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1.
An Escherichia coli arsRp::luc-based biosensor was constructed to measure the bioavailability of arsenic (As) in soil. In previous induction experiments, it produced a linear response (R 2?=?0.96, P?<?0.01) to As from 0.05 to 5 μmol/L after a 2-h incubation. Then, both chemical sequential extraction, Community Bureau of Reference recommended sequential extraction procedures (BCR-SEPs) and E. coli biosensor, were employed to assess the impact of different long-term fertilization regimes containing N, NP, NPK, M (manure), and NPK?+?M treatments on the bioavailability of arsenic (As) in soil. Per the BCR-SEPs analysis, the application of M and M?+?NPK led to a significant (P?<?0.01) increase of exchangeable As (2–7 times and 2–5 times, respectively) and reducible As (1.5–2.5 times and 1.5–2.3 times, respectively) compared with the no fertilization treated soil (CK). In addition, direct contact assay of E. coli biosensor with soil particles also supported that bioavailable As in manure-fertilized (M and M?+?NPK) soil was significantly higher (P?<?0.01) than that in CK soil (7 and 9 times, respectively). Organic carbon may be the major factor governing the increase of bioavailable As. More significantly, E. coli biosensor-determined As was only 18.46–85.17 % of exchangeable As and 20.68–90.1 % of reducible As based on BCR-SEPs. In conclusion, NKP fertilization was recommended as a more suitable regime in As-polluted soil especially with high As concentration, and this E. coli arsRp::luc-based biosensor was a more realistic approach in assessing the bioavailability of As in soil since it would not overrate the risk of As to the environment.  相似文献   

2.
Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)4G-NH2] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M–S distances of 2.63 and 2.54 Å, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS2 bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)3 3? → M(II)(TRI LXC)3 ?. While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)2(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)3 ?, the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)2 + at pH < 5 followed by a two-proton dissociation step (pK a2) yielding M(II)(TRI LXC)3 ?. Pb(II)(TRI LXC)(HTRI LXC)2 + converts to Pb(II)(TRI LXC)3 ? at slightly lower pH values than the corresponding Cd(II)–peptide complexes. In addition, Pb(II) displays a lower pK a of binding to the “d”-substituted peptide, (TRI L12C, pK a2 = 12.0) compared with the “a”-substituted peptide, (TRI L16C, pK a2 = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K bind = 3.2 × 107 M?1) compared with that with TRI L16C (K bind = 1.2 × 107 M?1) at pH > 8.  相似文献   

3.
Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.  相似文献   

4.
5.
The R21(TC) factor, obtained by transduction of the R10(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R21(TC)+ transductants are infected with the F13 factor, the nontransferable R21(TC) factor acquires transmissibility by conjugation. R21(TC)+ conjugants of Escherichia coli K-12, to which only the R21(TC) factor was transmitted by cell-to-cell contact from an F′ R+ donor, were still unable to transfer their R21(TC) factor by conjugation. In crosses between Hfr and FE. coli K-12 strains containing R21(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.  相似文献   

6.
Uroporphyrinogen III (urogen III) was produced from 5-aminolevulinic acid (ALA), which is a common precursor of all metabolic tetrapyrroles, using thermostable ALA dehydratase (ALAD), porphobilinogen deaminase (PBGD), and urogen III synthase (UROS) of Thermus thermophilus HB8. The UROS-coding gene (hemD 2 ) of T. thermophilus HB8 was identified by examining the gene product for its ability to produce urogen III in a coupled reaction with ALAD and PBGD. The genes encoding ALAD, PBGD, and UROS were separately expressed in Escherichia coli BL21 (DE3). To inactivate indigenous mesophilic enzymes, the E. coli transformants were heated at 70 °C for 10 min. The bioconversion of ALA to urogen III was performed using a mixture of heat-treated E. coli transformants expressing ALAD, PBGD, and UROS at a cell ratio of 1:1:1. When the total cell concentration was 7.5 g/l, the mixture of heat-treated E. coli transformants could convert about 88 % 10 mM ALA to urogen III at 60 °C after 4 h. Since eight ALA molecules are required for the synthesis of one porphyrin molecule, approximately 1.1 mM (990 mg/l) urogen III was produced from 10 mM ALA. The present technology has great potential to supply urogen III for the biocatalytic production of vitamin B12.  相似文献   

7.

Background

Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility.

Results

To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library.

