High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system

A continuous fermentation process has been developed in Pichia pastoris (P. pastoris) with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in order to produce large quantities of recombinant human chitinase (rh-chitinase) for preclinical studies as a potential high-dose antifungal drug. Expression levels of about 200 to 400 mg/L have been demonstrated in fed-batch fermentations using strains with either the traditional methanol-inducible or the constitutive GAP promoter. Proteolytic degradation of the enzyme was typically seen in fed-batch fermentations. Continuous production of the enzyme by P. pastoris with the GAP promoter was demonstrated in a 1.5-L working volume fermentor using either glucose or glycerol as the carbon source. The fermentation could be extended for >1 month with a steady-state protein concentration of approximately 300 mg/L. Cell densities were >400 g/L wet cell weight (WCW) (approximately 100 g/L dry cell weight [DCW]) at a dilution rate (D) of 0.83 day(-1) or 1.2 volume exchanges per day (VVD). No proteolytic degradation of the enzyme was seen in the continuous fermentation mode.     

Growth-rate-independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325

Most recombinant proteins generated in filamentous fungi are produced in fed-batch cultures, in which specific growth rate normally decreases progressively with time. Because of this, such cultures are more suited to the production of products that are produced efficiently at low-growth rates (e.g., penicillin) than to products which are produced more efficiently at high-growth rates (e. g., glucoamylase). Fusarium venenatum A3/5 has been transformed (JeRS 325) to produce Aspergillus niger glucoamylase (GAM) under the control of the Fusarium oxysporum trypsin-like protease promoter. No glucoamylase was detected in the culture supernatant during exponential growth of F. venenatum JeRS 325 in batch culture. In glucose-limited chemostat cultures, GAM concentration increased with decrease in dilution rate, but the specific production rate of GAM (g GAM [g biomass](-1) h(-1)) remained approximately constant over the dilution-rate range 0.05 h to 0.19 h(-1), i.e., the recombinant protein was produced in a growth-rate-independent manner. The specific production rate decreased at dilution rates of 0.04 h(-1) and below. Specific production rates of 5.8 mg and 4.0 mg GAM [g biomass](-1) h(-1) were observed in glucose-limited chemostat cultures in the presence and absence of 1 g mycological peptone L(-1). Compared to production in batch culture, and for the same final volume of medium, there was no increase in glucoamylase production when cultures were grown in fed-batch culture. The results suggested that a chemostat operated at a slow dilution rate would be the most productive culture system for enzyme production under this trypsin-like promoter.     

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures to ethanol and sorbitol

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures were carried out at different operational conditions (aeration, feed rate and substrate concentration) to test their effects on the system productivity. In these fermentations, the main products were ethanol and sorbitol. Kinetic parameters were calculated using the experimental data. However, parameters in the sorbitol synthesis rate were estimated from data recorded in different experiments in order to avoid the effect of the simultaneous cell growth and ethanol synthesis. In this case, the crude cell extract was used as source of the enzyme responsible for the sorbitol synthesis. The highest degree of conversion of fructose into sorbitol obtained with the extract was equal to 71% in a sugar mixture with an initial concentration of 200 g/l. Results obtained in the fed-batch fermentations showed that aeration of the culture has a positive effect on the final biomass concentration. However, final ethanol concentration is lower under aerated conditions. The best sugar yields to biomass and ethanol were 0.032 and 0.411 g/g, respectively. On the other hand, the highest sorbitol yield in the fed-batch fermentations was 0.148 g/g.     

Production and separation of alpha-agarase from Altermonas agarlyticus strain GJ1B

This paper presents results on the production of alpha-agarase by a fermentation process and its separation using membrane microfiltration (MF). Optimization of fermentation conditions for alpha-agarase production using Altermonas agarlyticus grown on medium containing agar as a carbon source was done in batch, fed-batch and continuous fermentations. Continuous culture at a dilution rate of 0.03 h(-1) appeared to be best suited for production of alpha-agarase by this organism. At 0.03 h(-1) dilution rate, enzyme activity was 0.9 U/ml. Clarification of broth was done using a hollow-fibre microfiltration membrane. The influence of hydrodynamic parameters on permeate flux and enzyme activity was studied. The best performance was obtained with prefiltered fermentation broth. A stable permeate flux of about 250-270 ml/min.m2 and an enzyme retention rate between 0% and 25% was obtained at temperatures between 6 degrees C and 22 degrees C, transmembrane pressure of 100 mm Hg and fluid cross-flow velocity of 4 x 10(-2) m/s. From the experiments on concentration of fermentation broth, the best compromise between enzyme activity transmission and permeate flux was obtained at a concentration factor of 2.     

