scholarly journals Engineering Escherichia coli for Microbial Production of Butanone

2016 ◽  
Vol 82 (9) ◽  
pp. 2574-2584 ◽  
Author(s):  
Kajan Srirangan ◽  
Xuejia Liu ◽  
Lamees Akawi ◽  
Mark Bruder ◽  
Murray Moo-Young ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Batch Cultivation ◽  
Culture Conditions ◽  
Sleeping Beauty ◽  
Genetically Engineered ◽  
Content Type ◽  
Factors Affecting ◽  
Formation Pathway ◽  
E Coli ◽  
Biochemical Genetic

ABSTRACTTo expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway inEscherichia colifor heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producingE. colihost strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohologenesis under selected fermentative culture conditions were investigated. Using the engineeredE. colistrain for batch cultivation with 30 g liter−1glycerol as the carbon source, we achieved coproduction of 1.3 g liter−1butanone and 2.9 g liter−1acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform.

scholarly journals Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase

2014 ◽  
Vol 80 (21) ◽  
pp. 6828-6836 ◽  
Author(s):  
Yuta Miki ◽  
Yasuhisa Asano
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Biosynthetic Pathway ◽  
Culture Conditions ◽  
Culture Broth ◽  
Single Step ◽  
Content Type ◽  
E Coli ◽  
Pharmaceutical Industries ◽  
Aldoxime Dehydratase

ABSTRACTThe biosynthetic pathway for the production of phenylacetonitrile (PAN), which has a wide variety of uses in chemical and pharmaceutical industries, was constructed inEscherichia coliutilizing enzymes from the plant glucosinolate-biosynthetic and bacterial aldoxime-nitrile pathways. First, the single-step reaction to produceE,Z-phenylacetaldoxime (PAOx) froml-Phe was constructed inE. coliby introducing the genes encoding cytochrome P450 (CYP) 79A2 and CYP reductase fromArabidopsis thaliana, yielding theE,Z-PAOx-producing transformant. Second, this step was expanded to the production of PAN by further introducing the aldoxime dehydratase (Oxd) gene fromBacillussp. strain OxB-1, yielding the PAN-producing transformant. TheE,Z-PAOx-producing transformant also produced phenethyl alcohol and PAN as by-products, which were suggested to be the metabolites ofE,Z-PAOx produced byE. colienzymes, while the PAN-producing transformant accumulated only PAN in the culture broth, which suggested that the CYP79A2 reaction (the conversion ofl-Phe toE,Z-PAOx) was a potential bottleneck in the PAN production pathway. Expression of active CYP79A2 and concentration of biomass were improved by the combination of the autoinduction method, coexpression ofgroE, encoding the heat shock protein GroEL/GroES, N-terminal truncation of CYP79A2, and optimization of the culture conditions, yielding a >60-fold concentration ofE,Z-PAOx (up to 2.9 mM). The concentration of PAN was 4.9 mM under the optimized conditions. These achievements show the potential of this bioprocess to produce nitriles and nitrile derivatives in the absence of toxic chemicals.

scholarly journals Fed-Batch Cultivation and Adding Supplements to Increase Yield of β-1,3-1,4-Glucanase by Genetically Engineered Escherichia coli

Catalysts ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 269
Author(s):  
Lijuan Zhong ◽  
Zheng Liu ◽  
Yinghua Lu
Keyword(s):  
Escherichia Coli ◽  
Feeding Strategy ◽  
Batch Cultivation ◽  
Genetically Engineered ◽  
Fed Batch ◽  
Glucanase Activity ◽  
E Coli ◽  
Constant Rate ◽  
Engineered Escherichia Coli ◽  
Fed Batch Cultivation

