High cell density fed-batch fermentation for the production of recombinant <Emphasis Type="Italic">E. coli</Emphasis> K-12 ghost vaccine against streptococcal disease

The bacterial ghost system is a novel vaccine delivery method which provides versatile carrier functions for foreign antigens with excellent natural intrinsic adjuvant properties. In this study, ghost bacteria of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM were created for mass production of a Streptococcus iniae ghost vaccine. The optimal fed-batch process for high cell density culture of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM was developed using the nutrient feeding strategy with Riesenberg defined medium. Fermentation was conducted in four phases as follow: (1) initial batch phase, (2) fed-batch phase for high cell density culture, (3) thermal induction phase for the formation of ghost by the expression of lysis gene E, and (4) high temperature holding phase to increase ghost formation efficiency. The maximum ghost bacteria vaccine (GBV) was obtained from the fed-batch fermentation of 34.9 g dry cell weight (dcw)/L. The expression of antigen glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the ghost cell with a high temperature holding phase was confirmed with outer-membrane protein fractionation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results indicate no damage to the expressed antigen on the ghost cell surfaces even after the temperature was increased to 47°C for high efficiency ghost cell formation. Efficacy of the GBV was evaluated by the challenge test in which vaccinated Olive flounder were infected with live S. iniae. The E. coli K-12 host strain, E. coli K-12/pHCE vector control, and formalin-killed cell (FKC) -treated vaccine groups showed 100, 100, and 65% cumulative mortality, respectively. The GBV-treated groups showed 50% cumulative mortality with increased survival ratios. Hence, the immunoprotective efficacy of GBV against S. iniae was better than that of the FKC vaccine. Therefore, the GBV is proposed as an effective vaccine in aquaculture for the prevention of streptococcal disease.     

Effects of temperature shift strategies on human preproinsulin production in the fed-batch fermentation of recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis>

Preproinsulin is a well-known precursor of human insulin for the regulation of blood glucose levels. In this study, fed-batch fermentations of recombinantEscherichia coli JM109/pPT-MRpi were carried out for the overexpression of human preproinsulin. The expression of human preproinsulin was controlled by the temperature inducibleP2 promoter. The time-course profiles of fed-batch fermentation and SDS-PAGE analysis showed that human insulin expression was triggered by a culture temperature change from 30 to 37°C. Fermentation shift strategies, including the multi-step increase of temperature and the modulation of initiation time, were optimized to obtain high titers of cell mass and preproinsulin. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from 30 to 37°C for 2 h, gave the best results of 43.1 g/L of dry cell weight and 33.3% preproinsulin content, which corresponded to 2.0- and 1.2-fold increases, respectively, as compared to those of fed-batch culture at a constant temperature of 37°C.     

Effect of Sucrose Concentration on the Infectivity of ϕX 174 DNA to Protoplast of Escherichia coli

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS–PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg⁺², ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 μM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.     

Fimbriae and lipopolysaccharides are necessary for co-aggregation between Lactobacilli and Escherichia coli

Cells of Lactobacilli co-aggregated with Escherichia coli K-12 cells to form co-aggregates under mixed-culture conditions at 37?°C for 24?h. Co-aggregation was inhibited by sodium dodecyl sulfate but not by protease. E. coli deletion mutants of fimbriae formation and lipopolysaccharide (LPS) formation did not co-aggregate with Lactobacilli. These results showed that fimbriae and LPS are necessary for co-aggregation between Lactobacilli and E. coli.     

Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding

The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-d-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.     

Effect of oxygen limitation and starvation on the benzalkonium chloride susceptibility of Escherichia coli

Aims: To investigate the effect of oxygen limitation, glucose-starvation and temperature on the susceptibility of Escherichia coli towards the quaternary ammonium biocide benzalkonium chloride (BAC). Methods and Results: The effect of BAC on planktonic and sessile cells were investigated using the gfp-tagged E. coli K-12 strain MG1655[pOX38Km]. Increasing temperature from 10°C to 30°C increased the bactericidal effect of BAC for both starved and nonstarved E. coli under aerobic and anaerobic conditions. The lowest minimum bactericidal concentration was observed for cells in anaerobic media at 30°C (30 mg l⁻¹ BAC). Decreasing cell densities increased the decay rate for BAC-exposed cells for both starved and nonstarved E. coli. Biofilms of E. coli exposed to BAC in anaerobic medium showed a greater percentage of membrane-compromised cells than biofilms grown in aerobic medium. Image analyses of BAC-exposed biofilms showed that membrane-compromised cells were occasionally located in the interior structure of the biofilm microcolonies. Conclusions: Increasing temperatures and the absence of oxygen, and energy substrates increased the antimicrobial effect of BAC towards E. coli. Significance and Impact of the Study: The results are relevant for understanding the disinfection efficacy of quaternary ammonium compounds towards planktonic and sessile bacteria.     