Conclusion

Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3 × improved relative to that of the initially constructed biosensor (29%).  相似文献   

8.
As mercury (Hg) biosensors are sensitive to only intracellular Hg, they are useful in the investigation of Hg uptake mechanisms and the effects of speciation on Hg bioavailability to microbes. In this study, bacterial biosensors were used to evaluate the roles that several transporters such as the glutathione, cystine/cysteine, and Mer transporters play in the uptake of Hg from Hg-thiol complexes by comparing uptake rates in strains with functioning transport systems to strains where these transporters had been knocked out by deletion of key genes. The Hg uptake into the biosensors was quantified based on the intracellular conversion of inorganic mercury (Hg(II)) to elemental mercury (Hg(0)) by the enzyme MerA. It was found that uptake of Hg from Hg-cysteine (Hg(CYS)2) and Hg-glutathione (Hg(GSH)2) complexes occurred at the same rate as that of inorganic complexes of Hg(II) into Escherichia coli strains with and without intact Mer transport systems. However, higher rates of Hg uptake were observed in the strain with a functioning Mer transport system. These results demonstrate that thiol-bound Hg is bioavailable to E. coli and that this bioavailability is higher in Hg-resistant bacteria with a complete Mer system than in non-resistant strains. No difference in the uptake rate of Hg from Hg(GSH)2 was observed in E. coli strains with or without functioning glutathione transport systems. There was also no difference in uptake rates between a wildtype Bacillus subtilis strain with a functioning cystine/cysteine transport system, and a mutant strain where this transport system had been knocked out. These results cast doubt on the viability of the hypothesis that the entire Hg-thiol complex is taken up into the cell by a thiol transporter. It is more likely that the Hg in the Hg-thiol complex is transferred to a transport protein on the cell membrane and is subsequently internalized.  相似文献   

9.
The complete nucleotide sequence of a small cryptic plasmid pKST21 from Escherichia coli was determined. This plasmid is 1,460 bp long with an overall GC content of 51 %. Based on sequence analysis, the presence of two segments with different average GC density was observed. The segment with higher GC content revealed 98–90 % similarity to several small plasmids of E. coli and to pCR1 from Gram-positive Corynebacterium renale. Plasmid pKST21 possesses two conversely oriented open reading frames encoding proteins with a high degree of amino acid identity to Rep proteins involved in replication. ORF1 encodes replication protein similar to RepA protein of Bartonella tribocorum or Bacillus cereus plasmids or to the putative plasmid Rep protein from ecologically close Selenomonas ruminantium. ORF2 similarly encodes a replication protein, which shares 97 % homology with Rep protein from C. renale. Genetic diversity observed in plasmid pKST21 indicates a mosaic structure of the plasmid with different segments acquired from different sources. Deletion analysis showed that both fragments carrying the repA and repB genes are necessary for the replication of pKST21 in E. coli. The presence of plasmid with the same gene composition was revealed in 14 % of tested E. coli isolates from the rumen of sheep. All these strains produced identical ERIC-PCR profiles indicating isogenic origin of the strain and lack of horizontal gene transfer of pKST21 plasmid.  相似文献   

10.
We report the high-yield heterologous expression of bioactive θ-defensin RTD-1 inside Escherichia coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. RTD-1 is a small backbone-cyclized polypeptide with three disulfide bridges and a natural inhibitor of anthrax lethal factor protease. Recombinant RTD-1 was natively folded and able to inhibit anthrax lethal factor protease. In-cell expression of RTD-1 was very efficient and yielded ≈0.7 mg of folded RTD-1 per gram of wet E. coli cells. This approach was used to generate of a genetically-encoded RTD-1-based peptide library in live E. coli cells. These results clearly demonstrate the possibility of using genetically-encoded RTD-1-based peptide libraries in live E. coli cells, which is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide RTD-1 as a molecular scaffold.  相似文献   

11.
The benzoylformate decarboxylase gene (mdlC) from Pseudomonas putida was expressed in Escherichia coli BL21(DE3). The recombinant strain together with E. coli/pET30a-mdlB converted (S)-3-ethoxy-4-hydroxymandelic acid (S-EMA) into ethyl vanillin without ethyl vanillin degradation. 4 g ethyl vanillin/l was obtained from 10 g EMA/l within 12 h at 30 °C. This is the first report on the biotransformation of (S)-EMA to ethyl vanillin.  相似文献   

12.
The biological production of 3-hydroxypropionic acid (3-HP) has attracted significant attention because of its industrial importance. The low titer, yield and productivity, all of which are related directly or indirectly to the toxicity of 3-HP, have limited the commercial production of 3-HP. The aim of this study was to identify and select a 3-HP tolerant Escherichia coli strain among nine strains reported to produce various organic acids efficiently at high titer. When transformed with heterologous glycerol dehydratase, reactivase and aldehyde dehydrogenase, all nine E. coli strains produced 3-HP from glycerol but the level of 3-HP production, protein expression and activities of the important enzymes differed significantly according to the strain. Two E. coli strains, W3110 and W, showed higher levels of growth than the others in the presence of 25 g/L 3-HP. In the glycerol fed-batch bioreactor experiments, the recombinant E. coli W produced a high level of 3-HP at 460 ± 10 mM (41.5 ± 1.1 g/L) in 48 h with a yield of 31 % and a productivity of 0.86 ± 0.05 g/L h. In contrast, the recombinant E. coli W3110 produced only 180 ± 8.5 mM 3-HP (15.3 ± 0.8 g/L) in 48 h with a yield and productivity of 26 % and 0.36 ± 0.02 g/L h, respectively. This shows that the tolerance to and the production of 3-HP differ significantly among the well-known, similar strains of E. coli. The titer and productivity obtained with E. coli W were the highest reported thus far for the biological production of 3-HP from glycerol by E. coli.  相似文献   

