Origin and morphology of atypical forms ofStreptomyces granaticolor

Nonfilamentous forms ofStreptomyces granaticolor are formed in a medium with amino acids and glucose. They form filaments again after transfer to a medium with glucose and peptone. The nonfilamentous forms do not produce granaticin. Formation of nonfilamentous forms depends on the concentration of the inoculum, on the cultivation temperature and on the presence of simple sugars. Ultrathin sections revealed atypical septation in the nonmycelial forms and non-uniform accumulation of the wall material.     

A study of the growth condition and solubilization of phosphate from hydroxyapatite byPantoea agglomerans

The growth conditions ofPantoea agglomerans, a phosphate solubilizing organism, were studied in our laboratory to determine the optimal conditions.Pantoea agglomerans showed the highest growth rate at 30°C, pH 7.0 and 2 vvm, after 50 h cultivation. A certain relationship between pH and phosphate concentration, was evident when the glucose concentration in the medium was changed. Increasing glucose concentration increased the pH buffer action of the broth. At glucose concentrations higher than the optimum concentration of 0.2 M, the cell growth was retarded.P. agglomerans consumed glucose as a substrate to produce organic acids which caused the pH decrease in the culture medium. The phosphate concentration in the medium was increased by the presence of the organic acids, which solubilized insoluble phosphates such as hydroxyapatite.     

Corynebacterium glutamicum,a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro‐aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild‐type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L^?1) corresponded to the lower k_La value of 5 h^?1. Above 5 h^?1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L^?1. Taking into account this threshold, a fed‐batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild‐type strain was able to produce 93.6 g L^?1 of succinate, which is one of the highest concentration reported in the literature.     

Effect of the conditions for dynamic homogeneous batch stab cultivation on the reproductive process in Shigella sonnei bacteriophages

The use of the bacterial population, exponentially multiplying in the newly developed cultivation process, ensures the optimum conditions for the dynamic batch cultivation of S. sonnei bacteriophages. The yield of bacteriophage biomass has been found to depend on the concentration of bacterial cells, the oxygen saturation of the medium and the concentration of glucose and amino acids in the medium.     

Conditions with high intracellular glucose inhibit sensing through glucose sensor Snf3 in Saccharomyces cerevisiae

Gene expression in micro‐organisms is regulated according to extracellular conditions and nutrient concentrations. In Saccharomyces cerevisiae, non‐transporting sensors with high sequence similarity to transporters, that is, transporter‐like sensors, have been identified for sugars as well as for amino acids. An alternating‐access model of the function of transporter‐like sensors has been previously suggested based on amino acid sensing, where intracellular ligand inhibits binding of extracellular ligand. Here we studied the effect of intracellular glucose on sensing of extracellular glucose through the transporter‐like sensor Snf3 in yeast. Sensing through Snf3 was determined by measuring degradation of Mth1 protein. High intracellular glucose concentrations were achieved by using yeast strains lacking monohexose transporters which were grown on maltose. The apparent affinity of extracellular glucose to Snf3 was measured for cells grown in non‐fermentative medium or on maltose. The apparent affinity for glucose was lowest when the intracellular glucose concentration was high. The results conform to an alternating‐access model for transporter‐like sensors. J. Cell. Biochem. 110: 920–925, 2010. © 2010 Wiley‐Liss, Inc.     

A model-based framework for parallel scale-down fed-batch cultivations in mini-bioreactors for accelerated phenotyping

Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small-scale parallel cultivation devices. In this study, we combine mechanistic growth models with a parallel mini-bioreactor system to create a high-throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled-down approach, a model-based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large-scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale-down scheme, compared with the reference cultivations. In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale-down studies to select the most robust strains for bioprocess scale-up.     

