Cloning and expression of L-asparaginase gene in Escherichia coli

The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72h of cultivation and 5h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.     

Dissolved oxygen concentration affects the accumulation of HIV-1 recombinant proteins in Escherichia coli

A central problem in aerobic growth of any culture is the maintenance of dissolved oxygen concentration (DOC) above growth-limiting levels especially in high-cell density fermentations that are usually of the fed-batch type. Fermentor studies have been conducted to determine the influence of DOC on the production of heterologous proteins in Escherichia coli. The results demonstrated that there is a significant degree of product-to-product variation in the response of heterologous protein accumulation to DOC. For translational fusions of the human immunodeficiency virus-1 (HIV-1) proteins p24Gag and Env41, the imposition of a dissolved oxygen (DO) limitation resulted in 100 and 15% increases in the respective product yields. On the other hand, the imposition of a DO limitation had no effect on the production of a similar translational fusion of the HIV-1 protein p55Gag, and a large negative effect on the production of an influenza protein (C13). The stimulatory effects of DOC on p24Gag production were investigated further. The results of my studies suggested that the stimulatory effect observed at reduced agitation rates on p24Gag accumulation was owing to an oxygen effect and not a shear effect. Furthermore, the results of my investigations indicated that the effect a DOC had on the production of p24Gag was strongly influenced by the cell density at which the culture was induced.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.     

The Action of the Selenomorpholine Compounds on Escherichia coli Growth by Microcalorimetry

The action of three kinds of the selenomorpholine compounds on a strain ofEscherichia coli was studied by microcalorimetry. Differences in their capacities to affect the metabolism of this bacterium were observed. The extent and duration of the effect on the metabolism as judged from the rate constant (k) of Escherichia coli (in log phase) varied with the different drugs. The kinetics show that selenomorpholine compounds had an effect on the metabolism process of Escherichia coli. The k of Escherichia coli in the presence of the drugs increased with the increasing concentrations of the drugs (C) at low concentration; but at high concentration, the rate constant decreased with the increasing concentrations of the drugs. The experimental results reveal that the sequence of antibiotic activity of selenomorpholines is: N-selenomorpholinemethyl succinimide and its hydrochloride>N-(α-selenomorpholinebenzyl) succinimide. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass

Two new ethanologenic strains (FBR4 and FBR5) of Escherichia coli were constructed and used to ferment corn fiber hydrolysate. The strains carry the plasmid pLO1297, which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically. Each culture was serially transferred 10 times in anaerobic culture with sugar-limited medium containing xylose, but noselective antibiotic. An average of 93 and 95% of the FBR4 and FBR5 cells, respectively, maintained pLO1297 in anaerobic culture. The fermentation performances of the repeatedly transferred cultures were compared with those of cultures freshly revived from stock in pH-controlled batch fermentations with 10% (w/v) xylose. Fermentation results were similar for all the cultures. Fermentations were completed within 60 h and ethanol yields were 86–92% of theoretical. Maximal ethanol concentrations were 3.9–4.2% (w/v). The strains were also tested for their ability to ferment corn fiber hydrolysate, which contained 8.5% (w/v) total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). E. coli FBR5 produced more ethanol than FBR4 from the corn fiber hydrolysate. E. coli FBR5 fermented all but 0.4% (w/v) of the available sugar, whereas strain FBR4 left 1.6% unconsumed. The fermentation with FBR5 was completed within 55 h and yielded 0.46 g of ethanol/g of available sugar, 90% of the maximum obtainable. Author to whom all correspondence and reprint requests should be addressed. Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA im plies no approval of the product to the exclusion of others that may also be suitable.     

Growth of Escherichia coli under extremely low-frequency electromagnetic fields

The inflence of extremely low-frequency (ELF) electromagnetic fields on Escherichia coli cultures in submerse fermentation was studied. The fermentation processes were carried out recycling the culture medium externally through a stainless steel tube inserted in a magnetic field generator (solenoid). The exposure time and electromagnetic induction were varied in a range of 1 to 12 h and 0.010 to 0.10 T, respectively, according to a Box-Wilson Central Composite Designs of face centered with five central points. Growth of E. coli could be altered (stimulated or inhibited) under magnetic fieldinduced effects. E. coli culturesexposed at 0.1 T during 6.5 h exhibited changes in its viability compared to unexposed cells, which was 100 times higher than the control. The magnetic field generator associated with the cellular suspension recycle is a new way of magnetic treatment in fermentation processes and could be appropriate to industrial scale up.     

