Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system

The enzyme controlled substrate delivery cultivation technology EnBase(?) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.     

Model based optimization of the fed-batch production of a highly active transglutaminase variant in Escherichia coli

A process for the production of a thermostable variant of a microbial transglutaminase was developed. The transglutaminase variant produced, carried a single amino acid exchange (serine replaced by proline at position 2) and showed a nearly doubled specific activity of 46.1 Umg(-1) compared to the wild-type enzyme. Based on a model based optimization strategy, intracellular soluble production in Escherichia coli was optimized. After parameter identification and only two fed-batch cultivations, a space time yield of 1438 U(TG)L(-1)h(-1) was obtained which is 175% higher than the highest values published so far (extracellular production using Corynebacterium ammoniagenes). High carbon source concentrations during expression were found to increase the product formation. Prior to the fed-batch cultivation, the host strain was adapted from complex medium to minimal medium by serial dilution. Upon transfer to the minimal medium, initially the maximal growth rate dropped to 0.13 h(-1). After the six consecutive cultivations the rate increased to 0.47 h(-1) and the portion of the complex medium was reduced to 1 ppm. Using the adapted cells, temperature after induction and IPTG-concentration were investigated by satellite batch cultivation according to a Design of Experiment (DoE) plan. The product yield was strongly influenced by the temperature after induction but not by the inductor concentration. The highest specific activity of 1386 Ug(-1) bio dry mass was obtained at 29°C and 0.7 mM IPTG.     

Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.     

Size characterization of inclusion bodies by sedimentation field-flow fractionation

Sedimentation field-flow fractionation (sedFFF) was evaluated to characterize the size of Delta(4-23)TEM-beta-lactamase inclusion bodies (IBs) overexpressed in fed-batch cultivations of Escherichia coli. Heterologous Delta(4-23)TEM-beta-lactamase protein formed different sizes of IBs, depending upon the induction conditions. In the early phases of recombinant protein expression, induced with low concentrations of IPTG (isopropyl-beta-d-thiogalactoside), IB masses were larger than expected and showed heterogeneous size distributions. During cultivation, IB sizes showed a Gaussian distribution and reached a broad range by the end of the fed-batch cultivations. The obtained result proved the aptitude of sedFFF to rapidly assess the size distribution of IBs in a culture.     

Expression of xyloglucan endotransglycosylases of Gerbera hybrida and Betula pendula in Pichia pastoris

The plant enzyme xyloglucan endotransglycosylase (XET; EC 2.4.1.207, xyloglucan:xyloglucosyl transferase) participates in selective modification of plant cell walls during cell growth. XETs are potential catalysts in various applications. Here, sequences encoding two XETs from Gerbera hybrida and Betula pendula are reported. The encoded proteins, which are 51% identical at the amino acid level, were expressed in the yeast Pichia pastoris in secreted form with the aid of mating factor alpha signal sequence. XET production in shake flask cultivations was better at 22 degrees C than at 30 degrees C. Both the yield of protein of expected molecular mass and the XET activity improved at the lower temperature. Under all cultivation conditions studied, higher amounts of XET from B. pendula (BXET) were expressed than XET from G. hybrida (GXET). Both XET enzymes were produced in 16l fed-batch bioreactor cultures. GXET was produced in methanol-limited fed-batch cultivation in minimal medium, and BXET in temperature-limited fed-batch (TLFB) in minimal or complex medium. Production was highest in TLFB in complex medium. BXET was purified from the culture filtrate and characterized. Based on the specific activity of the purified protein, 60-70 mg l(-1) BXET was produced in the TLFB in complex medium.     

Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding

An automated glucose feeding strategy that avoids acetate accumulation in cultivations of Escherichia coli is discussed. We have previously described how a probing technique makes it possible to detect and avoid overflow metabolism using a dissolved oxygen sensor. In this article these ideas are extended with a safety net that guarantees that aerobic conditions are maintained. The method is generally applicable, as no strain-specific information is needed and the only sensor required is a standard dissolved oxygen probe. It also gives the highest feed rate possible with respect to limitations from overflow metabolism and oxygen transfer, thus maximizing bioreactor productivity. The strategy was implemented on three different laboratory-scale platforms and fed-batch cultivations under different operating conditions were performed with three recombinant strains, E. coli K-12 UL635, E. coli BL21(DE3), and E. coli K-12 UL634. In spite of disturbances from antifoam and induction of recombinant protein production, the method reproducibly gave low concentrations of acetate and glucose. The ability to obtain favorable cultivation conditions independently of strain and operating conditions makes the presented strategy a useful tool, especially in situations where it is important to get good results on the first attempt.     

Clavulanic acid degradation in Streptomyces clavuligerus fed-batch cultivations

Clavulanic acid (CA) is an important antibiotic that is produced by Streptomyces clavuligerus. CA is unstable and product degradation has turned out to have a major impact on product titers in fed-batch cultivations. Three different types of experiments have been used to elucidate CA degradation under fed-batch cultivation conditions. First, the influence of individual medium compounds was examined. Second, degradation was monitored during the exponential growth phase in batch cultivations. Third, CA degradation was studied in the supernatant of samples taken during a fed-batch. In addition, data from six fed-batch cultivations were studied to derive information about CA degradation during the production phase. These cultivations were based on a mineral medium, containing glycerol, glutamate, ammonium, and phosphate as the main nutrients. The ammonium concentration had a large influence on the degradation rate constant. In addition, either changes in the substrate availability or high concentrations of ammonium or glycerol cause a major increase in the degradation rate constant. Finally, a linear and a fuzzy logic model were made to predict CA degradation rates in these fed-batches.     

