Fed-batch Fermentation System Research Articles

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16

BackgroundExisting plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2.ResultsIn this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB− 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml.ConclusionThe new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.Graphical

Bioethanol Production from Sugarcane Molasses by Fed-Batch Fermentation Systems Using Instant Dry Yeast

Bioethanol Production from Sugarcane Molasses by Fed-Batch Fermentation Systems Using Instant Dry Yeast

Black glucose-releasing silicon elastomer rings for fed-batch operation allow measurement of the oxygen transfer rate from the top and optical signals from the bottom for each well of a microtiter plate

BackgroundIn industrial microbial biotechnology, fed-batch processes are frequently used to avoid undesirable biological phenomena, such as substrate inhibition or overflow metabolism. For targeted process development, fed-batch options for small scale and high throughput are needed. One commercially available fed-batch fermentation system is the FeedPlate®, a microtiter plate (MTP) with a polymer-based controlled release system. Despite standardisation and easy incorporation into existing MTP handling systems, FeedPlates® cannot be used with online monitoring systems that measure optically through the transparent bottom of the plate. One such system that is broadly used in biotechnological laboratories, is the commercial BioLector. To allow for BioLector measurements, while applying the polymer-based feeding technology, positioning of polymer rings instead of polymer disks at the bottom of the well has been proposed. This strategy has a drawback: measurement requires an adjustment of the software settings of the BioLector device. This adjustment modifies the measuring position relative to the wells, so that the light path is no longer blocked by the polymer ring, but, traverses through the inner hole of the ring. This study aimed at overcoming that obstacle and allowing for measurement of fed-batch cultivations using a commercial BioLector without adjustment of the relative measurement position within each well.ResultsDifferent polymer ring heights, colours and positions in the wells were investigated for their influence on maximum oxygen transfer capacity, mixing time and scattered light measurement. Several configurations of black polymer rings were identified that allow measurement in an unmodified, commercial BioLector, comparable to wells without rings. Fed-batch experiments with black polymer rings with two model organisms, E. coli and H. polymorpha, were conducted. The identified ring configurations allowed for successful cultivations, measuring the oxygen transfer rate and dissolved oxygen tension, pH, scattered light and fluorescence. Using the obtained online data, glucose release rates of 0.36 to 0.44 mg/h could be determined. They are comparable to formerly published data of the polymer matrix.ConclusionThe final ring configurations allow for measurements of microbial fed-batch cultivations using a commercial BioLector without requiring adjustments of the instrumental measurement setup. Different ring configurations achieve similar glucose release rates. Measurements from above and below the plate are possible and comparable to measurements of wells without polymer rings. This technology enables the generation of a comprehensive process understanding and target-oriented process development for industrial fed-batch processes.

EVOLUTION AT THE MICROFERMENTER LEVEL OF THE GROWTH DYNAMICS OF Saccharomyces cerevisiae AND Starmella bacillaris YEASTS WITH POTENTIAL FOR USE IN WINEMAKING AT THE PIETROASA WINERY

The grape surface hosts a complex community of yeast Saccharomyces and non-Saccharomyces species responsible for spontaneous alcoholic fermentation in wine industry. The yeast strains used for this study were isolated from ‘Tămâioasă Românească’ and ‘Busuioacă de Bohotin’ grape varieties from Pietroasa vineyard, and the isolates were identified through a molecular method. Identification of yeast strains through the BLASTn analysis of the 5.8S-ITS region revealed that PFE5 strain showed the best sequence match to Saccharomyces cerevisiae (98% similarity) and PFE15 strain to Starmerella bacillaris (99.78% similarity), respectively. In this first micro-pilot study, the differences between Sacharomyces and non-Saccharomyces yeasts in batch (for Starmella bacillaris) and fed-batch fermentation system (for S. cerevisae) and how these regimes influence the culture growth were assessed. The applied fed-batch process was capable for producing two times more S. cerevisae yeast biomass than Starmella bacillaris through a batch process. In addition, the yield of S. cerevisiae converting the substrate into biomass was 42.3%, almost double compared to the yield of Starmella bacillaris. Moreover, the cell wet weight (WCW) for S. cerevisae was 32.5 g/L and for Starmella bacillaris 15.35 g/L, respectively. Both yeast biomass will be used at Pietroasa winery for inoculation separately or mixed as co-culture for ‘Tămâioasă Românească’ and ‘Busuioacă de Bohotin’ grape juice.

