Steady-state Continuous Cultures Research Articles

Lysine synthesis control in Corynebacterium glutamicum RC 115 in mixed substrate (glucose-acetate) medium

The effect of acetate as a glucose co-substrate on growth, lysine synthesis and experimental lysine yield from carbon substrates by Corynebacterium glutamicum RC 115 was investigated. It was found that low amounts of acetate, injected with a glucose-acetate pulse into the steady-state continuous culture in bioreactor, caused a slight decrease in the specific rates of glucose uptake and bacterial growth, but a significant increase in the cell specific rate of lysine synthesis and an increase in lysine yield. In contrast, acetate injected in high amounts was followed by a drastic decrease in the values of these parameters. A strong increase in experimental lysine yield under the latter conditions was reached in the response to pyruvate addition. Therefore it was shown that acetate in low concentrations can be used as a glucose co-substrate to increase the cell specific rate of lysine synthesis and lysine yield by C. glutamicum RC 115. Pyruvate supplementation was found as a promising method to enhance lysine synthesis by bacterial cells grown in glucose-acetate media with an increased concentration of acetate.

Shear stress tolerance and biochemical characterization of Phaeodactylum tricornutum in quasi steady-state continuous culture in outdoor photobioreactors

A bubble column and two airlift photobioreactors (a draft-tube sparged vessel and a split-cylinder device) of the same general design (0.19 m column diameter, 2 m tall, 0.06 m 3 working volume) were evaluated for outdoor continuous culture of the microalga Phaeodactylum tricornutum at a dilution rate of 0.03 h −1. At a daily averaged irradiance (photosynthetically active) value of 900 μE m −2 s −1, all bioreactors attained a quasi steady-state biomass concentration of ∼1 kg m −3 and a biomass productivity of ∼0.3 kg m −3 per day when the aeration velocity was 0.01 m s −1. The microalgal cells were susceptible to aeration-associated hydrodynamic stress if the superficial aeration velocity exceeded 0.01 m s −1. Supplementing the culture medium with 0.02% or more carboxymethyl cellulose (CMC), allowed stable culture under conditions that had previously damaged the cells. The average elemental composition of the biomass was: 49.2% C, 6.3% H, 0.8% N, and 1.3% S. The chlorophylls, carotenoids, and pigments content of the biomass changed with irradiance within a given day. Low irradiance favored accumulation of the light capture pigments. Increasing daily irradiance led to accumulation of carbohydrates. Some of the carbohydrate accumulated during the day was consumed at night and partly converted to proteins. Eicosapentaenoic acid (EPA, 20:5n3) constituted between 27 and 30% of the total fatty acids present, or 2.6–3.1% of the dry biomass. The other main fatty acids present were palmetic acid (16:0), palmoleic acid (16:1n7), and myristic acid (14:0). On average, these three fatty acids constituted 16.9% (16:0), 14.0% (16:1n7) and 9.4% (14:0) of the total fatty acids present.

Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

The production of the extracellular alkaline protease Savinase® (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h −1, whereas the yield of Savinase® on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase® were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates.

Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp.

A phosphatase-overproducing Citrobacter sp. (NCIMB 40259) was grown in an air-lift reactor in steady-state continuous culture under limitation of carbon, phosphorus or nitrogen. Substantial biofilm formation, and the highest phosphatase activity, were observed under lactose limitation. However, the total amount of biofilm wet biomass and the phosphatase specific activity were reduced in phosphorus- or nitrogen-limited cultures or when glucose was substituted for lactose as the limiting carbon source. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) showed differences in cell and biofilm morphology in relation to medium composition. Electron microscopy suggested that the differences in biofilm formation may relate to differential expression of fimbriae on the cell surface.

Transcriptional repression of ADH2-regulated beta-xylanase production by ethanol in recombinant strains of Saccharomyces cerevisiae.

The regulation of endo-beta-(1,4)-xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2 gene from Trichoderma reesei under control of the promoter of the alcohol dehydrogenase II (ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l(-1). Pulses of 190-630 mmol ethanol l(-1) into aerobic glucose-limited steady-state continuous cultures reduced the xylanase activity about five-fold and showed that ethanol repressed the ADH2 promoter, as was evident from Northern blot analyses. Derepression of the ADH2-regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l(-1).