Amino acid fermentation byBacteroides melaninogenicus

Each of four strains ofBacteroides melaninogenicus grew well in a trypticaseyeast extract medium, without carbohydrate. Addition of glucose did not increase growth, and the sugar was fermented to only a limited extent. However, growth decreased when the trypticase concentration of the medium was reduced. These observations suggest that amino acid fermentation is of major importance in the energy metabolism ofB. melaninogenicus. Acetic, butyric and isovaleric acids were produced by all four strains. Two of the strains also formed propionic and isobutyric acids. Experiments using media containing either labeled glucose or labeled protein indicated that these acids are primarily derived from the proteinaceous substrates in the medium, rather than from glucose, indicating that amino acid fermentation byB. melaninogenicus is not subject to glucose repression. Resting-cell suspensions ofB. melaninogenicus possessed a limited ability to ferment free acids, as judged by the liberation of ammonia. However the organisms readily fermented amino acids when present as peptides, suggesting that peptides are more readily transported into the cell. Three of the 4 strains studied grew well when incubated under an atmosphere containing 2 to 4 % oxygen in cultures possessing a high surface to volume ratio. Hence not all strains ofB. melaninogenicus can be considered strict anaerobes.This investigation was supported in part by grant DE-02847 from the National Institute of Dental Research, and in part by a grant from the Colgate-Palmolive Co.     

Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis

Aims: Three precursor amino acids and adenosine triphosphate ( ATP) are necessary for fermentative production of glutathione. In this study, our aims were to develop a strategy to enhance glutathione production by adding three precursor amino acids coupled with ATP in high cell density (HCD) cultivation of Candida utilis. Methods and Results: A high-glutathione yeast strain, C. utilis WSH 02-08, was used in this study. Whole fermentative process for glutathione production was divided into two phases of cell growth and glutathione synthesis. Cells concentration was increased by HCD cultivation. Meanwhile, intracellular glutathione content was enhanced by the addition of three precursor amino acids. Concentrations of three precursor amino acids added at stationary phase were optimized by response surface methodology. Moreover, the addition of ATP 15 h after the addition of the three amino acids can further enhance glutathione production. Based on aforementioned phenomenon, a strategy of adding three precursor amino acids coupled with ATP was developed to enhance glutathione production. Conclusion: Without the addition of three precursor amino acids and the ATP, a total glutathione of 1123 mg l⁻¹ was achieved after 60-h cultivation. In comparison, addition of three precursor amino acid counterparts resulted in a total glutathione of 1841 mg l⁻¹. Moreover, by adding amino acids combined with ATP, a total glutathione of 2043 mg l⁻¹ was achieved after 72-h cultivation, increased by 81·9% and 11%, respectively, as compared with the control and the one without ATP addition. Significance and Impact of the Study: This is the first report on investigating changes of the intracellular three precursor amino acids and ATP, and γ-glutamylcysteine synthase activity in HCD cultivation of C. utilis for glutathione production. A strategy of combining addition of three precursor amino acids with ATP was developed to enhance glutathione production in C. utilis.     

Development of embryonic chick insulin cells in culture: Beneficial effects of serum-free medium, raised nutrients, and biomatrix

Summary A previous finding that insulin cells do not survive or differentiate in explants of embryonic avian pancreas cultured in collagen gel with a serum-containing medium has provided a model system for identification of conditions favorable for development of these cells. To this end, we here modify the substrate and the medium. The epithelial component of dorsal pancreatic buds of 5-d chick embryos was cultured for 7 d on Matrigel in serum-containing and in serum-free medium, the latter incorporating insulin, transferrin, and selenium, Endocrine cell types were distinguished by immunocytochemistry; insulin cell counts were expressed as a proportion of insulin plus glucagon cells. With serum-containing medium, Matrigel stimulated a significant increase in this proportion as compared with collagen gel—3.1% as against 0.2%; the serum-free medium further increased this proportion to 17.3%. Raising the level of essential amino acids approximately fivefold increased the latter figure somewhat (to 18.9%), but it was more than doubled (to 37.4%) by raising the glucose concentration from 10 mM to 20 mM. Raising the levels of amino acids and glucose simultaneously yielded a lesser increase (to 31.8%). Some cultures grown in collagen gel and serum-containing medium for 7 d were transferred to Matrigel and serum-free medium for a further 7 d. Insulin cell development recovered, indicating that progenitor cells had survived and were stimulated to develop by the improved conditions. This study indicates that components of the biomatrix and the medium (in particular, a raised glucose concentration) are important for the survival and differentiation of embryonic insulin cells.     

