SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

Requirement of de novo synthesis of pyruvate carboxylase in long-term succinic acid production in Corynebacterium glutamicum.

Protein turnover through de novo synthesis is critical for sustainable cellular functions. We previously found that glucose consumption rate in Corynebacterium glutamicum under anaerobic conditions increased at temperature higher than the upper limit of growth temperature. Here, we showed that production of lactic and succinic acids increased at higher temperature for long-term (48 h) anaerobic reaction in metabolically engineered strains. At 42 °C, beyond the upper limit of growth temperature range, biomass-specific lactic acid production rate was 8% higher than that at 30 °C, the optimal growth temperature. In contrast, biomass-specific succinic acid production rate was highest at 36 °C, 28% higher than that at 30 °C, although the production at 42 °C was still 23% higher than that at 30 °C. As enzymes are usually unstable at high temperatures, we investigated whether protein turnover of metabolic enzymes is required for the production of lactic and succinic acids under these conditions. Interestingly, when de novo protein synthesis was inhibited by addition of chloramphenicol, after 6 h, only succinic acid production was inhibited. Because glycolytic enzymes are involved in both lactic and succinic acids synthesis, enzymes in the anaplerotic pathway and the tricarboxylic acid (TCA) cycle leading to succinic acid synthesis were likely to be responsible for its decreased production. Among the five enzymes examined, the specific activity of only pyruvate carboxylase was drastically decreased after 48 h at 42 °C. Thus, the de novo synthesis of pyruvate carboxylase is required for long-term production of succinic acid. Graphical abstract KEY POINTS: • Long-term reaction for organic acids can be improved at temperature beyond ideal growth conditions. • De novo synthesis of pyruvate carboxylase is required for long-term succinic acid production.

Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures.

Recombinant yeast pyruvate carboxylase (PYC2) expression was previously shown to be an effective metabolic engineering strategy for reducing lactate formation in a number of relevant mammalian cell lines, but, in the case of CHO cells, did not consistently lead to significant improvement in terms of cell growth, product titer and energy metabolism efficiency. In the present study, we report on the establishment of a PYC2-expressing CHO cell line producing a monoclonal antibody and displaying a significantly altered lactate metabolism compared to its parental line. All clones exhibiting strong PYC2 expression were shown to experience a significant and systematic metabolic shift toward lactate consumption, as well as a prolonged exponential growth phase leading to an increased maximum cell concentration and volumetric product titer. Of salient interest, PYC2-expressing CHO cells were shown to maintain a highly efficient metabolism in fed-batch cultures, even when exposed to high glucose levels, thereby alleviating the need of controlling nutrient at low levels and the potential negative impact of such strategy on product glycosylation. In bioreactor operated in fed-batch mode, the higher maximum cell density achieved with the PYC2 clone led to a net gain (20%) in final volumetric productivity.

C4-dicarboxylic acid production by overexpressing the reductive TCA pathway.

As C4-dicarboxylic acids could replace C4-petrochemicals, the reductive tricarboxylic acid (TCA) pathway was overexpressed in Pichia pastoris for production of the C4-dicarboxylic acids. Three expression cassettes which carried the pyruvate carboxylase gene (pc), the cytoplasmic malate dehydrogenase gene (mdh1) and the retargeted fumarase gene (Tfum) were integrated into the chromosomal DNA of P. pastoris GS115 alone or jointly. Multicopy integrations were screened using quantitative PCR for C4-dicarboxylic acid overaccumulation. The results showed that the highest titer in 96 h of fumaric, malic and succinic acid (0.76, 42.28 and 9.42 g l(-1)) was obtained by co-expression of pc and mdh1 in P. pastoris. This is the first report about multiple genes engineered in P. pastoris for C4-dicarboxylic acid production. The strain Pp-PC-MDH1, moreover, has a significant potential to produce malic acid in aerobic conditions.

In vivo analysis of lung cancer metabolism: nothing like the real thing.

