Fed-batch optimization of alpha-amylase and protease-producing Bacillus subtilis using Markov chain methods

A stoichiometry-based model for the fed-batch culture of the recombinant bacterium Bacillus subtilis ATCC 6051a, producing extracellular alpha-amylase as a desirable product and proteases as undesirable products, was developed and verified. The model was then used for optimizing the feeding schedule in fed-batch culture. To handle higher-order model equations (14 state variables), an optimization methodology for the dual-enzyme system is proposed by integrating Pontryagin's optimum principle with fermentation measurements. Markov chain Monte Carlo (MCMC) procedures were appropriate for model parameter and decision variable estimation by using a priori parameter distributions reflecting the experimental results. Using a simplified Metropolis-Hastings algorithm, the specific productivity of alpha-amylase was maximized and the optimum path was confirmed by experimentation. The optimization process predicted a further 14% improvement of alpha-amylase productivity that could not be realized because of the onset of sporulation. Among the decision variables, the switching time from batch to fed-batch operation (t(s)) was the most sensitive decision variable.     

Fed-batch fermentation for the production of alpha-amylase by Bacillus amyloliquefaciens

Fed-batch cultures were performed to maximize the alpha-amylase activity in a bioreactor. Kinetic equations containing a catabolite repression effect were used to model the enzyme formation from Bacillus amyloliquefaciens. Fed-batch culture experiments were performed using maltose to implement the optimal feeding strategy. Optimal fed-batch culture based on sequential parameter estimation was performed successfully using off-line analysis while the fermentation was in progress. The enzyme activity from the fed-batch culture employing maltose was higher than that of the batch culture by 60%. Enzyme production using starch showed similar trends to those obtained using maltose.     

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.     

Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy

A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h).     

Morphologically structured model for antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis

A morphologically structured model is proposed to describe trends in biomass growth, substrate consumption, and antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis. Filamentous biomass is structured into three morphological compartments (apical, subapical, and hyphal), and the production of retamycin, a secondary metabolite, is assumed to take place in the subapical cell compartment. Model accounts for the effect of glucose as well as complex nitrogen source on both the biomass growth and retamycin production. Laboratory data from bench-scale batch and fed-batch fermentations were used to estimate some model parameters by nonlinear regression. The predictive capability of the model was then tested for additional fed-batch and continuous experiments not used in the previous fitting procedure. The model predictions show fair agreement to the experimental data. The proposed model can be useful for further studies on process optimization and control.     

Enhanced production of alpha-amylase in fed-batch cultures of Bacillus subtilis TN106[pAT5

Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid-encoded protein (alpha-amylase) are investigated in batch, continuous, and fed-batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of alpha-amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of alpha-amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed-batch experiments with constant volumetric feed rate demonstrate that alpha-amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable alpha-amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed-batch experiments, production of alpha-amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two-stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and alpha-amylase production in the second stage, in attaining increased bulk alpha-amylase activity is demonstrated. (c) 1993 John Wiley & Sons, Inc.     

Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol

Metabolic fluxes estimated from stable-isotope studies provide a key to understanding cell physiology and regulation of metabolism. A limitation of the classical method for metabolic flux analysis (MFA) is the requirement for isotopic steady state. To extend the scope of flux determination from stationary to nonstationary systems, we present a novel modeling strategy that combines key ideas from isotopomer spectral analysis (ISA) and stationary MFA. Isotopic transients of the precursor pool and the sampled products are described by two parameters, D and G parameters, respectively, which are incorporated into the flux model. The G value is the fraction of labeled product in the sample, and the D value is the fractional contribution of the feed for the production of labeled products. We illustrate the novel modeling strategy with a nonstationary system that closely resembles industrial production conditions, i.e. fed-batch fermentation of Escherichia coli that produces 1,3-propanediol (PDO). Metabolic fluxes and the D and G parameters were estimated by fitting labeling distributions of biomass amino acids measured by GC/MS to a model of E. coli metabolism. We obtained highly consistent fits from the data with 82 redundant measurements. Metabolic fluxes were estimated for 20 time points during course of the fermentation. As such we established, for the first time, detailed time profiles of in vivo fluxes. We found that intracellular fluxes changed significantly during the fed-batch. The intracellular flux associated with PDO pathway increased by 10%. Concurrently, we observed a decrease in the split ratio between glycolysis and pentose phosphate pathway from 70/30 to 50/50 as a function of time. The TCA cycle flux, on the other hand, remained constant throughout the fermentation. Furthermore, our flux results provided additional insight in support of the assumed genotype of the organism.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

Effects of substrate branching characteristics on kinetics of enzymatic depolymerizaion of mixed linear and branched polysaccharides: I. Amylose/amylopectin alpha-amylolysis

