Temperature gradient-based high-cell density fed-batch fermentation for the production of pyruvate oxidase by recombinant E. coli

Pyruvate oxidase (PyOD) is a very powerful enzyme for clinical diagnostic applications and environmental monitoring. Influences of temperature on cell growth, plasmid stability, and PyOD expression during the PyOD fermentation process by recombinant Escherichia coli were investigated. Based on the influences of temperature on the physiological metabolism, a novel high-cell density fed-batch cultivation with gradient temperature decrease strategy for effective PyOD production was achieved, under which the biomass (OD₆₀₀) of recombinant E. coli could reach to 71 and the highest PyOD activity in broth could reach to 3,307 U/L in 26?hr fermentation.     

Optimization of culture condition for the production of D-amino acid oxidase in a recombinant Escherichia coli

The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD₆₀₀ 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD₆₀₀ 81, respectively. By reproducing these optimized conditions in a 12-m³ fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD₆₀₀ 80.     

Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

Limited cell growth and the resulting low volumetric productivity of ethanologenic Escherichia coli KO11 in mineral salts medium containing xylose have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid pathway. The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis. During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol ml⁻¹ optical density at 550 nm [OD₅₅₀] unit⁻¹), and growth was limited. Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol ml⁻¹ OD₅₅₀ unit⁻¹), trehalose (9.9 nmol ml⁻¹ OD₅₅₀ unit⁻¹), and betaine (19.8 nmol ml⁻¹ OD₅₅₀ unit⁻¹). Two independent genetic modifications of E. coli KO11 (functional expression of Bacillus subtilis citZ encoding NADH-insensitive citrate synthase; deletion of ackA encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity. This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement.     

Fermentation and purification of microbial monomer 4-amminocinnamic acid to produce ultra-high performance bioplastics

Aromatic amines are base materials for generating super-engineering plastics such as polyamides and polyimides. Recombinant Escherichia coli ferments 4-aminocinnamic acid (4ACA) from glucose, and it can be derived to plastics of biomass origin with extreme thermal properties. Here, we scaled-up 4ACA production by optimizing microbial fermentation processes. The initial fermentation of 4-aminophenylalanine (4APhe) using E. coli generated the papABC genes of Pseudomonas fluorescens that produced 4APhe with a volumetric mass transfer coefficient (k_La) of 70 h⁻¹ in 115 L of culture broth, and 334 g of 4APhe at a final concentration of 2.9 g 4APhe L⁻¹. Crude 4APhe prepared from the fermentation broth was bioconverted to 4ACA by an E. coli strain producing phenylalanine ammonia lyase of the yeast Rhodotorula glutinis. The E. coli cells cultured under optimized conditions converted 4APhe to 4ACA at a rate of 0.65 g L⁻¹ 4ACA OD₆₀₀⁻¹. These processes resulted in the final derivation of 4.1 g L⁻¹ of 4ACA from glucose. The 4ACA was purified from the reaction as a hydrochloric acid salt and photodimerized to 4,4’-diaminotruxillic acid, which was polycondensed to produce bioaromatic polyimides. Large-scale 4ACA production will facilitate investigations of the physicochemical properties of biomass-derived aromatic polymers of 4ACA origin.     

High-Level Production of Human Leptin by Fed-Batch Cultivation of Recombinant Escherichia coli and Its Purification

Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obese gene coding for leptin was expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD₆₀₀), 30, 90, and 140. When cells were induced at an OD₆₀₀ of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.     

