Metabolic flux analysis for human therapeutic protein productions and hypothesis for new therapeutical strategies in medicine

This work may be considered as a model study for therapeutic protein production, and a theoretical approach to hypothesise new medical strategies to further applied medical questions. A comprehensive generalised metabolic reaction network of Bacillus licheniformis that considers 149 reaction fluxes and 106 metabolites was used in the mass flux balance-based stoichiometric model for the analysis of human leukocyte interferon (IFN-alpha(1)) and erythropoietin (EPO) production capacities of recombinant Bacillus species. The importance of cellular energetics on optimum performance was quantitatively assessed. The metabolic pathways leading to optimised IFN-alpha(1) and EPO overproduction were determined for the two carbon sources that have different reduction degrees (gamma), i.e. glucose (gamma=4.0) and citrate (gamma=3.0), and the variation of the fluxes were obtained. Metabolic capacity analyses showed that maximum IFN-alpha(1) and EPO synthesis rates were, respectively, 0.062 and 0.055 mmol g(-1) DWh(-1) at mu=0h(-1) when glucose uptake rate was 10 mmol g(-1) DWh(-1); and IFN-alpha(1) and EPO synthesis rates decreased, respectively, 1.70- and 1.75-fold when citrate was used as the carbon source. The flux distributions showed that the amino acid composition of the proteins influence the production. Leucine appears to be the most important amino acid for both IFN-alpha(1) and EPO production. Consequently, pyruvate seems to be the critical main branch point and B. pasteurii seems to be the favourable host for therapeutical protein production due to the high leucine uptake capacity. The results encourage the discussion on the potential strategies for improving production of IFN-alpha(1) and EPO, and further enable us to assert medical hypothesis in order to support the immune system of the human body against the deficiencies of the synthesis of IFN-alpha(1) and EPO in the human cells

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and alpha-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and alpha -amylase (AMY) on five carbon sources that have different reduction degrees (gamma), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose (gamma = 4.0), acetate (gamma = 4.0), and the TCA cycle organic-acids succinate (gamma = 3.5), malate (gamma = 3.0), and citrate (gamma = 3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates (mu) were calculated as 1.142 and 0.766 h(-1), respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191 mmol g(-1) DW h(-1) when glucose uptake rate was 10 mmol g(-1) DW h(-1) and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY

Mass flux balance-based model and metabolic pathway engineering analysis for serine alkaline protease synthesis by Bacillus licheniformis

A mass flux balance-based stoichiometric model of Bacillus licheniformis for the serine alkaline protease (SAP) fermentation process has been established. The model considers 147 reaction fluxes, and there are 105 metabolites that are assumed to be in pseudo-steady state. Metabolic flux distributions were obtained from the solution of the model based on the minimum SAP accumulation rate assumption in B. licheniformis in combination with the off-line extracellular analyses of the metabolites that were the sole carbon source citrate, dry cell, organic acids, amino acids, and SAP; variations in the intracellular fluxes were demonstrated for the three periods of the batch bioprocess. The flux distribution maps showed that the cells completed the TCA cycle and utilized the gluconeogenesis; pathway, pentose phosphate pathway, and anaplerotic reactions throughout the fermentation; however the glycolysis pathway was inactive in all the periods of the fermentation. The flux values toward SAP increased throughout the bioprocess and slightly decreased in the last period; however, SRP selectivity values were almost the same in Periods II and III and higher than Period I. The diversions in the pathways and certain metabolic reactions depending on the bioprocess periods are also presented and the results indicated that the intracellular amino acid fluxes played an important role in the SAP fermentation process. (C) 1999 Elsevier Science Inc. All rights reserved

Metabolic network analysis for human therapeutic protein productions: Effects of the P/O ratio

The metabolic fluxes through the central carbon pathways in the bioprocesses for human leukocyte interferon (IFN-alpha(1)) and erythropoietin (EPO) overproductions by recombinant Bacillus sp, carrying the related human genes were determined separately, on two alternative carbon sources, i.e. glucose and citrate, which have different reduction degrees. In addition, the influence of the P/O ratio on the cell growth, IFN-alpha(1) and EPO productions were investigated. Thus, the potential influence of increased energy coupling in oxidative phosphorylation is also presented. In parallel to the increase in P/O ratio, with each substrate tested, cell growth and total ATP generation rates increased significantly. The interactions between specific cell growth and protein synthesis rates in the transition periods were analysed for the production of IFN-alpha(1) and EPO. The influence of the P/O ratio on metabolic flux distributions was significant at high specific growth rates; further, with the decrease in the growth rate in the transition period, the IFN-alpha(1) and EPO synthesis fluxes increased. During the product synthesis period (mu = 0 h(-1)) for both substrate, the P/O ratio influences neither the EPO synthesis nor the IFN-alpha(1) synthesis significantly. The potential strategies for improving IFN-alpha(1) and EPO productions are discussed

Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: Use of citric acid as the carbon source

The effects of oxygen transfer on serine alkaline protease (SAP) production by Bacillus licheniformis on a defined medium with C-c = 9.0 kg m(-3) citric acid as sole carbon source were investigated in 3.5 dm(3) batch bioreactor systems. The concentrations of the product (SAP) and by-products, i.e., neutral protease, amylase, amino acids, and organic acids were determined in addition to SAP activities. Ar Q(o)/V = 1 vvm airflow rate, the effect of agitation rate on DO concentration, pH, product, and by-product concentrations and SAP activity were investigated at N = 150, 500, and 750 min(-1); these are named as low-(LOT), medium-(MOT), and high oxygen transfer (HOT) conditions. LOT conditions favor biomass concentration; however, substrate consumption was highest at HOT conditions. MOT was optimum for maximum SAP activity which was 441 U cm(-3) at t = 37 h. The total amino acid concentration was maximum in LOT and minimum in MOT conditions; lysine had the highest concentration under all oxygen transfer conditions. Among organic acids, acetic acid had the highest concentration and its concentration increased with oxygen transfer rate. The oxygen transfer coefficient increases with the agitation rate and the oxygen consumption rate increased almost linearly with the biomass concentration. (C) 1998 Elsevier Science Inc

Bioreaction network flux analysis for industrial microorganisms: A review

This review focuses on the analysis of the bioreaction networks of the microorgansims used in fermentation processes, by metabolic flux analysis that is the novel tool of biochemical reaction engineering, required for the quantitative analysis of cell metabolism which is under the control of the cell physiology. Metabolic flux analysis (MFA) is based on calculation of intracellular reaction network rates through various reaction pathways either theoretical or by using elaborate experimental data on uptake, excretion, secretion rates, biosynthetic requirements with metabolic stoichiometry - by solving the mass-balance-based mathematical model developed for the bioreaction network components, either at pseudo-steady state or at steady-state. MFA describes the interactions between the cell and the bioreactor with proper emphasis on the metabolic state and the metabolic process in order to fine-tune the bioreactor performance. This analysis can be used to find the critical branch points and bottlenecks in the overall flux distributions, for modifying the medium composition, for improving the bioreactor operation conditions, moreover for calculating the theoretical metabolic capacities of the microorganism, and for selecting the host microorganism. The methodology of the metabolic flux analysis is provided briefly; thereafter, a comprehensive overview of the state-of-the-art pertaining to cell growth and synthesis of biomolecules in the organisms, i.e. Candida lipolytica, Propionibacterium, Candida utilis, Escherichia coli, Corynebacterium glutamicum, Corynebacterium melassecola, Brevibacterium flavum, Penicillium chrysogenum, Saccharomyces cerevisiae, Streptomyces lividans, Aspergilus niger, Aspergilus oryzae, Bacillus subtilis, Bacillus licheniformis, Ashbya gossypii, Lactococcus lactis, Clostridium acetobutylicum, Torulopsis glabrata, Zymomonas mobilis, Haemophilus influenzae, Streptomyces coelicolor, hybridoma cells, baby hamster kidney cells, Chinese hamster ovary cells, and human liver cells, are given in order to understand the cellular metabolism and the physiology to improve the cellular activities of the cells

Serine alkaline protease overproduction capacity of Bacillus licheniformis

A comprehensive metabolic network that considers 147 reaction fluxes and 105 metabolites is used in a mass-Aux-balance-based stoichiometric model for Bacillus licheniformis for serine alkaline protease (SAP) overproduction. The theoretical capacity analysis leading to optimized SAP overproduction was carried out by using a linear constrained optimization technique for several specific growth rates and the variation of the fluxes were calculated by fixing the sole carbon source citrate's uptake rate at 10 mmol/gDW/h. The theoretical data-based capacity analysis was conducted by using the model in combination with the off-line extracellular analyses of the dry cell and the metabolites that were citrate, organic acids, amino acids, and SAP; and the variations in the intracellular fluxes were obtained for the three periods of the batch bioprocess. The flux distribution maps of the analyses showed that the tricarboxylic acid cycle was active and the cells utilized the gluconeogenesis pathway, the pentose phosphate pathway, and the anaplerotic reactions; nevertheless, the glyoxylate shunt and the glycolysis pathway were inactive. The theoretical capacity analysis showed that SAP synthesis flux increased with the decrease in the specific growth rate, and was the highest at mu = 0 h(-1) as 0.0260 mmol/gDW/h. Both in the theoretical capacity and the theoretical data-based capacity analyses, among the fluxes towards the amino acid groups, aspartic acid group had the highest value and aromatic acid group had the lowest flux value; the flux distributions are similar. The Aux values towards SAP was maximum in Period II, whereas it was minimum in Period I. In Period II of the theoretical data-based capacity analysis, the fluxes of alanine and valine are higher than the other amino acid fluxes; and the pyruvate branch point seems to be the potential metabolic engineering site. The results reveal that SAP production can theoretically be increased 1.09, 16.68, and 7.21 folds, respectively, in Periods I, II, and III. The diversions in the pathways and certain metabolic reactions depending on the bioprocess periods and potential strategies for improving SAP production are also discussed. (C) 2000 Elsevier Science Inc. All rights reserved

