Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

Energy substrates and the completion of spontaneous meiotic maturation

This study was carried out to examine how different combinations of pyruvate and glucose affect spontaneous meiotic maturation of cumulus-cell-enclosed mouse oocytes (CEO) to metaphase II (MII). Most experiments used an open system in which oocytes were cultured in 1 ml medium in plastic tubes. Initial experiments examined the dose response effects of pyruvate or glucose alone in the presence or absence of 2 mM glutamine. When medium lacked both pyruvate and glucose, more than 91% of the oocytes died in glutamine-free medium during 15 h of culture; viability was restored with the addition of glutamine, but only 11% of the CEO reached MII. In the absence of glutamine, 62-68% of oocytes completed maturation in 0.23-2.3 mM pyruvate, while 44-60% MII was observed in 0.55-27.8 mM glucose. The addition of glutamine to these cultures had a general suppressive effect on the completion of maturation. When glucose was added to pyruvate-containing cultures, the combination of 1 mM pyruvate/5.5 mM glucose was most effective in supporting maturation (about 90% MII), with little effect of glutamine. No further increase in maturation was observed when glucose was increased five-fold (to 27.8 mM). The positive effect of glucose was in part attributed to stimulation of glycolysis and increased production of pyruvate, since a reduced culture volume (8 microl), which allows the accumulation of secreted pyruvate, improved maturation in glucose-containing, but not pyruvate-containing, medium, and FSH, which stimulates glycolysis, increased progression to MII in glucose-containing, but not pyruvate-containing, medium. Yet these results also suggest that glucose has a beneficial effect on maturation apart from simple provision of pyruvate. The pyruvate effect was directly on the oocyte, because denuded oocytes responded more effectively than CEO to this energy substrate. The highest percentage of MII oocytes (96-97%) occurred in microdrop cultures containing glucose but lacking glutamine. These results indicate that glutamine supports oocyte viability but is not an adequate energy source for the completion of spontaneous meiotic maturation and may be detrimental. In addition, while pyruvate and glucose alone can each support meiotic progression of CEO to MII, optimal maturation requires the provision of both substrates to the culture medium when a large volume (1 ml) is used. It is concluded that careful attention to specific energy substrate supplementation and culture volume is important to optimise spontaneous meiotic maturation in vitro.     

Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.     

Removal of inhibitors of bacterial growth by dialysis culture.

Dialysis culture was used to investigate the extent to which growth inhibition in bacterial cultures may be caused by accumulation of metabolites. Escherichia coli B was grown in a glucose/salts medium. A concentrated nutrient solution was pumped at a constant rate into the growing culture to ensure that growth was not limited by exhaustion of nutrients. In this way the only difference between growth conditions in dialysis and non-dialysis cultures was the transfer of dialysable metabolites from the culture vessel to the reservoir in the dialysis culture system. By adjusting the glucose concentration in the feed and maintaining a constant rate of feeding, glucose-limited growth could be achieved. Under these conditions, with oxygen in excess, bacterial yields of 140 to 150 g dry wt l-1 were obtained in dialysis culture compared with 30 to 40 g l-1 in non-dialysis culture. The high yields in dialysis culture depended on the removal of end-products of glucose metabolism. Growth inhibition was demonstrated to be the result of the combined influence of acetate, lactate, pyruvate, succinate, propionate and isobutyrate in concentrations found at the end of growth in non-dialysis cultures of Escherichia coli B.     

Metabolism of resorcinylic compounds by bacteria: orcinol pathway in Pseudomonas putida.

Enrichment cultures yielded two strains of Pseudomonas putida capable of growth with orcinol (3,5-dihydroxytoluene) as the sole source of carbon. Experiments with cell suspensions and cell extracts indicate that orcinol is metabolized by hydroxylation of the benzene ring followed successively by ring cleavage and hydrolyses to give 2 mol of acetate and 1 mol of pyruvate per mol of orcinol as shown: orcinol leads to 2,3,5-trihydroxytoluene leads to 2,4,6-trioxoheptanoate leads to acetate + acetylpyruvate leads to acetate + pyruvate. Evidence for this pathway is based on: (i) high respiratory activities of orcinol-grown cells towards 2,3,5-trihydroxytoluene; (ii) transient accumulation of a quinone, probably 2-hydroxy-6-methyl-1,4-benzoquinone, during grouth with orcinol; (iii) formation of pyruvate and acetate from orcinol, 2,3,5-trihydroxytoluene, and acetylpyruvate catalyzed by extracts of orcinol, but not by succinate-grown cells; (iv) characterization of the product of oxidation of 3-methylcatechol (an analogue of 2,3,5-trihydroxytoluene) showing that oxygenative cleavage occurs between carbons bearing methyl and hydroxyl substituents; (v) transient appearance of a compound having spectral properties similar to those of acetylpyruvate during 2,3,5-trihydroxytoluene oxidation by extracts of orcinol-grown cells. Orcinol hydroxylase exhibits catalytic activity when resorcinol or m-cresol is substituted for orcinol; hydroxyquinol and 3-methylcatechol are substrates for the ring cleavage enzyme 2,3,5-trihydroxytoluene-1,2-oxygenase. The enzymes of this pathway are induced by growth with orcinol but not with glucose or succinate.     

