scholarly journals Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1201
Author(s):  
Márcia R. Couto ◽  
Joana L. Rodrigues ◽  
Lígia R. Rodrigues
Keyword(s):  
Glucose Dehydrogenase ◽  
Cell Extract ◽  
Capra Hircus ◽  
In Vitro Assays ◽  
Uridine Diphosphate ◽  
Phylogenetic Groups ◽  
Biotechnological Production ◽  
E Coli

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.

scholarly journals Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

2006 ◽  
Vol 80 (17) ◽  
pp. 8329-8344 ◽  
Author(s):  
Jamie Ashby ◽  
Emmanuel Boutant ◽  
Mark Seemanpillai ◽  
Adrian Sambade ◽  
Christophe Ritzenthaler ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Cell Extract ◽  
Transport Processes ◽  
In Vitro Assays ◽  
Protein Functions ◽  
Intercellular Spread

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

scholarly journals A comparative study of GT27 Family N-acetylgalactosaminyltransferases

2021 ◽  
Author(s):  
Biprajit Sanyal
Keyword(s):  
Half Life ◽  
Homo Sapiens ◽  
Effective Means ◽  
Therapeutic Proteins ◽  
In Vitro Assays ◽  
Future Research ◽  
E Coli ◽  
Glycosylation Sites

Therapeutic proteins face short half lives in vivo. Their high costs and associated toxicity effects of increasing dosage warrant exploration of methods to increase serum half-life. These proteins can be produced with native or engineered glycosylation sites, which has been shown to be an effective means of prolonging serum half-life. Engineered E. coli represents an economical route of production. I have been able to produce, purify and test the activity of three N-acetylgalactosaminyltransferase isoform 2 in Escherichia coli and show glycosylation on peptides derived from Interleukin 29. I followed the activity of these enzymes on three candidate therapeutic proteins via lectin blotting. Data suggest the Homo sapiens orthologue of GalNAcT2 is the most efficient enzyme in the in vitro assays with all candidate therapeutic protein substrates displaying the Tn-antigen. Future research should investigate continuous assays for precise results as well as assaying native peptides as opposed to non-native ones.

scholarly journals Systematic identification of metabolites controlling gene expression in E. coli

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Martin Lempp ◽  
Niklas Farke ◽  
Michelle Kuntz ◽  
Sven Andreas Freibert ◽  
Roland Lill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
In Vitro Assays ◽  
E Coli ◽  
A Genome ◽  
Concentration Changes ◽  
Systematic Identification ◽  
Genome Scale

Abstract Metabolism controls gene expression through allosteric interactions between metabolites and transcription factors. These interactions are usually measured with in vitro assays, but there are no methods to identify them at a genome-scale in vivo. Here we show that dynamic transcriptome and metabolome data identify metabolites that control transcription factors in E. coli. By switching an E. coli culture between starvation and growth, we induce strong metabolite concentration changes and gene expression changes. Using Network Component Analysis we calculate the activities of 209 transcriptional regulators and correlate them with metabolites. This approach captures, for instance, the in vivo kinetics of CRP regulation by cyclic-AMP. By testing correlations between all pairs of transcription factors and metabolites, we predict putative effectors of 71 transcription factors, and validate five interactions in vitro. These results show that combining transcriptomics and metabolomics generates hypotheses about metabolism-transcription interactions that drive transitions between physiological states.

scholarly journals Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli

2022 ◽  
Vol 10 (1) ◽  
pp. 119
Author(s):  
Bram Van den Bergh
Keyword(s):  
Drosophila Melanogaster ◽  
In Vitro Assays ◽  
Antibiotic Tolerance ◽  
In Vivo Models ◽  
Antibiotic Development ◽  
E Coli ◽  
Germ Free ◽  
Flexible System

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics’ efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

scholarly journals Expression of Codon-Optimized Plant GlycosyltransferaseUGT72B14inEscherichia coliEnhances Salidroside Production

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Feiyan Xue ◽  
Huili Guo ◽  
Yingying Hu ◽  
Ran Liu ◽  
Lina Huang ◽  
...  
Keyword(s):  
Codon Optimization ◽  
Batch Cultivation ◽  
Plant Secondary Metabolite ◽  
Uridine Diphosphate ◽  
Wild Type ◽  
E Coli ◽  
Similar Kinetic ◽  
Fed Batch Cultivation