The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·1⁻¹ on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol·1⁻¹. The specific antibody production rate had a maximum value at about 25 mmol·1⁻¹ glutamine concentration.     

Regulation of branched-chain amino acid catabolism: glucose limitation enhances the component of isovalerylspiramycin for the bitespiramycin production

4′′-O-isovalerylspiramycins are the major components of bitespiramycin complex consisting of a group of 4′′-O-acylated spiramycins. The availability of isovaleryl group, usually in vivo derived from leucine, one of the branched-chain amino acids, affects the content of isovaleryispiramycin significantly. In this study, the effect of glucose on the activity of branched-chain α-keto acid dehydrogenase (BCKDH), which catalyzed the rate-limiting as well as the first irreversible reaction oxidative decarboxylation for branched-chain amino acids degradation, and isovaleryispiramycin biosynthesis was investigated. In the initial glucose concentration experiment, when the residual glucose concentration in the medium declined to 2–4 g/L, the BCKDH activity rose rapidly, and glucose deprivation and the summit of BCKDH activity appeared nearly at the same time. After a delay of about 6 h, the maximal isovalerylspiramycin content was observed. However, the shortage of glucose at the later production phase resulted in the marked decrease in BCKDH activity and isovaleryispiramycin content. In the fermentation in a 50 L fermentor, glucose feeding at the late production phase helped to maintain the residual glucose concentration between 0 and 1 g/L, leading to the high level of BCKDH activity and thus isovalerylspiramycin content. These suggested that glucose concentration could be used as a key parameter to regulate BCKDH activity and isovaleryispiramycin biosynthesis in the bitespiramycin production.     

Structured model for cell growth and enzyme production by recombinant Escherichia coli

Summary A structured cell model has been developed to describe the cultivation of recombinant Escherichia coli K12 with multicopy plasmid under the control of a lambda P_R-promoter and a temperature-sensitive lambda cI 857 repressor. The model, based on measurements of a batch culture in a stirred tank reactor, allows statements to be made on the time variation of intracellular processes. Based on cell regulation, the substrate transfer into the cell was considered to be the rate-limiting step for substrate utilization. The model describes substrate utilization, cell growth, and product formation by means of a system of time-dependent, coupled, and partly non-linear differential equations. The solution of these equations allows calculation of the time variation of the concentrations of substrates (glucose and amino acids), dissolved oxygen and cells in the broth as a function of time and the process parameters.Offprint requests to: K. Schügerl     

Peculiarities of amino acid transport inSchizosaccharomyces pombe: Effects of growth medium

Transport systems for amino acids in the wild-type strain ofSchizosaccharomyces pombe are not constitutive. During growth on different media no transport of acidic, neutral and basic amino acids is detectable. To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose. The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth. After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used.l-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.     

Changes in the metabolome of Saccharomyces cerevisiae associated with evolution in aerobic glucose-limited chemostats

The effect of culture age on intra- and extracellular metabolite levels as well as on in vitro determined specific activities of enzymes of central carbon metabolism was investigated during evolution for over 90 generations of Saccharomyces cerevisiae CEN.PK 113-7D in an aerobic glucose/ethanol-limited chemostat at a specific dilution rate of 0.052 h(-1). It was found that the fluxes of consumed (O2, glucose/ethanol) and secreted compounds (CO2) did not change significantly during the entire cultivation period. However, morphological changes were observed, leading to an increased cellular surface area. During 90 generations of chemostat growth not only the residual glucose concentration decreased, also the intracellular concentrations of trehalose, glycolytic intermediates, TCA cycle intermediates and amino acids were found to have decreased with a factor 5-10. The only exception was glyoxylate which showed a fivefold increase in concentration. In addition to this the specific activities of most glycolytic enzymes also decreased by a factor 5-10 during long-term cultivation. Exceptions to this were hexokinase, phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase of which the activities remained unchanged. Furthermore, the concentrations of the adenylate nucleotides as well as the energy charge of the cells did not change in a significant manner. Surprisingly, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH), malate synthase (MS) and isocitrate lyase (ICL) increased significantly during 90 generations of chemostat cultivation. These changes seem to indicate a pattern where metabolic overcapacities (for reversible reactions) and storage pools (trehalose, high levels of amino acids and excess protein in enzymes) are lost during the evolution period. The driving force is proposed to be a growth advantage in the absence of these metabolic overcapacities.     

Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells

Mammalian cells have the ability to proliferate under different nutrient environments by utilizing different combinations of the nutrients, especially glucose and the amino acids. Under the conditions often used in in vitro cultivation, the cells consume glucose and amino acids in great excess of what is needed for making up biomass and products. They also produce large amounts of metabolites with lactate, ammonia, and some non-essential amino acids such as alanine as the most dominant ones. By controlling glucose and glutamine at low levels, cellular metabolism can be altered and can result in reduced glucose and glutamine consumption as well as in reduced metabolite formation. Using a fed-batch reactor to manipulate glucose at a low level (as compared to a typical batch culture), cell metabolism was altered to a state with substantially reduced lactate production. The culture was then switched to a continuous mode and allowed to reach a steady-state. At this steady-state, the concentrations of cells and antibody were substantially higher than a control culture that was initiated from a batch culture without first altering cellular metabolism. The lactate and other metabolite concentrations were also substantially reduced as compared to the control culture. This newly observed steady-state was achieved at the same dilution rate and feed medium as the control culture. The paths leading to the two steady-states, however, were different. These results demonstrate steady-state multiplicity. At this new steady-state, not only was glucose metabolism altered, but the metabolism of amino acids was altered as well. The amino acid metabolism in the new steady-state was more balanced, and the excretion of non-essential amino acids and ammonia was substantially lower. This approach of reaching a more desirable steady-state with higher concentrations of cells and product opens a new avenue for high-density- and high-productivity-cell culture.     

A robust soft sensor based on artificial neural network for monitoring microbial lipid fermentation processes using Yarrowia lipolytica

Microbial oils produced by Yarrowia lipolytica offer an environmentally friendly and sustainable alternative to petroleum as well as traditional lipids from animals and plants. The accurate measurement of fermentation parameters, including the substrate concentration, dry cell weight, and lipid accumulation, is the foundation of process control, which is indispensable for industrial lipid production. However, it remains a great challenge to measure the complex parameters online during the lipid fermentation process, which is nonlinear, multivariate, and characterized by strong coupling. As a type of AI technology, the artificial neural network model is a powerful tool for handling extremely complex problems, and it can be employed to develop a soft sensor to monitor the microbial lipid fermentation process of Y. lipolytica. In this study, we first analyzed and emphasized the volume of sodium hydroxide and dissolved oxygen concentration as central parameters of the fermentation process. Then, a soft sensor based on a four-input artificial neural network model was developed, in which the input variables were fermentation time, dissolved oxygen concentration, initial glucose concentration, and additional volume of sodium hydroxide. This provides the possibility of online monitoring of dry cell weight, glucose concentration, and lipid production with high accuracy, which can be extended to similar fermentation processes characterized by the addition of bases or acids, as well as changes of the dissolved oxygen concentration.     

Profile of urinary amino acids and their post-translational modifications (PTM) including advanced glycation end-products (AGEs) of lysine,arginine and cysteine in lean and obese ZSF1 rats

