scholarly journals A Synthetic Approach for Biosynthesis of Miquelianin and Scutellarin A in Escherichia coli

2019 ◽  
Vol 9 (2) ◽  
pp. 215 ◽  
Author(s):  
Ramesh Prasad Pandey ◽  
Ha Young Jung ◽  
Prakash Parajuli ◽  
Thi Huyen Trang Nguyen ◽  
Puspalata Bashyal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vector System ◽  
Synthetic Approach ◽  
Uridine Diphosphate ◽  
Acid Biosynthesis ◽  
Glucokinase Gene ◽  
E Coli ◽  
Starting Point ◽  
Good Starting Point ◽  
L 62

Grapevine (Vitis vinifera) glycucuronosyltransferase (VvGT5) specifically catalyzes flavonol-3-O-glucuronosylation and the blue flowers of Veronica persica (Lamiales, Scrophulariaceae) uridine diphosphate (UDP)-dependent glycosyltransferase (UGT88D8) as flavonoid 7-O-specific glucuronosyltransferases, were chosen, codon optimized, and employed to synthesize the high valued flavonoids glucuronoids, miquelianin and scutellarin A in Escherichia coli. A single vector system was constructed to overexpress entire UDP-glucuronic acid biosynthesis pathway genes, along with a glucokinase gene in Escherichia coli BL21 (DE3). The newly generated E. coli BL21 (DE3) piBR181-glk.pgm2.galU.ugd.UGT88D8 strain produced 12 mg/L (28 µmol/L) of scutellarin A from apigenin, representing only 14% of maximum conversion percentage. Similarly, the strain E. coli BL21 (DE3) piBR181-glk.pgm2.galU.ugd.VvGT5 produced 30 mg/L (62 µmol/L) of miquelianin, representing a 31% conversion of quercetin. This production profile is a good starting point for further host engineering, and for production of respective compounds.

scholarly journals Improving Microbial Biogasoline Production in Escherichia coli Using Tolerance Engineering

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Jee Loon Foo ◽  
Heather M. Jensen ◽  
Robert H. Dahl ◽  
Kevin George ◽  
Jay D. Keasling ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Production Capacity ◽  
Short Chain ◽  
Methionine Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Improve Production ◽  
Starting Point ◽  
Microbial Tolerance

ABSTRACT Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production. IMPORTANCE The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.

scholarly journals Importance of Hand Hygiene during the Harvesting of Strawberries

2015 ◽  
Vol 25 (3) ◽  
pp. 380-384 ◽  
Author(s):  
Angela Shaw ◽  
Amanda Svoboda ◽  
Beatrice Jie ◽  
Aura Daraba ◽  
Gail Nonnecke
Keyword(s):  
Escherichia Coli ◽  
Hand Hygiene ◽  
Transfer Rate ◽  
Direct Transfer ◽  
Foodborne Outbreak ◽  
E Coli ◽  
Worker Training ◽  
Pig Skin ◽  
Starting Point ◽  
Foodborne Outbreaks

Foodborne outbreaks linked to poor hygiene indicate a need for education on the importance of hand hygiene during harvesting of fruit. This study simulated two potential scenarios (laboratory and field) that would lead to the transfer of Escherichia coli O157:H7 from hands to strawberries (Fragaria ×ananassa), harvesters, and field plants. The potential of direct transfer of E. coli O157:H7 from contaminated “pig skin” hands to strawberries was shown in Scenario 1. The potential of E. coli O157:H7 being transferred from contaminated hands to strawberries during harvesting was shown in all treatments up to the 100th strawberry tested with a transfer rate of 71% (1 berry) to 45% (100 berries) of E. coli. Scenario 2 mimicked the “bacteria” transfer from the worker’s contaminated hands to the workers’ clothing and to the field with the use of glowing lotion. It was shown that contaminated hands can transfer “bacteria” to the worker’s clothing, shoes, the picked strawberries, and the strawberry plants, weeds and straw mulch within the field (average spread of 50.25 ft from starting point). The transfer rate varied from worker to worker. Hand hygiene and proper worker training are essential for food-safe harvesting to avoid foodborne outbreak events.

Residu Antibiotik Serta Keberadaan Escherichia Coli Penghasil ESBL pada Daging Ayam Broiler di Pasar Kota Purwokerto

2021 ◽  
Vol 20 (2) ◽  
pp. 137-142
Author(s):  
Anriani Puspita Karunia Ning Widhi ◽  
Imam Nafi Yana Saputra
Keyword(s):  
Escherichia Coli ◽  
Broiler Chickens ◽  
Broiler Chicken ◽  
Chicken Meat ◽  
Primary Data ◽  
Antibiotic Residues ◽  
Growth Promoters ◽  
E Coli ◽  
Extended Spectrum ◽  
Starting Point

