scholarly journals High-titer production of aromatic amines in metabolically engineered Escherichia coli

2022 ◽  
Author(s):  
Taiwei Yang ◽  
Peiling Wu ◽  
Yang Zhang ◽  
Jifeng Yuan
Keyword(s):  
Escherichia Coli ◽  
Aromatic Amine ◽  
Aromatic Amines ◽  
High Titer ◽  
Coli Strain ◽  
Acid Decarboxylase ◽  
Amino Acid Decarboxylase ◽  
E Coli ◽  
Shake Flasks ◽  
Byproduct Formation

Aromatic amines are widely used in the pharmaceutical industry. Here, we reported the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine, and phenylethylamine. Firstly, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with an increased shikimate (SHK) pathway flux toward L-tyrosine or L-phenylalanine synthesis. We found that glycerol served as a better carbon source than glucose, resulting in 940 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), formate acetyltransferase (pflB), phosphate acetyltransferase (pta), and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation byproduct formation. The tyramine level was further increased to 1.965 g/L in shake flasks, corresponding to 2.1 times improvement compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 mg/L dopamine and 555 mg/L phenethylamine. In summary, we have demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy in improving aromatic amine productions, and achieved the highest aromatic amine titers in E. coli under shake flasks reported to date.

scholarly journals Regulation of Expression of the TIR-Containing Protein C Gene of the Uropathogenic Escherichia coli Strain CFT073

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 549
Author(s):  
Julia Ittensohn ◽  
Jacqueline Hemberger ◽  
Hannah Griffiths ◽  
Maren Keller ◽  
Simone Albrecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein C ◽  
Interleukin 1 ◽  
Coli Strain ◽  
Uropathogenic Escherichia Coli ◽  
Reporter Construct ◽  
Regulation Of Expression ◽  
E Coli ◽  
Strain Cft073 ◽  
Myeloid Differentiation Factor

The uropathogenic Escherichia coli strain CFT073 causes kidney abscesses in mice Toll/interleukin-1 receptor domain-containing protein C (TcpC) dependently and the corresponding gene is present in around 40% of E. coli isolates of pyelonephritis patients. It impairs the Toll-like receptor (TLR) signaling chain and the NACHT leucin-rich repeat PYD protein 3 inflammasome (NLRP3) by binding to TLR4 and myeloid differentiation factor 88 as well as to NLRP3 and caspase-1, respectively. Overexpression of the tcpC gene stopped replication of CFT073. Overexpression of several tcpC-truncation constructs revealed a transmembrane region, while its TIR domain induced filamentous bacteria. Based on these observations, we hypothesized that tcpC expression is presumably tightly controlled. We tested two putative promoters designated P1 and P2 located at 5′ of the gene c2397 and 5′ of the tcpC gene (c2398), respectively, which may form an operon. High pH and increasing glucose concentrations stimulated a P2 reporter construct that was considerably stronger than a P1 reporter construct, while increasing FeSO4 concentrations suppressed their activity. Human urine activated P2, demonstrating that tcpC might be induced in the urinary tract of infected patients. We conclude that P2, consisting of a 240 bp region 5′ of the tcpC gene, represents the major regulator of tcpC expression.

scholarly journals Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli during Initial Contact with Cattle Colonic Explants

2020 ◽  
Vol 8 (11) ◽  
pp. 1662
Author(s):  
Zachary R. Stromberg ◽  
Rick E. Masonbrink ◽  
Melha Mellata
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Bacterial Colonization ◽  
Coli Strain ◽  
Fold Change ◽  
Initial Contact ◽  
Lon Protease ◽  
E Coli ◽  
Log2 Fold Change ◽  
Colonization Factors

Foodborne pathogens are a public health threat globally. Shiga toxin-producing Escherichia coli (STEC), particularly O26, O111, and O157 STEC, are often associated with foodborne illness in humans. To create effective preharvest interventions, it is critical to understand which factors STEC strains use to colonize the gastrointestinal tract of cattle, which serves as the reservoir for these pathogens. Several colonization factors are known, but little is understood about initial STEC colonization factors. Our objective was to identify these factors via contrasting gene expression between nonpathogenic E. coli and STEC. Colonic explants were inoculated with nonpathogenic E. coli strain MG1655 or STEC strains (O26, O111, or O157), bacterial colonization levels were determined, and RNA was isolated and sequenced. STEC strains adhered to colonic explants at numerically but not significantly higher levels compared to MG1655. After incubation with colonic explants, flagellin (fliC) was upregulated (log2 fold-change = 4.0, p < 0.0001) in O157 STEC, and collectively, Lon protease (lon) was upregulated (log2 fold-change = 3.6, p = 0.0009) in STEC strains compared to MG1655. These results demonstrate that H7 flagellum and Lon protease may play roles in early colonization and could be potential targets to reduce colonization in cattle.