The aim of this study was to analyze the major influence factors of culture medium on the expression level of β-1,3-1,4-glucanase, and to further develop an optimized process for the extracellular production of β-glucanase at a bioreactor scale (7 L) with a genetically engineered Escherichia coli (E. coli) JM109-pLF3. In this study, batch cultivation and fed-batch cultivation including the constant rate feeding strategy and the DO-stat (DO: Dissolved Oxygen) feeding strategy were conducted. At a 7 L bioreactor scale for batch cultivation, biomass reached 3.14 g/L and the maximum β-glucanase activity was 506.94 U/mL. Compared with batch cultivation, the addition of glycerol, complex nitrogen and complete medium during fed-batch cultivation increased the production of biomass and β-1,3-1,4-glucanase. The maximum biomass and β-glucanase activity, which were 7.67 g/L and 1680 U/mL, respectively, that is, 2.45 and 3.31 times higher than those obtained with batch cultivation, were obtained by feeding a complex nitrogen source at a constant rate of 1.11 mL/min. Therefore, these nutritional supplements and strategies can be used as a reference to enhance the production of other bioproducts from E. coli.

scholarly journals The Intimin-Like Protein FdeC Is Regulated by H-NS and Temperature in Enterohemorrhagic Escherichia coli

2014 ◽  
Vol 80 (23) ◽  
pp. 7337-7347 ◽  
Author(s):  
Donna M. Easton ◽  
Luke P. Allsopp ◽  
Minh-Duy Phan ◽  
Danilo Gomes Moriel ◽  
Guan Kai Goh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Culture Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Infection Model ◽  
Content Type ◽  
E Coli ◽  
Uremic Syndrome ◽  
Animal Hosts ◽  
And Function

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) is a Shiga-toxigenic pathogen capable of inducing severe forms of enteritis (e.g., hemorrhagic colitis) and extraintestinal sequelae (e.g., hemolytic-uremic syndrome). The molecular basis of colonization of human and animal hosts by EHEC is not yet completely understood, and an improved understanding of EHEC mucosal adherence may lead to the development of interventions that could disrupt host colonization. FdeC, also referred to by its IHE3034 locus tag ECOK1_0290, is an intimin-like protein that was recently shown to contribute to kidney colonization in a mouse urinary tract infection model. The expression of FdeC is tightly regulatedin vitro, and FdeC shows promise as a vaccine candidate against extraintestinalE. colistrains. In this study, we characterized the prevalence, regulation, and function offdeCin EHEC. We showed that thefdeCgene is conserved in both O157 and non-O157 EHEC and encodes a protein that is expressed at the cell surface and promotes biofilm formation under continuous-flow conditions in a recombinantE. colistrain background. We also identified culture conditions under which FdeC is expressed and showed that minor alterations of these conditions, such as changes in temperature, can significantly alter the level of FdeC expression. Additionally, we demonstrated that the transcription of thefdeCgene is repressed by the global regulator H-NS. Taken together, our data suggest a role for FdeC in EHEC when it grows at temperatures above 37°C, a condition relevant to its specialized niche at the rectoanal junctions of cattle.

scholarly journals Determining Thermal Inactivation of Escherichia coli O157:H7 in Fresh Compost by Simulating Early Phases of the Composting Process

2011 ◽  
Vol 77 (12) ◽  
pp. 4126-4135 ◽  
Author(s):  
Randhir Singh ◽  
Jinkyung Kim ◽  
Marion W. Shepherd ◽  
Feng Luo ◽  
Xiuping Jiang
Keyword(s):  
Escherichia Coli ◽  
Thermal Inactivation ◽  
Weibull Model ◽  
Model Parameters ◽  
Pathogen Inactivation ◽  
Content Type ◽  
Factors Affecting ◽  
E Coli ◽  
Pathogen Survival ◽  
Heating Up

