Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures

At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.     

Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed‐batch CHO cell culture

Cell metabolism can vary considerably over the course of a typical fed‐batch antibody production process. However, the intracellular pathway alterations associated with various phases of growth and antibody production have yet to be fully elucidated using industrially relevant production hosts. Therefore, we performed ¹³C labeling experiments and metabolic flux analysis (MFA) to characterize CHO cell metabolism during four separate phases of a fed‐batch culture designed to closely represent industrial process conditions. First, we found that peak specific growth rate was associated with high lactate production and minimal TCA cycling. Conversely, we found that lactate metabolism switched from net production to net consumption as the culture transitioned from peak growth to peak antibody production. During the peak antibody production phase, energy was primarily generated through oxidative phosphorylation, which was also associated with elevated oxidative pentose phosphate pathway (oxPPP) activity. Interestingly, as TCA cycling and antibody production reached their peaks, specific growth rate continued to diminish as the culture entered stationary phase. However, TCA cycling and oxPPP activity remained high even as viable cell density began to decline. Overall, we found that a highly oxidative state of metabolism corresponded with peak antibody production, whereas peak cell growth was characterized by a highly glycolytic metabolic state. Biotechnol. Bioeng. 2013; 110: 2013–2024. © 2013 Wiley Periodicals, Inc.     

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal‐fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse‐chase LC‐MS‐based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine‐derived fluxes: Oxidation of glucose‐derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.     

谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析

以一株由自然界筛选获得的能够利用糖质原料直接产L-丝氨酸的谷氨酸棒杆菌Corynebacterium glutamicum SYPS-062为研究对象,考察了一碳单元循环中的辅因子—叶酸和维生素B12对菌株生长、蔗糖消耗及L-丝氨酸生成的影响,同时对处于对数生长期的菌株进行了代谢流量分析。结果发现,添加扰动因子叶酸和维生素B12对磷酸戊糖途径(HMP)碳流影响较大,碳源主要用于细胞生长及合成能量,而流向目的产物L-丝氨酸的碳流减少。同时在添加维生素B12时,增大了G3P节点的L-丝氨酸合成途径的分流比,但造成三羧酸循环(TCA)的流量不足,需要大量回补,从而限制了产物合成速率的进一步提高。     

Flux balance analysis of CHO cells before and after a metabolic switch from lactate production to consumption

Mammalian cell cultures typically exhibit an energy inefficient phenotype characterized by the consumption of large quantities of glucose and the concomitant production of large quantities of lactate. Under certain conditions, mammalian cells can switch to a more energy efficient state during which lactate is consumed. Using a metabolic model derived from a mouse genome scale model we performed flux balance analysis of Chinese hamster ovary cells before and after a metabolic switch from lactate production (in the presence of glucose) to lactate consumption (after glucose depletion). Despite a residual degree of freedom after accounting for measurements, the calculated flux ranges and associated errors were narrow enough to enable investigation of metabolic changes across the metabolic switch. Surprisingly, the fluxes through the lower part of the TCA cycle from oxoglutarate to malate were very similar (around 60 µmol/gDW/h) for both phases. A detailed analysis of the energy metabolism showed that cells consuming lactate have an energy efficiency (total ATP produced per total C‐mol substrate consumed) six times greater than lactate producing cells. Biotechnol. Bioeng. 2013; 110: 660–666. © 2012 Wiley Periodicals, Inc.     

Insights into pH‐induced metabolic switch by flux balance analysis

Lactate accumulation in mammalian cell culture is known to impede cellular growth and productivity. The control of lactate formation and consumption in a hybridoma cell line was achieved by pH alteration during the early exponential growth phase. In particular, lactate consumption was induced even at high glucose concentrations at pH 6.8, whereas highly increased production of lactate was obtained at pH 7.8. Consequently, constraint‐based metabolic flux analysis was used to examine pH‐induced metabolic states in the same growth state. We demonstrated that lactate influx at pH 6.8 led cells to maintain high fluxes in the TCA cycle and malate‐aspartate shuttle resulting in a high ATP production rate. In contrast, under increased pH conditions, less ATP was generated and different ATP sources were utilized. Gene expression analysis led to the conclusion that lactate formation at high pH was enabled by gluconeogenic pathways in addition to facilitated glucose uptake. The obtained results provide new insights into the influence of pH on cellular metabolism, and are of importance when considering pH heterogeneities typically present in large scale industrial bioreactors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:347–357, 2015     

Fundamental Escherichia coli biochemical pathways for biomass and energy production: creation of overall flux states

We have previously shown that the metabolism for most efficient cell growth can be realized by a combination of two types of elementary modes. One mode produces biomass while the second mode generates only energy. The identity of the four most efficient biomass and energy pathway pairs changes, depending on the degree of oxygen limitation. The identification of such pathway pairs for different growth conditions offers a pathway-based explanation of maintenance energy generation. For a given growth rate, experimental aerobic glucose consumption rates can be used to estimate the contribution of each pathway type to the overall metabolic flux pattern. All metabolic fluxes are then completely determined by the stoichiometries of involved pathways defining all nutrient consumption and metabolite secretion rates. We present here equations that permit computation of network fluxes on the basis of unique pathways for the case of optimal, glucose-limited Escherichia coli growth under varying levels of oxygen stress. Predicted glucose and oxygen uptake rates and some metabolite secretion rates are in remarkable agreement with experimental observations supporting the validity of the presented approach. The entire most efficient, steady-state, metabolic rate structure is explicitly defined by the developed equations without need for additional computer simulations. The approach should be generally useful for analyzing and interpreting genomic data by predicting concise, pathway-based metabolic rate structures.     

