Improved Thermostability and Acetic Acid Tolerance of
            <i>Escherichia coli</i>
            via Directed Evolution of Homoserine
            <i>o</i>
            -Succinyltransferase

Mordukhova, Elena A.; Lee, Heesoon; Pan, Jae-Gu

doi:10.1128/aem.00654-08

Cited by 37 publications

(33 citation statements)

References 22 publications

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“…Previously, we found that stabilized mutants of the MetA protein demonstrate an increased tolerance to acetic acid (24). Here, we hypothesized that the acetate-tolerant MetE mutants might improve the thermal stability of E. coli cells.…”

Section: Resultsmentioning

confidence: 99%

“…The amplified sequence was then transfected into freshly prepared E. coli WE ⌬metE(pKD46) cells via electroporation, as described previously (23). The WE⌬metE strain was obtained through P1vir transduction of a kanamycin resistance construct from the JW3805 ⌬metE donor strain into the WE strain (24). The transformed cells were finally incubated at 37°C in M9 minimal medium plates supplemented with glucose to select clones containing a functional metE gene.…”

Section: Methodsmentioning

confidence: 99%

“…To test the combined effect of the stabilized MetA and MetE enzymes on the growth of the WE strain in sodium acetate medium and at the higher temperature of 45°C, the metA-Y229 gene, encoding the thermal-tolerant Y229 MetA mutant (28), was inserted into the chromosome of the WE-214A strain instead of the wild-type metA gene. We previously found that stabilized MetA mutants display enhanced acetate tolerance (24). The Y229-214A double mutant MetA-MetE strain and the single mutant MetA (Y229) and MetE (WE-214A) strains were cultivated in minimal M9 glucose medium in the presence of sodium acetate (20 mM) or at 45°C, and they were compared with the control WE strain harboring the wild-type metA and metE genes ( Fig.…”

Section: Mete Engineering For Acetate and Thermal Tolerancementioning

confidence: 99%

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

Mordukhova

Pan

2013

Appl Environ Microbiol

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Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in L-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (T m ) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by L-methionine.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Mete Engineering For Acetate and Thermal Tolerancementioning

confidence: 99%

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

Mordukhova

Pan

2013

Appl Environ Microbiol

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“…Deletions and point mutations in metJ are known to increase methionine levels in E. coli (47,48); the metJ[ΔE91] mutation likely causes a similar effect by elevating transcription of the metABFIKNR genes (Table S2). Interestingly, elevating methionine biosynthesis or supplementing with methionine also compensates for other stresses in E. coli, including acetate, organic acids, nitrosating agents, and heat shock (49)(50)(51). MetA, which catalyzes the first step in methionine biosynthesis, is known to aggregate during heat stress (51), suggesting that stress-induced MetA aggregation may reduce functional MetA and resultant methionine levels.…”

Section: Discussionmentioning

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.

130

121

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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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“…Escherichia coli, being the most extensively studied bacterium and also a popularly utilized host cell for producing pharmaceutically important recombinant proteins, is known to be unable to grow at a temperature higher than 46.5°C (4)(5)(6). It has been widely reported that heterologous overexpression of certain exogenous molecular chaperones (7)(8)(9)(10)(11) or an endogenous transcriptional regulator (12) is able to significantly increase the viability of E. coli cells undergoing heat shock treatment at lethal temperatures (around 50°C).…”

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confidence: 99%

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50 C Conceivably by Maintaining Cell Envelope Integrity

Ezemaduka

Shi

et al. 2014

Journal of Bacteriology

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It is essential for organisms to adapt to fluctuating growth temperatures. Escherichia coli, a model bacterium commonly used in research and industry, has been reported to grow at a temperature lower than 46.5°C. Here we report that the heterologous expression of the 17-kDa small heat shock protein from the nematode Caenorhabditis elegans, CeHSP17, enables E. coli cells to grow at 50°C, which is their highest growth temperature ever reported. Strikingly, CeHSP17 also rescues the thermal lethality of an E. coli mutant deficient in degP, which encodes a protein quality control factor localized in the periplasmic space. Mechanistically, we show that CeHSP17 is partially localized in the periplasmic space and associated with the inner membrane of E. coli, and it helps to maintain the cell envelope integrity of the E. coli cells at the lethal temperatures. Together, our data indicate that maintaining the cell envelope integrity is crucial for the E. coli cells to grow at high temperatures and also shed new light on the development of thermophilic bacteria for industrial application.T emperature is considered the most important single environmental factor that profoundly affects the structure and function of biomolecules. Each organism in nature has evolved to live at a certain optimal temperature range. Nonetheless, effective mechanisms have also been evolved for organisms to survive under nonoptimal temperature conditions, typically termed heat shock response (1, 2) and cold shock response (3). It is of great value to understand such mechanisms of living organisms for both unveiling the nature of life and exploring biotechnological application.Escherichia coli, being the most extensively studied bacterium and also a popularly utilized host cell for producing pharmaceutically important recombinant proteins, is known to be unable to grow at a temperature higher than 46.5°C (4-6). It has been widely reported that heterologous overexpression of certain exogenous molecular chaperones (7-11) or an endogenous transcriptional regulator (12) is able to significantly increase the viability of E. coli cells undergoing heat shock treatment at lethal temperatures (around 50°C). It is also well established that preincubating E. coli cells (13,14) and other organisms (reviewed in reference 15) at a sublethal temperature (e.g., 42°C) significantly increases the thermotolerance of the treated organisms at lethal temperatures. However, all these alternations usually would not allow the modified cells to permanently survive and even grow under such lethal temperatures. In two attempts at selecting heat-resistant phenotypes by using extensive experimental evolution, E. coli mutant strains that are able to grow at up to 48°C (16) or 48.5°C (5) were obtained, with growth at the latter temperature being partially related to the high level of expression of the molecular chaperone GroEL/GroES. In contrast, thermophilic bacteria have been found to grow effectively at optimal temperatures much higher than 50°C (17)(18)(19)(20). They are known...

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Improved Thermostability and Acetic Acid Tolerance of Escherichia coli via Directed Evolution of Homoserine o -Succinyltransferase

Cited by 37 publications

References 22 publications

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

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

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

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50 C Conceivably by Maintaining Cell Envelope Integrity

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