ABSTRACTA three-strain mixture ofEscherichia coliO157:H7 was inoculated into fresh dairy compost (ca. 107CFU/g) with 40 or 50% moisture and was placed in an environmental chamber (ca. 70% humidity) that was programmed to ramp from room temperature to selected composting temperatures in 2 and 5 days to simulate the early composting phase. The survivingE. coliO157:H7 population was analyzed by direct plating and enrichment. Optimal and suboptimal compost mixes, with carbon/nitrogen (C/N) ratios of 25:1 and 16:1, respectively, were compared in this study. In the optimal compost mix,E. coliO157:H7 survived for 72, 48, and 24 h in compost with 40% moisture and for 72, 24, and 24 h with 50% moisture at 50, 55, and 60°C, respectively, following 2 days of come-up time (rate of heating up). However, in the suboptimal compost mix, the pathogen survived for 288, 72, and 48 h in compost with 40% moisture and for 240, 72, 24 h in compost with 50% moisture at the same temperatures, respectively. Pathogen survival was longer, with 5 days of come-up time compared with 2 days of come-up. Overall,E. coliO157:H7 was inactivated faster in the compost with 50% moisture than in the compost with 40% at 55 and 60°C. Both moisture and come-up time were significant factors affecting Weibull model parameters. Our results suggest that slow come-up time at the beginning of composting can extend pathogen survival during composting. Additionally, both the C/N ratio and the initial moisture level in the compost mix affect the rate of pathogen inactivation as well.

scholarly journals AnEscherichia coliNitrogen Starvation Response Is Important for Mutualistic Coexistence withRhodopseudomonas palustris

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Alexandra L. McCully ◽  
Megan G. Behringer ◽  
Jennifer R. Gliessman ◽  
Evgeny V. Pilipenko ◽  
Jeffrey L. Mazny ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Starvation ◽  
Physiological State ◽  
Rhodopseudomonas Palustris ◽  
Genetically Engineered ◽  
Individual Species ◽  
Starvation Response ◽  
Content Type ◽  
E Coli ◽  
Stable Coexistence

ABSTRACTMicrobial mutualistic cross-feeding interactions are ubiquitous and can drive important community functions. Engaging in cross-feeding undoubtedly affects the physiology and metabolism of individual species involved. However, the nature in which an individual species' physiology is influenced by cross-feeding and the importance of those physiological changes for the mutualism have received little attention. We previously developed a genetically tractable coculture to study bacterial mutualisms. The coculture consists of fermentativeEscherichia coliand phototrophicRhodopseudomonas palustris. In this coculture,E. colianaerobically ferments sugars into excreted organic acids as a carbon source forR. palustris. In return, a genetically engineeredR. palustrisstrain constitutively converts N2into NH4+, providingE. coliwith essential nitrogen. Using transcriptome sequencing (RNA-seq) and proteomics, we identified transcript and protein levels that differ in each partner when grown in coculture versus monoculture. When in coculture withR. palustris,E. coligene expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disruptingE. coliNtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at lowR. palustrisNH4+excretion levels. Destabilization of the nitrogen starvation regulatory network resulted in variable growth trends and, in some cases, extinction. Our results highlight that alternative physiological states can be important for survival within cooperative cross-feeding relationships.IMPORTANCEMutualistic cross-feeding between microbes within multispecies communities is widespread. Studying how mutualistic interactions influence the physiology of each species involved is important for understanding how mutualisms function and persist in both natural and applied settings. Using a bacterial mutualism consisting ofRhodopseudomonas palustrisandEscherichia coligrowing cooperatively through bidirectional nutrient exchange, we determined that anE. colinitrogen starvation response is important for maintaining a stable coexistence. The lack of anE. colinitrogen starvation response ultimately destabilized the mutualism and, in some cases, led to community collapse after serial transfers. Our findings thus inform on the potential necessity of an alternative physiological state for mutualistic coexistence with another species compared to the physiology of species grown in isolation.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli

2020 ◽  
Vol 14 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Maryam Ahankoub ◽  
Gashtasb Mardani ◽  
Payam Ghasemi-Dehkordi ◽  
Ameneh Mehri-Ghahfarrokhi ◽  
Abbas Doosti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Performance ◽  
Human Cancer ◽  
Genetically Engineered ◽  
Hazardous Substances ◽  
E Coli ◽  
Spiked Soil ◽  
Engineered Escherichia Coli ◽  
Genetically Manipulated ◽  
Hplc Measurement

Background: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. Objective: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. Methods: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. Results: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. Conclusion: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document