Production of recombinant ghost bacterial vaccine against streptococcal disease of olive flounder

The simple fed-batch fermentation was carried out to produce E. coli XL1-Blue/pHCE-InaN-GAPDH Ghost 37 SDM as a ghost bacterial vaccine (GBV). The fermentation was carried out in four phases of batch fermentation (phase 1), fed-batch fermentation with intermittent feeding strategy (phase 2), thermal induction by temperature increase to 42 °C for the expression of lysis E gene (GBV formation, phase 3) and high temperature holding phase to increase the efficiency of GBV formation (phase 4). After the high temperature holding phase at 47 °C, efficiency of the GBV formation reached 99.7% with the culture OD₆₀₀ of 57.9. The maximum GBV of 22 g dcw/l was obtained. The protective efficacy of GBV was determined by a challenge test to immunized olive flounder using live Streptococcus iniae. In 14 days of challenge test, the positive and E. coli strain control groups showed 100% cumulative mortalities. Test groups immunized by formalin killed cell (FKC) vaccine, GBV with 42 °C and 47 °C heat shock showed 66%, 54% and 54% of cumulative mortalities, respectively. These results suggest that GBV showed the effectiveness for the protection from the streptococcal infection and had higher potential to induce protective antibodies than FKC vaccine.     

Characterization of a recombinant cold-adapted purine nucleoside phosphorylase and its application in ribavirin bioconversion

Ribavirin is a broad-spectrum antiviral drug and can be produced by enzymatic synthesis by purine nucleoside phosphorylase (PNP). In this study, we describe the application of such a cold-adapted XmPNP in ribavirin bioconversion which showed approximately 15°C lower optimum temperature and 1.80-fold higher catalytic efficiency (k_cat/K_m) at 37°C within substrate inosine than homolog in E. coli. By contrast, E. coli (XmPNP) took only 12 h to reach maximum substrate conversion rate (70%) under its optimum temperature (50°C) by using recombinant strain cell as enzyme source, but E. coli (EcPNP) did at 24 h. These results suggest cold-adapted PNP is one attractive candidate for ribavirin bioconversion and other nucleoside medications to improve the catalytic efficiency.     

Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli

Aims: The cell‐surface display of Cex, which encodes xylanase and exoglucanase from Cellulomonas fimi, was constructed on Escherichia coli using PgsA as the anchor protein. Characterization of the cell‐surface display of Cex was performed. Methods and Results: PgsA was fused to the N‐terminus of Cex and six histidines were utilized as spacers between the targeting and anchor proteins. Successful cell‐surface display of Cex was demonstrated by Western blot and immunofluorescence analyses on E. coli C41 (DE3). According to the time‐course analysis, the xylanase activity of Cex was achieved at 49 U g^?1 dry cell weight after 12 h culture at 37°C. The optimal temperature and pH ranges of the cell‐surface displayed protein with whole‐cell were broader than the corresponding ranges of the purified form. Further determination of thermostability indicated that the half‐life of cell‐surface displayed Cex was 1·6 times longer than that of purified Cex at 60°C. Conclusions: We have successfully developed the cell‐surface display of xylanase on E. coli. The cell‐surface display can enhance the stability of xylanase against changes in temperature and has the potential of becoming a whole‐cell biocatalyst for industrial applications, such as biobleaching of paper and production of renewable energy. Significance and Impact of the Study: The results demonstrated that the cell‐surface display of xylanase embedded in the cell membrane is more stable than that of the purified enzyme. Thus, to improve the stability of heterologous proteins production, cell‐surface display using the PgsA anchor protein as a tool can be considered in E. coli.     

Advanced tool for characterization of microbial cultures by combining cytomics and proteomics

Flow cytometry approaches are applicable to recover sub-populations of microbial cultures in a purified form. To examine the characteristics of each sorted cell population, Omics technologies can be used for comprehensively monitoring cellular physiology, adaptation reactions, and regulated processes. In this study, we combined flow cytometry and gel-free proteomic analysis to investigate an artificial mixed bacterial culture consisting of Escherichia coli K-12 and Pseudomonas putida KT2440. Therefore, a filter-based device technique and an on-membrane digestion protocol were combined in conjunction with liquid chromatography and mass spectrometry. This combination enabled us to identify 903 proteins from sorted E. coli K-12 and 867 proteins from sorted P. putida KT2440 bacteria from only 5 × 10⁶ cells of each. Comparative proteomic analysis of sorted and non-sorted samples was done to prove that sorting did not significantly influence the bacterial proteome profile. We further investigated the physicochemical properties, namely M _r, pI, hydropathicity, and transmembrane helices of the proteins covered. The on-membrane digestion protocol applied did not require conventional detergents or urea, but exhibited similar recovery of all protein classes as established protocols with non-sorted bacterial samples.     

