 0.5 wt%. This toxic effect results mostly from acetic acid dissociation inside microbial cells, causing a decrease of intracellular pH and metabolic disturbance by the anion, among other deleterious effects. These microbial inhibition mechanisms enable acetic acid to be used as a preservative, although its usefulness is limited by the emergence of highly tolerant spoilage strains. Several biotechnological processes are also inhibited by the accumulation of acetic acid in the growth medium including production of bioethanol from lignocellulosics, wine making, and microbe-based production of acetic acid itself. To design better preservation strategies based on acetic acid and to improve the robustness of industrial biotechnological processes limited by this acid's toxicity, it is essential to deepen the understanding of the underlying toxicity mechanisms. In this sense, adaptive responses that improve tolerance to acetic acid have been well studied in Escherichia coli and Saccharomyces cerevisiae. Strains highly tolerant to acetic acid, either isolated from natural environments or specifically engineered for this effect, represent a unique reservoir of information that could increase our understanding of acetic acid tolerance and contribute to the design of additional tolerance mechanisms. In this article, the mechanisms underlying the acetic acid tolerance exhibited by several bacterial strains are reviewed, with emphasis on the knowledge gathered in acetic acid bacteria and E. coli. A comparison of how these bacterial adaptive responses to acetic acid stress fit to those described in the yeast Saccharomyces cerevisiae is also performed. A systematic comparison of the similarities and dissimilarities of the ways by which different microbial systems surpass the deleterious effects of acetic acid toxicity has not been performed so far, although such exchange of knowledge can open the door to the design of novel approaches aiming the development of acetic acid-tolerant strains with increased industrial robustness in a synthetic biology perspective.

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Section: Inhibition Of the Methionine Biosynthetic Pathwaymentioning

confidence: 98%

Adaptation and tolerance of bacteria against acetic acid

Trček

Mira

Jarboe

2015

Appl Microbiol Biotechnol

195

100

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“…In the methionine biosynthesis pathway, MetA (EC2.3.1.46, homocysteine O-succinyltransferase) is the first enzyme that catalyzes the transfer of succinate from succinyl-coenzyme A to L-homoserine [16] , [17] and MetE (EC 2.1.1.14, cobalamin-independent homocysteine transmethylase) catalyzes the final step to form methionine from L-homocysteine [18] , [19] . Mordukhova and coworkers mutated the MetA and MetE enzymes and found that the introduction of thermostable mutants of MetA and/or MetE to E. coli strains improved the cell growth in M9 glucose medium (pH 6.0) supplemented with 20 mM sodium acetate [20] , [21] . Sandoval et al tested the alleviating effect of specific amino acids and/or pyrimidine bases on the acetate toxicity and found that supplementation of four amino acids (arginine, methionine, threonine and lysine) and two pyrimidines (cytosine and uracil) to the minimal growth medium led to restoration of the specific growth rate at different levels [12] .…”

Section: Introductionmentioning

confidence: 99%

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

Wang

et al. 2014

PLoS ONE

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Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5–8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0±0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

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“…Notably, the concentrations of almost all glycolytic metabolites and of most nucleic acid-related metabolites were decreased in strain N1-4 cultivated at 37°C for 24 h. The levels of most amino acids (except tryptophan and arginine) were decreased in cells cultured at 37°C, compared to cells cultured at 30°C, but the differences were most prominent at an earlier time point of 12 h (Table S1). Only in the case of E. coli is growth actively inhibited by the excess of an amino acid intermediate (homocysteine) (18,19). We instead hypothesized that impaired growth of strain N1-4 at 37°C reflected depletion of one or more primary metabolites that became limiting factors for cell growth and butanol production at the elevated temperature.…”

Section: Discussionmentioning

confidence: 99%

Adenine Addition Restores Cell Viability and Butanol Production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) Cultivated at 37°C

Kiyoshi

Kawashima

Nobuki

et al. 2017

Appl Environ Microbiol

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We have developed butanol-producing consolidated bioprocessing from cellulosic substrates through coculture of cellulolytic clostridia and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4. However, the butanol fermentation by strain N1-4 (which has an optimal growth temperature of 30°C) is sensitive to the higher cultivation temperature of 37°C; the nature of this deleterious effect remains unclear. Comparison of the intracellular metabolites of strain N1-4 cultivated at 30°C and 37°C revealed decreased levels of multiple primary metabolites (notably including nucleic acids and cofactors) during growth at the higher temperature. Supplementation of the culture medium with 250 mg/liter adenine enhanced both cell growth (with the optical density at 600 nm increasing from 4.3 to 10.2) and butanol production (increasing from 3.9 g/liter to 9.6 g/liter) at 37°C, compared to those obtained without adenine supplementation, such that the supplemented 37°C culture exhibited growth and butanol production approaching those observed at 30°C in the absence of adenine supplementation. These improved properties were based on the maintenance of cell viability. We further showed that adenine supplementation enhanced cell viability during growth at 37°C by maintaining ATP levels and inhibiting spore formation. This work represents the first demonstration (to our knowledge) of the importance of adenine-related metabolism for clostridial butanol production, suggesting a new means of enhancing target pathways based on metabolite levels. IMPORTANCE Metabolomic analysis revealed decreased levels of multiple primary metabolites during growth at 37°C, compared to 30°C, in C. saccharoperbutylacetonicum strain N1-4. We found that adenine supplementation restored the cell growth and butanol production of strain N1-4 at 37°C. The effects of adenine supplementation reflected the maintenance of cell viability originating from the maintenance of ATP levels and the inhibition of spore formation. Thus, our metabolomic analysis identified the depleted metabolites that were required to maintain cell viability. Our strategy, which is expected to be applicable to a wide range of organisms, permits the identification of the limiting metabolic pathway, which can serve as a new target for molecular breeding. The other novel finding of this work is that adenine supplementation inhibits clostridial spore formation. The mechanism linking spore formation and metabolomic status in butanol-producing clostridia is expected to be the focus of further research.

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Evolved Cobalamin-Independent Methionine Synthase (MetE) Improves the Acetate and Thermal Tolerance of Escherichia coli

Cited by 30 publications

References 38 publications

Adaptation and tolerance of bacteria against acetic acid

Adaptation and tolerance of bacteria against acetic acid

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

Adenine Addition Restores Cell Viability and Butanol Production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) Cultivated at 37°C

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