Effect of cycle time on fungal morphology, broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon source

For many years, high broth viscosity has remained a key challenge in large-scale filamentous fungal fermentations. In previous studies, we showed that broth viscosity could be reduced by pulsed addition of limiting carbon during fed-batch fermentation. The objective in this study was to determine how changing the frequency of pulsed substrate addition affects fungal morphology, broth rheology, and recombinant enzyme productivity. To accomplish this, a series of duplicate fed-batch fermentations were performed in 20-L fermentors with a recombinant glucoamylase producing strain of Aspergillus oryzae. The total cycle time for substrate pulsing was varied over a wide range (30-2,700 s), with substrate added only during the first 30% of each cycle. As a control, a fermentation was conducted with continuous substrate feeding, and in all fermentations the same total amount of substrate was added. Results show that the total biomass concentration remained relatively unaltered, while a substantial decrease in the mean projected area of fungal elements (i.e., average size) was observed with increasing cycle time. This led to reduced broth viscosity and increased oxygen uptake rate. However, high values of cycle time (i.e., 900-2,700 s) showed a significant increase in fungal conidia formation and significantly reduced recombinant enzyme productivity, suggesting that the fungi channeled substrate to storage compounds rather than to recombinant protein. In addition to explaining the effect of cycle time on fermentation performance, these results may aid in explaining the discrepancies observed on scale-up to larger fermentors.     

Pulsed addition of limiting-carbon during Aspergillus oryzae fermentation leads to improved productivity of a recombinant enzyme

Fungal morphology in many filamentous fungal fermentations leads to high broth viscosity which limits oxygen mass transfer, and often results in reduced productivity. The objective in this study was to determine if a simple, fed-batch, process strategy-pulsed addition of limiting-carbon source-could be used to reduce fungal broth viscosity, and increase productivity of an industrially relevant recombinant enzyme (glucoamylase). As a control, three Aspergillus oryzae fed-batch fermentations were carried out with continuous addition of limiting-carbon. To determine the effect of pulse-feeding, three additional fermentations were carried out with limiting-carbon added in 90-second pulses, during repeated five-minute cycles. In both cases, overall carbon feed-rate was used to control dissolved oxygen concentration, such that increased oxygen availability led to increased addition of limiting-carbon. Pulse-fed fermentations were found to have smaller fungal mycelia, lower broth viscosity, and improved oxygen mass transfer. As a result, more carbon was added to pulse-fed fermentations that led to increased enzyme productivity by as much as 75%. This finding has significant implications for the bioprocessing industry, as a simple process modification which is likely to cost very little to implement in most production facilities, has the potential to substantially increase productivity.     

Stable production of hyaluronic acid in Streptococcus zooepidemicus chemostats operated at high dilution rate

Hyaluronic acid is routinely produced through fermentation of both Group A and C streptococci. Despite significant production costs associated with short fermentations and removal of contaminating proteins released during entry into stationary phase, hyaluronic acid is typically produced in batch rather than continuous culture. The main reason is that hyaluronic acid synthesis has been found to be unstable in continuous culture except at very low dilution rates. Here, we investigated the mechanisms underlying this instability and developed a stable, high dilution rate (0.4 h-1) chemostat process for both chemically defined and complex media operating for more than 150 h of production. In chemically defined medium, the product yield was 25% higher in chemostat cultures than in conventional batch culture when arginine or glucose was the limiting substrate. In contrast, glutamine limitation resulted in higher ATP requirements and a yield similar to that observed in batch culture. In complex, glucose-limited medium, ATP requirements were greatly reduced but biomass synthesis was favored over hyaluronic acid and no improvement in hyaluronic acid yield was observed. The successful establishment of continuous culture at high dilution rate enables both commercial production at reduced cost and a more rational characterization and optimization of hyaluronic acid production in streptococci.     

The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells

Protein hydrolysates as substitutes for serum havebeen employed by many in cell culture mediumformulation, especially with the shift to low proteinor protein-free media. More recently, vegetablehydrolysates have also been added as nutritionalsupplements to fortify the amino acid content in smallpeptide form for batch and fed-batch fermentations. Several of these new hydrolysates (peptones of soy,rice, wheat gluten etc.) were tested as protein-freemedium supplements for the production of a recombinanttherapeutic protein. Multiple peptone-supplemented,continuous perfusion bioreactor experiments wereconducted, varying dilution rates and basal mediumcomposition over the various runs. Cell specificrates and product quality studies were obtained forthe various peptones and compared with peptone-freemedium. The potential for peptones to decreaseintrinsic and proteolytic degradation of the productwas also investigated.It was found that peptones confer a nutritionalbenefit, especially at low dilution rates, for therecombinant BHK cell line used in this investigation.The specific productivity increased 20–30% comparedto the peptone-free controls. However, this benefitwas also fully delivered by using fortified medium inplace of the peptone-enriched media. Therefore, whilepeptones may be considered as useful medium additiveswhen development time is limited, their addition maybe avoided by systematic medium development ifpermitted by the time line of the project.     

