scholarly journals Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

2015 ◽  
Vol 81 (15) ◽  
pp. 5103-5114 ◽  
Author(s):  
Michael E. Pyne ◽  
Murray Moo-Young ◽  
Duane A. Chung ◽  
C. Perry Chou
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Efficient Production ◽  
Chromosomal Gene ◽  
Content Type ◽  
Pcr Screening ◽  
Colony Pcr ◽  
Marker Recycling ◽  
Dna Deletions ◽  
Pcr Products

ABSTRACTTo date, most genetic engineering approaches coupling the type IIStreptococcus pyogenesclustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements inEscherichia colican be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and “scar”-less.

scholarly journals Molecular Characterization of Resistance to Extended-Spectrum Cephalosporins in Clinical Escherichia coli Isolates from Companion Animals in the United States

2011 ◽  
Vol 55 (12) ◽  
pp. 5666-5675 ◽  
Author(s):  
Bashar W. Shaheen ◽  
Rajesh Nayak ◽  
Steven L. Foley ◽  
Ohgew Kweon ◽  
Joanna Deck ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Regulatory Region ◽  
Companion Animals ◽  
The United States ◽  
Conjugative Plasmid ◽  
Chromosomal Gene ◽  
Content Type ◽  
E Coli ◽  
Extended Spectrum

ABSTRACTResistance to extended-spectrum cephalosporins (ESC) among members of the familyEnterobacteriaceaeoccurs worldwide; however, little is known about ESC resistance inEscherichia colistrains from companion animals. Clinical isolates ofE. coliwere collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009.E. coliisolates (n= 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC ≥ 16 μg/ml) and extended-spectrum-β-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal geneampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried theblaCTX-M-1-group β-lactamase genes in addition to one or more of the following β-lactamase genes:blaTEM,blaSHV-3,blaCMY-2,blaCTX-M-14-like, andblaOXA-1.DifferentblaTEMsequence variants were detected in some isolates (n= 40). Three isolates harbored ablaTEM-181gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal geneampC, one of which was a novel insertion of adenine between bases −28 and −29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of β-lactamase genes increased the MICs (≥16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs amongE. colistrains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.

scholarly journals P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

2016 ◽  
Vol 82 (7) ◽  
pp. 1992-2002 ◽  
Author(s):  
Pyung-Gang Lee ◽  
Joonwon Kim ◽  
Eun-Jung Kim ◽  
EunOk Jung ◽  
Bishnu Prasad Pandey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reaction System ◽  
Gut Bacteria ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Efficient Production ◽  
Cell Reaction ◽  
Whole Cell ◽  
Content Type ◽  
Postmenopausal Symptoms

ABSTRACT(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis fromSlackia isoflavoniconvertensintoEscherichia coliBL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis oftrans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

scholarly journals Molecular Characterization of UpaB and UpaC, Two New Autotransporter Proteins of Uropathogenic Escherichia coli CFT073

2011 ◽  
Vol 80 (1) ◽  
pp. 321-332 ◽  
Author(s):  
Luke P. Allsopp ◽  
Christophe Beloin ◽  
Glen C. Ulett ◽  
Jaione Valle ◽  
Makrina Totsika ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Characterization ◽  
Host Responses ◽  
Upec Strain ◽  
Content Type ◽  
Pcr Screening ◽  
E Coli ◽  
K 12 ◽  
Encoding Genes

ABSTRACTUropathogenicEscherichia coli(UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that theupaBandupaCAT-encoding genes are common inE. coli. TheupaBandupaCgenes were cloned and characterized in a recombinantE. coliK-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073,upaBis expressed whileupaCis transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted itsupaB(but notupaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of theupaBgene in CFT073 significantly reduced early colonization of the bladder.

