Metabolic Flux Analysis (MFA) is a system biology technique that uses experimental data and mathematical models to quantitatively analyze the flow of metabolites in intracellular metabolic networks. The basic principle includes the representation of intracellular metabolic reactions through metabolic networks, and it is assumed that the input and output fluxes of metabolites are balanced under steady state. The key steps of MFA include constructing metabolic networks, collecting experimental data ( such as isotope labeling experiments ), model calibration and flux calculation, and result verification and analysis. MFA is widely used in metabolic engineering, disease research, system biology, agriculture and food science, such as optimizing microbial cell factories, studying cancer cell metabolic reprogramming, revealing metabolic network regulation mechanisms, and improving crop yield and stress resistance.
Microbial metabolic engineering
Microbial metabolic engineers use MFA to optimize the metabolic pathway of bacteria or yeast to improve the yield of target products (such as antibiotics, enzymes, amino acids, etc.). Through MFA, researchers can regulate metabolic flow, optimize the expression of genome and enzymes, and then improve production efficiency.
Cyanobacteria such as Synechocystis 6803 grow through a series of complex metabolic pathways, including EMP pathway, OPP pathway, ED pathway, CBB cycle, PK pathway and TCA cycle. Compared with other bacteria, the metabolic network of cyanobacteria is more complex and highly redundant, and the light mixed nutrition model can effectively utilize sugar and carbon dioxide to achieve rapid growth, which has great potential for bio-manufacturing. The application of 13C mfa in the analysis of photosynthetic mixed nutrition metabolism of cyanobacteria (Synechocystis sp. 6803) was reviewed. Under the condition of light mixed nutrition growth, different studies reported different flux distributions. For example, the fluxes of malic enzyme and OPP (pentose phosphate pathway) show different changes in different experiments. In some studies, the fluxes of these pathways are almost non-existent, while others are high. The effect of CO2 supply mode on flux is also significantly different. Some experiments used high concentration of NaHCO3, while this study used atmospheric CO2, which affected the uptake ratio of CO2 to sugar. The mechanism of light mixed nutrition allows cells to produce ATP and NADPH by light, but there is usually no NADPH limitation. For CBB cycle, cells mainly maintain the supply of ATP and NADPH through GAP recovery. In the process of NADPH regeneration, OPP pathway flux provides some support, but its contribution is far less than CBB cycle. In this study, a 13C mfa method was developed, which can fully describe the core carbon network of Synechocystis sp. under light mixed nutrition. Through GC/MS analysis, complete metabolic flux data can be obtained with less investment in experiments. It was found that in the process of mixed nutrition growth, glucose almost completely provided power for CBB cycle, which maximized the fixation of CO2. During the CBB cycle operation, the shunting in glycolytic pathway is mainly PGI and OPP, while the flux of ED is not found. The defective mutants of ED pathway showed that OPP flux decreased significantly, CBB circulating flux increased, and glycogen formation increased. The mutant showed reduced adaptability under fluctuating light conditions. It is also found that the secondary regulation through ED pathway may provide some GAP for the regeneration of CBB cycle, and may further regulate the activity of OPP pathway through KDPG (Schulze D et al., 2022).
In vivo flux distribution of Synechocystis 6803 ∆eda during photomixotrophic growth of glucose and CO2 (Schulze D et al., 2022).
Oily microorganisms such as filamentous fungi and microalgae can synthesize healthy polyunsaturated fatty acids (such as GLA, EPA and DHA), and they have important biological significance in lipid accumulation. The study also revealed that pyruvate, a glycolytic product, was converted into acetyl coenzyme A after entering mitochondria, and this metabolite needed to be transported to the cytosol through citrate transport system for lipid synthesis. Oily microorganisms such as filamentous fungi and microalgae can synthesize healthy polyunsaturated fatty acids (such as GLA, EPA and DHA), and they have important biological significance in lipid accumulation. The study also revealed that pyruvate, a glycolytic product, was converted into acetyl coenzyme A after entering mitochondria, and this metabolite needed to be transported to the cytosol through citrate transport system for lipid synthesis. In this study, the metabolic flux of genetically engineered Mucor circinelloides (M. circinelloides) strain was analyzed by 13C labeling. The results showed that the flux of tricarboxylic acid (TCA) was lower than that of the control strain, while the flux of glyoxylic acid (GOX) was higher than that of the control strain. In contrast, mt gene knockout strains showed the opposite phenomenon, with higher TCA circulating flux and lower GOX circulating flux. GOX cycle may be more effective than TCA cycle in the synthesis of malate and oxaloacetic acid. The flux of PP pathway of mt overexpression strain is high, which indicates that this pathway may play an important role in providing NADPH needed for fatty acid synthesis. Studies have shown that malate dehydrogenase is not the limiting factor in fatty acid synthesis. Malic acid transporter regulates PP pathway by promoting the transport of acetyl coenzyme A, which provides necessary NADPH for lipid synthesis. It shows that the overexpression of malate transporter can promote the synthesis of fatty acids by changing the metabolic direction of TCA cycle and GOX cycle, and increase the supply of NADPH through PP pathway, which drives the accumulation of lipids. Therefore, malate transporter plays a key role in lipid synthesis (Wang L et al., 2019).