Transcriptional repression of -regulated ?-xylanase production by ethanol in recombinant strains of

The regulation of endo-β-(1,4)-xylanase production by two different strains of Saccharomyces cerevisiae, each transformed with the XYN2 gene from Trichoderma reesei under control of the promoter of the alcohol dehydrogenase II (ADH2) gene of S. cerevisiae, was investigated. In batch culture, the rate of xylanase production was severely reduced by the pulse addition of 390 mmol ethanol l−1. Pulses of 190–630 mmol ethanol l−1 into aerobic glucose-limited steady-state continuous cultures reduced the xylanase activity about five-fold and showed that ethanol repressed the ADH2 promoter, as was evident from Northern blot analyses. Derepression of the ADH2-regulated xylanase gene occurred at ethanol concentrations below approximately 50 mmol l−1.

Kinetics and metabolism of cellulose degradation at high substrate concentrations in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium.

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h(-1), biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose x liter(-1) the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose x liter(-1), respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose x liter(-1). Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119-130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD+, and q(NADH produced)/q(NADH used) ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y(max)X/S) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y(max)ATP) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327-335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells(-1) x h(-1) could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells(-1) x h(-1); (ii) while the NADH/NAD+ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.

Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture

The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.

Oxygen availability and growth of Escherichia coli W3110: Dynamic responses to limitation and starvation

It is well known that oxygen availability can have a profound effect upon the growth of an Escherichia coli culture with respect to acetate excretion or lower product yield. The current investigation seeks to determine the dynamic responses of steady state continuous cultures to abrupt changes in the oxygen supply. This should yield information regarding the behaviour of such cells as they circulate through areas of low and high oxygen availability in an industrial-scale bioreactor. It was found that a decoupling of catabolism and anabolism occurred following sudden switches both from and to oxygen limitation. It also appeared that the imposed growth rate had an effect on the speed of recovery of the system following any such changes.

Global adaptations resulting from high population densities in Escherichia coli cultures.

The scope of population density effects was investigated in steady-state continuous cultures of Escherichia coli in the absence of complications caused by transient environmental conditions and growth rates. Four distinct bacterial properties reflecting major regulatory and physiological circuits were analyzed. The metabolome profile of bacteria growing at high density contained major differences from low-density cultures. The 10-fold-elevated level of trehalose at higher densities pointed to the increased role of the RpoS sigma factor, which controls trehalose synthesis genes as well as the general stress response. There was an eightfold difference in RpoS levels between bacteria grown at 10(8) and at 10(9) cells/ml. In contrast, the cellular content of the DNA binding protein H-NS, controlling many genes in concert with RpoS, was decreased by high density. Since H-NS and RpoS also influence porin gene expression, the influence of population density on the intricate regulation of outer membrane composition was also investigated. High culture densities were found to strongly repress ompF porin transcription, with a sharp threshold at a density of 4.4 x 10(8) cells/ml, while increasing the proportion of OmpC in the outer membrane. The density-dependent regulation of ompF was maintained in rpoS or hns mutants and so was independent of these regulators. The consistently dramatic changes indicate that actively growing, high-density cultures are at least as differentiated from low-density cultures as are exponential- from stationary-phase bacteria.

Kinetic analysis of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch points for distribution of carbon fluxes inside and outside cells as revealed by steady-state continuous culture.

During the growth of Clostridium cellulolyticum in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by C. cellulolyticum was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphoglucomutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen biosynthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosynthesis reached a maximum during the exponential growth phase. Glycogen synthesis in C. cellulolyticum was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262-3269, 1999).

Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation

The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media. Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate. In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h-1 whereas in synthetic medium it was 10% at 0.035 h-1. In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4%. At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose. Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose. The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media. These data suggest that, in C. cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process. In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C. cellulolyticum.

Growth-rate-dependent bacteriochlorophyll c / d ratio in the antenna of Chlorobium limicola strain UdG6040

The green sulfur bacterium Chlorobium limicola UdG6040 exhibited a significant change in the spectral properties of its antenna when transferred from batch culture to a sulfide-limited chemostat. In steady-state continuous cultures, the in vivo absorption maximum of the culture changed to shorter wavelengths according to the dilution rate. The maximum difference observed was of 15 nm when cells were growing at 0.087 h–1. HPLC analyses revealed that the observed spectral change was caused by a partial enrichment of the original BChl c-containing antenna with BChl d molecules together with a change in the homolog composition of both pigments. The relative amount of BChl d reached a maximum value of 50% when cells were growing at 0.087 h–1. The content of BChl d decreased to less than the 22% when the dilution rate was diminished to 0.015 h–1. An unbalance of pigment synthesis at high dilution rates is suspected to be responsible of the changes observed in the antenna composition. Chlorosomes isolated from Chl. limicola UdG6040 growing at 0.070 h–1 contain organised pools of BChl c and BChl d in equal amounts.

