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 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 inEscherichia colienables rapid bioconversion of model lignin monomers

2017 ◽  
Author(s):  
Sonya M. Clarkson ◽  
Donna M. Kridelbaugh ◽  
James G. Elkins ◽  
Adam M. Guss ◽  
Joshua K. Michener
Keyword(s):  
Escherichia Coli ◽  
Sole Source ◽  
Biofuel Production ◽  
List Type ◽  
Cellulosic Biofuels ◽  
E Coli ◽  
Gene Pathway ◽  
Cellulosic Biofuel ◽  
Fuels And Chemicals ◽  
Heterologous Pathway

AbstractCellulosic biofuel production yields a substantial lignin byproduct stream that currently has few applications. Biological conversion of lignin compounds into chemicals and fuels has the potential to improve the economics of cellulosic biofuels, but few microbes are able both to catabolize lignin and generate valuable products. WhileEscherichia colihas been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we have engineeredE. colito catabolize a model lignin monomer, protocatechuate, as the sole source of carbon and energy, via heterologous expression of a nine-gene pathway fromPseudomonas putidaKT2440. We next used experimental evolution to select for mutations that increased growth with PCA more than two-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 lignin bioconversions.HighlightsA heterologous pathway for PCA catabolism was transferred to Escherichia coli.Evolution identified a mutation that increased growth with PCA by 2.5-fold.Optimization plus further engineering allowed efficient catabolism of 4-HB

scholarly journals Rapid Identification of Different Escherichia coli Sequence Type 131 Clades

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yasufumi Matsumura ◽  
Johann D. D. Pitout ◽  
Gisele Peirano ◽  
Rebekah DeVinney ◽  
Taro Noguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epidemiological Studies ◽  
Rapid Identification ◽  
Sequence Type ◽  
Fluoroquinolone Resistance ◽  
Pcr Assay ◽  
Content Type ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
Clade C

ABSTRACT Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-β-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with bla CTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

scholarly journals Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

2014 ◽  
Vol 80 (10) ◽  
pp. 3276-3282 ◽  
Author(s):  
Matthew S. Wong ◽  
Mai Li ◽  
Ryan W. Black ◽  
Thao Q. Le ◽  
Sharon Puthli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fermentation Process ◽  
Safety Issue ◽  
Specific Productivity ◽  
Content Type ◽  
E Coli ◽  
Pathway Gene ◽  
Fuels And Chemicals ◽  
Glycerol Uptake ◽  
The Cost

ABSTRACTGlycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol byEscherichia colithat presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genesdhaKLM,gldA, andglpK, as well as the ethanol pathway geneadhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used theadhEandicdpromoters fromE. coliin our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of anE. coli-based glycerol-to-ethanol process.

scholarly journals Rapid Identification of Major Escherichia coli Sequence Types Causing Urinary Tract and Bloodstream Infections

2014 ◽  
Vol 53 (1) ◽  
pp. 160-166 ◽  
Author(s):  
M. Doumith ◽  
M. Day ◽  
H. Ciesielczuk ◽  
R. Hope ◽  
A. Underwood ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Bloodstream Infections ◽  
Epidemiological Studies ◽  
Rapid Identification ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding ◽  
Signature Sequences ◽  
Sequence Types

Escherichia colisequence types (STs) 69, 73, 95, and 131 are collectively responsible for a large proportion ofE. coliurinary tract and bloodstream infections, and they differ markedly in their antibiotic susceptibilities. Here, we describe a novel PCR method to rapidly detect and distinguish these lineages. Three hundred eighteen publishedE. coligenomes were compared in order to identify signature sequences unique to each of the four major STs. The specificities of these sequences were assessedin silicoby seeking them in an additional 98 genomes. A PCR assay was designed to amplify size-distinguishable fragments unique to the four lineages and was validated using 515E. coliisolates of known STs. Genome comparisons identified 22 regions ranging in size from 335 bp to 26.5 kb that are unique to one or more of the four predominantE. coliSTs, with two to 10 specific regions per ST. These regions predominantly harbor genes encoding hypothetical proteins and are within or adjacent to prophage sequences. Most (13/22) were highly conserved (>96.5% identity) in the genomes of their respective ST. The new assay correctly identified all 142 representatives of the four major STs in the validation set (n= 515), with only two ST12 isolates misidentified as ST95. Compared with MLST, the assay has 100% sensitivity and 99.5% specificity. The rapid identification of major extraintestinalE. coliSTs will benefit future epidemiological studies and could be developed to tailor antibiotic therapy to the different susceptibilities of these dominant lineages.

scholarly journals Identification of a Formate-Dependent Uric Acid Degradation Pathway inEscherichia coli

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Yumi Iwadate ◽  
Jun-ichi Kato
Keyword(s):  
Escherichia Coli ◽  
Uric Acid ◽  
Formate Dehydrogenase ◽  
Sole Source ◽  
Degradation Pathway ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Purine Catabolism ◽  
E Coli ◽  
Acid Degradation

ABSTRACTPurine is a nitrogen-containing compound that is abundant in nature. In organisms that utilize purine as a nitrogen source, purine is converted to uric acid, which is then converted to allantoin. Allantoin is then converted to ammonia. InEscherichia coli, neither urate-degrading activity nor a gene encoding an enzyme homologous to the known urate-degrading enzymes had previously been found. Here, we demonstrate urate-degrading activity inE. coli. We first identifiedaegAas anE. coligene involved in oxidative stress tolerance. An examination of gene expression revealed that bothaegAand its paralogygfTare expressed under both microaerobic and anaerobic conditions. TheygfTgene is localized within a chromosomal gene cluster presumably involved in purine catabolism. Accordingly, the expression ofygfTincreased in the presence of exogenous uric acid, suggesting thatygfTis involved in urate degradation. Examination of the change of uric acid levels in the growth medium with time revealed urate-degrading activity under microaerobic and anaerobic conditions in the wild-type strain but not in theaegA ygfTdouble-deletion mutant. Furthermore, AegA- and YgfT-dependent urate-degrading activity was detected only in the presence of formate and formate dehydrogenase H. Collectively, these observations indicate the presence of urate-degrading activity inE. colithat is operational under microaerobic and anaerobic conditions. The activity requires formate, formate dehydrogenase H, and eitheraegAorygfT. We also identified other putative genes which are involved not only in formate-dependent but also in formate-independent urate degradation and may function in the regulation or cofactor synthesis in purine catabolism.IMPORTANCEThe metabolic pathway of uric acid degradation to date has been elucidated only in aerobic environments and is not understood in anaerobic and microaerobic environments. In the current study, we showed thatEscherichia coli, a facultative anaerobic organism, uses uric acid as a sole source of nitrogen under anaerobic and microaerobic conditions. We also showed that formate, formate dehydrogenase H, and either AegA or YgfT are involved in uric acid degradation. We propose that formate may act as an electron donor for a uric acid-degrading enzyme in this bacterium.

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 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 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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