High-level poly(β-hydroxybutyrate) production in recombinantEscherichia coliin sugar-free, complex medium

1995 ◽  
Vol 41 (13) ◽  
pp. 216-221 ◽  
Author(s):  
S. Kalousek ◽  
W. Lubitz
Keyword(s):  
Transcriptional Control ◽  
Promoter Sequence ◽  
Dry Mass ◽  
Luria Bertani ◽  
Translational Initiation ◽  
E Coli ◽  
Phb Production ◽  
In The Wild ◽  
Lac Promoter ◽  
Phbc Gene

The poly(β-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50–80% PHB/cell dry mass when the cells are grown in Luria–Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria–Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the β-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter–operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria–Bertani medium without glucose supplement.Key words: PHB production, engineered phbCAB operon.

scholarly journals Enhanced Functional Expression of the Polyhydroxyalkanoate Synthase Gene from Cupriavidus Necator A-04 Using a Cold-Shock Promoter for Efficient Poly(3-Hydroxybutyrate) Production in Escherichia Coli

2020 ◽  
Author(s):  
Thanawat Boontip ◽  
Rungaroon Waditee-Sirisattha ◽  
Kohsuke Honda ◽  
Suchada Chanprateep Napathorn
Keyword(s):  
Sequence Similarity ◽  
Constitutive Expression ◽  
Functional Expression ◽  
Dry Mass ◽  
Cupriavidus Necator ◽  
Yield Coefficient ◽  
16S Rrna Sequence ◽  
E Coli ◽  
Phb Production ◽  
Low Expression

Abstract Background: The present study attempted to increase PHB production by improving the functional expression of the PhaC gene using various types of promoters, and the effects on PhaC activity in terms of PHB productivity, yield coefficient (YP/S) and molecular weights were investigated.Results: Here, the PHB biosynthesis operon of Cupriavidus necator A-04, isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO and pUC19 (constitutive expression) and transformed into E. coli JM109. To alter the expression of phaCAB biosynthesis genes, we optimized parameters in flask experiments to obtain high expression of soluble PhaCA−04 protein with high YP/S and PHB productivity. pColdTF-phaCABA−04-expressing E. coli produced 2.5 ± 0.1 g/L (90.6±4.3%) PHB in 24 h, similar to pColdI-phaCABA−04-expressing E. coli. The amounts of phaC protein and PHB produced from pColdTF-phaCABA−04 and pColdI-phaCABA−04 were significantly higher than those from other promoters. Cultivation in a 5-L fermenter led to PHB production of 7.9±0.7 g/L with 90.0±2.3% PHB content in the cell dry mass (DCM), a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.26 g PHB/(L⋅h) using pColdTF-phaCABA−04. The PHB from pColdTF-phaCABA−04 had MW 5.79 × 105 Da, MN 1.86 × 105 Da and PDI 3.11 and the film exhibited high transparency, Young’s modulus and tensile strength, possibly due to the TF chaperones. Interestingly, when pColdI-phaCABA−04-expressing E. coli was used to produce PHB from crude glycerol and compared with constitutive pUC19-nativeP-phaCABA−04-expressing E. coli, the amounts of PHB were similar, but MW 1.1 × 106 Da, MN 2.6 × 105 Da and PDI 4.1 were obtained from constitutive pUC19-nativeP-phaCABA−04-expressing E. coli, indicating that slow and low expression could prolong and maintain phaC polymerization activity.Conclusions: The cspA promoter in a cold-inducible vector can improve PhaCA−04 expression levels, and TF chaperones show obvious effects on enhancing PhaCA−04 solubility. The high level of phaCA−04 resulted in a high amount of PHB, but the chain termination reaction of PhaC polymerization occurred faster than that with the retarded and low expression of phaCA−04 by the constitutive promoter pUC19, which in turn resulted in a low amount of PHB with a high molecular weight.

scholarly journals Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Suchada Chanprateep Napathorn ◽  
Sirirat Visetkoop ◽  
Onruthai Pinyakong ◽  
Kenji Okano ◽  
Kohsuke Honda
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Dry Mass ◽  
Inducible Expression ◽  
Rrna Gene ◽  
E Coli ◽  
Inducible Expression System ◽  
Phb Production ◽  
Phb Synthase ◽  
Coa Reductase

