scholarly journals Construction of a plasmid addiction system using grpE as selection marker in Escherichia coli and its application in phloroglucinol biosynthesis

2021 ◽  
Author(s):  
Ji-ming Wang ◽  
Xiao Men ◽  
Yu-jin Cao ◽  
Hai-bo Zhang
Keyword(s):  
Escherichia Coli ◽  
Essential Gene ◽  
High Ratio ◽  
Selection Marker ◽  
Acetyl Coa Carboxylase ◽  
Expression Systems ◽  
Mineral Salts ◽  
E Coli ◽  
Commodity Chemicals ◽  
The Stability

Abstract Microbial synthesis of commodity chemicals often be conducted in recombinant plasmid-based expression systems, in which plasmids play pivotal roles on productivity. The recombinant plasmids always encounter instability, leading to losses in product recovery of entire process. To maintain the stability of plasmids, several mechanisms have been evolved. Plasmid addition system, selectively killing plasmid-free cells, is regard as a useful strategy to improve the proportion of plasmid-containing cells. In this study, a novel plasmid addition system using an essential gene grpE that encodes a molecular cochaperone as selection marker, avoiding use of antibiotics, was constructed in Escherichia coli . The solo copy of grpE gene on the chromosome was knocked out and relocated on multicopy plasmids. The generated strains can maintain high ratio of plasmid-harboring cells without antibiotics supplementation in mineral salts media and exhibit improved cell growth and increased tolerance to phloroglucinol. Using this system in phloroglucinol synthesis, it could significantly increase the phloroglucinol titer from 0.75 g/L to 1.26 g/L, which was further increased to 1.78 g/L when biotin-[acetyl-CoA-carboxylase] ligase BirA was overexpressed. It can be expected that this system will be a powerful tool for microbial manufacture of important chemicals in E . coli .

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease

2012 ◽  
Vol 446 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Anders Tryggvesson ◽  
Frida M. Ståhlberg ◽  
Axel Mogk ◽  
Kornelius Zeth ◽  
Adrian K. Clarke
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Terminal Sequence ◽  
Core Complex ◽  
Clp Protease ◽  
E Coli ◽  
Loop Region ◽  
N Terminus ◽  
Specific Association ◽  
The Stability

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

High-Fidelity Translation of Recombinant Human Hemoglobin in Escherichia coli

1998 ◽  
Vol 64 (5) ◽  
pp. 1589-1593 ◽  
Author(s):  
Michael J. Weickert ◽  
Izydor Apostol
Keyword(s):  
Escherichia Coli ◽  
Error Rates ◽  
High Fidelity ◽  
Human Hemoglobin ◽  
Heterologous Proteins ◽  
Expression Systems ◽  
High Level Expression ◽  
E Coli ◽  
Translation Error ◽  
High Level

ABSTRACT Coexpression of di-α-globin and β-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed ≤0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of ≤0.001, which is not different from typical translation error rates found for E. coli proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to ∼20% of the level of E. colisoluble proteins also resulted in equivalent translational fidelity.

scholarly journals Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8320-8329 ◽  
Author(s):  
Eva-Maria Borst ◽  
Gabriele Hahn ◽  
Ulrich H. Koszinowski ◽  
Martin Messerle
Keyword(s):  
Escherichia Coli ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Reading Frame ◽  
E Coli ◽  
Artificial Chromosomes ◽  
Internal Repeat ◽  
The Stability

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

scholarly journals Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2341-2351 ◽  
Author(s):  
Mario Juhas ◽  
Daniel R. Reuß ◽  
Bingyao Zhu ◽  
Fabian M. Commichau
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Essential Gene ◽  
Building Blocks ◽  
Essential Genes ◽  
Minimal Cell ◽  
E Coli ◽  
Cell Factories ◽  
Gene Sets ◽  
K 12

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell ‘chassis’. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

scholarly journals YhdJ, a Nonessential CcrM-Like DNA Methyltransferase of Escherichia coli and Salmonella enterica

2007 ◽  
Vol 189 (11) ◽  
pp. 4325-4327 ◽  
Author(s):  
Sarah E. Broadbent ◽  
Roberto Balbontin ◽  
Josep Casadesus ◽  
Martin G. Marinus ◽  
Marjan van der Woude
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Essential Gene ◽  
Gene Products ◽  
E Coli ◽  
Dna Adenine Methyltransferase

ABSTRACT The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the α-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.

scholarly journals Titration and Conditional Knockdown of the prfB Gene in Escherichia coli: Effects on Growth and Overproduction of the Recombinant Mammalian Selenoprotein Thioredoxin Reductase

2006 ◽  
Vol 73 (2) ◽  
pp. 432-441 ◽  
Author(s):  
Olle Rengby ◽  
Elias S. J. Arnér
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Essential Gene ◽  
Thioredoxin Reductase ◽  
Lower Growth ◽  
E Coli ◽  
Native Promoter ◽  
Lower Growth Rate ◽  
The Cost ◽  
Translational Termination

ABSTRACT Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable PBAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a PBAD-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.

scholarly journals Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase

1994 ◽  
Vol 302 (3) ◽  
pp. 881-887 ◽  
Author(s):  
A Chapman-Smith ◽  
D L Turner ◽  
J E Cronan ◽  
T W Morris ◽  
J C Wallace
Keyword(s):  
Escherichia Coli ◽  
Exchange Chromatography ◽  
Carrier Protein ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
E Coli ◽  
Biotin Ligase ◽  
Domain Peptide

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.

scholarly journals Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1972 ◽  
Vol 126 (5) ◽  
pp. 1081-1090 ◽  
Author(s):  
S. E. Halford ◽  
M. J. Schlesinger ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Steady State ◽  
Analytical Ultracentrifugation ◽  
Kinetic Studies ◽  
Product Formation ◽  
E Coli ◽  
Enzyme Subunits ◽  
Self Association ◽  
The Stability

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of Pi to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

scholarly journals Different Processing of an mRNA Species inBacillus subtilis and Escherichia coli

2000 ◽  
Vol 182 (3) ◽  
pp. 689-695 ◽  
Author(s):  
Martin Persson ◽  
Elisabeth Glatz ◽  
Blanka Rutberg
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Inverted Repeat ◽  
Fusion Transcript ◽  
Temperature Sensitive ◽  
Rnase Iii ◽  
Transcription Terminator ◽  
E Coli ◽  
Steady State Levels ◽  
The Stability

ABSTRACT Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds toglpD leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of theglpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD. The TS phenotype is due to temperature-dependent degradation of theglpD mRNA. In the presence of GlpP, theglpD mRNA is stabilized. glpDleader-lacZ fusions were integrated into the chromosomes ofB. subtilis and Escherichia coli. Determination of steady-state levels of fusion mRNA in B. subtilis showed that the stability of the fusion mRNA is determined by theglpD leader part. Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA inB. subtilis and E. coli revealed significant differences. A glpD leader-lacZ fusion transcript that was unstable in B. subtilis was considerably more stable in E. coli. GlpP, which stabilizes the transcript in B. subtilis, did not affect its stability in E. coli. Primer extension analysis showed that theglpD leader-lacZ fusion transcript is processed differently in B. subtilis and in E. coli. The dominating cleavage site in E. coli was barely detectable in B. subtilis. This site was shown to be a target ofE. coli RNase III.

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