Production of glutaconic acid in a recombinant Escherichia coli strain

The assembly of six genes that encode enzymes from glutamate-fermenting bacteria converted Escherichia coli into a glutaconate producer when grown anaerobically on a complex medium. The new anaerobic pathway starts with 2-oxoglutarate from general metabolism and proceeds via (R)-2-hydroxyglutarate, (R)-2-hydroxyglutaryl-coenzyme A (CoA), and (E)-glutaconyl-CoA to yield 2.7 ± 0.2 mM (E)-glutaconate in the medium.     

Biosynthesis of somatotropin in a recombinant strain of Escherichia coli

A recombinant Escherichia coli K-12 strain was grown in the regime of chemostat with glucose limitation at a different flow rate and in the regime of turbidostat. The stability of its population and the dynamics of somatotropin biosynthesis were studied. The plasmid-containing strain became less stable as the flow rate in the fermenter dropped down, which was due, apparently, to a greater limitation. The level of somatotropin biosynthesis was higher at a low dilution rate (D = 0.075, 0.17 and 0.34 h-1). Possible factors responsible for this phenomenon are discussed.     

Quantification of metabolic limitations during recombinant protein production in Escherichia coli

Escherichia coli is one of the major microorganisms for recombinant protein production because it has been best characterized in terms of molecular genetics and physiology, and because of the availability of various expression vectors and strains. The synthesis of proteins is one of the most energy consuming processes in the cell, with the result that cellular energy supply may become critical. Indeed, the so called metabolic burden of recombinant protein synthesis was reported to cause alterations in the operation of the host's central carbon metabolism.To quantify these alterations in E. coli metabolism in dependence of the rate of recombinant protein production, ¹³C-tracer-based metabolic flux analysis in differently induced cultures was used. To avoid dilution of the ¹³C-tracer signal by the culture history, the recombinant protein produced was used as a flux probe, i.e., as a read out of intracellular flux distributions. In detail, an increase in the generation rate rising from 36 mmol_ATP g_CDW⁻¹ h⁻¹ for the reference strain to 45 mmol_ATP g_CDW⁻¹ h⁻¹ for the highest yielding strain was observed during batch cultivation. Notably, the flux through the TCA cycle was rather constant at 2.5 ± 0.1 mmol g_CDW⁻¹ h⁻¹, hence was independent of the induced strength for gene expression. E. coli compensated for the additional energy demand of recombinant protein synthesis by reducing the biomass formation to almost 60%, resulting in excess NADPH. Speculative, this excess NADPH was converted to NADH via the soluble transhydrogenase and subsequently used for ATP generation in the electron transport chain. In this study, the metabolic burden was quantified by the biomass yield on ATP, which constantly decreased from 11.7 g_CDW mmol_ATP⁻¹ for the reference strain to 4.9 g_CDW mmol_ATP⁻¹ for the highest yielding strain. The insights into the operation of the metabolism of E. coli during recombinant protein production might guide the optimization of microbial hosts and fermentation conditions.     

Production of human epidermal growth factor by an ampicillin resistant recombinant Escherichia coli strain

Summary The effect of medium composition and initial glucose concentration on production of hEGF by recombinant E. coli cells was investigated. Optimum hEGF production was observed in a yeast extract/acid hydrolysed casein/salts media containing an initial glucose concentration of 10 g.l^-1. A maximum hEGF titer of 250 mg.l^-1 was obtained in this medium after 32 h in laboratory fermenters with pH, temperature, agitation and aeration set at 6.8, 30°C, 500 rpm and 2 vvm, respectively.     

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

Microbial sensor for penicillins using a recombinant strain of Escherichia coli.

E. coli harboring the multicopy plasmid pBR327, which codes for beta-lactamase synthesis, was immobilized on acetylcellulose membranes. These were placed in the vicinity of a flat pH electrode, thus constituting a penicillin biosensor based on the detection of changes in pH using a reference pH electrode. A response time of 8 min was obtained with at least 2 mg of cells cm-2 of membrane. A linear detection range between 5 and 30 mM of penicillin was achieved. Buffering capacity decreased the sensitivity, but to a lower extent as when compared with probes using purified enzyme. The sensor was completely stable for at least 13 days and proved to be useful for penicillin estimation in complex media such as fermentation broths and milk.     

Modeling and observer design for recombinant Escherichia coli strain

A mathematical model for recombinant bacteria which includes foreign protein production is developed. The experimental system consists of an Escherichia Coli strain and plasmid pIT34 containing genes for bioluminescence and production of a protein, β-galactosidase. This recombinant strain is constructed to facilitate on-line estimation and control in a complex bioprocess. Several batch experiments are designed and performed to validate the developed model. The design of a model structure, the identification of the model parameters and the estimation problem are three parts of a joint design problem. A nonlinear observer is designed and an experimental evaluation is performed on a batch fermentation process to estimate the substrate consumption.     