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA–04 and pColdTF-phaCABA–04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA–04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA–04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 534-537
Author(s):  
S Mitra ◽  
B C Pal ◽  
R S Foote
Keyword(s):  
Escherichia Coli ◽  
Dna Methyltransferase ◽  
Wild Type ◽  
E Coli ◽  
Methylguanine Dna Methyltransferase ◽  
Synthetic Dna ◽  
In The Wild ◽  
Low Levels ◽  
Enzyme Molecules ◽  
Dna Substrate

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

Effect offurmutation on acid-tolerance response and in vivo virulence of avian septicemicEscherichia coli

2002 ◽  
Vol 48 (5) ◽  
pp. 458-462 ◽  
Author(s):  
Chengru Zhu ◽  
Musangu Ngeleka ◽  
Andrew A Potter ◽  
Brenda J Allan
Keyword(s):  
Parent Strain ◽  
Acid Tolerance ◽  
Wild Type ◽  
Acid Tolerance Response ◽  
E Coli ◽  
Regulator Protein ◽  
In The Wild ◽  
Tolerance Response ◽  
Wild Type Parent

The Fur (ferric uptake regulator) protein is a master regulator of iron metabolism in gram-negative bacteria. In the present study, the effect of a partial deletion of the fur gene on the acid-tolerance response and in vivo virulence of avian Escherichia coli was examined. The fur mutant was unable to trigger the acid-tolerance response as observed in the wild-type parent strain. However, the mutant was as virulent as the wild-type parent strain when tested in 1-day-old chickens by subcutaneous inoculation. These data indicate that the fur gene is involved in the acid-tolerance response but not involved in the virulence of E. coli, as detected by the ability to cause septicemia in our experimental infection.Key words: E. coli, fur, acid-tolerance response.

scholarly journals Photothermal-assisted antibacterial application of GO-Ag against clinical isolated MDR E. coli

2019 ◽  
Author(s):  
Yuqing Chen ◽  
Wei Wu ◽  
Zeqiao Xu ◽  
Cheng Jiang ◽  
Shuang Han ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Near Infrared ◽  
Antibacterial Effect ◽  
Luria Bertani ◽  
Fluorescent Imaging ◽  
Photothermal Effect ◽  
Antibacterial Efficiency ◽  
E Coli ◽  
Morphology Characterization

Abstract Background: Treatment of multidrug-resistant (MDR) bacterial infection is a great challenge in public health. Herein, we provide a solution to this problem with the use of graphene oxide-silver (GO-Ag) nanocomposites as anti-bacterial agent. Methods: Following established protocols, silver nanoparticles were grown on graphene oxide sheets. Then, a series of in-vitro studies were conducted to validate the antibacterial efficiency of the GO-Ag nanocomposites against clinical MDR Escherichia coli (E. coli) strains. Firstly, minimum inhibitory concentrations (MICs) of different antimicrobials were tested against MDR E. Coli strains. Then, bacteria viability assessments were conducted with different nanomaterials in Luria-Bertani (LB) broth. Afterwards, photothermal irradiation was conducted on MDR E. coli with lower GO-Ag concentration. At last, fluorescent imaging and morphology characterization using scanning electron microscope (SEM) were done to find the possible cause of antibacterial effect. Results: GO-Ag nanocomposites showed the highest antibacterial efficiency among tested antimicrobials. Synergetic antibacterial effect was observed in GO-Ag nanocomposites treated group. The remained bacteria viabilities were 4.4% and 4.1% respectively for different bacteria strains with GO-Ag concentration at 14.0 µg mL-1. In addition, GO-Ag nanocomposites have strong absorption in the near-infrared field and can convert the electromagnetic energy to heat. With the use of this photothermal effect, effective sterilization could be achieved using GO-Ag nanocomposites concentration as low as 7.0 µg mL-1. Fluorescent imaging and morphology characterization were used to analyze bacteria living status, which uncovered that bacteria integrity was disrupted after GO-Ag nanocomposites treatment. Conclusions: GO-Ag nanocomposites are proved to be efficient antibacterial agent against multi-drug resistant E. coli. Their strong antibacterial effect arises from inherent antibacterial property and photothermal effect that provides aid for bacteria killing.

