Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains

Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k(d)) and transport (k(t)) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k(d) and k(t). The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.     

Change in Bacillus anthracoides spores and their content of dipicolinic acid during germination]

The content of dipicolinic acid (DPA) was assayed in the spores of Bacillus anthracoides 96 during various stages of its growth. The content of DPA was ca. 10.7 per cent of the dry biomass weight in the seven-day-old culture containing 96 to 99 per cent of the spores in a "starvation" medium. The morphology of the culture was modified, and the content of DPA in the spores fell to 3.6 per cent half an hour after the inoculation into the medium favourable for the growth (MPA). During the following one to four hours of the germination, the refraction index of the spores and the content of DPA in them decreased (the content of DPA to 2 per cent).     

Effect of penicillin precursors on antibiotic biosynthesis in various strains]

The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant Escherichiacoli Strains

Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k_d) and transport (k_t) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k_d and k_t. The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.     

Docosahexaenoic acid ethyl esters from Isochrysis galbana

In order to produce docosahexaenoic acid (DHA), a culture of the microalgal strain Isochrysis galbana was implemented. In Erlenmeyer flasks, a natural seawater medium, the Provasoli 1/3 medium, was compared to the classical Jones medium for DHA production. The Provasoli 1/3 medium stimulated growth (0.44 d(-1)), but influenced DHA accumulation negatively (0.240 pg cell(-1)). However, DHA production per liter of culture medium were of the same order of magnitude with both media (0.961 mg l(-1)). In a 2-l bioreactor, DHA production per liter of culture medium did not increase significantly between 4 and 8 days of culture. With a view to optimize DHA productivity, cells should be harvested at the end of exponential phase i.e. after 4 days of culture. Two strategies were then attempted to produce DHA ethyl esters. First, lipids from I. galbana were submitted to lipase-catalyzed transesterification with ethanol. Secondly, fatty acids from I. galbana were submitted to lipase catalyzed esterification with ethanol. In both cases, lipase from Candida antarctica was shown to be the best candidate, among the five tested, with conversion yields of 20 and 60% after 24 h of transesterification and esterification respectively.     

The use of the protoplast method for the selection of oleandomycin producers

The use of protoplasting with subsequent reversion to the cellular form in improvement of the oleandomycin-producing organism provided a 110% increase in the range of culture variation with respect to the antibiotic production property. A regenerant with a potency of 12 to 20 per cent higher than that of the initial strain which produced 30 per cent lower amounts of dark pigments of melanin nature was isolated. Repeated protoplasting and regeneration of the regenerant provided a very low regeneration frequency i.e. 0.0002%. The potency of all the secondary regenerants was low.     

Effects of Physical and Chemical Factors on Induction Frequency of the Pollen Plantlet and Changes in Starch Formation in Anthers

Physical and chemical facttors affected distinctly induction frequency of the pollen plantlets from anther culture in vitro of Lycium. Experimental results showed that the anthers with their pollen grains at the uninucleate stage when the nuclei were centrally situated, were the best material for the anther culture. The different proportions of various hormones have affected the embryoid formation. When the MS medium was supplemented with 6-BAP (1 ppm) and NAA (0.1 ppm), the induction frequency was increased ( 16.9% ). When 3–15 per cent of sucrose was added in medium, the embryoids were induced and 15 per cent was the optimum. The callus of the filament was inhibited by the increase of the sucrose concentration. Before inoculation the anthers were pretreated at 3–5℃ for 4 days, the frequency of embryoid formation was efficiently increased in comparision with those of untreated anthers. The induction frequency of normal anthers was only 2.8 per cent, but that of the anthers pretreated was 3–9 per cent, the highest was 8.9 per cent .The changes of substances in anther pretreated and in normal anther was compared by means of histochemistry. Under normal conditions, there were a lot of starch accumulated in the inner wall Of the anthers and the distribution of the cytoplasm and the staining of the protein were even: In the anther pretreated, the starch grains have disappeared and the cytoplasm has condensed and the staining of the protein wasn't even. The differences may be related to induction, frequency of the anther culture.     

Effect of the oxidation-reduction potential of the medium on the production of gentamycin

The medium redox potential had an effect on gentamicin production by Micromonospora purpurea v. violacea, strain VNIIA 7R. The Eh influence was shown to be statistically reliable when the results were expressed in relative units against the control. In the laboratory experiments with low volumes of the medium the Eh increase by more than 170 per cent induced inhibition of gentamicin biosynthesis while the Eh increase by 108 to 168 per cent induced stimulation of the activity.     

Recombinant Protein Production by the Baculovirus-Insect Cell System in Basal Media Without Serum Supplementation

The production of β-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the β-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted β-galactosidase production, their removal by medium replacement on post-infection day 1 gave a β-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing β-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.     