High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer

Expression of a recombinant pyranose oxidase (P2O) from the basidiomycete Trametes ochracea has been increased 10-fold in shaking flask cultures of Escherichia coli BL21(DE3) harboring plasmid pSE33 by optimizing the composition of the culture medium using an experimental design approach. Inexpensive lactose was used as a medium component and inducer of expression of the P2O gene, which is under the control of a trc promoter. The expression system was studied in detail in batch and fed-batch cultivations with the aim to improve the expression level of active recombinant protein and to minimize the formation of inclusion bodies. In batch cultivations, the highest specific P2O activity of 0.9 U (mg of soluble protein)(-1) was measured in oxygen-limited cultures grown at 25 degrees C. The highest overall volumetric productivity of 33 mg of active P2O per liter and hour (corresponding to 345U (L h)(-1)) has been determined in a high-density fed-batch process with a feed-forward exponential feeding strategy. During the fed-batch process, lactose was added intermittently to the culture. A final biomass concentration of 33 g L(-1) (based on cell dry weight) was obtained. Compared to shaking flask cultures in not optimized culture media, the overall volumetric P2O productivity has been improved by a factor of 110 using the fed-batch strategy and the optimized culture medium. Recombinant P2O was expressed in the cytoplasm with 9% of the total soluble protein being active P2O. In terms of physical and enzyme kinetic properties, the purified recombinant P2O was found to be similar to the previously published data of P2O isolated from its original host.     

Cultivation of recombinant E. coli and production of fusion protein in 60-1 bubble column and airlift tower loop reactors.

E. coli K12 with multicopy plasmid (lambda PR-promoter and temperature-sensitive lambda cI 857 repressor) was cultivated in 60-l bubble column and airlift tower loop reactors. The medium composition, cell concentration, and intracellulary enzyme activity were monitored on-line during batch, fed-batch, and continuous cultivations. The specific growth rates, cell mass yield coefficients, plasmid stabilities, productivities of the amount of active fusion protein (beta-galactosidase activity), concentrations and yields of acetic acid, and volumetric oxygen transfer coefficient were evaluated for different medium compositions and cultivation conditions. The enzyme activity was also monitored during the temperature induction. The results evaluated in the 60-l bubble column and airlift tower loop reactors are compared with those evaluated in a 1-1 stirred-tank reactor.     

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli

Abstract: The production of the fusion protein staphylococcal protein A/E. coli β-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g 1^-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g 1^-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g 1^-1.     

High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium

A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E. coli that underwent filamentation. Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E. coli.     

Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli.

Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.     

Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3)

Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-beta- D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l(-1)), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.     

High-cell-density cultivation of Escherichia coli.

High-cell-density cultivations of Escherichia coli in glucose-mineral-salt media produce more than 100 g dry cells litre-1 in special fed-batch modes with feeding of glucose and ammonia only. The specific growth rate can be adjusted to allow optimum recombinant protein generation.     

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His₆ tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L⁻¹ could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.     

Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation

Industrial enzymes are often produced by filamentous fungi in fed-batch cultivations. During cultivation, the different morphological forms displayed by the fungi have an impact on the overall production. The morphology of a recombinant lipase producing Aspergillus oryzae strain was investigated during fed-batch cultivations. During the exponential batch phase of the fed-batch cultivations, the average hyphal length increased as did the number of tips per hyphal element. Most striking was the finding that the diameter of the hyphal elements increased with an average factor of 1.5 during the batch phase from 2.8–2.9 up to 4.0–4.4 μm. The diameter of the hyphal elements remained constant, around 4 μm, after the feed was started. However, the diameter of the immediate hyphal tip, where the enzyme secretion is thought to take place, increased dramatically with up to a factor 2.5 during the feeding period. The expression of the recombinant lipase was induced by the feeding with maltose, and an increase in lipase activity was seen in parallel to a swelling of the tips. The results indicate that the two events are linked as a return to normal growth was observed upon cessation of production due to oxygen limitations.     

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.     

High-level production of bacillus subtilis glycine oxidase by fed-batch cultivation of recombinant Escherichia coli Rosetta (DE3)

A fed-batch process for the high cell density cultivation of Escherichia coli Rosetta (DE3) and the production of the recombinant protein glycine oxidase (GOX) from Bacillus subtilis was developed. GOX is a deaminating enzyme that shares substrate specificity with d-amino acid oxidase and sarcosine oxidase and has great biotechnological potential. The B. subtilis gene coding for GOX was expressed in E. coli Rosetta under the strong inducible T7 promotor of the pET28a vector. Exponential feeding based on the specific growth rate and a starvation period for acetate utilization was used to control cell growth, acetate production, and reconsumption and glucose consumption during fed-batch cultivation. Expression of GOX was induced at three different cell densities (20, 40, and 60 g . L(-1)). When cells were induced at intermediate cell density, the amount of GOX produced was 20 U . g(-1) cell dry weight and 1154 U . L(-1) with a final intracellular protein concentration corresponding to approximately 37% of the total cell protein concentration. These values were higher than those previously published for GOX expression and also represent a drastic decrease of 26-fold in the cost of the culture medium.