Interactions between Escherichia coli and the plant‐parasitic nematode Meloidogyne javanica

Aims: To determine the potential of the plant‐parasitic nematode Meloidogyne javanica to serve as a temporary reservoir for Escherichia coli. Methods and Results: The adhesion to and persistence of E. coli on the surface of M. javanica were evaluated at different times and temperatures. A pure culture of green fluorescent protein (GFP) tagged E. coli was mixed with ca. 1000 J2 M. javanica for 2 h at 25°C. The nematodes were then washed and the rate of the adhesion of the bacteria to the nematodes was determined by counting the viable nematode‐associated E. coli, and by fluorescence microscopy. A dose‐dependent adhesion rate was observed only at a bacterium to nematode ratio of 10⁴–10⁶ : 1. The adhesion of E. coli to the nematodes was also tested over a 24 h‐period at 4°C, 25°C and 37°C. At 4°C and 37°C, maximal adhesion was observed at 5 h; whereas at 25°C, maximal adherence was observed at 8 h. Survival experiments showed that the bacteria could be detected on the nematodes for up to 2 weeks when incubated at 4°C and 25°C, but not at 37°C. Conclusions: Under laboratory conditions, at 4°C and 25°C, M. javanica could serve as a temporary vector for E. coli for up to 2 weeks. Significance and Impact of the Study: These findings support the hypothesis that, in the presence of high concentrations of E. coli, M. javanica might serve as a potential vehicle for the transmission of food‐borne pathogens.     

Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi

Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5° to 20°C or 30°C and from 28° to 34°C. The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature. The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams. The rates of synthesis, after transfer of cells from 5° to 30°C, 5° to 20°C, and 28° to 34°C, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively. Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins. From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E. coli CSP [15], the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly. Received: 22 November 1995 / Accepted: 22 December 1995     

Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

Background  

The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30°C as compared to 37°C in E. coli K12 hosts. Since OmpT levels are higher at 37°C than at 30°C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold.     

Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in <Emphasis Type="BoldItalic">Escherichia coli</Emphasis>

A plant-specific biogenic amine, serotonin, was produced by heterologous expression of two key biosynthetic genes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), in Escherichia coli. The native T5H, a cytochrome P450 enzyme, was unable to be functionally expressed in E. coli. Through a series of N-terminal deletions or additions of tagging proteins, we generated a functional T5H enzyme construct (GST∆37T5H) in which glutathione S transferase (GST) was translationally fused with the N-terminal 37 amino acid deleted T5H. Dual expression of GST∆37T5H and TDC using a pCOLADuet-1 E. coli vector produced serotonin at concentrations of approximately 24 mg l⁻¹ in the culture medium and 4 mg l⁻¹ in the cells. An optimum temperature of approximately 20°C was required to achieve peak serotonin production in E. coli because the low induction temperature gave rise to the highest soluble expression of GST∆37T5H.     

The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid

Shufflon DNA rearrangement selects one of seven PilV proteins with different C-terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64. The PilV proteins determine the recipient specificity in liquid matings. A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA′, -C, and -C′ proteins, while E. coli B was recognized only by the PilVA′ protein. To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E. coli K-12 waa (rfa) mutants and E. coli B transformants as recipient cells. E. coli K-12 waa mutants lack receptors for specific PilV proteins. E. coli B cells carrying waaJ or waaJKL genes of E. coli K-12 were recognized by donors expressing the PilVC′ protein or the PilVC and -C′ proteins, respectively, in addition to the PilVA′ protein. Addition of E. coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings. We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. Received: 2 September 1999 / Accepted: 18 November 1999     

Production of hepatitis B core antigen in a stirred tank bioreactor: The influence of temperature and agitation

The influence of temperature and agitation on the growth ofEscherichia coli expressing hepatitis B core antigen (HBcAg) in stirred tank bioreactor were investigated. The highest specific growth rate forE. coli (0.844 h⁻¹) was achieved at a temperature of 37°C and an agitation speed of 250 rpm. The activation energy for the growth of theE. coli strain W3110IQ in the stirred tank bioreactor was estimated to be 11 kcal/mol. The highest protein yield was achieved at a temperature of 44°C and an agitation speed of 250 rpm. The relative protein concentration at 44°C is 30 and 6% higher compared to that at 30 and 37°C, respectively.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress

Aims: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. Methods and Results: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55–70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE? system to measure net K⁺ fluxes. No difference in K⁺ fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K⁺ loss from the heat‐treated E. coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K⁺ flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K⁺ flux disruption was not clearly observed until 68°C was reached. Conclusions: No changes in net K⁺ flux from heat‐stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K⁺ fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two‐stage process enabled evaluation of the lethality of heat‐treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. Significance and Impact of the Study: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture‐dependent test offers an alternative system to culture‐based methods.     

Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production

Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical example of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms (Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered l-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L l-threonine by fed-batch culture, resulting in an overall yield of 0.284 g l-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass.