Improved production of the non-native cofactor F420 in Escherichia coli

AbstractThe deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

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Dynamic regulation of extracellular ATP in Escherichia coli

Biochemical Journal ◽

10.1042/bcj20160879 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1395-1416 ◽

Cited By ~ 7

Author(s):

Cora Lilia Alvarez ◽

Gerardo Corradi ◽

Natalia Lauri ◽

Irene Marginedas-Freixa ◽

María Florencia Leal Denis ◽

...

Keyword(s):

Escherichia Coli ◽

Atpase Activity ◽

Atp Release ◽

Membrane Vesicles ◽

Fold Increase ◽

Extracellular Atp ◽

Outer Membrane Vesicles ◽

Dynamic Regulation ◽

E Coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

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Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.345.134 ◽

2011 ◽

Vol 345 ◽

pp. 134-138 ◽

Cited By ~ 1

Author(s):

Li Hui Lv ◽

Xue Gang Luo ◽

Meng Ni ◽

Xiao Lan Jing ◽

Nan Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Streptococcus Pneumoniae ◽

Expression System ◽

High Yield ◽

Gene Synthesis ◽

Post Translational Modification ◽

Iptg Induction ◽

E Coli ◽

Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

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Increased Type 1 Fimbrial Expression among Commensal Escherichia coli Isolates in the Murine Cecum following Catabolic Stress

Infection and Immunity ◽

10.1128/iai.67.2.745-753.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 745-753 ◽

Cited By ~ 29

Author(s):

Barbara A. Hendrickson ◽

Jun Guo ◽

Robert Laughlin ◽

Yimei Chen ◽

John C. Alverdy

Keyword(s):

Escherichia Coli ◽

Intestinal Epithelium ◽

Tissue Injury ◽

Fold Increase ◽

E Coli ◽

Type 1 Fimbriae ◽

Colon Cell ◽

Colon Cell Line

ABSTRACT Although indigenous bacteria intimately colonize the intestinal mucosa, under normal conditions the intestinal epithelial cell is free of adherent bacteria. Nonetheless, commensal bacteria such asEscherichia coli adhere to and translocate across the intestinal epithelium in association with a number of pathologic states including hemorrhagic shock, immunosuppression, traumatic tissue injury, and lack of enteral feedings. The adhesins involved in the adherence of indigenous E. coli to the intestinal epithelium in vivo following catabolic stress are unknown. We have developed a mouse model to study the bacterial adhesins which mediate the increased intestinal adherence of E. coliafter partial hepatectomy and short-term starvation. Our studies demonstrated that hepatectomy and starvation in the mouse were associated with a 7,500-fold increase in the numbers of E. coli bacteria adhering to the cecum. In addition, erythrocyte agglutination studies, as well as immunostaining of fimbrial preparations and electron micrographs of the bacteria, revealed that surface type 1 fimbriae were more abundant in the commensal E. coli harvested from the ceca of the stressed mice. These E. coli isolates adhered to a mouse colon cell line and injected cecal loops in a mannose-inhibitable manner, which suggests a role for type 1 fimbriae in the adherence of the E. coli isolates to the cecum in vivo following host catabolic stress.

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Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

BMC Biotechnology ◽

10.1186/s12896-021-00732-4 ◽

2022 ◽

Vol 22 (1) ◽

Author(s):

Aida Bakhshi Khalilvand ◽

Saeed Aminzadeh ◽

Mohammad Hossein Sanati ◽

Fereidoun Mahboudi

Keyword(s):

Escherichia Coli ◽

Cell Density ◽

Shake Flask ◽

High Cell Density ◽

Fold Increase ◽

Soluble Expression ◽

High Yield ◽

High Cell ◽

E Coli ◽

High Cell Density Fermentation

Abstract Background SHuffle is a suitable Escherichia coli (E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced in E. coli as inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7 E. coli expressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation. Results Factors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2 concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor. Conclusion The proposed media was suitable for high cell density fermentation of E. coli SHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

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A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00662-17 ◽

2017 ◽

Vol 83 (14) ◽

Cited By ~ 12

Author(s):

Mariela P. Mezzina ◽

Daniela S. Álvarez ◽

Diego E. Egoburo ◽

Rocío Díaz Peña ◽

Pablo I. Nikel ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Sources ◽

Added Value ◽

Heterologous Proteins ◽

Bacterial Strains ◽

Content Type ◽

E Coli ◽

The Road ◽

Product Titer ◽

Protein Group

ABSTRACT The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against several kinds of stress. PhaP enhances growth and poly(3-hydroxybutyrate) synthesis in polymer-producing recombinant strains and reduces the formation of inclusion bodies during overproduction of heterologous proteins. In this work, the heterologous expression of this phasin in E. coli was used as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which both GroELS and PhaP were observed to exert a beneficial effect that varied depending on the chemical tested. Lastly, the potential of PhaP and GroEL to enhance the accumulation of ethanol or 1,3-propanediol was analyzed in recombinant E. coli. Strains that overexpressed either groEL or phaP had increased growth, reflected in a higher final biomass and product titer than the control strain. Taken together, these results add a novel application to the already multifaceted phasin protein group, suggesting that expression of these proteins or other chaperones can be used to improve the production of biofuels and other chemicals. IMPORTANCE This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the PhaP phasin and the well-known chaperone GroEL were used to increase the biosynthesis of the biotechnologically relevant compounds ethanol and 1,3-propanediol in recombinant E. coli. These findings open the road for the use of these proteins for the manipulation of bacterial strains to optimize the synthesis of diverse bioproducts from renewable carbon sources.

