scholarly journals Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Daisuke Koma ◽  
Takahiro Kishida ◽  
Eisuke Yoshida ◽  
Hiroyuki Ohashi ◽  
Hayato Yamanaka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Aromatic Compound ◽  
Aromatic Compounds ◽  
High Yield ◽  
Rational Approach ◽  
Chromosome Engineering ◽  
Phenyllactic Acid ◽  
Content Type ◽  
E Coli ◽  
Free Strains

ABSTRACT Many phenylalanine- and tyrosine-producing strains have used plasmid-based overexpression of pathway genes. The resulting strains achieved high titers and yields of phenylalanine and tyrosine. Chromosomally engineered, plasmid-free producers have shown lower titers and yields than plasmid-based strains, but the former are advantageous in terms of cultivation cost and public health/environmental risk. Therefore, we engineered here the Escherichia coli chromosome to create superior phenylalanine- and tyrosine-overproducing strains that did not depend on plasmid-based expression. Integration into the E. coli chromosome of two central metabolic pathway genes (ppsA and tktA) and eight shikimate pathway genes (aroA, aroB, aroC, aroD, aroE, aroGfbr, aroL, and pheAfbr), controlled by the T7lac promoter, resulted in excellent titers and yields of phenylalanine; the superscript “fbr” indicates that the enzyme encoded by the gene was feedback resistant. The generated strain could be changed to be a superior tyrosine-producing strain by replacing pheAfbr with tyrAfbr. A rational approach revealed that integration of seven genes (ppsA, tktA, aroA, aroB, aroC, aroGfbr, and pheAfbr) was necessary as the minimum gene set for high-yield phenylalanine production in E. coli MG1655 (tyrR, adhE, ldhA, pykF, pflDC, and ascF deletant). The phenylalanine- and tyrosine-producing strains were further applied to generate phenyllactic acid-, 4-hydroxyphenyllactic acid-, tyramine-, and tyrosol-producing strains; yield of these aromatic compounds increased proportionally to the increase in phenylalanine and tyrosine yields. IMPORTANCE Plasmid-free strains for aromatic compound production are desired in the aspect of industrial application. However, the yields of phenylalanine and tyrosine have been considerably lower in plasmid-free strains than in plasmid-based strains. The significance of this research is that we succeeded in generating superior plasmid-free phenylalanine- and tyrosine-producing strains by engineering the E. coli chromosome, which was comparable to that in plasmid-based strains. The generated strains have a potential to generate superior strains for the production of aromatic compounds. Actually, we demonstrated that four kinds of aromatic compounds could be produced from glucose with high yields (e.g., 0.28 g tyrosol/g glucose).

scholarly journals Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Sonya M. Clarkson ◽  
Richard J. Giannone ◽  
Donna M. Kridelbaugh ◽  
James G. Elkins ◽  
Adam M. Guss ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Aromatic Compound ◽  
Aromatic Compounds ◽  
Sole Source ◽  
Rapid Identification ◽  
Content Type ◽  
E Coli ◽  
Aromatic Compound Degradation ◽  
Fuels And Chemicals ◽  
Heterologous Pathway

ABSTRACT The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of lignin-derived aromatics. IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.

scholarly journals Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

2012 ◽  
Vol 78 (17) ◽  
pp. 6203-6216 ◽  
Author(s):  
Daisuke Koma ◽  
Hayato Yamanaka ◽  
Kunihiko Moriyoshi ◽  
Takashi Ohmoto ◽  
Kiyofumi Sakai
Keyword(s):  
Escherichia Coli ◽  
Aromatic Compounds ◽  
Phenylacetic Acid ◽  
Shikimate Pathway ◽  
Phenyllactic Acid ◽  
Content Type ◽  
E Coli ◽  
Dehydrogenase Gene ◽  
Hydroxyphenylacetic Acid ◽  
By Products

ABSTRACTEscherichia coliwas metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) fromCupriavidus necatorwas introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) fromAzospirillum brasilenseand the phenylacetaldehyde dehydrogenase gene (feaB) fromE. coliwere introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereasipdCand the alcohol dehydrogenase gene (adhC) fromLactobacillus breviswere introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases inE. coliencoded by theyqhD,yjgB, andyahKgenes. Cointroduction and cooverexpression of each gene withipdCin the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of theyahKgene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion offeaB,pheA, and/ortyrAgenes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.

scholarly journals Complete Draft Genome Sequence of Escherichia coli KRX, a Strain for Efficient Cloning and High-Yield Expression of Proteins under Control of the T7 RNA Polymerase