Metabolic adaptation of MDCK cells to different growth conditions: effects on catalytic activities of central metabolic enzymes

Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed-batch cultivations these toxic by-products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin-Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high-throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six-well plates in serum-containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium suggesting that more glucose-6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD(+)-dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.     

Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.     

Interpretation of metabolic flux maps by limitation potentials and constrained limitation sensitivities

Two new concepts, "Limitation Potential" and "Constraint Limitation Sensitivity" are introduced that use definitions derived from metabolic flux analysis (MFA) and metabolic network analysis (MNA). They are applied to interpret a measured flux distribution in the context of all possible flux distributions and thus combine MFA with MNA. The proposed measures are used to quantify and compare the influence of intracellular fluxes on the production yield. The methods are purely based on the stoichiometry of the network and constraints that are given from irreversible fluxes. In contrast to metabolic control analysis (MCA), within this approach no information about the kinetic mechanisms are needed. A limitation potential (LP) is defined as the reduction of the reachable (theoretical) maximum by a measured flux. Measured fluxes that strongly narrow the reachable maximum are assumed to be limiting as the network has no ability to counterbalance the restriction due to the observed flux. In a second step, the sensitivity of the reduced maximum is regarded. This measure provides information about the necessitated changes to reach higher yields. The methods are applied to interpret the capabilities of a network based on measured fluxes for a L-phenylalanine producer. The strain was examined by a series of experiments and three flux maps of the production phase are analyzed. It can be shown that the reachable yield is drastically reduced by the measured efflux into the TCA cycle, while the oxidative pentose-phosphate pathway only plays a secondary role on the reachable maximum.     

乳酸钠促进法夫酵母虾青素合成代谢流作用分析

【目的】研究乳酸钠(一种糖代谢产物)的加入对法夫酵母JMU-VDL668发酵过程中细胞生长和虾青素合成的影响。【方法】分别在摇瓶和7 L发酵罐实验基础上,采用代谢通量分析的方法分析添加乳酸钠对法夫酵母菌株JMU-VDL668合成虾青素代谢流的影响。【结果】在7 L发酵罐实验中添加乳酸钠,虾青素产量最高可达17.70 mg/L,与对照组相比提高26%。代谢通量分析表明,乳酸钠可以调节丙酮酸、乙酰辅酶A节点处的代谢通量分布,乳酸在乳酸脱氢酶的作用下可以直接进入代谢网络的后半程,乙酰辅酶A的通量和进入TCA循环的通量得到了显著加强。【结论】乳酸钠的加入提供了更多的乙酰辅酶A等前体物质和能量供给,因此促进了虾青素的合成。     

Optimizing performance of semi‐continuous cell culture in an ambr15™ microbioreactor using dynamic flux balance modeling

The ambr bioreactors are single‐use microbioreactors for cell line development and process optimization. With operating conditions for large‐scale biopharmaceutical production properly scaled down, microbioreactors such as the ambr15? can potentially be used to predict the effect of process changes such as modified media or different cell lines. While there have been some recent studies evaluating the ambr15? technology as a scale‐down model for fed‐batch operations, little has been reported for semi‐continuous or continuous operation. Gassing rates and dilution rates in the ambr15? were varied in this study to attempt to replicate performance of a perfusion process at the 5 L scale. At both scales, changes to metabolite production and consumption, and cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15? bioreactor that produced metabolic shifts and specific metabolic and protein production rates that are characteristic of the corresponding 5 L perfusion process. A dynamic flux balance (DFB) model was employed to understand and predict the metabolic changes observed. The DFB model predicted trends observed experimentally, including lower specific glucose consumption and a switch from lactate production to consumption when dissolved CO₂ was maintained at higher levels in the broth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:420–431, 2018     

A dynamic metabolic flux balance based model of fed‐batch fermentation of bordetella pertussis

A mathematical model based on a dynamic metabolic flux balance (DMFB) is developed for a process of fed‐batch fermentation of Bordetella pertussis. The model is based on the maximization of growth rate at each time interval subject to stoichiometric constraints. The model is calibrated and verified with experimental data obtained in two different bioreactor experimental systems. It was found that the model calibration was mostly sensitive to the consumption or production rates of tyrosine and, for high supplementation rates, to the consumption rate of glutamate. Following this calibration the model correctly predicts biomass and by‐products concentrations for different supplementation rates. Comparisons of model predictions to oxygen uptake and carbon emission rates measurements indicate that the TCA cycle is fully functional. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 520–531, 2013     