Hydrolysis reactions of homopolysaccharides, which differ in their degree of branching, and mixtures of linear and branched polymers were carried out with alpha-amylase. The branching structures of both the original amylopectin substrate and the cluster domains of amylopectin, obtained by ethanol precipitation of the products of the action of alpha-amylase, were characterized via enzymatic digestion with debranching enzyme (i.e., isoamylase), followed by the fractions of the resulting products using gel filtration chromatography. The structural properties (i.e., molecular weight, molecular weight distribution, and branching characteristics) of the resulting products during depolymerization of amylose, amylopectin and their mixtures via alpha-amylase were characterized by size exclusion chromatography coupled with a low angle laser right scattering (SEC/LALLS) technique. It was determined that substrate branching characteristics strongly influence both the observed enzymatic activity as well as the enzyme's action pattern. A simplified kinetic model that represents the hydrolysis reactions of amylose and amylopectin mixtures via endo-acting alpha-amylase is proposed. We found that that reaction kinetics (i.e., enzyme affinity) was also governed by the substrate's conformation in solution. The relationships between the mass fraction of branched polymers and the kinetic parameters during alpha-amylolysis were compared with those predicted by the kinetic model. Excellent agreement was found between the model predictions and the experimental observations. The results reported here imply and interrelationship between enzyme action and polymeric substrate structural properties. (c) 1994 John Wiley & Sons, Inc.     

Production of fungal alpha-amylase by Saccharomyces kluyveri in glucose-limited cultivations

Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. Alpha-amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 micro plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active alpha-amylase in both cases. The S. cerevisiae 2 micro plasmid was found to be stable in S. kluyveri as evaluated by a constant alpha-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted alpha-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h(-1) (4.8-5.7 mg (g dry weight)(-1)). At a dilution rate of 0.3 h(-1) the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l(-1) and the alpha-amylase concentration reached 320 mg l(-1). Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.     

Stoichiometric balance based macrokinetic model for Penicillium chrysogenum in fed-batch fermentation

A macrokinetic model describing mycelium growth and product formation in fed-batch fermentation of Penicillium chrysogenum is proposed on the basis of intracellular metabolic pathway networks found in the literature. The model is composed of the stoichiometric balance equations for carbon source, ATP, NADH and pyruvate. A regulator model is used as an auxiliary part to simulate the lag phase at the beginning of product synthesis. Combining the macrokinetic model with the bioreactor model, one yields the relationship between manipulating variables, i.e., the substrate feeding rate, and the other state variables such as biomass, substrate and product concentrations. The rolling parameter identification is then applied to online identify the most sensitive model parameters dealing with the model mismatch caused by time-variant kinetic changes. Model validation was performed with the data of 22 batches from industrial-scale penicillin cultivation. Finally, the predictions of the product concentration, up to 24 h ahead, are carried out with an average prediction error of less than 3.5%.     

SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus alpha-amylase

The Bacillus stearothermophilus alpha-amylase (amyS) is a heat-stable monomeric exoenzyme which catalyses random hydrolysis of 1,4-alpha-glucosidic linkages in polyglucosans. The Bacillus alpha-amylase was engineered for use as an intracellular (AmyS(Delta S)) as well as a secreted reporter protein (SAMY; secreted alpha-amylase) in mammalian cells. The 5' end of amyS containing the prokaryotic secretion signal was either deleted (amyS(Delta S)) or replaced by a murine immunoglobulin secretion signal. SAMY was cloned under control of the cytomegalovirus promoter (P(CMV)) in a mammalian expression vector or the promoter of the human elongation factor 1 alpha (P(EF1 alpha)) in a lentiviral expression context. A variety of mammalian and human cell lines growing as monolayers, in suspension or as three-dimensional spheroids were transfected/transduced with SAMY- or amyS(Delta S)-encoding expression/lentiviral vectors and alpha-amylase activity was measured in cell lysates and culture supernatants. These experiments showed that SAMY and AmyS(Delta S) were either secreted or remained intracellular as highly sensitive reporter enzymes. SAMY expression and detection was fully compatible with established SEAP (human secreted alkaline phosphatase) and u-PA(LMW) (low molecular weight urokinase-type plasminogen activator) reporter systems and could be used to quantify expression of up to three independent genes in one culture supernatant.     

Kinetics of alpha-amylase secretion in Aspergillus oryzae.

