scholarly journals Arsenic Methylation and Volatilization by ArseniteS-Adenosylmethionine Methyltransferase in Pseudomonas alcaligenes NBRC14159

2015 ◽  
Vol 81 (8) ◽  
pp. 2852-2860 ◽  
Author(s):  
Jun Zhang ◽  
Tingting Cao ◽  
Zhu Tang ◽  
Qirong Shen ◽  
Barry P. Rosen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Main Product ◽  
Inorganic Arsenic ◽  
Biogeochemical Cycle ◽  
Content Type ◽  
Arsenic Methylation ◽  
Inorganic As ◽  
Pseudomonas Alcaligenes ◽  
Microbial Methylation

ABSTRACTInorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation byPseudomonas alcaligenesNBRC14159.P. alcaligeneswas able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arseniteS-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). WhenP. alcaligenes arsMgene (PaarsM) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III).PaarsMwas expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression ofPaarsMin an As-hypersensitive strain ofEscherichia coliconferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.

scholarly journals Two New Unspecific Peroxygenases from Heterologous Expression of Fungal Genes in Escherichia coli

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Dolores Linde ◽  
Andrés Olmedo ◽  
Alejandro González-Benjumea ◽  
María Estévez ◽  
Chantal Renau-Mínguez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Aromatic Compounds ◽  
Ad Hoc ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Veratryl Alcohol ◽  
Cytochrome P450 Monooxygenases ◽  
Content Type ◽  
Fungal Genes

ABSTRACT Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds. IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.

scholarly journals Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

2016 ◽  
Vol 82 (20) ◽  
pp. 6167-6173 ◽  
Author(s):  
Meenu Katoch ◽  
Rabia Mazmouz ◽  
Rocky Chau ◽  
Leanne A. Pearson ◽  
Russell Pickford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Uv Radiation ◽  
Active Ingredient ◽  
Stress Factors ◽  
Heterologous Production ◽  
Content Type ◽  
E Coli

ABSTRACTMycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacteriumCylindrospermum stagnalePCC 7417 revealed a new gene cluster with homology to MAA synthase fromNostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylAtomylE), compared to the four found in other MAA gene clusters. Heterologous expression ofmylAtomylEinEscherichia coliresulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced inE. coliand structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods.IMPORTANCEMycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs inE. coliis also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.

scholarly journals Genome Sequence of Escherichia coli Stbl4, a Versatile Genetic Tool for Heterologous Expression

2021 ◽  
Vol 10 (40) ◽  
Author(s):  
Lina Assad ◽  
Karolis Matjošaitis ◽  
Harald Gross
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Genome Sequence ◽  
Laboratory Strain ◽  
Gene Clusters ◽  
F Plasmid ◽  
Draft Sequence ◽  
Content Type ◽  
Genetic Tool ◽  
Large Gene

Escherichia coli Stbl4 is widely used as a laboratory strain for heterologous expression of large gene clusters. Since no genome sequence has been publicly available, we here report the draft sequence of Stbl4, including its F-plasmid. It should serve as a useful reference for researchers working with Stbl4.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Draft Genome Sequences of Escherichia coli Strains FP2 and FP3, Isolated from the Canada Goose (Branta canadensis)

2018 ◽  
Vol 7 (9) ◽  
Author(s):  
Allison L. Denny ◽  
Susan E. Arruda
Keyword(s):  
Escherichia Coli ◽  
Draft Genome ◽  
Branta Canadensis ◽  
Canada Goose ◽  
Genome Sequences ◽  
Content Type

Draft genomes of two strains of Escherichia coli, FP2 and FP3, isolated from the feces of the Canada goose (Branta canadensis), were sequenced. Genome sizes were 5.26 Mb with a predicted G+C content of 50.54% (FP2) and 5.07 Mb with a predicted G+C content of 50.41% (FP3).

scholarly journals Ecological Effect of Ceftaroline-Avibactam on the Normal Human Intestinal Microbiota

2015 ◽  
Vol 59 (8) ◽  
pp. 4504-4509 ◽  
Author(s):  
Mamun-Ur Rashid ◽  
Staffan Rosenborg ◽  
Georgios Panagiotidis ◽  
Karin Söderberg-Löfdal ◽  
Andrej Weintraub ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Intestinal Microbiota ◽  
Ecological Effect ◽  
New Combination ◽  
Plasma Samples ◽  
Ceftaroline Fosamil ◽  
Content Type ◽  
Human Intestinal Microbiota ◽  
Normal Human ◽  
Lactamase Inhibitor

ABSTRACTCeftaroline-avibactam is a new combination of the antibiotic ceftaroline with a novel non-β-lactam β-lactamase inhibitor, avibactam. The purpose of the present study was to investigate the effect of ceftaroline-avibactam on the human intestinal microbiota. Fourteen healthy volunteers received ceftaroline-avibactam (600 mg ceftaroline fosamil and 600 mg avibactam) intravenously over 2 h every 8 h on days 1 to 6 and as a single dose on day 7. Fecal samples were collected on day −1 (within 24 h of the first infusion on day 1) and on days 2, 5, 7, 9, 14, and 21.Escherichia colinumbers decreased during the study and normalized on day 21. An increased number ofKlebsiellabacteria appeared on day 14 and normalized on day 21. The number of other enterobacteria decreased during the study, and the number of enterococci decreased from days 2 to 7 and normalized on day 9.Candidanumbers increased from days 5 to 9 and normalized after day 14. The number of lactobacilli decreased during the study and recovered on day 14. The number of bifidobacteria decreased on day 2 and normalized on day 21. The number ofBacteroidesbacteria was unchanged.Clostridium difficilenumbers decreased on days 7 and 9 and increased on days 14 and 21. A toxigenicC. difficilestrain was detected in one volunteer on day 21 with no reported adverse events. Plasma samples were collected on days −1, 2, 5, and 7. Ceftaroline and avibactam concentrations were 0 to 34.5 mg/liter and 0 to 61.6 mg/liter, respectively, in plasma and 0 to 35.4 mg/kg and 0 to 98.5 mg/kg, respectively, in feces. (This study is registered in the European Clinical Trials Database [https://eudract.ema.europa.eu/] under number EudraCT 2012 004921-25.)

scholarly journals Autotransporter Protein-Encoding Genes of Diarrheagenic Escherichia coli Are Found in both Typical and Atypical Enteropathogenic E. coli Strains

2012 ◽  
Vol 79 (1) ◽  
pp. 411-414 ◽  
Author(s):  
Afonso G. Abreu ◽  
Vanessa Bueris ◽  
Tatiane M. Porangaba ◽  
Marcelo P. Sircili ◽  
Fernando Navarro-Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Virulence Markers ◽  
Protein Encoding ◽  
Encoding Genes ◽  
Specific Virulence

ABSTRACTAutotransporter (AT) protein-encoding genes of diarrheagenicEscherichia coli(DEC) pathotypes (cah,eatA,ehaABCDJ,espC,espI,espP,pet,pic,sat, andtibA) were detected in typical and atypical enteropathogenicE. coli(EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

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