肌苷发酵过程中后期的代谢流节点分析

应用代谢流分析方法,实验测定副产物的积累速率,将数据输入计算机,应用MArllAB软件计算肌苷发酵中后期代谢流分布。通过分析代谢网络中重要的节点,提出了优化肌苷生物合成途径的建议。     

Dynamic metabolic flux analysis (DMFA): a framework for determining fluxes at metabolic non-steady state

Metabolic flux analysis (MFA) is a key tool for measuring in vivo metabolic fluxes in systems at metabolic steady state. Here, we present a new method for dynamic metabolic flux analysis (DMFA) of systems that are not at metabolic steady state. The advantages of our DMFA method are: (1) time-series of metabolite concentration data can be applied directly for estimating dynamic fluxes, making data smoothing and estimation of average extracellular rates unnecessary; (2) flux estimation is achieved without integration of ODEs, or iterations; (3) characteristic metabolic phases in the fermentation data are identified automatically by the algorithm, rather than selected manually/arbitrarily. We demonstrate the application of the new DMFA framework in three example systems. First, we evaluated the performance of DMFA in a simple three-reaction model in terms of accuracy, precision and flux observability. Next, we analyzed a commercial glucose-limited fed-batch process for 1,3-propanediol production. The DMFA method accurately captured the dynamic behavior of the fed-batch fermentation and identified characteristic metabolic phases. Lastly, we demonstrate that DMFA can be used without any assumed metabolic network model for data reconciliation and detection of gross measurement errors using carbon and electron balances as constraints.     

Effect of light fluctuations on photosynthesis and metabolic flux in Synechocystis sp. PCC 6803

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds. In the present study, we investigated the metabolic response of Synechocystis sp. PCC 6803 to light fluctuations using ¹³C-metabolic flux analysis. The light intensity and duty ratio were adjusted such that the total number of photons or the light intensity during the low-light phase was equal. Light fluctuations affected cell growth and photosynthetic activity under the experimental conditions. However, metabolic flux distributions and cofactor production rates were not affected by the light fluctuations. Furthermore, the estimated ATP and NADPH production rates in the photosystems suggest that NADPH-consuming electron dissipation occurs under fluctuating light conditions. Although we focused on the water–water cycle as the electron dissipation path, no growth effect was observed in an flv3-disrupted strain under fluctuating light, suggesting that another path contributes to electron dissipation under these conditions.     

Stoichiometric model and metabolic flux analysis for Leptospirillum ferrooxidans

A metabolic model for Leptospirillum ferrooxidans was developed based on the genomic information of an analogous iron oxidizing bacteria and on the pathways of ferrous iron oxidation, nitrogen and CO₂ assimilation based on experimental evidence for L. ferrooxidans found in the literature. From this metabolic reconstruction, a stoichiometric model was built, which includes 86 reactions describing the main catabolic and anabolic aspects of its metabolism. The model obtained has 2 degrees of freedom, so two external fluxes were estimated to achieve a determined and observable system. By using the external oxygen consumption rate and the generation flux biomass as input data, a metabolic flux map with a distribution of internal fluxes was obtained. The results obtained were verified with experimental data from the literature, achieving a very good prediction of the metabolic behavior of this bacterium at steady state. Biotechnol. Bioeng. 2010;107:696–706. © 2010 Wiley Periodicals, Inc.     

Metabolic flux analysis of CHO cell metabolism in the late non‐growth phase

Chinese hamster ovary (CHO) cell cultures are commonly used for production of recombinant human therapeutic proteins. Often the goal of such a process is to separate the growth phase of the cells, from the non‐growth phase where ideally the cells are diverting resources to produce the protein of interest. Characterizing the way that the cells use nutrients in terms of metabolic fluxes as a function of culture conditions can provide a deeper understanding of the cell biology offering guidance for process improvements. To evaluate the fluxes, metabolic flux analysis of the CHO cell culture in the non‐growth phase was performed by a combination of steady‐state isotopomer balancing and stoichiometric modeling. Analysis of the glycolytic pathway and pentose phosphate pathway (PPP) indicated that almost all of the consumed glucose is diverted towards PPP with a high NADPH production; with even recycle from PPP to G6P in some cases. Almost all of the pyruvate produced from glycolysis entered the TCA cycle with little or no lactate production. Comparison of the non‐growth phase against previously reported fluxes from growth phase cultures indicated marked differences in the fluxes, in terms of the split between glycolysis and PPP, and also around the pyruvate node. Possible reasons for the high NADPH production are also discussed. Evaluation of the fluxes indicated that the medium strength, carbon dioxide level, and temperature with dissolved oxygen have statistically significant impacts on different nodes of the flux network. Biotechnol. Bioeng. 2011; 108:82–92. © 2010 Wiley Periodicals, Inc.