Cancer metabolism research
In cancer research, MFA can be used to analyze the metabolic characteristics of cancer cells. Cancer cells often change their metabolic patterns and increase the activity of some metabolic pathways (such as glycolysis) to promote rapid proliferation. Through MFA, researchers can reveal the unique metabolic flow pattern of cancer cells and provide potential targets for early diagnosis and treatment of cancer.
Intracellular metabolic flux cannot be measured directly, and it usually depends on isotope labeling experiments, combined with complex mathematical and computational analysis methods. Isotope-assisted metabolic flux analysis (iMFA) is used to study the metabolic reprogramming of various cells (such as macrophages, brown fat, platelets, etc.) and reveal their metabolic changes in different physiological states. Studies have shown that after MPLA activates macrophages, malate metabolism is temporarily redirected to avoid entering damaged mitochondria, and at the same time, glycolysis is increased to provide energy needed for antibacterial. Under cold stimulation, brown adipose tissue increases the intake of glucose and fatty acids, promotes glycolysis and β oxidation, and TCA cycle is active. Resting platelets depend on glycolysis, while activated platelets increase glycogen decomposition and redirect products to TCA cycle. Cancer cells adjust mitochondrial metabolism in high lactic acid environment and increase anaerobic drug metabolic flow, while hepatocellular carcinoma undergoes exogenous citrate metabolism in hypoxia environment. In the aspect of antibiotic therapy, iMFA revealed the influence of betaquinoline (BDQ) on Mycobacterium tuberculosis (Mtb), and found that it can activate specific metabolic compensation reactions, such as enhancing anaerobic metabolism through phosphoenolpyruvate carboxylase (PEPCK) and increasing ATP production through pyruvate kinase (PykA). In the aspect of viral infection, iMFA was used to study the metabolic changes of amniotic cell-derived cell lines infected with adenovirus type 5. It was found that viral infection promoted reductive carboxylation, increased PPP flux and promoted lipogenesis. These metabolic changes contribute to virus replication and provide potential targets for treatment and vaccine development. In addition, iMFA is also used to study how cells cope with the loss of genetic function, such as metabolic changes caused by mitochondrial DNA mutation. Studies have shown that mutant cells compensate for the lack of mitochondrial metabolic function by changing the metabolic pathways of glutamine and glucose. Similar analysis is also applied to tumor cells, revealing how metabolic compensation can enhance the drug resistance of cells and help to understand the key drivers of cancer metabolic reprogramming (Moiz B et al., 2022).
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Plant metabolism research
In the study of plant metabolism, MFA helps to reveal the metabolic regulation of plants when they grow, develop and cope with environmental changes (such as light, temperature, nutrition, etc.). It can help to understand how plants respond to stress through metabolic pathways and optimize crop growth and yield.
Photosynthetic leaves are the main organs of carbon assimilation in plants, which can transform atmospheric carbon dioxide (CO2) into organic metabolic intermediates through Calvin Benson cycle (CBC), and then synthesize metabolites needed by plants, such as polysaccharides, lipids, protein amino acids and nucleotides of DNA and RNA. TAG content in most leaves is low, but they play a key role in membrane lipid homeostasis and fatty acid release during abiotic stress. In this study, the relationship between central carbon metabolism and fatty acid biosynthesis, especially in high fatty acid yield (HLP) tobacco strains, was discussed by instantaneous 13CO2 labeling and unsteady mfa (INST-MFA). The synthesis of fatty acids in chloroplast matrix may increase the release of CO2, which is recaptured by RuBisCO to help it perform carboxylation. Although the photorespiration rate is low in HLP plants, these processes show that RuBisCO still responds to the change of CO2 concentration. The results showed that carbon metabolism in HLP leaves enhanced fatty acid synthesis through multiple channels, thus promoting the accumulation of TAG. By engineering transgenic plants to increase fatty acid synthesis, incorporate it into TAG biosynthesis, stabilize lipid droplets and minimize TAG turnover, the production and accumulation of TAG in oilseeds and leaves can be significantly improved. By analyzing ADP- glucose and starch isotopes, it is found that unlabeled ADP- glucose is transformed into starch in metabolism, and this process has a high degree of intercellular difference. Especially in HLP leaves, the distribution of starch decreased and the accumulation of lipid increased, indicating that the carbon distribution of plants changed. Especially, 13CO2 labeling experiment revealed that HLP leaves enhanced fatty acid biosynthesis through central carbon metabolism, and NADP- malic enzyme played an important role in the production of pyruvate in plastids. The activity of NADP- malic enzyme in HLP leaves was significantly enhanced, which promoted the production of plastid pyruvate, thus enhancing the biosynthesis of fatty acids. Compared with wild-type tobacco (WT), the study showed that the metabolic pattern of starch and lipid in HLP tobacco leaves changed significantly, which enabled the leaves to be transformed into lipid storage tissues, thus supporting the accumulation of high TAG levels. In old tobacco leaves, non-instantaneous starch pool, as a carbon reserve, can provide a carbon source for lipid synthesis (Chu KL et al., 2022).
