首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13C metabolic flux analysis (13C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13C-MFA using the optimal tracers [1,2-13C]glucose, [1,6-13C]glucose, [1,2-13C]xylose and [5-13C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-13C]glucose and [U-13C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO2 originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH2, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.  相似文献   

2.
13C metabolic flux analysis (13C-MFA) is a widely used tool for quantitative analysis of microbial and mammalian metabolism. Until now, 13C-MFA was based mainly on measurements of isotopic labeling of amino acids derived from hydrolyzed biomass proteins and isotopic labeling of extracted intracellular metabolites. Here, we demonstrate that isotopic labeling of glycogen and RNA, measured with gas chromatography-mass spectrometry (GC-MS), provides valuable additional information for 13C-MFA. Specifically, we demonstrate that isotopic labeling of glucose moiety of glycogen and ribose moiety of RNA greatly enhances resolution of metabolic fluxes in the upper part of metabolism; importantly, these measurements allow precise quantification of net and exchange fluxes in the pentose phosphate pathway. To demonstrate the practical importance of these measurements for 13C-MFA, we have used Escherichia coli as a model microbial system and CHO cells as a model mammalian system. Additionally, we have applied this approach to determine metabolic fluxes of glucose and xylose co-utilization in the E. coli ΔptsG mutant. The convenience of measuring glycogen and RNA, which are stable and abundant in microbial and mammalian cells, offers the following key advantages: reduced sample size, no quenching required, no extractions required, and GC-MS can be used instead of more costly LC-MS/MS techniques. Overall, the presented approach for 13C-MFA will have widespread applicability in metabolic engineering and biomedical research.  相似文献   

3.
In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and 13C-metabolic flux analysis (13C-MFA). Here, cells were grown in parallel cultures with [1-13C]glucose and [U-13C]glucose as tracers and 13C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of 13C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for 13C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased 13C-flux measurements in C. acetobutylicum.  相似文献   

4.
13C-Metabolic flux analysis (13C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by 13C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for 13C-MFA. The best single tracers were doubly 13C-labeled glucose tracers, including [1,6-13C]glucose, [5,6-13C]glucose and [1,2-13C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6-13C]glucose and [1,2-13C]glucose. Combined analysis of [1,6-13C]glucose and [1,2-13C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1-13C]glucose +20% [U-13C]glucose.  相似文献   

5.
Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by 13C-metabolic flux analysis (13C-MFA). Principal component analysis of 13C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. 13C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism.  相似文献   

6.
Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several “proof of principle” studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by 13C metabolic flux analysis (13C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report 13C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of 12C and 13C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism’s preferred mode under nitrogen-fixing conditions. The 13C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism’s distinct metabolic features under nitrogen-fixing and -non-fixing conditions.  相似文献   

7.
13C-Metabolic flux analysis (13C-MFA) is a powerful model-based analysis technique for determining intracellular metabolic fluxes in living cells. It has become a standard tool in many labs for quantifying cell physiology, e.g., in metabolic engineering, systems biology, biotechnology, and biomedical research. With the increasing number of 13C-MFA studies published each year, it is now ever more important to provide practical guidelines for performing and publishing 13C-MFA studies so that quality is not sacrificed as the number of publications increases. The main purpose of this paper is to provide an overview of good practices in 13C-MFA, which can eventually be used as minimum data standards for publishing 13C-MFA studies. The motivation for this work is two-fold: (1) currently, there is no general consensus among researchers and journal editors as to what minimum data standards should be required for publishing 13C-MFA studies; as a result, there are great discrepancies in terms of quality and consistency; and (2) there is a growing number of studies that cannot be reproduced or verified independently due to incomplete information provided in these publications. This creates confusion, e.g. when trying to reconcile conflicting results, and hinders progress in the field. Here, we review current status in the 13C-MFA field and highlight some of the shortcomings with regards to 13C-MFA publications. We then propose a checklist that encompasses good practices in 13C-MFA. We hope that these guidelines will be a valuable resource for the community and allow 13C-flux studies to be more easily reproduced and accessed by others in the future.  相似文献   