Salidroside, a plant secondary metabolite inRhodiola, has been demonstrated to have several adaptogenic properties as a medicinal herb. Due to the limitation of plant source, microbial production of salidroside by expression of plant uridine diphosphate glycosyltransferase (UGT) is promising. However, glycoside production usually remains hampered by poor expression of plant UGTs in microorganisms. Herein, we achieved salidroside production by expression ofRhodiolaUGT72B14 inEscherichia coli(E. coli) and codon optimization was accordingly applied.UGT72B14expression was optimized by changing 278 nucleotides and decreasing the G+C content to 51.05% without altering the amino acid sequence. The effect of codon optimization on UGT72B14 catalysis for salidroside production was assessed bothin vitroandin vivo.In vitro, salidroside production by codon-optimized UGT72B14 is enhanced because of a significantly improved protein yield (increased by 4.8-fold) and an equivalently high activity as demonstrated by similar kinetic parameters (KMandVmax), compared to that by wild-type protein.In vivo, both batch and fed-batch cultivation using the codon-optimized gene resulted in a significant increase in salidroside production, which was up to 6.7 mg/L increasing 3.2-fold over the wild-typeUGT72B14.

STUDIES ON MECHANISM OF REPAIR OF ULTRAVIOLET-IRRADIATED VIRAL AND BACTERIAL DNA IN VIVO AND IN VITRO

1965 ◽  
Vol 43 (7) ◽  
pp. 1207-1219 ◽  
Author(s):  
Emanuel Riklis
Keyword(s):  
Uv Irradiation ◽  
Soluble Fraction ◽  
Cell Extract ◽  
Host Cells ◽  
Bacterial Dna ◽  
E Coli ◽  
Thymine Dimers ◽  
K 12

The formation of thymine dimers [Formula: see text] from adjacent intrastrand thymines by ultraviolet (UV) irradiation in DNA was studied under different conditions. When thymine-2-C14 DNA was exposed in quartz tubes to 0.5 × 106 ergs/mm2 ultraviolet irradiation, two photoproducts were formed: "a" + [Formula: see text]. The concentration formed in dry DNA was only about [Formula: see text] of that formed in wet DNA.The survival of T1 phage in the dark in the resistant (uvr+) and the sensitive (uvr−) mutants of E. coli K-12 after UV irradiation of the phage in the wet and dry state is markedly dependent on the state of the phage during irradiation. Survival of T1 phage, when UV irradiated dry, was the same in the sensitive as in the resistant host cells, over a wide range of UV doses, while a marked difference in sensitivity existed when it was UV irradiated wet. Similar survival was obtained also by photoreactivation. These results correspond with the notion that thymine dimers are involved both in photoreactivation and in dark (host cell) reactivation.Thymine-requiring E. coli K-12 cells were mutated to a radioresistant strain uvr+ (AB 2416) and a radiosensitive strain uvr− (AB 2419).Irradiation of cells with a dose of 1000 ergs/mm2, followed by incubation of the cells in the dark in enriched M9 media, stopped by addition of 5% trichloroacetic acid, hydrolysis of the acid-insoluble fraction and the acid-soluble fraction in trifluoroacetic acid at 175 °C, and separation of the products by paper chromatography, showed that the two irradiation products "a" + [Formula: see text], which were formed in the bacterial DNA, are excised from the DNA in the uvr+ strain and appear in the acid-soluble fraction. No such excision occurred upon incubation of the radiosensitive strain uvr−.Incubation of the cells under light showed that photoreactivation prevails in the radiosensitive strain, i.e. disappearance of "a" + [Formula: see text] from the DNA, without their appearance in the acid-soluble fraction, while dark reactivation prevailed in the uvr+ strain, a result that indicates a stronger affinity of the dark reactivating system to UV-irradiated DNA. Cell extracts prepared by breaking the cells in a French press in Tris buffer, pH 7.5, plus 10−3 M Mg++ plus 10−3 M mercapto-ethanol showed a similar mechanism; the two irradiation products were excised from DNA by a uvr+ cell extract and not by a uvr− cell extract. Extract of uvr+ cells brought about excision of photoproducts from DNA of the uvr− cell extract.The results suggest that an enzyme, capable of excising thymine dimers, is present in the radioresistant cell as part of the system of repair of DNA from UV irradiation, and its mechanism is demonstrated, both in vivo and in vitro.

scholarly journals Glucosylation of (±)-Menthol by Uridine-Diphosphate-Sugar Dependent Glucosyltransferases from Plants

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5511
Author(s):  
Elisabeth Kurze ◽  
Victoria Ruß ◽  
Nadia Syam ◽  
Isabelle Effenberger ◽  
Rafal Jonczyk ◽  
...  
Keyword(s):  
Water Solubility ◽  
Tea Plant ◽  
Sugar Residue ◽  
Resonance Spectroscopy ◽  
Uridine Diphosphate ◽  
Biotechnological Production ◽  
Physiological Properties ◽  
Aroma Components

