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

Wang, Hengwei; Wang, Fengqing; Wang, Wei; Xueling, Yao; Wei, Dongzhi; Cheng, Hairong; Deng, Zixin

doi:10.1371/journal.pone.0112777

Cited by 41 publications

(22 citation statements)

References 45 publications

(77 reference statements)

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“…According to Novy and Morris (2001), Glucose contained in the culture medium will increase the protein expression in pET30 system. This finding also similar with Wang et al (2014), that mutant TC110 will grew quickly and produced much less acetate in 2×LB broth medium with 2% glucose.…”

Section: Protein Expressionsupporting

confidence: 87%

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

Indriawati

Volkandari

Margawati

2019

Si.Pet.

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There are several factors influencing the protein expression. The study was aimed to increase the yield of protein recombinant JSU-pET by applying combination of temperature and medium culture composition. This research was composed in a 2x2 factorial design with two level temperatures of 37oC and 25oC and 2 medium compositions of LB and mLB (modified). Escherichia coli bearing JSU-pET plasmid were grown in two applied media and incubated in two applied temperatures. The cells were harvested by centrifugation the pellets were then lysed. The lysed cells were centrifuged, the supernatant then purified by Ni-NTA Resin Agarose. The purified JSU protein recombinant was characterized and quantified by spectrophotometer. The Result showed that the JSU protein recombinant was successfully expressed on the right size (37kDa) in all treatments even though the JSU-pET showed sharper band in m LB at 37oC. Statistically the protein yield was not significant different (P>0.05), however culture temperature of 25oC showed higher yield (0.618±0.095 vs 0.704±0.094) than cultured at 37oC (0.598±0.137 vs 0.553±0.041), respectively either cultured in LB or mLB. This finding suggests that a lower culture temperature could increase protein yield both either in LB or modified LB.

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Section: Protein Expressionsupporting

confidence: 87%

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

Indriawati

Volkandari

Margawati

2019

Si.Pet.

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“…Studies of the pH effect have gone beyond inhibition of growth. During challenge of E. coli BL21 at a variety of acetate concentrations, the intracellular acetate concentration was reduced in half when cells were grown at pH 7.5 instead of 6.5 (Wang et al 2014), confirming that acetate internal accumulation depends of the diffusion of the undissociated form. The external pH had a roughly linear influence on the proton motive force and total transmembrane chemical gradient of Streptococcus bovis during acetic acid challenge (Russell 1991b).…”

Section: External Phmentioning

confidence: 74%

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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“…With the evolution of computer simulation technology, many researchers have investigated enzyme developments based on the calculated parameter change of substrate-docked enzyme modeling before and after in silico mutation. 39,40,47,48,49,50,51,52 A good design in the substrate-binding site for improving enzymatic function can be applied to other enzymes as well. In this study, we focused on ccMA as a precursor compound of 1,3-butadiene.…”

Section: Discussionmentioning

confidence: 99%

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant

Mori

Noda

Shirai

et al. 2020

Preprint

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The C4 unsaturated compound 1,3-butadiene is an important monomer in synthetic rubber and engineering plastic production. However, microorganisms cannot directly produce 1,3-butadiene using glucose as a renewable carbon source via biological processes. This study constructed a novel artificial metabolic pathway for 1,3-butadiene production from glucose in Escherichia coli by combining the cis,cis-muconic acid (ccMA)-producing pathway and tailored ferulic acid decarboxylase mutants. The rational enzyme design of the substrate-binding site by computational simulation improved 1,3-butadiene-producing ccMA decarboxylation with a small mutant library. We found that changing dissolved oxygen and controlling pH were important factors for 1,3-butadiene production. Using dissolved oxygen–stat fed-batch fermentation in a 1-L jar fermenter, we could produce 2.1 g L− 1 of 1,3-butadiene. These results indicated that using a rational enzyme design, we can produce unnatural/nonbiological compounds (those that are made from petroleum and cannot be produced by microorganisms) using glucose as a renewable carbon source.

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

Cited by 41 publications

References 45 publications

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

Adaptation and tolerance of bacteria against acetic acid

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant

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