Efficient 1,3-dihydroxyacetone biosynthesis in Gluconobacter oxydans using metabolic engineering and a fed-batch strategy

1,3-Dihydroxyacetone (DHA) is a commercially important chemical and widely used in cosmetics, pharmaceuticals, and food industries as it prevents excessive water evaporation, and provides anti-ultraviolet radiation protection and antioxidant activity. Currently, the industrial production of DHA is based on a biotechnological synthetic route using Gluconobacter oxydans. However, achieving higher production requires more improvements in the synthetic process. In this study, we compared DHA synthesis levels in five industrial wild-type Gluconobacter strains, after which the G. oxydans WSH-003 strain was selected. Then, 16 dehydrogenase genes, unrelated to DHA synthesis, were individually knocked out, with one strain significantly enhancing DHA production, reaching 89.49 g L−1 and 42.27% higher than the wild-type strain. By optimizing the culture media, including seed culture and fermentation media, DHA production was further enhanced. Finally, using an established fed-batch fermentation system, DHA production reached 198.81 g L−1 in a 5 L bioreactor, with a glycerol conversion rate of 82.84%.Graphical

A cell engineering approach to enzyme-based fed-batch fermentation

BackgroundA fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation.ResultsA putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent.ConclusionsThis study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation.

Scaling-up production of cost-effective and eco-friendly bio-fertilizer and its application on Barley green fodder via IoT hydroponic system

BackgroundPlant-associated microbes (endophytes) have a significant relationship to enhance plant growth and crop productivity by producing proficient bioactive metabolites. Since endophytes promoted plant growth either directly by releasing active metabolites such as phytohormones or indirectly by suppressing the growth of phytopathogens, so, in this work, biomass yield of local endophytic Trichoderma harzianum was maximized at shake-flask scale and scaled up via 7-L Bioflo310 fermenter using continuous exponential fed-batch fermentation mode. Subsequently, the effect of these cells as bio-fertilizer was assessed using two-barley grain genotypes (Russian and Egyptian seeds) via an intelligent hydroponic system based on Internet of Things (IoT). ResultsTo reduce the cost of a biomass production line, agro-waste media containing potato, onion, garlic, pea, and cabbage peels were chosen as the culturing medium. The pea peel medium was found to be the best producer of biomass (2.2 g/L). The cultivation factors were evaluated to improve this biomass yield. The results showed that the maximum biomass production (4.9 g/L) was reported by adjusting the medium pH at 5.0 that inoculated with 10% of spore suspension, then incubated at 30°C, and 200 rpm. Then, this biomass yield was scaled up kinetically (505.4 g/L) by using exponential fed-batch fermentation mode via a 7-L bioreactor. The stimulation impacts of this endophytic T. harzianum on the growth of different barley genotypes (Russian and Egyptian seeds) were determined using a controlled hydroponic chamber. The total chlorophyll, carotenoid, and carbohydrate amounts in treated Russian showed the proficient stimulation percentage (81.05, 80, 40.8%) compared to the Egyptian barley groups (76.39, 73.5, 25.9%) respectively. Also, the maximum carbohydrate content (83.95 ± 1.7%) was recorded in the case of Russian barley. ConclusionVia this work, the optimal combination conditions for the cost-effective biomass production of endophytic T. harzianum were designed industrially via a fed-batch fermentation system using the cheapest culturing medium. Furthermore, by applying this promising bio-fertilizer, the total cost of barley production via an IoT hydroponic growing system was reduced. Besides, these animal diets (sprouted barley) could be produced in 3 cycles per month.

Evaluation of Fed-Batch Fermentation for Production of Polyhydroxybutyrate With a Banana Pulp Juice Substrate From an Agro Industrial By-Product

Pollution resulting from the persistence of plastics in the environment has driven the development of substitutes for these materials through fermentation processes using agro-industrial wastes. Polyhydroxybutyrate (PHB) is a rapidly biodegradable material with chemical and mechanical properties comparable to those of some petroleum-derived plastics. PHB accumulates intracellularly as an energy reserve in a wide variety of microorganisms exposed to nutritionally imbalanced media. The objective of this study was to evaluate the use of a banana waste product as a carbon source for PHB production. PHB was extracted by acid methanolysis and detected by gas chromatography-mass spectrometry. Eleven bacterial strains with potential for PHB production were evaluated by in vitro fermentation in a culture broth containing fructose as the carbon source and limited nitrogen. A 22 central composite rotational design was applied to optimize the concentrations of banana juice and ammonium chloride needed to maximize the PHB-producing biomass concentration. The process was then carried out in a 3 L fed-batch fermentation system that included an initial stage of biomass growth. Banana juice was used as the carbon source and fructose pulses were added to maintain the test sugar concentrations of 30, 40, and 50 g/L. The control strain, Cupriavidus necator (ATCC 17699), produced 2.816 g/L of PHB, while productivity of the most promising isolate, C. necator (CR-12), was 0.495 g/L. Maximum biomass production was obtained using 5% banana juice and 2 g/L ammonium chloride. PHB production was not detected in fed-batch fermentations supplemented with 30 or 40 g/L of fructose, while the mean PHB production in fermentations with 50 g/L of fructose was 1.3 g/L.