Metabolic flux analyses for serine alkaline protease production

The intracellular metabolic fluxes through the central carbon pathways in Bacillus licheniformis in serine alkaline protease (SAP) production were calculated to predict the potential strategies for increasing the performance of the bacilli, by using two optimization approaches, i.e. the theoretical data-based (TDA) and the theoretical data-based capacity (TDC) analyses based on respectively minimum in-vivo SAP accumulation rate and maximum SAP synthesis rate assumptions, at low-, medium, and high-oxygen transfer conditions. At all periods of low-oxygen transfer condition, in lag and early exponential periods of medium-oxygen transfer (MOT) condition, and SAP synthesis period of high-oxygen transfer (HOT) condition, the TDA and TDC analyses revealed that SAP overproduction capacity is almost equal to the observed SAP production due to the regulation effect of the oxygen transfer. In the growth and early SAP synthesis period and in SAP synthesis period at MOT condition the calculated results of the two analyses reveal that SAP synthesis rate of the microorganism can be increased 7.2 and 16.7 folds, respectively; whereas, in the growth and early SAP synthesis period at HOT condition it can be increased 12.6 folds. The diversions in the biochemical reaction network and the influence of the oxygen transfer on the performance of the bacilli were also presented. The results encourage the application of metabolic engineering for lifting the rate limitations and for improving the genetic regulations in order to increase the SAP production. (C) 2000 Elsevier Science Inc. All rights reserved

Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: Effects of the oxygen transfer rate

The metabolic fluxes through the central carbon pathways in the bioprocess for serine alkaline protease (SAP) production by Bacillus licheniformis were calculated by the metabolic flux-based stoichiometric model based on the proposed metabolic network that contains 102 metabolites and 133 reaction fluxes using the time profiles of citrate, dry cell, organic acids, amino acids, and SAP as the constraints. The model was solved by minimizing the SAP accumulation rate in the cell, The effects of the oxygen-transfer rate (OTR) on the metabolic fluxes were investigated in a defined medium where citrate was used as the sole carbon source. The centra I pathways were active for the growth and the SAP synthesis in all the periods of the bioprocess at low (LOT), medium (MOT), and high (HOT) oxygen-transfer conditions. The flux partitioning in the TCA cycle at alpha-ketoglutarate towards glutamate group and at oxalacetate (OA) toward aspartic acid group amino acids were dependent on the OTR. The flux of the anaplerotic reaction that connects the TCA cycle either from malate or OA to the gluconeogenesis pathway via the main branch point pyruvate (Pyr) was also influenced by the OTR. With the decrease in the OTR, the intracellular flux values after glycerate 3-phosphate (PG3) in the gluconeogenesis pathway and the specific growth rate decreased. The total ATP-generation rate increased with the increase in OTR. The pathway towards the aspartic acid family amino acids which is important for sporulation that precedes the SAP synthesis were all active throughout the bioprocess. Metabolic flux analysis results at LOT, MOT, and HOT conditions encourage the design of an oxygen-transfer strategy in the bioreactor; moreover, asparagine synthetase or aspartate kinase could be the potential metabolic engineering sites due to the low value of the flux from the branch point aspartate toward asparagine. (C) 1999 John Wiley & Sons, Inc

Regulatory effects of alanine-group amino acids on serine alkaline protease production by recombinant Bacillus licheniformis

Influences of the concentration and addition time of alanine-group amino acids, i.e. alanine, leucine and valine, on serine alkaline protease (SAP) synthesis were investigated by Bacillus licheniformis (DSM 1969) carrying pHV1431::subC in a defined medium to identify the amino acids creating intracellular reaction-rate limitation in SAP production. While the precursors of alanine-group amino acids, pyruvate and alanine, did not affect SAP production considerably within the range 0-15 mM, the addition of leucine decreased both SAP production and cell formation, because of the inhibition of valine synthesis. Although valine inhibits reactions starting with pyruvate towards 2-oxo-isovalerate, due to conversion of valine into 2-oxoisovalerate and from 2-oxo-isovalerate to leucine, valine did not inhibit leucine synthesis. Val (7.5 mM) supply at t = 0 h increased SAP activity to an activity of 1070 units - cm-' which was 1.3-fold higher than that of the reference production medium. The highest cell growth yield on substrate (Y-X/S) was obtained as 0.24 g . g(-1) with the supply of alanine; and the highest product formation yield on substrate was obtained as 0.134 units . g(-1) with the supply of valine. By using the results obtained, strategies for increasing SAP production and complex medium design were also discussed