13.
Natural and modified nucleoside-5′-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.  相似文献   

14.
A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na2, 0.5 mM ATP, 5 mM Mg2+, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.  相似文献   

15.
A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester. The top layer copolymer was 47 μm thick whereas the bottom layer of copolymer plus E. coli cells was 30 μm thick. The immobilized E. coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II). The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range. The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl2 in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal. Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl2. Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture. The copolymer patches with immobilized cells were stable at −20°C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating. Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl2. Received 05 November 1998/ Accepted in revised form 08 April 1999  相似文献   

16.
A yeast strain MJ2 that was found to produce a higher amount of γ-aminobutyric acid (GABA) was isolated from the surface of kiwi. Phylogenetic analysis based on the ITS sequence and morphological, biochemical studies indicated that it may belong to Saccharomyces cerevisiae. Under optimum conditions in Czapek’s broth medium with 0.5 % monosodium glutamate, it produced GABA at a concentration of 5.823 g/L after 48 h. A full-length glutamate decarboxylase gene (Scgad) was cloned by PCR amplification. The open reading frame (ORF) of the Scgad gene was composed of 1,755 nucleotides and encoded a protein (585 amino acids) with a predicted molecular weight of 65.897 kDa. The deduced amino acids sequence of Scgad shows 100 %, 65 % and 62 % similarity with S. cerevisiae, Candida glabrata and Kluyveromyces lactis GAD in the polypeptide level, respectively. The Scgad gene was expressed in Escherichia coli BL21 (DE3) cells, and the expression was confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. The results suggested that the S. cerevisiae GAD (ScGAD) was successfully encoded in E. coli BL21 (DE3) cells. Furthermore, the enzyme activity of ScGAD encoded in E. coli BL21 (DE3) had been significantly enhanced using artificial neural network linked with genetic algorithm (ANN-GA) method.  相似文献   

17.
Concentrations of toxic metals viz. mercury (Hg), cadmium (Cd) and lead (Pb) were evaluated in four species of fishes (Sardinella longiceps, Selaroides leptolepis, Epinephelus quoyanus and Lethrinus lentjan), one species of shrimp (Penaeus semisulcatus) and one species of crab (Portunus sanguinolentus) sampled from Thoothukudi, Keelakarai and Veerapandian pattinam of Gulf of Mannar, Southeast coast of India. Results revealed accumulation of these metals in the following order Hg > Cd > Pb. Hg concentration was found to be higher in Po. sanguinolentus followed by E. quoyanus, Pe. semisulcatus and L. lentjan however, the same was absent in Sa. longiceps and Se. leptolepis. Cd concentration was recorded in decreasing order in Po. sanguinolentus > Pe. semisulcatus > L. lentjen > E. quoyanus > Sa. longiceps > Se. leptolepis. Pb was detectable only in four species. Results of One-way ANOVA revealed significant variations (p < 0.05) in accumulation of Cd in Sa. longiceps, Se. leptolepis and Pe. semisulcatus and Hg in E. quoyanus, L. lentjan and Po. sanguinolentus. Variations noted in Pb were not statistically significant throughout.  相似文献   

18.
To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11 mg/mL) of 32a-ManAR2 was higher than that (1.46 mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7 ml/mg s. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25 °C), lower IPTG concentration (0.01–0.05 mM), and Triton X-100 supplement (0.1 %) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10 L fermentor. The productivity of the total soluble mannanase reached 9284.64 U/mL with the extracellular rate of 74 % at 46 h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date.  相似文献   

19.
In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD600 of 2–10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD600 of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al2O3 as catalyst in toluene with the yield of 96 %.  相似文献   

20.
A recombinant arginase was generated for a whole-cell biotransformation system to convert l-arginine to l-ornithine in Escherichia coli. The gene ARG1 coding arginase from Bos taurus liver was synthesized and expressed in E. coli BL21 (DE3) via pETDuet-1. The recombinant arginase was used to catalyze l-arginine to l-ornithine and urea. The reaction was optimal at pH 9.5 and 37 °C. Manganese (10?5 M) and Emulsifier OP-10 [0.033 % (v/v)] could promote arginase activity. In a scale up study, l-arginine conversion rate reached 98 % with a final concentration of 111.52 g l-ornithine/l.  相似文献   

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