8.
We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and 13C-metabolic flux analysis (13C-MFA) with [1,6-13C]glucose, [5-13C]xylose, and [1,6-13C]glucose+[5-13C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90 °C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81 °C, the maximum growth rate on glucose and xylose was 0.44 and 0.46 h−1, respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. 13C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, 13C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, 13C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain.  相似文献   

9.
The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO2 concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO2 in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO2 values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO2. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO2 concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO2 accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO2 might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.  相似文献   

10.
Elephant seals maintain rates of endogenous glucose production (EGP) typical of post-absorptive mammals despite enduring prolonged periods of food deprivation concurrent with low rates of glucose oxidation. These high rates of EGP suggest extensive glucose recycling during fasting. We investigated lactate metabolism in fasting elephant seals to assess its role in glucose recycling. Whole-animal glucose and lactate fluxes were measured as the rates of appearance of glucose and lactate (Ra gluc and Ra lac, respectively) using a primed constant infusion of [U-14C] lactate and [6-3H] glucose, and we calculated the minimum contribution of lactate to gluconeogenesis (GNG lac). Ra lac was high compared to resting values in other species (3.21 ± 0.71 mmol min?1* kg?1), did not change between 14 ± 1 and 31 ± 8 days of fasting and varied directly with Ra glu. The minimum GNG lac was 44.6 ± 6.0 % of EGP, varied directly with plasma lactate levels, and did not change over the fast. Ra lac and Ra glu both varied directly with plasma insulin concentrations. These data suggest that lactate is the predominant gluconeogenic precursor in fasting elephant seals and that high rates of glucose recycling through Cori cycle activity contribute to the maintenance of EGP during fasting. High levels of Cori cycle activity and EGP may be important components of metabolic adaptations that maintain glucose production while avoiding ketosis during extended fasting or are related to sustained metabolic alterations associated with extended breath-holds in elephant seals.  相似文献   

11.
12.
Vibrio natriegens is a fast-growing, non-pathogenic bacterium that is being considered as the next-generation workhorse for the biotechnology industry. However, little is known about the metabolism of this organism which is limiting our ability to apply rational metabolic engineering strategies. To address this critical gap in current knowledge, here we have performed a comprehensive analysis of V. natriegens metabolism. We constructed a detailed model of V. natriegens core metabolism, measured the biomass composition, and performed high-resolution 13C metabolic flux analysis (13C-MFA) to estimate intracellular fluxes using parallel labeling experiments with the optimal tracers [1,2−13C]glucose and [1,6−13C]glucose. During exponential growth in glucose minimal medium, V. natriegens had a growth rate of 1.70 1/h (doubling time of 24 min) and a glucose uptake rate of 3.90 g/g/h, which is more than two 2-fold faster than E. coli, although slower than the fast-growing thermophile Geobacillus LC300. 13C-MFA revealed that the core metabolism of V. natriegens is similar to that of E. coli, with the main difference being a 33% lower normalized flux through the oxidative pentose phosphate pathway. Quantitative analysis of co-factor balances provided additional insights into the energy and redox metabolism of V. natriegens. Taken together, the results presented in this study provide valuable new information about the physiology of V. natriegens and establish a solid foundation for future metabolic engineering efforts with this promising microorganism.  相似文献   

13.
Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-13C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and 13C-labeling dynamics of intracellular metabolites using non-stationary 13C-metabolic flux analysis (13C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.  相似文献   

14.
Improved design of metabolic flux estimation using mixed label 13C labeling experiments and identifiability analysis motivated re-examination of metabolic fluxes during anaerobic fermentation in the Escherichia coli. Comprehensive metabolic flux maps were determined by using a mixture of differently labeled glucose and compared to conventional flux maps obtained using extracellular measurements and comprehensive metabolic flux maps obtained using only U-13C glucose as the substrate. As expected, conventional flux analysis performs poorly in comparison to 13C-MFA, especially in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Identifiability analysis indicated and experiments confirmed that a mixture of 10% U-l3C glucose, 25% 1-13C glucose, and 65% naturally labeled glucose significantly improved the statistical quality of all calculated fluxes in the PP pathway, the EMP pathway, the anaplerotic reactions, and the tricarboxylic acid cycle. Modifying the network topology for the presence and absence of the Entner-Doudoroff pathway and the glyoxylate shunt did not affect the value or quality of estimated fluxes significantly. Extracellular measurement of formate production was necessary for the accurate estimation of the fluxes around the formate node.  相似文献   