scholarly journals Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

2007 ◽  
Vol 73 (24) ◽  
pp. 7814-7818 ◽  
Author(s):  
T. Hanai ◽  
S. Atsumi ◽  
J. C. Liao
Keyword(s):  
Escherichia Coli ◽  
Secondary Alcohol ◽  
Clostridium Beijerinckii ◽  
Synthetic Pathway ◽  
E Coli ◽  
Coa Transferase ◽  
Secondary Alcohol Dehydrogenase ◽  
Acetoacetate Decarboxylase ◽  
Shake Flasks ◽  
K 12

ABSTRACT A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.

scholarly journals Construction of an artificial biosynthetic pathway for hyperextended archaeal membrane lipids in the bacterium Escherichia coli

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ryo Yoshida ◽  
Hisashi Hemmi
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Membrane Lipids ◽  
Extreme Environments ◽  
Halophilic Archaea ◽  
Coli Strain ◽  
Glycerol Moiety ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Aeropyrum Pernix

Abstract Archaea produce unique membrane lipids, which possess two fully saturated isoprenoid chains linked to the glycerol moiety via ether bonds. The isoprenoid chain length of archaeal membrane lipids is believed to be important for some archaea to thrive in extreme environments because the hyperthermophilic archaeon Aeropyrum pernix and some halophilic archaea synthesize extended C25,C25-archaeal diether-type membrane lipids, which have isoprenoid chains that are longer than those of typical C20,C20-diether lipids. Natural archaeal diether lipids possessing longer C30 or C35 isoprenoid chains, however, have yet to be isolated. In the present study, we attempted to synthesize such hyperextended archaeal membrane lipids. We investigated the substrate preference of the enzyme sn-2,3-(digeranylfarnesyl)glycerol-1-phosphate synthase from A. pernix, which catalyzes the transfer of the second C25 isoprenoid chain to the glycerol moiety in the biosynthetic pathway of C25,C25-archaeal membrane lipids. The enzyme was shown to accept sn-3-hexaprenylglycerol-1-phosphate, which has a C30 isoprenoid chain, as a prenyl acceptor substrate to synthesize sn-2-geranylfarnesyl-3-hexaprenylglycerol-1-phosphate, a supposed precursor for hyperextended C25,C30-archaeal membrane lipids. Furthermore, we constructed an artificial biosynthetic pathway by introducing 4 archaeal genes and 1 gene from Bacillus subtilis in the cells of Escherichia coli, which enabled the E. coli strain to produce hyperextended C25,C30-archaeal membrane lipids, which have never been reported so far.

Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli

2019 ◽  
Vol 29 (1-6) ◽  
pp. 91-100
Author(s):  
Dorna Khoobbakht ◽  
Shohreh Zare Karizi ◽  
Mohammad Javad  Motamedi ◽  
Rouhollah Kazemi ◽  
Pooneh Roghanian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Subunit Vaccine ◽  
Fusion Gene ◽  
Intestinal Epithelial Cells ◽  
High Titer ◽  
Chimeric Protein ◽  
Amino Acid Sequences ◽  
Intestinal Epithelial ◽  
E Coli

Enterotoxigenic <i>Escherichia coli</i> (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing <i>cooD</i> and <i>cotD</i> genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of <i>E. coli</i> in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in <i>E. coli</i>BL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

scholarly journals Evaluation of Physiological Effects Induced by Manuka Honey Upon Staphylococcus aureus and Escherichia coli

2019 ◽  
Vol 7 (8) ◽  
pp. 258 ◽  
Author(s):  
Patricia Combarros-Fuertes ◽  
Leticia M. Estevinho ◽  
Rita Teixeira-Santos ◽  
Acácio G. Rodrigues ◽  
Cidália Pina-Vaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Membrane Potential ◽  
Membrane Integrity ◽  
Efflux Pumps ◽  
Coli Strain ◽  
Antibacterial Action ◽  
Manuka Honey ◽  
E Coli ◽  
Action Mechanisms

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary effects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. Effects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using different fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH effect on efflux pumps was also studied in an E. coli strain with an over-expression of several efflux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these effects displayed slight differences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block efflux pump activity in a dose-dependent fashion in the E. coli strain.