scholarly journals Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

2011 ◽  
Vol 77 (9) ◽  
pp. 2905-2915 ◽  
Author(s):  
Claire R. Shen ◽  
Ethan I. Lan ◽  
Yasumasa Dekishima ◽  
Antonino Baez ◽  
Kwang Myung Cho ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Driving Forces ◽  
High Titer ◽  
Catalytic Efficiency ◽  
Anaerobic Growth ◽  
High Yield ◽  
Efficient Production ◽  
Acetyl Coa ◽  
Content Type ◽  
Irreversible Reaction

ABSTRACT1-Butanol, an important chemical feedstock and advanced biofuel, is produced byClostridiumspecies. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway inEscherichia colito provide an irreversible reaction catalyzed bytrans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

scholarly journals Molecular Characterization ofblaNDM-5Carried on an IncFII Plasmid in an Escherichia coli Isolate from a Nontraveler Patient in Spain

2014 ◽  
Vol 59 (1) ◽  
pp. 659-662 ◽  
Author(s):  
Cristina Pitart ◽  
Mar Solé ◽  
Ignasi Roca ◽  
Angely Román ◽  
Asunción Moreno ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Female Patient ◽  
Urine Culture ◽  
Sequence Type ◽  
Content Type ◽  
Pcr Screening ◽  
Coli Isolate ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Indian Origin

ABSTRACTA carbapenem-resistantEscherichia coliisolate (sequence type 448 [ST448]) was recovered from a urine culture of a female patient with no recent record of traveling. PCR screening identified the presence ofblaNDM-5,blaTEM-1,blaOXA-1,blaCMY-42, andrmtB. blaNDM-5was carried in a conjugative IncFII-type plasmid (90 kb) together withblaTEM-1andrmtB. The genetic environment ofblaNDM-5showed a structure similar to those of pMC-NDM and pGUE-NDM, identified in Poland and France inE. coliof African and Indian origin, respectively.

scholarly journals Production of Ophthalmic Acid Using EngineeredEscherichia coli

2018 ◽  
Vol 84 (7) ◽  
Author(s):  
Tomokazu Ito ◽  
Maiko Tokoro ◽  
Ran Hori ◽  
Hisashi Hemmi ◽  
Tohru Yoshimura
Keyword(s):  
Escherichia Coli ◽  
Synthetic Medium ◽  
Product Inhibition ◽  
Efficient Production ◽  
Biosynthetic Enzymes ◽  
Phosphate Binding ◽  
Content Type ◽  
Food Industries ◽  
E Coli ◽  
Pharmaceutical Application

ABSTRACTOphthalmic acid (OA;l-γ-glutamyl-l-2-aminobutyryl-glycine) is an analog of glutathione (GSH;l-γ-glutamyl-l-cysteinyl-glycine) in which the cysteine moiety is replaced byl-2-aminobutyrate. OA is a useful peptide for the pharmaceutical and/or food industries. Herein, we report a method for the production of OA using engineeredEscherichia colicells.yggS-deficientE. coli, which lacks the highly conserved pyridoxal 5′-phosphate-binding protein YggS and naturally accumulates OA, was selected as the starting strain. To increase the production of OA, we overexpressed the OA biosynthetic enzymes glutamate-cysteine ligase (GshA) and glutathione synthase (GshB), desensitized the product inhibition of GshA, and eliminated the OA catabolic enzyme γ-glutamyltranspeptidase. The production of OA was further enhanced by the deletion ofmiaAandridAwith the aim of increasing the availability of ATP and attenuating the unwanted degradation of amino acids, respectively. The final strain developed in this study successfully produced 277 μmol/liter of OA in 24 h without the formation of by-products in a minimal synthetic medium containing 1 mM each glutamate, 2-aminobutyrate, and glycine.IMPORTANCEOphthalmic acid (OA) is a peptide that has the potential for use in the pharmaceutical and/or food industries. An efficient method for the production of OA would allow us to expand our knowledge about its physiological functions and enable the industrial/pharmaceutical application of this compound. We demonstrated the production of OA usingEscherichia colicells in which OA biosynthetic enzymes and degradation enymes were engineered. We also showed that unique approaches, including the use of a ΔyggSmutant as a starting strain, the establishment of an S495F mutation in GshA, and the deletion ofridAormiaA, facilitated the efficient production of OA inE. coli.