Latar belakang: Pemenuhan kebutuhan pangan asal ternak yang berasal dari unggas memiliki angka konsumsi yang cukup tinggi salah satunya yaitu ayam broiler. Untuk memenuhi tingginya permintaan terhadap ayam broiler pada pakandiberifeed additiveserta antibiotic growth promotor (AGP) dalam bentuk antibiotik untuk mempercepat pertumbuhan dan daya tahan tubuh. Pemanfaatan antibiotik yang tidak bijak akan menimbulkan residu antibiotik serta resistensi Escherichia coli penghasil Extended Spectrum β-lactamase (ESBL).Metode: Penelitian ini termasuk penelitian deskriptif. Data primer diperoleh dari hasil screeningtest. Data ditunjukkan dalam tabel dangambar yang dibahas secara deskriptif. Variabel yang diamati residu antibiotik tetrasiklin serta E. coli penghasil ESBL pada daging ayam broiler.Hasil: Temuan terhadap residu antibiotik tetrasiklin pada daging ayam broiler yaitu sebesar 7,14% dengan rerata zona hambat 12,13 mm, sedangkan hasil identifikasi E. coli penghasil ESBL pada daging ayam broiler diperoleh hasil sebesar 71,4%.Simpulan:Residu antibiotik tetrasiklin dalam daging ayam broiler masih tergolong aman dapat dikonsumsi namun, temuanE. coli penghasil ESBL pada daging ayam broiler menimbulkan masalah kesehatan, baik bagi kesehatan hewan maupun manusia, serta dapat menimbulkan resistensi terhadap antibiotik. ABSTRACT Title: Broiler Chicken Meat Sold at Purwokerto's Market Has Antibiotic Residues and Escherichia Coli That Produces EsblBackground: Poultry, of which broiler chickens are one example, is a food source with a relatively high consumption rate.Feed additives and antibiotic growth promoters (AGP) in the form of antibiotics in the ration are given to increase broilers’ growth and endurance to meet the high demand for them. It is important to note that unwise antibiotic use results in the buildup of antibiotic residues and resistance to the Extended Spectrum Beta-lactamase(ESBL)produced by Escherichia coli bacterium.Method: To collect primary data for this descriptive study, the screening test results were employed as a starting point. Next, the information was presented in the form of tables and figures.Result: According to this study, broiler chickens had a tetracycline residue of 7.14 percent, with an inhibitory zone mean of 12.13 mm, and 71.4 percent of ESBL-producing E. coli.Conclusion:According to the study, broiler chicken meat with tetracycline residue is still safe to consume. E. coli, which is known to develop an antibiotic-resistant strain of E. coli ESBL) can cause serious health problems in both humans and animals.

scholarly journals Metabolic engineering Escherichia coli for efficient production of icariside D2

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xue Liu ◽  
Lingling Li ◽  
Jincong Liu ◽  
Jianjun Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Carbon Sources ◽  
Gene Expression Pattern ◽  
Fine Tuning ◽  
Cell Factory ◽  
Efficient Production ◽  
Uridine Diphosphate ◽  
Microbial Cell Factory ◽  
E Coli

Abstract Background Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. Results We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. Conclusions We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

scholarly journals Escherichia coli: Physiological Clues Which Turn On the Synthesis of Antimicrobial Molecules

2020 ◽  
Vol 7 (4) ◽  
pp. 184
Author(s):  
Sarah-Jo Paquette ◽  
Tim Reuter
Keyword(s):  
Escherichia Coli ◽  
Bile Salt ◽  
Carbon Sources ◽  
Mitigation Strategies ◽  
E Coli ◽  
Safety And Health ◽  
Physiological Properties ◽  
Microbiological Methods ◽  
Starting Point ◽  
Gastro Intestinal Tract

Zoonotic pathogens, like Shiga toxin-producing Escherichia coli (STEC) are a food safety and health risk. To battle the increasing emergence of virulent microbes, novel mitigation strategies are needed. One strategy being considered to combat pathogens is antimicrobial compounds produced by microbes, coined microcins. However, effectors for microcin production are poorly understood, particularly in the context of complex physiological responses along the gastro-intestinal tract (GIT). Previously, we identified an E. coli competitor capable of producing a strong diffusible antimicrobial with microcin-associated characteristics. Our objective was to examine how molecule production of this competitor is affected by physiological properties associated with the GIT, namely the effects of carbon source, bile salt concentration and growth phase. Using previously described liquid- and agar-based assays determined that carbon sources do not affect antimicrobial production of E. coli O103F. However, bile salt concentrations affected production significantly, suggesting that E. coli O103F uses cues along the GIT to modulate the expression of antimicrobial production. Furthermore, E. coli O103F produces the molecule during the exponential phase, contrary to most microcins identified to date. The results underscored the importance of experimental design to identify producers of antimicrobials. To detect antimicrobials, conventional microbiological methods can be a starting point, but not the gold standard.

scholarly journals Homocysteine Toxicity in Escherichia coli Is Caused by a Perturbation of Branched-Chain Amino Acid Biosynthesis

2005 ◽  
Vol 187 (13) ◽  
pp. 4362-4371 ◽  
Author(s):  
Nina L. Tuite ◽  
Katy R. Fraser ◽  
Conor P. O'Byrne
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Biosynthetic Pathway ◽  
Amino Acid Biosynthesis ◽  
Inhibitory Effects ◽  
Acid Biosynthesis ◽  
Threonine Deaminase ◽  
E Coli ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACT In Escherichia coli the sulfur-containing amino acid homocysteine (Hcy) is the last intermediate on the methionine biosynthetic pathway. Supplementation of a glucose-based minimal medium with Hcy at concentrations greater than 0.2 mM causes the growth of E. coli Frag1 to be inhibited. Supplementation of Hcy-treated cultures with combinations of branched-chain amino acids containing isoleucine or with isoleucine alone reversed the inhibitory effects of Hcy on growth. The last intermediate of the isoleucine biosynthetic pathway, α-keto-β-methylvalerate, could also alleviate the growth inhibition caused by Hcy. Analysis of amino acid pools in Hcy-treated cells revealed that alanine, valine, and glutamate levels are depleted. Isoleucine could reverse the effects of Hcy on the cytoplasmic pools of valine and alanine. Supplementation of the culture medium with alanine gave partial relief from the inhibitory effects of Hcy. Enzyme assays revealed that the first step of the isoleucine biosynthetic pathway, catalyzed by threonine deaminase, was sensitive to inhibition by Hcy. The gene encoding threonine deaminase, ilvA, was found to be transcribed at higher levels in the presence of Hcy. Overexpression of the ilvA gene from a plasmid could overcome Hcy-mediated growth inhibition. Together, these data indicate that in E. coli Hcy toxicity is caused by a perturbation of branched-chain amino acid biosynthesis that is caused, at least in part, by the inhibition of threonine deaminase.

scholarly journals Biochemical traits that Escherichia coli evolved during its adaption to the gastrointestinal tract of humans

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Selection Pressure ◽  
Human Gastrointestinal Tract ◽  
Human Gut ◽  
Multiple Sequence ◽  
E Coli ◽  
Starting Point ◽  
Altered Gene ◽  
Different Strains

What biochemical tricks did Escherichia coli obtained or evolved during its time as a commensal in the human gastrointestinal system? E. coli is a natural symbiont of the human gastrointestinal tract. Thus, through evolutionary timescales, the bacterium must have co-evolved with humans with conditions in the gastrointestinal tract serving as selection pressure for the evolution of a variety of biochemical and physiological adaptations. These adaptations came about through mutations that arise in the genome, and thus, could be retrospectively profiled to understand the differing evolutionary pressure that selected for specific traits in the bacterium useful for its survival in the changing conditions of the human gut. Using sequenced and annotated genome information of different strains of E. coli as a guide and starting point, possibility exists to use a combination of bioinformatics, biochemical, and genetic approaches to decipher the biochemical tricks that E. coli evolved or pick up during its time as a commensal in the human gastrointestinal tract. Specifically, sequenced genomes serve as a molecular fossil from which we could obtain imprints of the various evolutionary events that impact on the bacterium. Adaptations to changing conditions could also be deciphered through analysis of single nucleotide polymorphism (SNPs). Comparison of the profiled mutations and altered gene sequences between different E. coli strains with different co-evolutionary history with the human gut might help reveal the different length of time in which different E. coli strains have co-evolved with humans. More importantly, multiple sequence alignment and phylogenetic analysis could also reveal which genes first evolve due to selection pressure exerted on the bacterium by fluctuating environmental conditions in the human gut. Genetic knockdowns of the putative genes would help indicate the overall essentiality of the genes to the physiology and functioning of the modern E. coli bacterium. The approach outlined should help answer some of the most fundamental questions regarding the evolution of different E. coli strains as well as how natural selection exerts its influence on the physiology of a commensal organism with respect to host adaptation.

scholarly journals Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside

2020 ◽  
Vol 21 (16) ◽  
pp. 5752
Author(s):  
Wenju Shu ◽  
Hongchen Zheng ◽  
Xiaoping Fu ◽  
Jie Zhen ◽  
Ming Tan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Low Cost ◽  
Expression System ◽  
Steviol Glycosides ◽  
Fusion Partner ◽  
Uridine Diphosphate ◽  
Heterologous Proteins ◽  
E Coli ◽  
Novel Strategy

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae

2001 ◽  
Vol 355 (2) ◽  
pp. 431-435 ◽  
Author(s):  
Daniel R. SYLVESTER ◽  
Emilio ALVAREZ ◽  
Arun PATEL ◽  
Kapila RATNAM ◽  
Howard KALLENDER ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Substrate Inhibition ◽  
Coli Strain ◽  
Cell Protein ◽  
Binding Motif ◽  
Gram Positive ◽  
E Coli ◽  
Starting Point ◽  
Good Starting Point

The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.

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