Plant secondary metabolites are natural compounds produced by plants to adapt to the environment, survive and reproduce. The plant kingdom can synthesize more than 200,000 kinds of natural products, which play an important role in defense (such as insect resistance and disease resistance) and reproduction (such as attracting pollinators). Many secondary metabolites have high value in medicine, nutrition and health care, spices and colorants, so they have become important targets of metabolic engineering. Accurate understanding of how plants use carbon and energy is more critical than theoretical requirements, which helps to measure the efficiency of metabolic pathways. As a quantitative method, MFA can estimate the flow of carbon in metabolic network. Steady-state MFA: By measuring the isotopic abundance of metabolites, the metabolic flux of plants is analyzed under steady-state conditions. Steady-state means that the isotope abundance of metabolites remains stable. This method can help to quantify the metabolic pathways including metabolic cycles. However, in autotrophic metabolism, the application of steady-state MFA is limited because the assimilation of CO2 may introduce carbon with natural abundance. INST-MFA: By applying isotopically labeled CO2 to plants and measuring the dynamic isotope labeling of metabolites, combined with analytical techniques such as GC-MS and LC-MS/MS, the photosynthetic autotrophic flux can be accurately quantified. INST-MFA is suitable for analyzing the changes of metabolic flux in metabolic processes such as photosynthesis, and can overcome some limitations of steady-state MFA. Dynamic MFA: It is similar to INST-MFA, but it focuses on the study of the isotope abundance of metabolites after the addition of exogenous isotope-labeled precursors, and it is necessary to monitor the change of cell size in the experiment. This method can capture the time variation of metabolites more accurately and is often used to analyze the flux in the secondary metabolic network. Metabolic channel and compartment metabolite pool: MFA analysis can also reveal the transfer mode of metabolites between different reaction areas, which is helpful to identify the existence of metabolite channel and inactive metabolite pool. Through these analyses, we can better understand the dynamic changes of metabolic reactions between different compartments in cells (Shih ML et al., 2020).
System biology
MFA is widely used in systems biology to construct and understand complex metabolic networks. By quantitatively analyzing the metabolic flow in cells or organisms, MFA can help reveal the dynamics of metabolic pathways and provide insights on how metabolic networks respond to external changes (such as environmental conditions, nutritional status, drug treatment, etc.).
Platelets are seedless cells that can quickly form blood clots to stop bleeding after blood injury. Recent studies have shown that platelets also play an important role in many chronic diseases (such as diabetes, atherosclerosis, etc.), and their metabolic changes may become potential biomarkers. The study of platelet metabolism usually observes the response of activation, storage, aging and specific reaction or pathway inhibitors, and adopts various techniques (such as radiation labeling, respirometry, extracellular flux analysis, etc.) to study the changes of metabolic pathways. However, due to the complexity of metabolic process, these measurements are sometimes difficult to explain, especially when analyzing extracellular flux. In this study, 13C-MFA(13C isotope metabolic flow analysis) was used to quantitatively measure the central metabolic flux of resting and thrombin-activated human platelets. The results show that the metabolic feature of resting platelets is that glucose is completely converted into lactic acid by glycolysis, and TCA flux is roughly equal to glycolysis flux. Pentose phosphate pathway has the smallest contribution, accounting for about 37% of the total flux. However, after thrombin activation, platelet metabolism changed significantly, especially glucose uptake increased, and glycolysis, TCA cycle and pentose phosphate pathway flux all increased significantly. The glycolytic flux of thrombin-activated platelets is three times that of resting state, and some glycolytic products enter TCA cycle, reflecting the transformation of metabolic pathway. After thrombin activation, the metabolic activity of platelets increased significantly. The flux of glycolysis increased by 3.4 to 4.4 times, resulting in a significant increase in lactic acid production. The flux through pentose phosphate oxidation pathway increased by 45%, and the TCA circulation flux increased by 14%. The absolute increase of glucose oxidation has also been reflected, and the production of CO2 has increased significantly, with an overall increase of 26%. This study further clarified the glycolytic phenotype of platelets, and discussed how thrombin activated platelets can adapt to higher energy demand by increasing glucose oxidation (Sake CL et al.,2022).