Multi-Path Optimization for Efficient Production of 2'-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative.

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

Side-by-Side Comparison of Clean and Biomass-Derived, Impurity-Containing Syngas as Substrate for Acetogenic Fermentation with Clostridium ljungdahlii

Syngas, the product of biomass gasification, can play an important role in moving towards the production of renewable chemical commodities, by using acetogenic bacteria to ferment those gaseous mixtures. Due to the complex and changing nature of biomass, the composition and the impurities present in the final biomass-derived syngas will vary. Because of this, it is important to assess the impact of these factors on the fermentation outcome, in terms of yields, productivity, and product formation and ratio. In this study, Clostridium ljungdahlii was used in a fed-batch fermentation system to analyze the effect of three different biomass-derived syngases, and to compare them to equivalent, clean syngas mixtures. Additionally, four other clean syngas mixtures were used, and the effects on product ratio, productivity, yield, and growth were documented. All biomass-derived syngases were suitable to be used as substrates, without experiencing any complete inhibitory effects. From the obtained results, it is clear that the type of syngas, biomass-derived or clean, had the greatest impact on product formation ratios, with all biomass-derived syngases producing more ethanol, albeit with lesser total productivity.

Fed-Batch Production of a Fermented Beverage Containing Vitamin B12

Production of fermented functional foods containing micronutrients is required for their health beneficial properties. The impact of 11 process variables on vitamin B12 production in a dairy beverage containing propionic acid was investigated. Propionibacterium freudenreichii ssp. shermanii was applied in a 3-l fermentor in the fed-batch fermentation system. The most suitable conditions for vitamin B12 production were achieved by 5% v/v inoculum size containing Propionibacterium freudenreichii (without Lactobacillus acidophilus) and continuous feeding of lactose with the rate of 0.04 l/h at 36°C in a medium containing 25 g/L molasses, 10 g/L corn steep liquor, at pH=6.5, after 96 h fermentation. Maximum vitamin concentration (30 mg/L) and productivity (7.5 mg/L.day) were obtained in trial 9. Organoleptic properties of the fermented beverage were also acceptable for panelists and no significant difference was observed between samples and control during 6 days refrigerated storage.

CORRELATION BETWEEN AERATION AND ERGOSTEROL PRODUCTION BY YEASTS

The present study represents an extension of ergosterol production by Saccharomyces cerevisiae fermentation processes using oxygen-vector by establishing the influence of the analyzed main factors, such as aeration efficiency and hydrocarbon concentration. The study has been developed for batch and fed-batch fermentation systems and has been focused on the variation of ergosterol content inside the yeast cells during the fermentation cycle in correlation with hydrocarbon volumetric fraction, COV, glucose concentration, CG, and air superficial velocity, vS. Moreover, the variation of ergosterol content has been discussed in relation to the oxygen mass transfer coefficient, kLa. The experimental results obtained in both fermentation systems were quantified in two mathematical correlations describing the influences of the mentioned main parameters on ergosterol concentration, CE. These two equations have the general expression, with the experiments, the average deviations being .5.94% for batch fermentation and .4.18% for fed-batchfermentation.

PRODUCTION OF MEDIUM CHAIN LENGTH POLYHYDROXYALKANOATES FROM Cupriavidus necator WITH BEESWAX HYDROLYZATES AS CARBON SOURCE

This study demonstrates that using beeswax hydrolyzates (Hw), short- or medium-chain-length polyhydroxyalkanoates (scl-mcl-PHA) can be obtained by a three stages fed-batch fermentation system. The fed batch fermentations were conducted with a strain of Cupriavidus necator . Glucose and ammonium sulphate as carbon and nitrogen sources, respectively, were used in a first and second culture stages. In the third stage (production of scl-mcl-PHA) a fresh medium was fed with Hw as a carbon source at a concentration of 5 g/L, obtaining 8.91 g/L of intracellular scl-mcl-PHA and 13.64 g/L of biomass, which resulted in 65.3 % w/w of scl-mcl-PHA. Using nuclear magnetic resonance, the presence of scl and mcl monomers, 3-hydroxybutyrate (3HB) and 3-hydroxydecanoate (3HD) was determined. The percentage of comonomer incorporation different to 3HB was 4.6 mol %. A glass transition temperature (T g ) of –50 oC and a melting temperature (T m ) of 79.8 oC were determined for the scl-mcl-PHA (3HB-co-3HDD).