15.
Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO2 fixation in the biosphere and in the development of biological processes – alone or in cocultures, under both autotrophic and mixotrophic conditions – for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using 13C-metabolic flux analysis (13C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our 13C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. 13C-MFA during mixotrophic growth using the parallel tracers [1–13C]fructose, [1,2–13C]fructose, [1,2,3–13C]fructose, and [U–13C]asparagine found that externally supplied CO2 contributed the majority of carbon consumed. All internally-produced CO2 from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.  相似文献   

16.
Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U-14C]lactate, [1-14C]glucose, or [6-14C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with Vmax = 360 μmol/g protein/h and Km = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO2. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO2 and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.  相似文献   

17.
Abstract: The present study determined the metabolic fate of [U-13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mMα-ketoisocaproate (α-KIC). When astrocytes were incubated with 0.2 mM [U-13C]glutamate, 64.1% of the 13C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-13C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [13C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [13C]glutamate into aspartate in astrocytes incubated in the presence of α-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [13C]glutamate into the 1,2,3-13C3-isotopomer of lactate in cells incubated in the presence of α-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of α-KIC. Altogether more of the [13C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of α-KIC than in control cells. Overall, the results demonstrate that the presence of α-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.  相似文献   

18.
At present, 13C-MFA is a primary method for quantitatively characterizing intracellular carbon fluxes in cells in vivo under steady-state conditions. The method has been successfully used to investigate both the fundamental characteristics of prokaryotic and eukaryotic cell metabolism and to improve producer strains for more than twenty years. This publication is the last in a set of reviews that describe various aspects of the method. Here, the authors highlight recent achievements that involved using 13C-MFA to elucidate bacterial metabolism. Analyses of well-characterized bacterial model strains revealed that central metabolism robustness is provided by a set of alternative metabolic pathways; these analyses also helped develop a better understanding of the physiological significance of these pathways and identified previously unknown functions of well-studied metabolic pathways. Several examples of 13C-MFA-based fundamental investigations of poorly characterized bacteria are also analyzed. In applied investigations, flux analysis of strains that produce amino acids, vitamins and antibiotics indicated targets for modifications, suggested unconventional metabolic engineering approaches, and, most importantly, confirmed their utility. In the last section of this article, 13C-MFA prospects, including the monitoring of the dynamics of metabolic flux distribution during culture growth, are discussed.  相似文献   

19.
Radioactive and stable isotopes have been applied for decades to elucidate metabolic pathways and quantify carbon flow in cellular systems using mass and isotope balancing approaches. Isotope-labeling experiments can be conducted as a single tracer experiment, or as parallel labeling experiments. In the latter case, several experiments are performed under identical conditions except for the choice of substrate labeling. In this review, we highlight robust approaches for probing metabolism and addressing metabolically related questions though parallel labeling experiments. In the first part, we provide a brief historical perspective on parallel labeling experiments, from the early metabolic studies when radioisotopes were predominant to present-day applications based on stable-isotopes. We also elaborate on important technical and theoretical advances that have facilitated the transition from radioisotopes to stable-isotopes. In the second part of the review, we focus on parallel labeling experiments for 13C-metabolic flux analysis (13C-MFA). Parallel experiments offer several advantages that include: tailoring experiments to resolve specific fluxes with high precision; reducing the length of labeling experiments by introducing multiple entry-points of isotopes; validating biochemical network models; and improving the performance of 13C-MFA in systems where the number of measurements is limited. We conclude by discussing some challenges facing the use of parallel labeling experiments for 13C-MFA and highlight the need to address issues related to biological variability, data integration, and rational tracer selection.  相似文献   

20.
13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled 13C-glucose, a commonly used isotope for 13C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (13C) or unlabeled (12C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between 12C and 13C-glucose as a carbon source for E. coli growth.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号