Continuous Production of Thermostable beta-Amylase with Clostridium thermosulfurogenes: Effect of Culture Conditions and Metabolite Levels on Enzyme Synthesis and Activity

A beta-amylase-overproducing mutant of Clostridium thermosulfurogenes was grown in continuous culture on soluble starch to produce thermostable beta-amylase. Enzyme productivity was reasonably stable over periods of weeks to months. The pH and temperature optima for beta-amylase production were pH 6.0 and 60 degrees C, respectively. Enzyme concentration was maximized by increasing biomass concentration by using high substrate concentrations and by maintaining a low growth rate. beta-Amylase concentration reached 90 U ml at a dilution rate of 0.07 h in a 3% starch medium. A further increase in enzyme activity levels was limited by acetic acid inhibition of growth and low beta-amylase productivity at low growth rates.     

Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.     

Optimized nikkomycin production by fed-batch and continuous fermentation

We performed fed-batch and continuous fermentations to extend the time of maximal nikkomycin production by Streptomyces tendae Tü 901/S 2566. This was achieved by the fed-batch culture technique. Furthermore, high productivity was obtained at slow growth rates in a continuous fermentation process. Different dilution rates with and without carbon limitation were done and the results were compared. Correspondence to : T. Schüz     

Pullulan from peat hydrolyzate fermentation kinetics

The Luedeking-Piret equation was used to fit the kinetic data of pullulan fermentations from peat hydrolyzate substrate. In batch mode, the kinetic parameters m, n, alpha, and beta varied as a function of fermentation conditions: aeration rate, agitation speed, and temperature. In constant-feed fed-batch mode, the parameters Varied according to the feed rates. In peat hydrolyzate medium, the polysaccharide synthesis was strongly growth associated in batch and continuous fermentations but entirely growth associated in fedbatch fermentations. The fed-batch mode of fermentation with an appropriate feed rate is more advantageous with respect to batch and continuous fermentations. Therefore, if the fermentation is started batchwise and then followed by fed-batch mode at a constant feed rate, the overall polysaccharide productivity (g pullulan/L h) is significantly higher than those obtained with batch or continuous fermentations using the same total medium volume.     

Activities of plasma membrane-bound enzymes isolated from roots of spruce (Picea abies) grown in the presence of aluminium

In batch cultures of Petunia hybrida cv. Rosy Morn Fertile. one respiratory peak is usually observed shortly after subculturing. However, two types of peak respiration could be distinguished, one connected with the dilution process and one with sugar addition at low biomass concentrations. The dilution peak was observed when cells were diluted in medium without sugar, in the presence or absence of mannitol. The sugar peak occurred only after previous dilution of the cells and not when sugar is added at high biomass concentrations Apparently the existence of a dilute suspension is a prerequisite for the induction of the peak. The presence of sugar is not a prerequisite for the increased respiratory activity but it is necessary lor growth: however, growth is possible without the increase in respiration, as was shown by the addition of sugar to a culture with a high biomass concentration. The peak caused by dilution either in the presence or absence of sugar showed no significant differences in height. The height of the peak caused by sugar addition to a previously diluted cell suspension was correlated with the sugar concentration. The respiratory peak disappeared long before the end of the growth period; this decline of the respiratory rates was not connected to sugar or oxygen limitation. In a continuous culture of Petunia hybrida growing at low biomass concentration, the respiration was always at the high level as observed during the peak of batch culture. Growing at lower biomass concentrations might be more expensive for plant cell suspensions.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Production of the macrolide antibiotic tylosin in cyclic fed-batch culture

Tylosin-producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate-glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (q(tylosin), mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high q(tylosin) had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed-batch fermentations with glutamate and glucose being fed to the culture in cyclic square-wave profiles with methyloleate in excess showed several-fold increase in final q(tylosin) and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed-batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when alpha-ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of q(tylosin) up to 240 h.     

Recombinant protein production in cultures of an Escherichia coli trp − strain

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp ^– derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-l batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l. Correspondence to: F. Bolivar