Production of the hybrid protein staphylococcal protein A/Escherichia coli β-galactosidase with E. coli

We have investigated the cultivation of an Escherichia coli strain producing the hybrid protein SpA-βgal. The hybrid protein consists of protein A from Staphylococcus aureus and β-galactosidase from E. coli with retained biological activity of both protein A and β-galactosidase. The expression was controlled by the temperature regulated P_R promoter from phage lambda. By late induction of the product synthesis it was possible to circumvent the problem with plasmid instability. The amount of produced SpA-βgal corresponded to approximately 1256 of the cell dry weight. In shake flask cultures most of the hybrid protein was found in an insoluble form and typical inclusion bodies were observed. However, the major part of the protein could be produced in a soluble and biological active form under controlled conditions in a reactor.     

Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli

The stability of the fusion protein staphylococcal protein A-E. coli β-galactosidase (SpA-βgal) produced in E. coli has been studied both in cell disintegrate and in purified preparations. SpA-βgal was degraded by a proteolytic cleavage between the two functional parts of the molecule, resulting in one β-galactosidase tetramer and four protein A molecules. Intermediates were detected, namely β-galactosidase containing three, two and one protein A. The β-galactosidase was stable with respect to enzyme activity and molecular weight, while protein A was further degraded. In cell disintegrate the half-life of SpA-βgal was found to be 6 h at 20°C and 1.5 h at 37°C. The protease responsible for initial proteolytic cleavage of SpA-βgal was shown to be cell debris associated.     

Efficient release of thermostable β-galactosidase by glycine addition and thermal treatment of Escherichia coli cells

Efficient release of thermostable β-galactosidase from a recombinant Escherichia coli by the addition of glycine to the culture broth and subsequent thermal treatment was investigated. The enzyme release rate was strongly dependent on glycine concentration. The enzyme release rate was almost proportional to glycine concentrations up to 2% in phosphate buffer; however, inactivation of the enzyme was not observed following incubation for up to 3 h at 70°C even in the presence of 10% glycine. In a preliminary experiment, severe thermal inactivation was observed in the presence of polyethylene glycol (PEG), but glycine was able to suppress the inactivation. Thermal treatment of the cell suspension was effective for the improvement of the enzyme release rate. In the absence of glycine, the enzyme release rate was low at 37 and 45°C, even though the initial release rate was high at 0.5 h and 60°C. The combination of thermal treatment and addition of glycine to the cell suspension significantly improved the initial enzyme release rate and the amount of enzyme released to the extracellular fraction at 37 and 45°C was as high as that at 60°C during a 2-h incubation.     

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.     

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar -galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular -galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of -galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

Production of γ-aminobutyric acid in Escherichia coli by engineering MSG pathway

Abstract

The compound γ-aminobutyric acid (GABA) has many important physiological functions. The effect of glutamate decarboxylases and the glutamate/GABA antiporter on GABA production was investigated in Escherichia coli. Three genes, gadA, gadB, and gadC were cloned and ligated alone or in combination into the plasmid pET32a. The constructed plasmids were transformed into Escherichia coli BL21(DE3). Three strains, E. coli BL21(DE3)/pET32a-gadA, E. coli BL21(DE3)/pET32a-gadAB and E. coli BL21(DE3)/pET32a-gadABC were selected and identified. The respective titers of GABA from the three strains grown in shake flasks were 1.25, 2.31, and 3.98?g/L. The optimal titer of the substrate and the optimal pH for GABA production were 40?g/L and 4.2, respectively. The highest titer of GABA was 23.6?g/L at 36?h in batch fermentation and was 31.3?g/L at 57?h in fed-batch fermentation. This study lays a foundation for the development and use of GABA.     