Cancer cells exhibit altered metabolism compared with that of the surrounding tissue. There is hope that these reprogrammed metabolic pathways in tumors hold the key to advances for both cancer imaging and therapy. Translation of observations in cultured cancer cells to live tumors, however, has proven to be highly complex, and robust methods to analyze metabolic activity in primary human tumors are sorely needed. In this issue of the JCI, Sellers et al. use perioperative administration of isotope-labeled glucose to lung cancer patients to differentiate metabolic pathways between tumors and benign lung. They identify pyruvate carboxylation, a reaction that enables glucose-derived carbon to replenish TCA cycle intermediates, as a key component of anabolic metabolism in tumor cells.

Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation.

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Systematic design and functional analysis of artificial microRNAs.

Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2-8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and functionally analysed over 200 artificial miRNAs for simultaneous repression of pyruvate carboxylase and glutaminase by selecting all seed matches shared by their 3' untranslated regions. Although we identified multiple miRNAs that repressed endogenous protein expression of both genes, seed-based artificial miRNA design was highly inefficient, as the majority of miRNAs with even perfect seed matches did not repress either target. Moreover, commonly used target prediction programs did not substantially discriminate effective artificial miRNAs from ineffective ones, indicating that current algorithms do not fully capture the features important for artificial miRNA targeting and are not yet sufficient for designing artificial miRNAs. Our analysis suggests that additional factors are strong determinants of the efficacy of miRNA-mediated target repression and remain to be discovered.

Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

BACKGROUND: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. METHODS: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. RESULTS: The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. CONCLUSION: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase.

Succinic acid is a specialty chemical having numerous applications in industrial, pharmaceutical and food uses. One of the major challenges in the succinate fermentation process is eliminating the formation of byproducts. In this study, we describe eliminating byproduct formate and improving succinate productivity by reengineering a high succinate producing E. coli strain SBS550MG-Cms243(pHL413Km). The NAD(+)-dependent formate dehydrogenase gene (fdh1) of Candida boidinii was coexpressed with Lactococcus lactis pyruvate carboxylase (pycA) under the control of Ptrc and PpycA promoters in plasmid pHL413KF1. The newly introduced fdh1 converts 1 mol of formate into 1 mol of NADH and CO2. The reengineered strain SBS550MG-Cms243(pHL413KF1) retains the reducing power of formate through an increase in NADH availability. In anaerobic shake flask fermentations, the parent strain SBS550MG-Cms243(pHL413Km) consumed 99.86 mM glucose and produced 172.38 mM succinate, 16.16 mM formate and 4.42 mM acetate. The FDH bearing strain, SBS550MG-Cms243(pHL413KF1) consumed 98.43 mM glucose and produced 171.80 mM succinate, 1mM formate and 5.78 mM acetate. Furthermore, external formate supplementation to SBS550MG(pHL413KF1) fermentations resulted in about 6% increase in succinate yields as compared to SBS550MG(pHL413Km). In an anaerobic fed-batch bioreactor process, the average glucose consumption rate, succinate productivity, and byproduct formate concentration of SBS550MG(pHL413Km) was 1.40 g/L/h, 1g/L/h, and 17 mM, respectively. Whereas, the average glucose consumption rate, succinate productivity and byproduct formate concentration of SBS550MG(pHL413KF1) was 2 g/L/h, 2 g/L/h, 0-3 mM respectively. A high cell density culture of SBS550MG(pHL413KF1) showed further improvement in succinate productivity with a higher glucose consumption rate. Reduced levels of byproduct formate in succinate fermentation broth would provide an opportunity for reducing the cost associated with downstream processing, purification, and waste disposal.

Metabolic engineering of Torulopsis glabrata for malate production.

The yeast Torulopsis glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate: (i) overexpression of pyruvate carboxylase and malate dehydrogenase; (ii) identification of the bottleneck in malate production by model iNX804; (iii) simultaneous overexpression of genes RoPYC, RoMDH and SpMAE1. Using these strategies, 8.5gL(-1) malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest that T. glabrata CCTCC M202019 is a promising candidate for industrial malate production.

[Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002].

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase.