Heat-shock and stringent responses have overlapping protease activity in Escherichia coli

The cellular response of a heat-shocked controlled chemostat of Escherichia coli JM105 [pSH101] was characterized and compared to that of a similar culture induced by isopropyl-β-d-thiogalactopyranoside (IPTG). The proteases elicited by the IPTG pulse were previously shown to be upregulated by the stringent stress response and were shown here to be upregulated by heat shock, although to a lesser extent. Owing to the apparent overlap between these responses, a relaxed mutant (rel ⁻, devoid of the stringent response; JM109) was examined for its response to both a chemically imposed stringent response and to IPTG induction in controlled chemostats. There was no significant upregulation of protease activity under either imposed stress. More important, a nine-fold increase of chloramphenicol acetyltransferase (CAT) activity was found for the IPTG-induced relaxed mutant culture. Additionally, the responses from heat shock and IPTG induction were examined in batch cultures. The culture that was simultaneously IPTG-induced and heat-shocked was observed to have the highest CAT activity as well as the most rapid loss in activity after a maximum. Control experiments indicated that the heat shock did not affect loss of CAT activity; instead, the loss of activity correlated with the amount of CAT synthesized. Furthermore, an increase in CAT expression was found during heat shock. Results indicated that heat shock and, alternatively, the use of stringent response-mutant hosts could both be used to facilitate increased recombinant protein yields in the E. coli expression system.     

Chromosome segregation in Escherichia coli division: a free energy-driven string model

Although the mechanisms of eukaryotic chromosome segregation and cell division have been elucidated to a certain extent, those for bacteria remain largely unknown. Here we present a computational string model for simulating the dynamics of Escherichia coli chromosome segregation. A novel thermal-average force field accounting for stretching, bending, volume exclusion, friction and random fluctuation is introduced. A Langevin equation is used to simulate the chromosome structural changes. The mechanism of chromosome segregation is thereby postulated as a result of free energy-driven structural optimization with replication introduced chromosomal mass increase. Predictions of the model agree well with observations of fluorescence labeled chromosome loci movement in living cells. The results demonstrate the possibility of a mechanism of chromosome segregation that does not involve cytoskeletal guidance or advanced apparatus in an E. coli cell. The model also shows that DNA condensation of locally compacted domains is a requirement for successful chromosome segregation. Simulations also imply that the shape-determining protein MreB may play a role in the segregation via modification of the membrane pressure.     

Study on eruption of heat for Escherichia coli B aroused by lanthanum nitrate and its mechanism

Biological effect of rare-earth lanthanum nitrate on the growth of Escherichia coli B was studied using the calorimetric method. There were exceptional changes on the growth thermogenic curves for high concentrations of lanthanum nitrate. For example, the peak high, the total quantity of heat (Q) of cultures and the growth rate constants (k) are evidently increased when compared with normal E. coli B cultures. When the concentration of lanthanum nitrate was at 300 mg/L and 500 mg/L, and Q of the cultures reached 3.89 and 2.54 times of normal cultures, respectively. The survivability of cells and the biomass of the cultures were measured using biological methods and the results show that the growth and multiplication of cells were inhibited and that the biomass decreased at high concentration of lanthanum nitrate. These revealed that the inhibiting cells discharged more quantity of heat than the normal growing cells. We named this phenomenon as “eruption of heat”. It was suggested that the mechanism for the eruption of heat was that La³⁺ ion damages the outer cell membrane and increases its permeability and the proton-electron potential energy across the cell membrane was reduced or couldn’t even be initiated. Energy could not be translated into ATP effectively in the course of oxidative phosphorylation resulting in heat release. So, the growth of the cells was inhibited due to scarceness of energy ATP. __________ Translated from Acta Chimica Sinica, 2007, 65(10): 917–922 [译自: 化学学报]     

Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli

An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.     

Effects of acetate on the growth and fermentation performance of Escherichia coli KO11

Escherichia coli KO11, in which the genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) encoding the ethanolpathway from Zymomonas mobili were inserted into the chromosome, has been shown to metabolize all major sugars that are consituents of hemicellulosic hydrolysates to ethanol, in anaerobic conditions. However, the growth and fermentation performance of this recombinant bacteria may be affected by acetic acid a potential inhibitor present in hemicellulose hydrolysates in a range of 2.0–15.0 g/L. It was observed that acetate affected the growth of E. coli KO11, prolonging the lag phase and inducing loss of biomass production and reduction of growth rate. At lower pH levels, the sensitivity to acetic acid was enhanced owing to the increased concentration of the protonated species. On the other hand, the recombinant bacteria showed a high tolerance to acetic acid regarding fermentative performance. In Luria broth medium with glucose or xylose as a single sugar source, it was observed that neither yield nor productivity was affected by the addition of acetate in a range of 2.0–12.0 g/L, suggesting some uncoupling of the growth vs ethanol production.     

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.     