Modelling of the protonophoric uncoupling by phenoxyacetic acid of the plasma membrane potential of Penicillium chrysogenum

Physiological effects of phenoxyacetic acid, the penicillin V side-chain precursor, on steady-state continuous cultures of Penicillium chrysogenum have been studied both theoretically and experimentally. Theoretical calculations show that at an extracellular pH of 6.50, phenoxyacetic acid has negligible influence on the growth energetics due to protonophoric uncoupling of membrane potentials by passive diffusive uptake. On the other hand, when the extracellular pH is lowered to 5.00, a severe maintenance-related uncoupling effect of phenoxyacetic acid is calculated. These findings were confirmed experimentally by steady-state continuous cultivations with a high-yielding penicillin strain of P. chrysogenum performed on a chemically defined and glucose-limited medium at pH 6.50 and pH 5.00, both with and without phenoxyacetic acid present. The yield and maintenance coefficients were determined from steady-state measurements of the specific uptake rates of glucose and oxygen and the specific production rate of carbon dioxide as functions of the specific growth rate. Combining these data with a simple stoichiometric model for the primary metabolism of P. chrysogenum allows quantitative information to be extracted on the growth energetics in terms of ATP spent in maintenance- and growth-related processes, i.e. mATP and YxATP. The increased maintenance-related ATP consumption when adding phenoxyacetic acid at pH 5.00 agrees with the theoretical calculations on the uncoupling effect of phenoxyacetic acid. When YxATP is compared with earlier reported values for the theoretical ATP requirement for biosynthesis of P. chrysogenum, i.e. YxATP,growth, it is found that YxATP,growth is only 40–50% of YxATP, which stresses that a large amount of ATP is wasted in turnover of macromolecules, leaks, and futile cycles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 761–767, 1998.

Modelling of the protonophoric uncoupling by phenoxyacetic acid of the plasma membrane potential ofPenicillium chrysogenum

Physiological effects of phenoxyacetic acid, the penicillin V side-chain precursor, on steady-state continuous cultures of Penicillium chrysogenum have been studied both theoretically and experimentally. Theoretical calculations show that at an extracellular pH of 6.50, phenoxyacetic acid has negligible influence on the growth energetics due to protonophoric uncoupling of membrane potentials by passive diffusive uptake. On the other hand, when the extracellular pH is lowered to 5.00, a severe maintenance-related uncoupling effect of phenoxyacetic acid is calculated. These findings were confirmed experimentally by steady-state continuous cultivations with a high-yielding penicillin strain of P. chrysogenum performed on a chemically defined and glucose-limited medium at pH 6.50 and pH 5.00, both with and without phenoxyacetic acid present. The yield and maintenance coefficients were determined from steady-state measurements of the specific uptake rates of glucose and oxygen and the specific production rate of carbon dioxide as functions of the specific growth rate. Combining these data with a simple stoichiometric model for the primary metabolism of P. chrysogenum allows quantitative information to be extracted on the growth energetics in terms of ATP spent in maintenance- and growth-related processes, i.e. mATP and YxATP. The increased maintenance-related ATP consumption when adding phenoxyacetic acid at pH 5.00 agrees with the theoretical calculations on the uncoupling effect of phenoxyacetic acid. When YxATP is compared with earlier reported values for the theoretical ATP requirement for biosynthesis of P. chrysogenum, i.e. YxATP, growth, it is found that YxATP,growth is only 40-50% of YxATP, which stresses that a large amount of ATP is wasted in turnover of macromolecules, leaks, and futile cycles.

Maintenance requirements in Bacillus sphaericus 1593M under dual substrate limitations estimated at zero growth rate in a total cell retention culture

The maintenance requirement(s) exhibited by Bacillus sphaericus1593M was clearly established from the data on the steady state continuous cultures wherein the growth yield was found to be increasing with increase in dilution rate (4). In the present study we have attempted to estimate the maintenance coefficient of B. sphaericus 1593M by achieving zero growth rate in a total cell retention culture (TCRC). Zero growth rate was achieved at a cell density of 5.57 g l−1 which remained constant reflecting a steady state in the TCRC. Based on the substrate flux calculations, maintenance coefficient was found to be 0.087 h−1. Further, using this data of maintenance coefficient, steady state cell density and substrate profiles in continuous cultures of B. sphaericus 1593M were predicted by simulation, which showed a close relationship to that of the experimental data.

Simple generic model for dynamic experiments with Saccharomyces cerevisiae in continuous culture: decoupling between anabolism and catabolism.