There is an imperative need for expression systems allowing the efficient and robust manufacturing of high quality glycoproteins. In the present work, HEK-293 cells stably expressing interferon-α2b were further engineered with the insertion of the yeast pyruvate carboxylase 2 gene. In batch cultures, marked reductions in lactate and ammonia production were observed compared to the parental cell clone. Although the maximum specific growth rate remained unchanged, the altered metabolism led to a 2-fold increase in maximum cell density and 33% increase in the integral of viable cell concentration and interferon production yield. The underlying metabolic changes were further investigated using various (13)C-labeled substrates and measuring the resulting lactate mass isotopomer distributions. Simultaneous metabolite and isotopomer balancing allowed the accurate determination of key intracellular fluxes. Such detailed and quantitative knowledge about the central carbon metabolism of the cells is instrumental to further support the development of high-yield fed-batch processes.

Pyruvate carboxylase is expressed in human skeletal muscle.

Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable by streptavidin blot and describe the presence of considerable amounts of PC in cultured human myotubes and in human muscle tissue.

[Expression of heterogenous pyruvate carboxylase in Escherichia coli with lactose as inducer and its effect on succinate production].

Escherichia coli strain DC1515, deficient in glucose phosphotransferase (ptsG), lactate dehydrogenase (ldhA) and pyruvate:formate lyase (pflA), is a promising candidate for the fermentative production of succinate. To further improve the succinate producing capability of DC1515, we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase (pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515. We used lactose as a substitute of IPTG to induce pyc. We optimized the culture conditions such as the lactose addition time, the lactose concentration and the culture temperature after induction for succinate production. We also explored the effect of lactose supplement during the fermentation. The results showed that pyc can be expressed under lactose induction in the fermentative medium with 15 g/L glucose due to the deficient of ptsG in DC1515. Under optimized conditions, the final succinate concentration reached to 15.17 g/L, which was 1.78-fold higher than that of control strain. If complementing lactose twice to the concentration of 1 g/L during the fermentation, the final succinate concentration could further reach to 17.54 g/L. This work might provide valuable information for gene expression in E. coli strains using lactose as inducer for succinate production in a glucose-medium. Due to the reduced cost, E. coli is becoming a more promising strain for succinate production through fermentation.

Expression of pyruvate carboxylase in cultured oligodendroglial, microglial and ependymal cells.

The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the "distal class" of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves.

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

Feed restriction induces pyruvate carboxylase but not phosphoenolpyruvate carboxykinase in dairy cows.

The ability of dairy cattle to adapt to changes in nutrient intake requires appropriately responsive expression of several key genes in liver. Holstein cows were used in 2 experiments to determine the effect of short-term feed restriction on expression of mRNA for gluconeogenic and ureagenic enzymes in liver. In experiment 1, cows were fed a total mixed diet for ad libitum intake for a 5-d period followed by 5 d of 50% of their previous 5-d ad libitum intake followed by 10 d of ad libitum feeding. Liver biopsies and blood samples were obtained on d 5, 10, and 20 of the experiment, the last day of each feeding period. Pyruvate carboxylase (PC) mRNA increased with feed restriction, but phosphoenolpyruvate carboxykinase (PEPCK) was unchanged. Expression of carbamoyl phosphate synthetase (CPS-I), argininosuccinate synthetase, and ornithine transcarbamylase mRNA were not altered by feed restriction; however, CPS-I mRNA expression tended to increase during realimentation. In experiment 2, cows were fed for ad libitum intake for 5 d and then fed 50% of previous intake for 5 d. Liver biopsy samples collected on d 5 and 10 were used for PC mRNA, PEPCK mRNA, and in vitro measure of gluconeogenesis from radiolabelled propionate and lactate. The data indicate expression of genes for key metabolic processes in liver of lactating cows is responsive to feeding level. Expression of PC mRNA is part of the adaptive response to feed intake restriction and is matched by increased capacity for gluconeogenesis from lactate.

Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabeling studies.

The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed.

Improving glucose and glutamine metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme.

Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.

Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum.

Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.