Industrial bioengineering
In the process of bio-manufacturing and fermentation, MFA can help optimize the production process. For example, when producing biofuels, drugs, enzymes or other biological products, MFA analysis can identify bottleneck reactions, improve metabolic pathways, and maximize product yield. MFA can also be used to monitor and control the metabolic flow direction during microbial fermentation and improve efficiency.
Some traditional vaccines, such as vaccinia, measles, mumps and influenza vaccines, still rely on primary materials such as embryonic eggs for production. However, the primary cell culture process is difficult to meet the needs of modern vaccine production, so a continuous suspension cell line such as AGE1.CR.pIX was developed. These cell lines come from muscovy ducks and adopt clear culture medium for virus transmission. In order to better understand the performance of this cell line in vaccine production, the researchers developed a metabolic stoichiometry model to analyze the central metabolism of AGE1.CR.pIX cells. Studies have shown that the metabolic flux distribution of avian cell line (AGE1.CR.pIX) is similar to that of mammalian cell lines (such as CHO and MDCK cells). In particular, the efficiency of glucose catabolism in cells is low, and the relationship between glycolysis and TCA cycle is weak. A notable feature is that glutamine plays a small role in energy production and precursor synthesis, resulting in a low extracellular ammonia concentration. The metabolic database of immortalized avian cell lines was expanded by measuring the concentrations of cellular and extracellular metabolites. The results showed that the biomass composition and growth characteristics of CR.pIX cells were similar to those of other transformed mammalian and insect cell lines. Through the analysis of flux variability, it was found that CR.pIX cells showed similar overflow metabolism to mammalian cells, mainly glucose was converted into lactic acid and alanine after ingestion. The relationship between TCA cycle and glycolysis is weak and depends on the inflow of amino acids (such as isoleucine, valine and aspartic acid). In addition, studies have shown that glutamine supplementation is not a necessary condition for the proliferation of CR.pIX cells. The hypothesis of low glutamine decomposition activity was confirmed by measuring enzyme activity, and the low dependence of CR.pIX cells on glutamine was verified by the passage experiment without glutamine medium. This discovery is of great significance to the fed-batch culture strategy that ammonia accumulation may reach toxic level (Lohr V et al.,2014).
References
- Schulze D, Kohlstedt M, Becker J, Cahoreau E, Peyriga L, Makowka A, Hildebrandt S, Gutekunst K, Portais JC, Wittmann C. "GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways." Microb Cell Fact. 2022;21(1):69. doi: 10.1186/s12934-022-01790-9
- Wang L, Zhang H, Zhang Y, Song Y. "13C metabolic flux analysis on roles of malate transporter in lipid accumulation of Mucor circinelloides." Microb Cell Fact. 2019;18(1):154. doi: 10.1186/s12934-019-1207-9
- Moiz B, Li A, Padmanabhan S, Sriram G, Clyne AM. "Isotope-Assisted Metabolic Flux Analysis: A Powerful Technique to Gain New Insights into the Human Metabolome in Health and Disease." Metabolites. 2022 ;12(11):1066. doi: 10.3390/metabo12111066
- Chu KL, Koley S, Jenkins LM, Bailey SR, Kambhampati S, Foley K, Arp JJ, Morley SA, Czymmek KJ, Bates PD, Allen DK. "Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves." Metab Eng. 2022;69:231-248. doi: 10.1016/j.ymben.2021.12.003
- Sake CL, Metcalf AJ, Meagher M, Di Paola J, Neeves KB, Boyle NR. "Isotopically nonstationary 13C metabolic flux analysis in resting and activated human platelets." Metab Eng. 2022 ;69:313-322. doi: 10.1016/j.ymben.2021.12.007
- Shih ML, Morgan JA. "Metabolic flux analysis of secondary metabolism in plants." Metab Eng Commun. 2020;10:e00123. doi: 10.1016/j.mec.2020.e00123
- Lohr V, Hädicke O, Genzel Y, Jordan I, Büntemeyer H, Klamt S, Reichl U. "The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis." BMC Biotechnol. 2014;14:72. doi: 10.1186/1472-6750-14-72