scholarly journals Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

2005 ◽  
Vol 187 (20) ◽  
pp. 6928-6935 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Haemophilus Influenzae ◽  
Control Region ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Class I ◽  
E Coli ◽  
Fnr Protein

ABSTRACT Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions −64.5 and −44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position −60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions −81.5 and −42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

CRISPR-Cas9 knockout of qseB induced asynchrony between motility and biofilm formation in Escherichia coli

2019 ◽  
Vol 65 (9) ◽  
pp. 691-702 ◽  
Author(s):  
Yi Gou ◽  
Weiqi Liu ◽  
Jing Jing Wang ◽  
Ling Tan ◽  
Bin Hong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Motility ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Signal Transmission ◽  
Bacterial Motility ◽  
Type I ◽  
E Coli ◽  
Real Time Quantitative Pcr ◽  
In The Wild

Generally, cell motility and biofilm formation are tightly regulated. The QseBC two-component system (TCS) serves as a bridge for bacterial signal transmission, in which the protein QseB acts as a response regulator bacterial motility, biofilm formation, and virulence. The mechanisms that govern the interaction between QseBC and their functions have been studied in general, but the regulatory role of QseB on bacterial motility and biofilm formation is unknown. In this study, the CRISPR-Cas9 system was used to construct the Escherichia coli MG1655ΔqseB strain (strain ΔqseB), and the effects of the qseB gene on changes in motility and biofilm formation in the wild type (WT) were determined. The motility assay results showed that the ΔqseB strain had higher (p < 0.05) motility than the WT strain. However, there was no difference in the formation of biofilm between the ΔqseB and WT strains. Real-time quantitative PCR illustrated that deletion of qseB in the WT strain downregulated expression of the type I pili gene fimA. Therefore, we might conclude that the ΔqseB induced the downregulation of fimA, which led to asynchrony between motility and biofilm formation in E. coli, providing new insight into the functional importance of QseB in regulating cell motility and biofilm formation.

scholarly journals Thymol tolerance in Escherichia coli induces morphological, metabolic and genetic changes

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Fatemah Al-Kandari ◽  
Rabeah Al-Temaimi ◽  
Arnoud H. M. van Vliet ◽  
Martin J. Woodward
Keyword(s):  
Antimicrobial Activity ◽  
Efflux Pump ◽  
Wide Spectrum ◽  
Genetic Changes ◽  
E Coli ◽  
Nmr Data ◽  
Stop Mutation ◽  
In The Wild ◽  
Cellular Integrity ◽  
Acrab Efflux Pump

Abstract Background Thymol is a phenolic compound used for its wide spectrum antimicrobial activity. There is a limited understanding of the antimicrobial mechanisms underlying thymol activity. To investigate this, E. coli strain JM109 was exposed to thymol at sub-lethal concentrations and after 16 rounds of exposure, isolates with a 2-fold increased minimal inhibitory concentration (MIC) were recovered (JM109-Thyr). The phenotype was stable after multiple sub-cultures without thymol. Results Cell morphology studies by scanning electron microscopy (SEM) suggest that thymol renders bacterial cell membranes permeable and disrupts cellular integrity. 1H Nuclear magnetic resonance (NMR) data showed an increase in lactate and the lactic acid family amino acids in the wild type and JM109-Thyr in the presence of thymol, indicating a shift from aerobic respiration to fermentation. Sequencing of JM109-Thyr defined multiple mutations including a stop mutation in the acrR gene resulting in a truncation of the repressor of the AcrAB efflux pump. AcrAB is a multiprotein complex traversing the cytoplasmic and outer membrane, and is involved in antibiotic clearance. Conclusions Our data suggests that thymol tolerance in E. coli induces morphological, metabolic and genetic changes to adapt to thymol antimicrobial activity.

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