L(-)-carnitine production using a recombinant Escherichia coli strain

The L(-)-carnitine production by biotransformation using the recombinant strain Escherichia coli pT7-5KE32 has been studied and optimized with crotonobetaine and D(+)-carnitine as substrates. A resting rather than a growing cells system for L(-)-carnitine production was chosen, crotonobetaine being the best substrate. High biocatalytic activity was obtained after growing the cells under anaerobic conditions at 37°C and with crotonobetaine or L(-)-carnitine as inducer. The growth incubation temperature (37°C) was high enough as to activate the heat-inducible λp_L promoter inserted in the plasmid pGP1-2. The best biotransformation conditions were with resting cells, under aerobiosis, with 4 g l⁻¹ and 100 mM biomass and substrate concentrations respectively. Under these conditions the biotransformation time (1 h) was shorter and the L(-)-carnitine yield (70%) higher than previously reported. Consequently productivity value (11.3 g l⁻¹h⁻¹) was highly improved when comparing with other published works. The resting cells could be reused until eight times maintaining product yield levels well over 50% that meant to increase ten times the L(-)-carnitine obtained per gram of biomass.     

Enhanced cytidine production by a recombinant Escherichia coli strain using genetic manipulation strategies

Cytidine is a nucleoside molecule that is widely used as a precursor for antiviral drugs. In this study, a cytidine-producing strain Cyt18 was developed from Escherichia coli K-12 through 3-step genetic manipulation strategies. Cytidine deaminase gene (cdd) was firstly deleted from the E. coli K-12 strain to develop Cyt10. Furthermore, homoserine dehydrogenase gene (thrA) was inactivated from the Cyt10 strain to develop Cyt12, in which the intracellular aspartate concentration was expected to be increased. The recombinant plasmid pMG1105 containing an pyrB-pyrA operon from Bacillus amyloliquefaciens CYTI was constructed and was introduced into Cyt12 to obtain the Cyt18 strain. Compared to the Cyt12 strain, the cytidine production by the recombinant strain Cyt18 was increased by ~3-fold (722.9 mg/l vs. 249.3 mg/l).     

Recovery of electric energy from formate by using a recombinant strain of Escherichia coli

Recombinant Escherichia coli cells were applied for the recovery of electric energy from formate. Initially, the fdh gene, which encodes formate dehydrogenase (FDH) of Mycobacterium vaccae, was introduced into E. coli cells to allow efficient degradation of formate. The constructed microbial fuel cell (MFC) with E. coli BW25113 cells carrying fdh gene showed appreciable generation of current density in the presence of formate as a substrate. Current density and polarization curves revealed that the performance of MFC under examined conditions was limited by the electron transfer from bulk liquid to the electrode surface; accordingly, agitation resulted in an increase in the current density and achieved a coulombic efficiency of 21.7 % on the basis of formate consumed. Thus, gene recombination enables E. coli cells to utilize formate as a fuel for MFC.     

Production of toluene cis-glycol by a recombinant Escherichia coli strain in a two-liquid phase culture system

Summary The recombinant Escherichia coli strain, TG2 (p1/1), can be used for biotransformation of toluene to toluene cis-glycol. However, toluene caused substantial inhibition of both growth and toluene cis-glycol production at concentrations above 0.04%. Tetradecane was found to be non-toxic at a phase ratio of 0.23 and provided protection against toluene toxicity. Thus, growth was still possible in the presence of 1.5% toluene. Furthermore, the yield of toluene cis-glycol was increased 1.7-fold compared with the yield in the absence of tetradecane.     

Expression of recombinant human nerve growth factor in Escherichia coli.

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons and may be of benefit in neurodegenerative diseases of humans. A method is described to obtain significant amounts of biologically active recombinant human NGF (rhNGF) in one step. RhNGF was expressed in E. coli and the majority of the protein accumulated in inclusion bodies. It was immunoprecipitated by a serum against mouse NGF. Solubilization of the inclusion bodies was done in 3M guanidine HCl and renaturation was effected by dilution and air oxidation in the presence of 6 microM CuSO4. Recoveries were 10-12 micrograms of rhNGF per ml of bacterial suspension. Its biological activity was tested in a bioassay system employing sympathetic chick embryo ganglia and was inhibited by the monoclonal antibody 27/21 against mouse NGF.     

Development of a biosensor for on-line detection of tributyltin with a recombinant bioluminescent Escherichia coli strain

A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli:: luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 micro M and 1.5 micro M TBT). High growth and dilution rates ( D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 micro M, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.     

Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain

Background  

The expression of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted E. coli strain.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant Escherichia coli strain

A recombinant plasmid (pMK57) was constructed by cloning the Bacillus stearothermophilus alpha-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed into Escherichia coli strain JM 103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced alpha-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more alpha-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much alpha-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.     

Evidence for rapid association-dissociation of ribonuclease T1 from a recombinant strain of Escherichia coli

On the basis of photon correlation experiments and computer simulations, we provide evidence for a rapid dimerization of the enzyme ribonuclease T1 isolated from an Escherichia coli overproducing strain. An attractive potential in addition to the usual repulsive hardcore and electrostatic potentials was found to be necessary for interpreting the concentration dependence of the diffusion coefficient of the enzyme. Computer searches of surface complementarity suggest that dimer formation of ribonuclease T1 takes place due to an extensive surface contact of approximately 700 A2. Energy minimization of the ribonuclease T1 dimer shows that large conformational changes are not induced upon self-association of the enzyme. The two molecules in the dimer are orientated back-to-back, and this is expected to lead to an active enzyme form.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.