Strategies for high titre plasmid DNA production in Escherichia coli DH5α

The production of plasmid pEGFP-N1 in Escherichia coli DH5α was optimised. A strategy evaluating different media components separately was not successful (OD < 2.5, low plasmid titres), a statistical approach via a Plackett Burman design (11 parameters) allowed some improvement (7 mg/L plasmid, OD₆₀₀ 8.5). Generally, high biomass did not correlate with high plasmid titres. When conditions were transferred to the bioreactor (batch operation) little improvement in plasmid titres (10 mg/L plasmid, OD₆₀₀ 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD₆₀₀ 50). By using an adaptive feeding strategy, plasmid titres could be increased to 50 mg/L. Finally, by combining a growth controlled (reduced temperature (35 °C), low dO₂) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 °C, glucose-/dO₂-limitation) we were reproducibly able to produce up to 250 mg/L of plasmid DNA in cultures that reached a final OD₆₀₀ of 80.     

Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH₄⁺ as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L⁻¹ product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-d-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.     

Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD₆₀₀) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD₆₀₀ up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.     

Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli

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

Biodegradation of methidathion by Serratia sp. in pure cultures using an orthogonal experiment design, and its application in detoxification of the insecticide on crops

An enrichment culture method was applied to the isolation of a bacterial strain responsible for biodegradation of methidathion residues, from a methidathion-treated orchard. The strain (SPL-2) was identified as Serratia sp. according to its physiological characteristics and 16S rRNA gene phylogenetic analysis. Serratia sp. was able to grow in a poor medium consisting of mineral salts and using methidathion as the sole carbon source at a concentrations of 50–150 mg/L. The effects of multifactors on degradation of methidathion in pure cultures by Serratia sp. were investigated using an orthogonal experimental design L₉ (3⁴). On the basis of range analysis and ANOVA results, the most significant factors were temperature and inoculum size. The optimal conditions for methidathion biodegradation in pure cultures were a temperature in 30 °C, an inoculum size of 10 %, pH?=?7 and an aeration rate of 200 rpm. Two different concentrations of strain SPL-2 fermenting liquids (OD₆₀₀?=?0.2 and OD₆₀₀?=?0.4) were prepared and applied to remove methidathion residues from agricultural products, and this process can be described by a first order rate model. In contrast to controls, the DT₅₀ of methidathion was shortened by 35.7 %, 8.2 % and by 62.3 %, 57.5 % on OD₆₀₀?=?0.2 and OD₆₀₀?=?0.4 treated haricot beans and peaches, respectively. These results suggest that the isolated bacterial strain may have potential for use in bioremediation of methidathion-contaminated crops.     

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD₆₀₀ of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD₆₀₀ of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD₆₀₀ of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.     

High cell density and high expression of recombinant human ApoA-IMilano in Escherichia coli by twice temperature-shifted induction

The effect of temperature on the formation of recombinant protein, apolipoprotein A-I_Milano was investigated in the present study. The temperature of the initial growth phase was set at 30°C, while temperature variation in induction phase was arranged in three modes. High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out. Experimental results showed that ApoA-I_Milano reached 4.8 g/L with the final cell density of OD₆₀₀, 150. It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product. The present study provides a basic procedure for the production of recombinant ApoA-I_Milano.     

Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD₆₀₀) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO₂ is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions.     

Increased production of cloned β-galactosidase in two-stage culture of Bacillus amyloliquefaciens

Bacillus amyloliquefaciens harboring recombinant plasmid pHG5, which encodes B. stearothermophilus β-galactosidase, was cultured in a jar fermentor. By feeding lactose a considerable concentration of the enzyme was produced, but the cells stopped growing at an OD₆₆₀ of about 30. On the other hand, the microorganism grew to a very high cell concentration with an OD₆₆₀ of around 110 with glucose as a carbon source, but the enzyme specific activity was a half of the maximum value with lactose. Based on these facts, B. amyloliquefaciens was first grown using glucose, and the carbon source was then switched to lactose to induce β-galactosidase production. By this two-step culture method, both good cell growth and high enzyme productivity were obtained.     

Influence of bacterial density during preculture on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato

An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar (Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD_600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and resuspended in a liquid cocultivation medium-I, adjusted to OD_600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency is stable and highly efficient.     