Improving culture performance and antibody production in CHO cell culture processes by reducing the Warburg effect

Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in the cell culture development to improve growth, productivity, and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl coenzyme A (acetyl‐CoA). The PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA), resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed‐batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by more than 3 days, and the final antibody titer increased by more than twofold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by approximately 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes, whereas fluxes in the tricarboxylic acid (TCA) cycle were not affected, suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis, only few proteins were identified as being differentially expressed, indicating that DCA acts on a posttranslational level. Antibody quality in terms of aggregation, charge variant, and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell‐specific productivity or antibody quality.     

Some biochemical properties of human lymphoblastoid Namalwa cells grown anaerobically

Using cultured lymphoblastoid Namalwa cells, we have demonstrated that humans cells can be grown for long periods in the absence of oxygen. Such anaerobic growth occurs at the same rate as that of aerobic cultures, but is characterized by a strict dependence on pyruvate. Cells in anaerobic culture undergo a severe loss of mitochondrial cytochromes, that is reversible on reaeration. Anaerobically grown cells show a two-fold increase in glucose consumption, consistent with anaerobic glycolysis providing the source of ATP for cellular maintenance and growth. The requirement for pyruvate is explained by the necessity for these cells to re-oxidise NADH derived from metabolic reactions. Detailed study of anaerobically grown human cells provides a new framework for investigating tissues depleted in mitochondrial functions, as occurs in mitochondrial diseases and the ageing process.     

Glucose requirement for induction by sodium butyrate of the glycoprotein hormone alpha subunit in HeLa cells

Butyric acid produces multiple effects on mammalian cells in culture, including alterations in morphology, depression of growth rate, increased histone acetylation, and modified production of various proteins and enzymes. The latter effect is exemplified by the induction in HeLa cells of the glycoprotein hormone alpha subunit by millimolar concentrations of the fatty acid. This report demonstrates that increased subunit accumulation in response to sodium butyrate is strikingly dependent on the presence of glucose (or mannose) in the growth medium. In contrast, basal levels of subunit synthesis are only marginally affected when the culture medium is supplemented with one of a variety of hexoses. An increase in the accumulation of HeLa alpha does not occur in medium containing pyruvate as the energy source, and sustained induction requires the simultaneous and continued presence of both glucose and butyrate. The effects of butyrate on HeLa cell morphology and subunit induction can be separated, since the latter is glucose-dependent while the former is not. Failure of butyrate to induce alpha in medium containing pyruvate does not result from restricted subunit secretion, since the levels of intracellular alpha are not increased disproportionately relative to those in the medium. The hexoses which support induction of HeLa alpha (glucose greater than or equal to mannose greater than galactose greater than fructose) are identical to those which have been shown previously to stimulate the glucosylation of lipid-linked oligosaccharides and enhance the synthesis of certain glycoproteins. Labeling of various glycosylation intermediates with [3H]mannose indicates that in glucose medium there is a decrease in the level of radioactivity associated with both dolicholpyrophosphoryl oligosaccharide and cellular glycoproteins and a concomitant increase in the fraction of label recovered in secreted glycoproteins. Butyrate also causes a decrease in [3H]mannose-labeled cellular glycoproteins and an increase in tritiated extracellular glycoproteins, particularly in glucose medium. Likewise, glucose stimulates the incorporation of [3H]glucosamine into immunoprecipitable alpha subunit relative to the bulk of HeLa-secreted glycoproteins, and this is further enhanced by butyrate. However, as demonstrated by lectin chromatography of conditioned media, a nonglycosylated subunit does not accumulate in pyruvate medium, either in the absence or presence of butyrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of uracil uptake in Escherichia coli by adenosine 3',5'-monophosphate.

Culture of a wild-type strain of Escherichia coli in the presence of cyclic AMP leads to an impairment of uracil uptake. Half maximum inhibition of uracil uptake was observed at 1.5 mM cyclic AMP. The effect seems to be specific since no inhibition was found in cultures supplemented with ATP, ADP or 5'-AMP. Similarly the inhibition was not observed in cultures of a mutant deficient in the cyclic AMP receptor protein. The inhibition in uracil uptake, found in bacteria cultured in the presence of cyclic AMP, is not a consequence of a reduction in the growth rate. On the other hand, this inhibition was observed only in cultures containing glucose or pyruvate as carbon source.     