Rational design and analysis of an Escherichia coli strain for high-efficiency tryptophan production.

L-tryptophan (L-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient L-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the L-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG fbr ). To further improve the production of L-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7g/L of L-trp with a conversion rate of 16.7% and a productivity of 1.6g/L/h in a 5L fed-batch fermentation system.

Continuous citric acid production in repeated-fed batch fermentation by Aspergillus niger immobilized on a new porous foam

The efficiency of current methods for industrial production of citric acid is limited. To achieve continuous citric acid production with enhanced yield and reduced cost, immobilized fermentation was employed in an Aspergillus niger 831 repeated fed-batch fermentation system. We developed a new type of material (PAF201), which was used as a carrier for the novel adsorption immobilization system. Hydrophobicity, pore size and concentration of carriers were researched in A. niger immobilization. The efficiency of the A. niger immobilization process was analyzed by scanning electron microscopy. Then eight-cycle repeated fed-batch cultures for citric acid production were carried out over 600 h, which showed stable production with maximum citric acid concentrations and productivity levels of 162.7 g/L and 2.26 g L−1 h−1, respectively. Compared with some other literatures about citric acid yield, PAF201 immobilization system is 11.3% higher than previous results. These results indicated that use of the new adsorption immobilization system could greatly improve citric acid productivity in repeated fed-batch fermentation. Moreover, these results could provide a guideline for A.niger or other filamentous fungi immobilization in industry.

Butanol production from organosolv treated spent mushroom substrate integrated with in situ biodiesel extraction

The aim of this study is to convert spent mushroom substrate (SMS), a by-product from industrial mushroom production, to high production of biobutanol via integration with in situ butanol removal strategy. The modified organosolv pretreatment under mild conditions (35 °C, without acid catalyst) could efficiently improve the sugar yield (9.7% Vs 41.7%) of SMS by reducing lignin content (20.2% Vs 10.5%). The batch fermentation results using the organosolv treated SMS enzymatic broth indicate that adding extractant of biodiesel could enhance butanol production (14.65 g/L) compared to the control batch (12.03 g/L). Moreover, no significant toxicity of biodiesel is observed on the growth of solventogenic Clostridium acetobutylicum. In the fed-batch fermentation system with sugar feeding, the maximum amount of butanol is obtained at 30.21 g/L. This study suggests that in situ butanol removal using biodiesel from SMS enzymatic hydrolysate has great potential for commercial butanol production.

Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system.

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed-batch fermentation system with high fidelity (R(2) 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L(-1) h(-1) , 3 μg mL(-1) and 40%, respectively. While 1711 IU mL(-1) nisin was produced by L. lactis N8 in control fed-batch fermentation, 5410 IU mL(-1) nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed-batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed-batch fermentation.

High‐yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli

Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h(-1), the cultures reached OD(600) values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L(-1) culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches.

OL-073 Evaluation of serum concentration of endothelium nitric oxide synthase in Helicobacter pylori positive people

Background: A recent IVI study in Kolkata, India, found that 24% of enteric fever episodes were due to S. paratyphi A. Estimates indicate that the incidence of enteric fever, in India, due to S. paratyphi A is 2.2 million cases per annum. Vi based vaccines only protect against typhoid fever, thus there is a need for a bivalent vaccine that protects against both S. typhi and S. paratyphi A. Methods: IVI transferred its high yield, high recovery technology for Vi and Vi conjugate production to Shantha Biotechnics in 2009. A high yielding fed batch fermentation system for Salmonella paratyphi A was first developed. The Lipopolysaccharide was separated from the cells using micro and ultrafiltration then detoxified by acid hydrolysis to release O specific polysaccharide (OSP) from the Lipid A. The OSP was further purified by precipitation of contaminating Lipid A, proteins and nucleic acid, concentrated by ultrafiltration then sterilized by 0.2mm filtration. The process developed uses equipment that is scalable and cGMP compliant thus it is anticipated that the technology transfer and scale up to a manufacturer will not be overly complicated. Results: OSP from S. paratyphi A has been successfully purified and the final bulk contains less than 1% of contaminating nucleic acid and protein, and very low endotoxin (by LAL) values. The purified OSP has been used to prepare a series of conjugates using Diphtheria Toxoid (DT) as the carrier protein. The conjugates are currently undergoing preclinical evaluation. Conclusion: We have developed a high yielding and efficient technologies for Vi and OSP production and for conjugation to these polysaccharides to DT. Our aim is to develop a cost effective bivalent conjugate vaccine for developing countries. This work was funded by the governments of Korea and Sweden.