scholarly journals Complete Genome Sequence of Escherichia coli Strain WG5

2018 ◽  
Vol 6 (2) ◽  
Author(s):  
Lejla Imamovic ◽  
Maria-Anna Misiakou ◽  
Eric van der Helm ◽  
Gianni Panagiotou ◽  
Maite Muniesa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Coli Strain ◽  
Somatic Coliphages ◽  
E Coli

ABSTRACT Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain.

scholarly journals Dynamic Mechanisms of the Bactericidal Action of an Al2O3-TiO2-Ag Granular Material on an Escherichia coli Strain

2015 ◽  
Vol 81 (20) ◽  
pp. 7135-7142 ◽  
Author(s):  
Marie-Anne Tartanson ◽  
Laurence Soussan ◽  
Matthieu Rivallin ◽  
Sophie Pecastaings ◽  
Cristian V. Chis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Granular Material ◽  
Morphological Changes ◽  
Membrane Damage ◽  
Coli Strain ◽  
Cell Integrity ◽  
Bactericidal Action ◽  
Intact Cells ◽  
Content Type ◽  
E Coli

ABSTRACTThe bactericidal activity of an Al2O3-TiO2-Ag granular material against anEscherichia colistrain was confirmed by a culture-based method. In particular, 100% of microorganisms were permanently inactivated in 30 to 45 min. The present work aimed to investigate the mechanisms of the bactericidal action of this material and their dynamics onEscherichia coliusing different techniques. Observations by transmission electron microscopy (TEM) at different times of disinfection revealed morphological changes in the bacteria as soon as they were put in contact with the material. Notably highlighted were cell membrane damage; cytoplasm detachment; formation of vacuoles, possibly due to DNA condensation, in association with regions exhibiting different levels of electron density; and membrane lysis. PCR and flow cytometry analyses were used to confirm and quantify the observations of cell integrity. The direct exposure of cells to silver, combined with the oxidative stress induced by the reactive oxygen species (ROS) generated, was identified to be responsible for these morphological alterations. From the first 5 min of treatment with the Al2O3-TiO2-Ag material, 98% ofE. coliisolates were lysed. From 30 min, cell viability decreased to reach total inactivation, although approximately 1% of permeableE. colicells and 1% of intact cells (105genomic units · ml−1) were evidenced. This study demonstrates that the bactericidal effect of the material results from a synergic action of desorbed and supported silver. Supported silver was shown to generate the ROS evidenced.

scholarly journals Preparation of gram quantities of a purified R-factor-mediated penicillinase from Escherichia coli strain W3310

1972 ◽  
Vol 130 (1) ◽  
pp. 55-62 ◽  
Author(s):  
J. Melling ◽  
G. K. Scott
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Coli Strain ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
E Coli ◽  
R Factor

Purified penicillinase, in gram quantities, has been prepared from Escherichia coli strain W3310 by using methods developed to handle large amounts of material. The final product had a specific enzyme activity of 3.08 units/μg of protein, which was over twice as high as that reported previously (Datta & Richmond, 1966). The purified enzyme was similar to that from E. coli strain TEM, but different in molecular weight and some other respects. The differences observed may be a result of the greater purity obtained.

Low Variability of Growth Parameters among Six O157:H7 and Non-O157:H7 Escherichia coli Strains

2014 ◽  
Vol 77 (11) ◽  
pp. 1988-1991
Author(s):  
I. FERNÁNDEZ-ESCUDERO ◽  
I. CARO ◽  
J. MATEO ◽  
J. TEJERO ◽  
E. J. QUINTO
Keyword(s):  
Escherichia Coli ◽  
Generation Time ◽  
Growth Parameters ◽  
Brain Heart Infusion ◽  
Lag Time ◽  
Coli Strain ◽  
Time Data ◽  
E Coli ◽  
Confidence Levels ◽  
Coefficients Of Variation

Five Escherichia coli O157:H7 strains and one nonpathogenic E. coli strain were used. All strains were cultured in brain heart infusion broth and were inoculated in 16-well disposable module cassettes of a Bactometer impedance system. Two initial concentrations were obtained in the wells: 1.37 × 103 and 1.36 × 105 CFU/ml. The impedance measurements were monitored for 72 h at 5, 10, or 15°C, 48 h at 20°C, and 24 h at 25, 30, 35, 40, 45, 50 or 55°C. The lag time and the generation time of each culture were calculated from the detection time data. The coefficients of variation between the strains' growth parameters were low (0.009 to 0.105 for generation time and 0.074 to 0.475 for lag time). An F test showed no significant differences between strains at 5 or 1% confidence levels.

Sign in / Sign up

Export Citation Format

Share Document