Acetoin production in growing Leuconostoc mesenteroides

Leuconostoc mesenteroides NCDO 518, provided with oxygen and pyruvate, preferentially used oxygen as accessory electron acceptor and converted pyruvate to acetoin. With glucose, 5.6 mM, as sole energy source only small amounts of acetoin were formed (0.08–0.21 mM). With glucose, 5.6 mM, and pyruvate, 20 mM, substantial amounts of acetoin were produced in growing, aerated cultures at pH 5 (2.8 mM, equivalent to 0.5 mol [mol glucose fermented]^–1). On exhaustion of glucose, growth ceased but metabolism of pyruvate continued with the formation of acetate and a little acetoin. In aerated cultures at pH 6 the general pattern was similar to that at pH 5 but less acetoin (0.6 mM) was formed during the growth phase and, after the exhaustion of glucose, pyruvate was converted very slowly to acetate only. Leuc. mesenteroides did not grow with pyruvate as sole energy source.     

Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations.     

Identification and Characterization of MAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.     

Impact of aeration on the metabolic end-products formed from glucose and galactose by Streptococcus lactis

Compared with cultures grown aerobically in batch culture with glucose, aerated cultures of lactic streptococci had a less homolactic type of metabolism when galactose was the carbohydrate source in batch cultures, or when glucose was limiting in chemostat cultures. Differences in end-products of sugar metabolism between aerated and unaerated cultures were observed. In addition to lactate, formate, acetate and ethanol were produced in anaerobic cultures, whereas acetate and acetoin were formed in aerated cultures. Acetate production in aerated cultures depended on lipoic acid, an essential cofactor of the pyruvate dehydrogenase complex. In a chemically defined medium with glucose as the energy substrate, lipoic acid (or acetate) was an essential growth factor. Formation of acetoin was inversely related to lipoic acid concentration in the growth medium. Although not observed in unaerated cultures, acetoin (and 2,3-butanediol) was produced in unaerated buffered suspensions metabolizing pyruvate. Aeration caused a modest increase in the activities of aP-acetolactate synthetase and phosphate acetyl trans-ferase, but it is unlikely that the increases were sufficient to account for the changes in end-products of sugar metabolism observed.     

Pathways of glucose catabolism and the origin and metabolism of pyruvate during calcium-induced conidiation ofPenicillium notatum

Experiments examined the metabolic basis of Ca²⁺-induced conidiation during the 12-h period following the addition of Ca²⁺ to 40-h vegetative cultures ofPenicillium notatum. Vegetative mycelium had enzymic capacity for three routes of glucose catabolism viz. the Embden-Meyerhof-Parnas (EMP), pentose phosphate (PP) and the Entner-Doudoroff (ED) sequences. Inhibitors of EMP enzymes restricted vegetative growth more than that associated with conidiation whilst arsenate augmented the limited capacity of lower levels of Ca²⁺ to promote conidiation. Arsenite (5.6 mmol · 1^–1) partially blocked the metabolism of pyruvate and caused its accumulation, which was also promoted by Ca²⁺ alone. Arsenite did not induce conidiation in vegetative cultures but when combined with Ca²⁺ it enhanced conidiation. Radiorespirometry and the analysis of accumulated pyruvate, promoted by arsenite, indicated that approximately 54% of carbon was catabolized via combined EMP/ED routes and 46% by the PP pathway and subsequently via a weakly functional TCA cycle. Calcium-induced cultures swung to a primarily ED (25%) and PP (75%) based catabolism with low substrate level phosphorylation, including a facility for a non-phosphorylative ED route, and further diminished oxidative TCA capacity. Pyruvate accumulation in Ca²⁺-induced cultures coincided with the decline in activity of pyruvate dehydrogenase and a reduced capacity for gluconeogenesis, with other enzymes of pyruvate metabolism showing altered activities. These changes in enzyme activities, pyruvate accumulation and its subsequent metabolism were related to growth rate and the developmental cycle, and are discussed in conjunction with the regulatory role of calcium.     

Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess.

In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.     

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.     

Accumulation of pyruvate by changing the redox status in Escherichia coli

Pyruvate was produced from glucose by Escherichia coli BW25113 that contained formate dehydrogenase (FDH) from Mycobacterium vaccae. In aerobic shake-flask culture (K (L) a?=?4.9?min(-1)), the recombinant strain produced 6.7?g pyruvate?l(-1) after 24?h with 4?g sodium formate?l(-1) and a yield of 0.34?g pyruvate?g?glucose(-1). These values were higher than those of the original strain (0.2?g?l(-1) pyruvate and 0.02?g pyruvate?g?glucose(-1)). Based on the reaction mechanism of FDH, the introduction of FDH into E. coli enhances the accumulation of pyruvate by the regeneration of NADH from NAD(+) since NAD(+) is a shared cosubstrate with the pyruvate dehydrogenase complex, which decarboxylates pyruvate to acetyl-CoA and CO(2). The oxygenation level was enough high to inactivate lactate dehydrogenase, which was of benefit to pyruvate accumulation without lactate as a by-product.