Production and pH-Dependent Bactericidal Activity of Lactocin S, a Lantibiotic from Lactobacillus sake L45

The amount of lactocin S activity in a growing culture depends on the growth stage of the bacteria, the pH of the medium, the presence of ethanol, and the aeration of the culture. We observed the highest levels of bacteriocin activity in the early stationary growth phase of cultures at 30 deg C. When Lactobacillus sake L45 was grown in a fermentor at pH 5, it produced 2,000 to 3,000 bacteriocin units per ml, which represented an 8- to 10-fold increase in bacteriocin production compared with production during batch culture fermentation. Less than 10% of this level of bacteriocin activity was observed during fermentation at pH 6.0. When 1% ethanol was included in the growth medium, a two- to fourfold increase in the bacteriocin yield was observed. Aerating the culture during growth almost completely eliminated the production of active bacteriocin. Our results also showed that lactocin S-mediated killing of target cells depended on the pH of the culture. The pH had to be less than 6 in order to obtain a bactericidal effect with lactocin S-sensitive cells. At pH values greater than 6, lactocin S had no apparent effect on sensitive cells.     

Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro

Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.     

Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway

The effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on phospholipid degradation was investigated in three cell lines of dissimilar origin, Madin-Darby canine kidney cells (MDCK), rat aorta smooth muscle cells (RASM), and bovine pulmonary artery endothelial cells (BPAE). In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into phosphatidylcholine (PC), TPA treatment (80 nM) in the absence or presence of ethanol (2%) in the culture medium resulted in either the rapid generation of [3H]phosphatidate (PA) or the sustained accumulation of [3H]phosphatidylethanol (PEt), respectively. Increases in [3H]PA and [3H]PEt were paralleled by quantitative decreases in cellular [3H]PC radioactivity. TPA-induced [3H]PEt formation occurred in a similar fashion, irrespective of the presence of Ca2+ in the culture medium. The experiments demonstrate that TPA elicits PC degradation by phospholipase D (PLD) in cells of diverse origin. Data from further experiments revealed a complex relationship between TPA-induced [3H]PA and [3H]diacylglycerol (DG) generation in the three cell lines that was suggestive of dual pathways for the generation of [3H]DG. Experiments to discern the pathways for TPA-induced, PC-derived DG were conducted by comparing the variation of [3H]PA and [3H]DG formation in the absence and in the presence of increasing ethanol concentrations in the culture medium. With increasing amounts of ethanol, the formation of [3H]PA decreased at the expense of [3H]PEt formation, and depending upon the pathway operable, the amount of [3H]DG formed was either decreased, indicative of indirect formation of DG via PA phosphohydrolase, or not modified, indicative of DG formation by a direct phospholipase C (PLC) pathway. Increasing the concentration of ethanol in the medium blocked TPA-induced [3H]DG generation in MDCK cells in a concentration-dependent manner, while the formation of [3H]PEt increased at the expense of [3H]PA formation. In BPAE cells the presence of ethanol likewise reduced TPA-elicited formation of DG. Conversely, in two smooth muscle cell lines, RASM and A-10, ethanol was without influence on TPA-induced formation of [3H]DG, although [3H]PEt was generated at the expense of [3H]PA. In RASM cells prelabeled with [3H]choline, TPA induced the release to the medium of [3H]choline and [3H]phosphocholine, indicative of both PLD and PLC activation. These results show that TPA elicits DG formation from PC in MDCK cells predominantly by an indirect pathway, whereas in arterial smooth muscle cells DG is formed in part by the direct action of PLC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein metabolism in the small intestine of the ethanol-fed rat

The effects of chronic ethanol feeding on the small intestine were investigated in young rats. Rats were fed a nutritionally-adequate liquid diet, containing 36 per cent of total energy as ethanol (treated, n = 7), or isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose (controls, n = 7). After six weeks the wet weight and total tissue contents of protein, RNA and DNA were significantly reduced by 21 per cent, 23 per cent, 16 per cent and 28 per cent respectively, (p less than 0.014). Rates of protein synthesis were measured with L[4(3H)]phenylalanine and fractional rates (defined as the percentage of constituent tissue protein synthesised each hour, i.e. ks, % h-1) were calculated from the specific radioactivity of free phenylalanine in both tissue homogenates and plasma. Ethanol-feeding reduced ks by approx 10 per cent (p less than 0.181). The amount of protein synthesized unit-1 RNA was also reduced by approx 15 per cent (p less than 0.059) but the amount of protein synthesis unit-1 DNA was unaffected by ethanol-feeding (p less than 1.000). In contrast, the absolute rates of protein synthesis were reduced by approximately 30 per cent (p less than 0.022). It was concluded that, as the small intestine contributes to approx. 20-25 per cent of whole body synthesis these results may have an important effect on whole body nitrogen homeostasis and may have implications for the gastrointestinal effects of ethanol seen during chronic alcoholic abuse.     

Study of the effect of protoplast formation on the antibiotic activity of erythromycin producers

The influence of protoplasting and regeneration on the strains of the erythromycin-producing organism differing by their origin was studied with respect to changes in the antibiotic production property. 223 regenerates of the erythromycin-producing culture were tested in several generations and it was shown that there was a marked change in the range of the variation by that property. 40 to 70 per cent of the variants in the IInd generation increased their levels of erythromycin biosynthesis by 20 to 60 per cent as compared to the intact cultures. However, in the subcultures the antibiotic production level decreased and by the IVth generation only 3 to 6 per cent of the variants preserved its increase by 10 to 20 per cent over the control level because of the culture high instability.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes.