Non-linear population dynamics in chemostats associated with live-dead cell cycling in Escherichia coli strain K12-MG1655

Bacterial populations conditionally display non-linear dynamic behaviour in bioreactors with steady inputs, which is often attributed to varying habitat conditions or shifting intracellular metabolic activity. However, mathematical modelling has predicted that such dynamics also might simply result from staggered birth, growth, and death events of groups of cells within the population, causing density oscillations and the cycling of live and dead cells within the system. To assess this prediction, laboratory experiments were performed on Escherichia coli strain K12-MG1655 grown in chemostats to first define fine-scale population dynamics over time (minutes) and then determine whether the dynamics correlate with live–dead cell cycles in the system. E. coli populations displayed consistent oscillatory behaviour in all experiments. However, close synchronisation between OD₆₀₀ and live–dead cell oscillations (within ~33–38 min cycles) only became statistically significant (p < 0.01) when pseudo-steady state operations approaching carrying capacity existed in the bioreactor. Specifically, live cells were highest at local OD₆₀₀ maxima and lowest at local OD₆₀₀ minima, showing that oscillations followed live–dead cell cycles as predicted by the model and also consistent with recent observations that death is non-stochastic in such populations. These data show that oscillatory dynamic behaviour is intrinsic in bioreactor populations, which has implications to process operations in biotechnology.     

High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium

To develop an economical industrial medium, untreated cane molasses (UCM) was tested as a carbon source for fermentation culturing of Escherichia coli. To test the industrial application of this medium, we chose a strain co-expressing a carbonyl reductase (PsCR) and a glucose dehydrogenase (BmGDH). Although corn steep liquor (CSL) could be used as an inexpensive nitrogen source to replace peptone, yeast extract could not be replaced in E. coli media. In a volume of 40 ml per 1-l flask, a cell concentration of optical density (OD₆₀₀) 15.1 and enzyme activities of 6.51 U/ml PsCR and 3.32 U/ml BmGDH were obtained in an optimized medium containing 25.66 g/l yeast extract, 3.88 g/l UCM, and 7.1% (v/v) CSL. When 3.88 g/l UCM was added to the medium at 6 h in a fed-batch process, the E. coli concentration increased to OD₆₀₀ of 24, and expression of both PsCR and BmGDH were twofold higher than that of a batch process. Recombinant cells from batch or fed-batch cultures were assayed for recombinant enzyme activity by testing the reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE). Compared to cells from batch cultures, fed-batch cultured cells showed higher recombinant enzyme expression, producing 560 mM CHBE in the organic phase with a molar yield of 92% and an optical purity of the (S)-isomer of >99% enantiomeric excess.     

Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride

Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).     

Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli

The overlap forward-primer-walk polymerase chain reaction method was used to synthesize the human tumor necrosis factor α (hTNF) gene in Escherichia coli cells. Growth curves for hTNF and pET23d vector cultures exhibited slower doubling rates than cultures containing the pET23d vector alone. Cell cultures transformed with hTNF reached peak densities (0.4-0.6 OD₆₀₀) 3 to 4 h post-induction, then decreased prior to growth recovery. This inhibition occurred in the BL21DE3 strain of E. coli, whereas no inhibition of growth and no expression of hTNF were observed in the JM109 strain of E. coli containing hTNF. Induced hTNF cultures hyperexpressed the hTNF-histidine fusion protein for the first 3 to 4 h of induction; subsequently, growth retardation was observed. Hyperexpression and continuous growth were observed in the extracellular expression system. Electron microscopy revealed that accumulation of hTNF inclusion bodies was apparent only in the intracellular expression system — no accumulation was observed with regard to the secretory system. The hTNF-pET23d vector was purified from cells expressing the fusion protein and from cells with recovered growth curves. Sequencing of the vector demonstrated the complete hTNF gene and T7 promoter in cells expressing the fusion protein and mutations of the T7 promoter site from recovered cells.