Heart failure with preserved ejection fraction (HFpEF) is associated with high mortality and has an increasing prevalence associated with the demographic change and limited therapeutic options. Underlying mechanisms are largely elusive and need to be explored to identify specific biomarkers and new targets, which mirror disease progression and intervention success. Obese ZSF1 (O-ZSF1) rats are a useful animal model, as they spontaneously develop hypertension, hyperlipidemia and glucose intolerance and finally HFpEF. The urinary profile of amino acids and their metabolites of post-translational modifications (PTM), including the advanced glycation end-products (AGEs) of lysine, arginine and cysteine, are poorly investigated in HFpEF and ZSF1 rats. The aim of the present study was to characterize the status of free amino acids and their metabolites of PTM and glycation in lean ZSF1 (L-ZSF1) and O-ZSF1 rats in urine aiming to find possible effects of glucose on the excretion of native and modified amino acids. In the urine of twelve L-ZSF1 and twelve O-ZFS1 rats collected at the age of 20 weeks, we measured the concentration of native and modified amino acids by reliable previously validated stable-isotope dilution gas chromatography-mass spectrometry (GC–MS) approaches. Serum glucose was 1.39-fold higher in the O-ZSF1 rats, while urinary creatinine concentration was 2.5-fold lower in the O-ZSF1 rats. We observed many differences in urinary amino acids excretion between L-ZSF1 and O-ZSF1 rats. The creatinine-corrected homoarginine excretion was twofold lower in the O-ZSF1 rats. We also observed distinct associations between the concentrations of serum glucose and urinary amino acids including their PTM and AGE metabolites in the L-ZSF1 and O-ZSF1 rats. Our study shows that PTM metabolites and AGEs are consistently lower in the L-ZSF1 than in the O-ZSF1 rats. Serum malondialdehyde (MDA) concentration was higher in the O-ZSF1 rats. These results suggest that hyperglycemia, hyperlipidemia and elevated oxidative stress in the O-ZSF1 rats favor PTM methylation of arginine and lysine and the glycation of lysine and cysteine. The area under the receiver operation characteristic (ROC) curve values were 0.996 for serum glucose, 0.951 for urinary creatinine, 0.939 for serum MDA, 0.885 for N^ε-carboxyethyl-lysine, 0.830 for carboxyethyl-cysteine, and 0.792 for monomethyl-lysine. Non-invasive measurement of methylation and glycation products of arginine, lysine and cysteine residues in proteins in urine of L-ZSF1 and O-ZSF1 rats may be useful in studying pathophysiology and pharmacology of HFpEF.

     

Cultivation of mesenchymal tissue with reference to antibody formationin vitro

The optimal conditions for the cultivation of rabbit lymphocytes in suspension and antibody formation by these cellsin vitro were studied. The lymphocytes were cultivated in medium in which serum was replaced by serum albumin containing the alpha globulin fraction. Several media were used, with different amounts and proportions of amino acids, vitamins and glucose. The cells survived best in medium in which a component with a high molecular weight was present and in which the glutamine concentration was 0.5–1 mM and the amount of glucose was 0.4–0.5%. Among the other factors determined, the initial pH had a significant influence on survival of the tissue, the optimal limits being 6.9–7.2. Under these conditions 55–60% of the cells survived during 120 hours’ cultivation. The conditions and factors influencing these findings are discussed. Study of the formation of antibodies againstBrucella suis in vitro showed that antibody formation increased only up to 48 hours, after which it remained at the same level.     

Regulation of formation of proteases inBacillus megaterium

Protease was formed only at the end of the growth phase and its synthesis continued in the stationary phase during the growth of the sporulatingBacillus megaterium KM Sp⁺ in complex media with amino acids or peptone and glucose. The enzyme was also not formed during the growth phase in the glucose containing mineral medium and was detected only later during the stationary phase, smaller quantities being observed than those formed in the complex medium. The addition of glucose at the beginning of the synthesis of protease inhibited the production of the enzyme. On the other hand, the addition of the mixture of amino acids under the same conditions stimulated its formation several fold. Cysteine blocked the synthesis of the enzyme unlike other amino acids. Within a certain range the stimulatory effect of amino acids was related to their concentration, being manifested only after a lag period of several hours. The ability to form protease dissappeared after the formation of refractile spores in sporangial cells. Preliminary communication published in Biochem. biophys. Res. Commun. 37: 233, 1969.     

Biosynthesis of fatty acids and sterols in relation to the antibiotic formation inOudemansiella mucida

The production of mucidin by the basidiomyceteOudemansiella mucida was negatively influenced by the application of D-glucitol as the main carbon source, the effect being independent of the growth rate of the mycelium. The rate of fatty acid synthesis was measured by incorporation of 1-¹⁴C-acetate. After 8 days of cultivation, the amount of fatty acids was approximately half that synthetized during cultivation on glucose. The specific rate of incorporation reached its maximum after seven days of cultivation. Incorporation of 2-¹⁴C-mevalonate into sterols was the same under the two sets of cultivation conditions. Acetate units from the degraded fatty acids are probably also utilized for antibiotic synthesis.