scholarly journals One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

2015 ◽  
Vol 81 (11) ◽  
pp. 3648-3654 ◽  
Author(s):  
Ryotaro Hara ◽  
Masashi Nakano ◽  
Kuniki Kino
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Maximum Yield ◽  
Test Tube ◽  
Efficient Production ◽  
Scale Production ◽  
Deficient Mutant ◽  
One Pot ◽  
Content Type ◽  
E Coli

ABSTRACTWe developed a novel process for efficient synthesis ofl-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation ofl-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesizel-THA. By using these recombinant enzymes,l-THA was obtained byl-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube.l-THA was obtained in a small amount with a molar yield of 0.076% by using intactEscherichia coliexpressing theasnOgene, and thus, two asparaginase-deficient mutants ofE. coliwere investigated. A remarkably increasedl-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of theasnOgene was enhanced by using the T7 promoter inE. coliinstead of thelacpromoter, thel-THA yield was significantly increased to 92%. By using a combination of theE. coliasparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently,l-THA was successfully obtained froml-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production ofl-THA.

scholarly journals Complete Sequence of pSAM7, an IncX4 Plasmid Carrying a NovelblaCTX-M-14bTransposition Unit Isolated from Escherichia coli and Enterobacter cloacae from Cattle

2013 ◽  
Vol 57 (9) ◽  
pp. 4590-4594 ◽  
Author(s):  
M. O. Stokes ◽  
M. AbuOun ◽  
S. Umur ◽  
G. Wu ◽  
S. R. Partridge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
United Kingdom ◽  
Enterobacter Cloacae ◽  
Complete Sequence ◽  
Human Isolate ◽  
Content Type ◽  
Pcr Screening ◽  
E Coli ◽  
The United Kingdom ◽  
Sequence Types

ABSTRACTThe same plasmid carryingblaCTX-M-14bwas identified from anEscherichia coliisolate and anEnterobacter cloacaeisolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three otherE. coliisolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.

General Taq PCR Master Mix -- CHEM 384/584 v2

2022 ◽  
Author(s):  
Ken Christensen
Keyword(s):  
Pcr Amplification ◽  
Blue Dye ◽  
Extension Rate ◽  
Dna Templates ◽  
Sample Handling ◽  
Pcr Screening ◽  
Colony Pcr ◽  
Hot Start ◽  
Direct Loading ◽  
Pcr Products

SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.

scholarly journals Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Ryotaro Hara ◽  
Kai Yamagata ◽  
Ryoma Miyake ◽  
Hiroshi Kawabata ◽  
Hisatoshi Uehara ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phylogenetic Analysis ◽  
Industrial Application ◽  
Bond Activation ◽  
Efficient Production ◽  
Content Type ◽  
Lysine Hydroxylation ◽  
Optimized Conditions ◽  
Molar Conversion

ABSTRACT Hydroxylation via C—H bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available l-lysine with l-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two l-lysine 3S-hydroxylases and four l-lysine 4R-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereoselective hydroxylation into either C-3 or C-4 positions of l-lysine, yielding (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant l-lysine hydroxylase-expressing Escherichia coli cells were used as a biocatalyst for l-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S,3S)-3-hydroxylysine was produced from 600 mM l-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S,4R)-4-hydroxylysine was produced from 300 mM l-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies. IMPORTANCE The present study identified six l-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of l-lysine hydroxylases was relatively narrow, we found that (2S,3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S,5R)-5-hydroxylysine was hydroxylated by both 3S- and 4R-hydroxylases. Moreover, the l-arginine hydroxylase VioC also hydroxylated l-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of l-lysine into (2S,3S)-3-hydroxylysine and (2S,4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic l-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

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