The dynamic behavior of a continuous culture of Saccharomyces cerevisiae subjected to a sudden increase in the dilution rate has been successfully modelled for anaerobic growth on glucose, and for aerobic growth on acetate, on ethanol, and on glucose. The catabolism responded by an immediate jump whereas biosynthesis did not. Thus catabolism was in excess to anabolism. The model considers the decoupling between biosynthesis and catabolism, both types of reactions being modelled by first-order kinetic expressions evolving towards maximal values. Yield parameters and maximal reaction rates were identified in steady state continuous cultures or during batch experiments. Only the time constant of biosynthesis regeneration, tauX, and the time constant of catabolic capacity regeneration, taucat, had to be identified during transient experiments. In most experiments tauX was around 3 h, and taucat varied between 2 and 2.5 h for the different metabolisms investigated.

Influence of bacteria on phytoplankton cell mortality with phosphorus or nitrogen as the algal-growth-limiting nutrient

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 14:271-280 (1998) - doi:10.3354/ame014271 Influence of bacteria on phytoplankton cell mortality with phosphorus or nitrogen as the algal-growth-limiting nutrient Corina P. D. Brussaard1,*, Roel Riegman2 1Department of Microbiology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway2Department of Biological Oceanography, Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg, Texel, The Netherlands *E-mail: corina.brussaard@uib.im.no The effects of bacteria on phytoplankton mortality were studied with phosphorus or nitrogen as the algal-growth-limiting nutrients. Experiments were performed with the diatom Ditylum brightwellii using batch cultures, steady state continuous cultures and batch-mode cultures which were starved for the limiting nutrient after being preconditioned in a chemostat. With phosphorus limiting algal growth, the specific death rates of D. brightwellii generally increased upon bacterial inoculation. Bacteria enhanced algal mortality very likely due to competition with D. brightwellii for the limiting phosphate. With nitrogen as the algal-growth-limiting nutrient, however, the presence of bacteria had either no pronounced effect on or led to a reduction of the specific death rates of D. brightwellii. Remineralized ammonium was probably partly utilized by N-starved cells of D. brightwellii, leading to reduced death rates of the algal cells. Bacteria thus indirectly prolonged survival of D. brightwellii populations under N starvation. This study shows that bacteria can affect phytoplankton survival, which in turn may influence algal species succession. The degree of bacterial influence on algal death kinetics depends largely on the type of nutrient limiting algal growth and on the culture conditions. Algal cell mortality · Ditylum brightwellii · Bacteria · N and P deficiency · Starvation Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 14, No. 3. Publication date: April 06, 1998 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1998 Inter-Research.

Influence of the Dissolved Oxygen Concentration on the Penicillin Biosynthetic Pathway in Steady-State Cultures of Penicillium chrysogenum

The influence the of dissolved oxygen concentration on penicillin biosynthesis was studied in steady-state continuous cultures of a high-yielding strain of Penicillium chrysogenum operated at a dilution rate of 0.05 h−1. The dissolved oxygen concentration was varied between 0.019 and 0.344 mM (corresponding to 7% and 131% air saturation at 1 bar) solely through manipulations of the inlet gas composition. At dissolved oxygen concentrations above 0.06–0.08 mM, a constant specific penicillin productivity of around 22 (μmol/g of DW) /h is maintained. At lower oxygen concentrations, the specific penicillin productivity decreases, and a value of 17 (μmol/g of DW) /h was obtained when the dissolved oxygen concentration was 0.042 mM. A further lowering of the dissolved oxygen concentration to 0.019 mM resulted in the loss of penicillin production. However, penicillin productivity was instantly recovered to its maximum value when the dissolved oxygen concentration was reset to a value above 0.08 mM. The specific formation rates of a number of typical byproducts of the penicillin production, i.e., δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine, isopenicillin N, 6-aminopenicillanic acid, 8-hydroxypenillic acid and 6-oxopiperidine-2-carboxylic acid, the δ-lactam form of α-aminoadipic acid, all increased with decreasing dissolved oxygen concentration. A simultaneous increase in the secretion of glutathione was observed, and a link between the secretion of δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine and glutathione is suggested. The intracellular pools of the pathway intermediates δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine and isopenicillin N increased respectively 2- and 3-fold when the dissolved oxygen concentration was lowered from 0.344 to 0.042 mM, whereas the intracellular pools of glutathione and cysteine decreased at low dissolved oxygen concentrations. On the basis of the intracellular pool measurements, metabolic control analysis is performed, and the flux control coefficients for the first two enzymes in the penicillin biosynthetic pathway, i.e., δ-(l-α-aminoadipyl) -l-cysteinyl-d-valine synthetase and isopenicillin N synthetase, are calculated at different dissolved oxygen concentrations. It is found that for low dissolved oxygen concentrations, the flux control is mainly exerted by the isopenicillin N synthetase, whereas a more even distribution of the flux control by the two enzymes is encountered at high dissolved oxygen concentrations.

Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-alpha-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 microM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.