Menthol is a cyclic monoterpene alcohol of the essential oils of plants of the genus Mentha, which is in demand by various industries due to its diverse sensorial and physiological properties. However, its poor water solubility and its toxic effect limit possible applications. Glycosylation offers a solution as the binding of a sugar residue to small molecules increases their water solubility and stability, renders aroma components odorless and modifies bioactivity. In order to identify plant enzymes that catalyze this reaction, a glycosyltransferase library containing 57 uridine diphosphate sugar-dependent enzymes (UGTs) was screened with (±)-menthol. The identity of the products was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Five enzymes were able to form (±)-menthyl-β-d-glucopyranoside in whole-cell biotransformations: UGT93Y1, UGT93Y2, UGT85K11, UGT72B27 and UGT73B24. In vitro enzyme activity assays revealed highest catalytic activity for UGT93Y1 (7.6 nkat/mg) from Camellia sinensis towards menthol and its isomeric forms. Although UGT93Y2 shares 70% sequence identity with UGT93Y1, it was less efficient. Of the five enzymes, UGT93Y1 stood out because of its high in vivo and in vitro biotransformation rate. The identification of novel menthol glycosyltransferases from the tea plant opens new perspectives for the biotechnological production of menthyl glucoside.

scholarly journals CELL WALL COMPOSITION AND VIRULENCE IN ESCHERICHIA COLI

1968 ◽  
Vol 128 (3) ◽  
pp. 399-414 ◽  
Author(s):  
Donald N. Medearis ◽  
Bruce M. Camitta ◽  
Edward C. Heath
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Uridine Diphosphate ◽  
E Coli ◽  
Endotoxin Activity ◽  
A Cell ◽  
The Difference ◽  
Definition Of

Uridine diphosphate galactose 4-epimerase and phosphomannose isomerase-deficient mutants of Escherichia coli O111:B4 were studied to test the hypothesis that in E. coli a specific relationship exists between O antigenicity, virulence, and capacity to resist phagocytosis. The first mutant, designated J-5, produces a cell wall lipopolysaccharide, the side chains of which do not contain galactose, glucose, N-acetylglucosamine, or colitose. The second mutant produces a cell wall lipopolysaccharide which lacks only colitose. The capacity of these various organisms to kill mice was strikingly different. E. coli O111 was 1000 times as virulent as J-5, and 100 times as virulent as L-2. The capacity of the organisms to kill mice was correlated with their ability to resist phagocytosis and to persist in the peritoneal cavity. The parent strain of O111 resisted phagocytosis by macrophages in vivo and polymorphonuclear leukocytes in vitro. The mutants did not, and the organism most deficient in the saccharide component of its LPS was most susceptible to phagocytosis and least virulent. These results were corroborated by growing the mutants in appropriately supplemented media which permitted the synthesis of complete LPS, reversed the susceptibility to phagocytosis, and restored virulence. Finally, serological reactivity was consistent with previous observations which had demonstrated that the O antigenicity of E. coli is determined by the saccharide composition of its cell wall lipopolysaccharide. Despite the difference in the capacity of the various log-phase organisms to kill mice when injected intraperitoneally, purified lipopolysaccharides extracted from them did not differ significantly in their capacity to kill or produce fever. Thus virulence was shown to be independent of endotoxin activity which in turn seemed to be unrelated to the saccharide composition of the cell wall LPS. Collectively, these data provide at least a partial molecular definition of virulence in E. coli by demonstrating that the presence or absence of specific sugars in its cell wall lipopolysaccharide is a determinant of its antiphagocytic capacity and its virulence.

scholarly journals A comparative study of GT27 Family N-acetylgalactosaminyltransferases

2021 ◽  
Author(s):  
Biprajit Sanyal
Keyword(s):  
Half Life ◽  
Homo Sapiens ◽  
Effective Means ◽  
Therapeutic Proteins ◽  
In Vitro Assays ◽  
Future Research ◽  
E Coli ◽  
Glycosylation Sites

Therapeutic proteins face short half lives in vivo. Their high costs and associated toxicity effects of increasing dosage warrant exploration of methods to increase serum half-life. These proteins can be produced with native or engineered glycosylation sites, which has been shown to be an effective means of prolonging serum half-life. Engineered E. coli represents an economical route of production. I have been able to produce, purify and test the activity of three N-acetylgalactosaminyltransferase isoform 2 in Escherichia coli and show glycosylation on peptides derived from Interleukin 29. I followed the activity of these enzymes on three candidate therapeutic proteins via lectin blotting. Data suggest the Homo sapiens orthologue of GalNAcT2 is the most efficient enzyme in the in vitro assays with all candidate therapeutic protein substrates displaying the Tn-antigen. Future research should investigate continuous assays for precise results as well as assaying native peptides as opposed to non-native ones.

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