Intracellular release of recombinant green fluorescent protein (gfp uv ) from Escherichia coli

The recombinant green fluorescent protein (gfp _uv ) was expressed by Escherichia coli DH5-α cells transformed with the plasmid pGFPuv. The gfp _uv was selectively permeabilized from the cells in buffer solution (25 mM Tris-HCl, pH 8.0), after freezing (−70°C for 15 h), by four freeze (−20°C)/thaw cycles interlaid by sonication. The average content of released gfp _uv (experiment 2) was 7.76, 34.58, 39.38, 12.90, and 5.38%, for the initial freezing (−70°C) and the first, second, third and fourth freeze/thaw cycles, respectively. Superfusion on freezing was observed between −11°C and −14°C, after which it reached −20°C at 0.83°C/min.     

Effect of ecological factors on conjugal transfer of chromium-resistant plasmid in Escherichia coli isolated from tannery effluent

The influence of total organic carbon (TOC), pH, and mating temperature on transfer of chromium-resistant plasmid between Escherichia coli strains in terms of variation in the number of transconjugants formed and variation in transfer frequency was investigated. In vitro transfer was studied in five chromate-tolerant E. coli strains isolated from tannery effluent using E. coli K12 J62 (Nal^r Lac⁻) as a recipient. Conjugal transfer of different selection markers was observed in three strains. The study was carried out in sterile wastewater. A gradual decrease was observed both in the number of transconjugants and in transfer frequencies as the concentration of TOC in the mating medium descended from 10,095 to 1.2 mg of C/L, obtaining the maximum values with a TOC concentration of 10,095 mg of C/L. The number of transconjugants and the transfer frequency were maximum at 30°C. However, neither the transfer frequency nor the transconjugant number varied significantly in the range of pHs assayed. The strains were also found resistant to different heavy metals and antibiotics. Curing of these strains resulted in loss of one or more resistance markers indicating the plasmid-borne resistance. It is inferred that plasmid transfer by conjugation occurs in wastewater bodies within a wide range of conditions.     

Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient

Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia entropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal σ⁷⁰ promotor. The pKS/PHB was the most efficient plasmid for phboperon expression among the three plasmids used: i.e., pKS⁻, pAED4, and pJM9131. It was observed that isopropyl-β-d-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkan cote content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.     

Construction of recombinant Escherichia coli strais for production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate)

Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC 19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry and medicine.     

Study on interaction between antibiotics and Escherichia coli DH5α by microcalorimetric method

A microcalorimetric technique based on the bacterial heat output was applied to evaluate the influence of antibiotics PIP (Piperacillin Sodium) and composite preparation of PIP and SBT (Sulbactam Sodium) on the growth of E. coli DH5α. The power–time curves of the growth metabolism of E. coli DH5α were studied using a TAM Air Isothermal Microcalorimeter at 37°C. By analyzing the power–time curves, the parameters such as growth rate constants (k), inhibitory ratio (I), the maximum heat power (P _m) and the time of the maximum heat power (t _m) were obtained. The results show that different concentrations of antibiotics affect the growth metabolism of E. coli DH5α. The PIP in the concentration range of 0–0.05 μg mL^–1 has a stimulatory effect on the E. coli DH5α growth, while the PIP of higher concentrations (0.05 –0.25 μg mL^–1) can inhibit its growth. It seems that the composite preparation composed of PIP and SBT cannot improve the inhibitory effect on E. coli DH5α as compared with the PIP.     

Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O181

Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E. coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E. coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The ¹H and ¹³C NMR chemical shift assignments of the OS were carried out using 1 D and 2 D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR ¹³C NMR glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.     

Electrophoretic Behavior Study of Bacteria of Pseudomonas aeruginosa, Edwardsiella tarda and Enteropathogenic Escherichia coli by Capillary Electrophoresis with UV and Fluorescence Detection

Capillary electrophoresis approaches have been utilized for the study of bacteria under specific experimental conditions. The main objective within our research work was to study electrophoretic behaviors of Pseudomonas aeruginosa by means of capillary electrophoresis with UV and fluorescence detection. Edwardsiella tarda and Enteropathogenic escherichia coli were also included in the study. The results showed that proper pretreatment (vortexing or sonication) for each bacterial sample before injection was necessary to disperse the clusters of cells, which is helpful to observe the single peaks and better peak shape of bacteria during electrophoresis. Apart from this, it was found that ionic strength of buffer affected mobilities of Pseudomonas aeruginosa as a result of increasing of buffer concentration from 25 mM to 150 mM. Moreover, sharp and single peaks were still observed without significant increase of noise in the concentration range. Eventually, mixtures of bacteria were well separated under optimized separation conditions with UV and fluorescence detection. In the mean time, comparison of concentration sensitivities for Pseudomonas aeruginosa by UV and fluorescence detection was made. Blue light emitting diode induced fluorescence detection was found to be more sensitive (8.5-fold higher) than UV detection with home-made fluorescence detection system. Generally, proposed CE methods for the analysis of bacteria could be potentially valuable for the monitoring of bacteria contamination in real life.