2017 ◽  
Vol 5 (39) ◽  
Author(s):  
Jan-Philipp Schwarzhans ◽  
Daniel Wibberg ◽  
Anika Winkler ◽  
Jörn Kalinowski ◽  
Karl Friehs
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Genetic Basis ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
High Yield ◽  
Content Type ◽  
E Coli ◽  
High Transformation ◽  
High Yields

ABSTRACT Escherichia coli KRX is a strain offering both a high transformation efficiency and the possibility to produce the target protein to high yields in one host, avoiding additional cloning steps. Here, the draft genome sequence of E. coli KRX is presented and provides the genetic basis for additional biotechnological applications.

scholarly journals Heterologous Expression ofPseudomonas putidaMethyl-Accepting Chemotaxis Proteins YieldsEscherichia coliCells Chemotactic to Aromatic Compounds

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Clémence Roggo ◽  
Estelle Emilie Clerc ◽  
Noushin Hadadi ◽  
Nicolas Carraro ◽  
Roman Stocker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Environmental Concern ◽  
Bacterial Chemotaxis ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Cell Accumulation ◽  
Chemotaxis Proteins

ABSTRACTEscherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars, and peptides. We envisioned modifying the chemotaxis specificity ofE. coliby expressing heterologous chemoreceptors fromPseudomonas putidaenabling attraction either to toluene or benzoate. ThemcpTgene encoding the type 40-helical bundle (40H) methyl-accepting chemoreceptor for toluene fromPseudomonas putidaMT53 and thepcaYgene for the type 40H receptor for benzoate and related molecules fromP. putidaF1 were expressed from thetrgpromoter on a plasmid in motile wild-typeE. coliMG1655.E. colicells expressing McpT accumulated in chemoattraction assays to sources with 60 to 200 μM toluene, although less strongly than the response to 100 μM serine, but statistically significantly stronger than that to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed inE. coliand yielded weak but distinguishable membranes and polar foci in 1% of cells.E. colicells expressing PcaY showed weak attraction to 0.1 to 1 mM benzoate, but 50 to 70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in theE. colichemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.IMPORTANCEBacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis ofEscherichia colihas been well studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds fromPseudomonas putidaat least partly functionally complement theE. colichemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants, like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds inE. coli.

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 1671. Diagnosis and management of Escherichia coli bacteriuria at a Veterans Affairs hospital

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S821-S821
Author(s):  
Niyati H Shah ◽  
Brooke K Decker ◽  
Brooke K Decker ◽  
Gaetan Sgro ◽  
Monique Y Boudreaux-Kelly ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Treatment ◽  
High Yield ◽  
Comorbid Conditions ◽  
Provider Education ◽  
Transmitted Infection ◽  
Physician Practices ◽  
E Coli ◽  
Diagnosis And Management ◽  
Tract Infections

Abstract Background The IDSA recommends against screening for and treating ASB in all patients except for those pregnant or undergoing urologic procedures. Nevertheless, antibiotic treatment of ASB is widespread. We conducted a retrospective analysis of physician practices in diagnosis and management of Escherichia coli (E. coli) ASB in a male Veteran population, and compared outcomes in ASB patients treated or not treated with antibiotics. Methods Patients with an E. coli positive urine culture during an ED visit or inpatient admission from 01/2017 to 12/2017 were screened. Patients admitted to the intensive care unit or diagnosed with a sexually transmitted infection, pyelonephritis, prostatitis, or epididymitis/orchitis were excluded. A total of 163 patients were included. Demographics, clinical comorbidities and severity of illness, and outcomes were compared in ASB patients managed with or without antibiotics. ANOVA and Chi-square or Fisher’s exact tests were utilized for comparing measurements. Results ASB was present in 92/163 patients. The majority (74%) of these patients were given antibiotics. Regardless of qSOFA score or alternate infection, there were no significant differences in outcomes between ASB patients treated or not treated with antibiotics: 3-month mortality (15% vs 21%; p = 0.53), emergence of newly resistant bacterial pathogens (7% vs 13%; p = 0.43), recurrent urinary tract infections (61% vs 50%; p = 0.72), clearance of urinary pathogens (75% vs 58%; p = 0.45), length of hospital stay (7 vs 6 days, p = 0.67). Factors that were predictive of physician treatment of ASB included patient comorbid conditions such as benign prostatic hyperplasia, pyuria, and the absence of hematuria. The incidence of adverse events with antibiotic treatment of ASB was low. Conclusion The rate of antibiotic treatment of E. coli ASB in male veterans is high. Outcomes do not differ among ASB patients managed with or without antibiotics. Future studies examining outcomes in patients prescribed antibiotics for multiple episodes of ASB may yield differences, particularly in emergence of resistant pathogens. Focusing on patients with comorbid conditions who are not critically ill would be a high yield target for provider education to reduce ASB treatment. Disclosures All Authors: No reported disclosures

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.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

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