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Overproduction of the Bacillus subtilis glutamyl-tRNA synthetase in its host and its toxicity to Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/w98-014 ◽

1998 ◽

Vol 44 (4) ◽

pp. 378-381 ◽

Cited By ~ 9

Author(s):

Martin Pelchat ◽

Lucille Lacoste ◽

Fu Yang ◽

Jacques Lapointe

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Recombinant Plasmid ◽

Specific Activity ◽

Shuttle Vector ◽

Fold Increase ◽

Trna Synthetase ◽

E Coli ◽

Downstream Region

The Bacillus subtilis glutamyl-tRNA synthetase (GluRS), encoded by the gltX gene, aminoacylates its homologous tRNAGlu and tRNAGln with glutamate. This gene was cloned with its sigmaA promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and B. subtilis. Transformation of B. subtilis with this recombinant plasmid (pMP411) led to a 30-fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation of E. coli with this plasmid gave no recombinants, but transformation with plasmids bearing an altered gltX was successful. These results indicate that the presence of B. subtilis glutamyl-tRNA synthetase is lethal for E. coli, probably because this enzyme glutamylates tRNA1Gln in vivo as it does in vitro.Key words: glutamyl-tRNA synthetase overproduction, Bacillus subtilis, toxicity, Escherichia coli.

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A Modular, Tn7-Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Applied and Environmental Microbiology ◽

10.1128/aem.01346-16 ◽

2016 ◽

Vol 82 (16) ◽

pp. 4931-4943 ◽

Cited By ~ 7

Author(s):

Dylan J. Shivak ◽

Keith D. MacKenzie ◽

Nikole L. Watson ◽

J. Alex Pasternak ◽

Brian D. Jones ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

High Throughput ◽

Constitutive Expression ◽

Bacterial Strains ◽

Efficient System ◽

Transposase Gene ◽

Content Type ◽

E Coli

ABSTRACTOur goal was to develop a robust tagging method that can be used to track bacterial strainsin vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies inSalmonellaandEscherichia coliand Tn7transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ70-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10−8to 10−10inSalmonella entericaserovar Typhimurium andE. coliAPEC O1, whereas the existing Tn7system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, andin vivoanimal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within theEnterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics.IMPORTANCEThis article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructsin vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system inSalmonellaandE. coli, and we predict that it will be widely applicable to other bacterial strains within theEnterobacteriaceae. This technology will allow for improvedin vivoanalysis of bacterial pathogens.

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Comparison of Carbon Nutrition for Pathogenic and Commensal Escherichia coli Strains in the Mouse Intestine

Infection and Immunity ◽

10.1128/iai.01386-07 ◽

2008 ◽

Vol 76 (3) ◽

pp. 1143-1152 ◽

Cited By ~ 216

Author(s):

Andrew J. Fabich ◽

Shari A. Jones ◽

Fatema Z. Chowdhury ◽

Amanda Cernosek ◽

April Anderson ◽

...

Keyword(s):

Escherichia Coli ◽

Single Cells ◽

Carbon Sources ◽

Bacterial Gene ◽

E Coli ◽

Acetylneuraminic Acid ◽

Plate Counts ◽

Mouse Intestine

ABSTRACT The carbon sources that support the growth of pathogenic Escherichia coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coli EDL933 grows primarily as single cells dispersed within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in vitro, using a mixture of the sugars known to be present in cecal mucus, and found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time. We conducted systematic mutation analyses of E. coli EDL933 and E. coli MG1655 by using lesions in the pathways used for catabolism of 13 mucus-derived sugars and five other compounds for which the corresponding bacterial gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic backgrounds was fed to streptomycin-treated mice, together with the respective wild-type parent strain, and their colonization was monitored by fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota.

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Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.66.6.2484-2490.2000 ◽

2000 ◽

Vol 66 (6) ◽

pp. 2484-2490 ◽

Cited By ~ 78

Author(s):

Hak Soo Seo ◽

Yeon Jong Koo ◽

Jae Yun Lim ◽

Jong Tae Song ◽

Chung Ho Kim ◽

...

Keyword(s):

Escherichia Coli ◽

Catalytic Efficiency ◽

Fold Increase ◽

Sequential Reaction ◽

E Coli ◽

Fusion Enzyme ◽

Bifunctional Fusion Enzyme ◽

Sequential Reactions

ABSTRACT To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The Km values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, thek cat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k cat/Km ). The Km and k cat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-β-d-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.

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Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽

10.1093/genetics/116.4.513 ◽

1987 ◽

Vol 116 (4) ◽

pp. 513-521

Author(s):

Nancy J Trun ◽

Thomas J Silhavy

Keyword(s):

Escherichia Coli ◽

Genetic Mapping ◽

Signal Sequence ◽

Illegitimate Recombination ◽

Mutant Phenotype ◽

E Coli ◽

Physical Location ◽

Sequence Deletion ◽

New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

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