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37 degrees C. This increased the concentration of the adenylic nucleotides ATP (+57 per cent, ADP (+39 per cent). Cyclic AMP was increased (+50 per cent) and the intracellular inorganic phosphate decreased (-22 per cent). These changes were accompaned by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bis-phosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD+/NADH ratio along with the increase of the ATP/ADP X PO4 ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD+/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Effect of a moderate intensity exercise training protocol on the metabolism of macrophages and lymphocytes of tumour-bearing rats

It is commonly accepted that moderate intensity exercise is beneficial to the immune system. We tested the influence of a moderate intensity training protocol (8 weeks) upon immune system function in Wistar tumour-bearing (TB) rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, together with some functional parameters (hydrogen peroxide production and lymphocyte proliferative response). These substrates were chosen since they represent the most important energetic and synthetic metabolites for these cellular types. The training protocol caused a decrease of 17.4 per cent in the production of H(2)O(2) by macrophages, as well as a decrease in glucose consumption (25 per cent) and lactate production (47.1 per cent), and an increase in the production of labelled CO(2) from the oxidation of [U-(14)C]-glucose, in TB rats. The training protocol was also able to induce changes in the maximal activity of some key enzymes in the metabolism of glucose and glutamine, a reduction of hexokinase (68.8 per cent) activity and an increase in the activity of citrate synthase (10.1 per cent) in TB rats. The training protocol increased the proliferative response of lymphocytes cultivated in the absence of mitogens (75 per cent), of those cultivated in the presence of ConA (38.2 per cent) and in the presence of LPS (45.0 per cent). These cells also showed an increase in the maximal activity of some key enzymes of the glycolytic and glutaminolytic pathways. Our data demonstrated that the training protocol was able to induce an increase in aerobic utilisation of both substrates in lymphocytes and macrophages. The training protocol was also able to prevent several changes in glucose and glutamine metabolism that are normally present in sedentary TB rats. These changes in immune cell metabolism induced by the training protocol were able to increase TB rat survival.     

Effect of glycerin on the biosynthesis of heliomycin by Streptomyces olivocinereus

Glycerol as the sole carbon source was added to the medium or biosynthesis of heliomycin by Streptomyces olivocinereus and the effect of its concentration on the culture growth and antibiotic production was studied. The culture growth and the amount of the antibiotic synthesized per 1 unit of the fermentation broth were limited by glycerol added in quantities of 0.05 to 1 per cent. Further increasing of the glycerol concentration had no significant effect on the culture growth and antibiotic biosynthesis. The amount of the antibiotic synthesized per 1 unit of the mycelial mass relatively slightly depended on the glycerol concentration. The rate of glycerol consumption by the young 24-hour culture in batch fermentations markedly exceeded that of glycerol consumption by the 48-hour culture. The younger mycelium significantly increased its rate of glycerol consumption when the initial concentration was increased whereas the rate of glycerol consumption by the more mature mycelium did not depend on the initial concentration of the carbon source (within 0.5-2 per cent). The rate of heliomycin biosynthesis practically slightly depended on the initial concentration of glycerol.     

Efficient preparation of pseudoalteromone A from marine Pseudoalteromonas rubra QD1-2 by combination of response surface methodology and high-speed counter-current chromatography: a comparison with high-performance liquid chromatography

Pseudoalteromone A (PA) is a cytotoxic and anti-inflammatory ubiquinone discovered recently from a marine bacterium Pseudoalteromonas sp. CGH2XX. In order to meet its sample supply for further in vivo pharmacological investigation, an efficient method was developed for the preparation of PA by combination of response surface methodology (RSM) and high-speed counter-current chromatography (HSCCC) from marine bacterium P. rubra QD1-2. First, optimization of culture conditions was studied by the RSM to enhance PA production. The results indicated that the optimal cultivation condition was peptone (2.21 g/l), yeast extract (3.125 g/l), glucose (0.125 g/l), KBr (0.02 g/l), inoculum size (6.5 %), medium volume (595 ml), initial pH value (7.0), temperature (28 °C). Under the optimized fermentation condition, PA production was 1.04 mg/l with 14.8-fold increase comparing to 0.07 mg/l under original standard fermentation condition. The PA production was further investigated using a 14-l jar fermenter. Compared to the flask culture, P. rubra QD1-2 offered 45 % increase of PA production at 1.51 mg/l. Then, a rapid and efficient method for the separation and purification of PA from crude culture extract was developed using HSCCC. The two-phase solvent system used for HSCCC separation was composed of n-hexane–ethyl acetate–methanol–water (5:5:9:5, v/v/v/v). The isolation was accomplished within 100 min, and the purity of PA was over 95 %. The recovery of the process was 93 %.