Pulse and pulse-chase experiments have been performed to study L-[(35)S] methionine incorporation and protein secretion kinetics in Aspergillus oryzae. Pulse experiments confirmed the mechanism of methionine uptake reported previously for Penicillium chrysogenum (Benko et al., 1967). Pulse-chase experiments were carried out to investigate the alpha-amylase secretion kinetics in A. oryzae. No unglycosylated alpha-amylase was detected neither intracellularly nor extracellularly demonstrating that glycosylation was not the rate controlling step in the secretory pathway. The pulse chase experiments indicated that there are two pools of intracellular alpha-amylase: a fast secreted and a slow secreted. The secretion of those two pools were described with a kinetic model, which was fitted to the pulse chase experiments.     

A combined metabolic/polymerization kinetic model on the microbial production of poly(3-hydroxybutyrate)

In the present work, an integrated dynamic metabolic/polymerization kinetic model is developed for the prediction of the intracellular accumulation profile and the molecular weight distribution of poly(3-hydroxybutyrate) (P(3HB) or PHB) produced in microbial cultures. The model integrates two different length/time scales by combining a polymerization kinetic model with a metabolic one. The bridging point between the two models is the concentration of the monomer unit (i.e. 3-hydroxybutyryl-CoA) produced during the central aerobic carbon metabolism. The predictive capabilities of the proposed model are assessed by the comparison of the calculated biopolymer concentration and number average molecular weight with available experimental data obtained from batch and fed-batch cultures of Alcaligenes eutrophus and Alcaligenes latus. The accuracy of the proposed model was found to be satisfactory, setting this model a valuable tool for the design of the process operating profile for the production of different polymer grades with desired molecular properties.     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

Modeling and simulation of fed-batch protein refolding process

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7 mg mL(-)(1), respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.     

Evaluation of two unstructured mathematical models for the penicillin G fedbatch fermentation

The mathematical model for the penicillin G fed-batch fermentation proposed by Heijnen et al. (1979) is compared with the model of Bajpai & Reuß (1980). Although the general structure of these models is similar, the difference in metabolic assumptions and specific growth and production kinetics results in a completely different behaviour towards product optimization. A detailed analysis of both models reveals some physical and biochemical shortcomings. It is shown that it is impossible to make a reliable estimation of the model parameters, only using experimental data of simple constant glucose feed rate fermentations with low initial substrate amount. However, it is demonstrated that some model parameters might be key factors in concluding whether or not altering the substrate feeding strategy has an important influence on the final amount of product.It is illustrated that feeding strategy optimization studies can be a tool in designing experiments for parameter estimation purposes.     

Microbial production of polyhydroxybutyrate with tailor-made properties: an integrated modelling approach and experimental validation

The microbial production of polyhydroxybutyrate (PHB) is a complex process in which the final quantity and quality of the PHB depend on a large number of process operating variables. Consequently, the design and optimal dynamic operation of a microbial process for the efficient production of PHB with tailor-made molecular properties is an extremely interesting problem. The present study investigates how key process operating variables (i.e., nutritional and aeration conditions) affect the biomass production rate and the PHB accumulation in the cells and its associated molecular weight distribution. A combined metabolic/polymerization/macroscopic modelling approach, relating the process performance and product quality with the process variables, was developed and validated using an extensive series of experiments and measurements. The model predicts the dynamic evolution of the biomass growth, the polymer accumulation, the consumption of carbon and nitrogen sources and the average molecular weights of the PHB in a bioreactor, under batch and fed-batch operating conditions. The proposed integrated model was used for the model-based optimization of the production of PHB with tailor-made molecular properties in Azohydromonas lata bacteria. The process optimization led to a high intracellular PHB accumulation (up to 95% g of PHB per g of DCW) and the production of different grades (i.e., different molecular weight distributions) of PHB.     

Production of alpha-amylase in fed-batch cultures of vgb+ and vgb- recombinant Escherichia coli: some observations.

Synthesis and excretion of Bacillus stearothermophilus alpha-amylase is analyzed in fed-batch cultivations of Escherichia coli JM103[pMK79] and E. coli JM103[pMK57], the former strain containing the plasmid-encoded Vitreoscilla hemoglobin (VHb) gene (vgb) and the latter strain being devoid of this gene. Fed-batch operation is observed to be substantially superior to batch operation as concerns the alpha-amylase production rate and the extent of excretion of the enzyme. Faster feeding of a nutrient medium (LB or M9) discourages synthesis of alpha-amylase. While synthesis of alpha-amylase in the vgb(-) strain is discouraged when oxygen availability is reduced, the reverse is the case with the vgb(+) strain, the promotion of alpha-amylase synthesis in the latter strain being linked to the synthesis of VHb. Increased availability of the principal carbon source (glucose) in a defined medium leads to overproduction of both alpha-amylase and VHb under oxygen limitation, which may be responsible for the segregational instability observed with the vgb(+) strain. The very high extents of excretion of alpha-amylase attained in fed-batch cultures are encouraging for downstream processing of the recombinant protein.