Production of secreted guar α-galactosidase by Lactococcus lactis

A plant -galactosidase gene was inserted in the expression vector pGKV259. The resulting plasmid pGAL2 consisted of the replication functions of the broad-host-range lactococcal plasmid pWV01, the lactococcal promoter P59, and the DNA sequences encoding the -amylase signal sequence from Bacillus amyloliquefaciens and the mature part of the -galactosidase from Cyamopsis tetragonoloba (guar). Lactococcus cells of strain MG1363 harbouring this vector produced the plant -galactosidase and secreted the enzyme efficiently as judged by Western blotting and activity assays. Expression levels of up to 4.3 mg extracellular -galactosidase g (dry weight) of biomass^–1 were achieved in standard laboratory batch cultures. The -galactosidase produced by Lactococcus was active on the chromogenic substrate 5-bromo-4-chloro-3-indolyl -d-galactopyranoside, the trisaccharide raffinose and on the galactomannan substrate, guar gum.     

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His(6) fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.     

The expression of β-galactosidase by Escherichia coli during continuous culture

We have used the technique of continuous culture to study the expression of β-galactosidase in Escherichia coli. In these experiments the cultures were grown on carbon-limited media in which half of the available carbon was supplied as glycerol, glucose, or glucose 6-phosphate, and the other half as lactose. Lactose itself provided the sole source of inducer for the lac operon. The steady-state specific activity of the enzyme passed through a maximal value as a function of dilution rate. Moreover, the rate at which activity was maximal (0.40 h^?1) and the observed specific activity of the enzyme at a given growth rate were found to be identical in each of the three media tested. This result was unexpected, since the steady-state specific activity can be shown to be equal to the differential rate of enzyme synthesis, and since it is known that glycerol, glucose, and glucose-6-P-cause different degrees of catabolite repression in batch culture. The differential rate of β-galactosidase synthesis was an apparently linear function of the rate of lactose utilization per milligram protein regardless of the composition of the input medium. That is, it is independent of the rate of metabolism of substrates other than lactose which are concurrently being utilized and the enzyme level appears to be matched to the metabolic requirement for it. If this relationship is taken to indicate the existence of a fundamental control mechanism, it may represent a form of attenuation of the rate of β-galactosidase synthesis which is independent of cyclic AMP levels.     

The control by respiration of the uptake of α-methyl glucoside in Escherichia coli K12

The uptake of methyl α-d-glucopyranoside (α-MG) by Escherichia coli K12 was decreased by the addition of substrates which stimulated the rate of oxygen consumption by the cells. The inhibition, which occurred only at non-saturating concentrations of α-MG, was not the result of a stimulation of the rate of exit of intracellular α-MG, and was abolished by the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide. Since those drugs inhibit energy conservation at the respiratory chain and did not alter significantly the rate of oxygen consumption under the conditions for the assay of α-MG uptake, it appears that the inhibition of the transport system by respirable substrates is mediated by some form of energy derived from respiration.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Effect of Inducers on the Production of 5‐Aminolevulinic Acid by Recombinant Escherichia coli

Abstract

Aminolevulinic acid (ALA) was produced by recombinant Escherichia coli BL21(DE3) (pET28‐A.R‐hemA) harboring the ALA synthase gene (hemA) from Agrobacterium radiobacter zju‐0121. The effects of inducers on the ALA synthase activity and ALA productivity were evaluated. The results indicated that a low isopropyl‐β‐D‐thiogalactoside (IPTG) concentration (0.05 mmol/L) was favorable for high expression of ALA synthase, which resulted in higher ALA productivity. For metabolic engineering applications, lactose was a better substitute of IPTG for active enzyme expression. When lactose concentration was 5 mmol/L, the specific ALA synthase activity and ALA productivity reached 16.7 nmol/(min · mg of protein) and 1.15 g/L, respectively, which were about 15% and 43% higher than those induced by IPTG.     

Ethanol-mediated gene-amplification in Escherichia coli enhances derepressed synthesis of β-galactosidase

Summary The presence of ethanol (5 % v/v), in nutrient medium, ehanced DNA synthesis per E. coli cell nearly 2.8-fold compared to that in control cells. At this concentration, the derepressed synthesis of -galactosidase per bacterium also increased about 3-fold. We, therefore, propose that the ethanol-mediated gene-amplification proportionately elevated the induced synthesis of -galactosidase.