Metabolic Flux Analysis (MFA) involves the measurement and analysis of metabolic reaction rate in biological system. By tracking the flow of metabolites in Mps , MFA enables scientists to build quantitative models to describe how cells allocate resources to different pathways. It relies on the use of isotope-labeled substrates (such as carbon or nitrogen isotopes) to track the movement of metabolites, so as to calculate metabolic flux, that is, the rate of transformation of metabolites in a particular metabolic reaction.
The goal of MFA is to obtain a detailed map of cell metabolism and understand how Mps interact and promote cell function. This can reveal key insights such as how bacteria respond to environmental changes, regulate growth and maintain steady state.
Importance of MFA in bacterial research
Bacteria are very diverse creatures and can adapt to a wide range of environments. They have complex and dynamic Mps, which can utilize different carbon sources, generate energy and synthesize necessary biomolecules. By performing MFA on bacteria, researchers can:
- Understand metabolic adaptation: bacteria can quickly adjust their Mps to cope with different environments and pressures. Through MFA, researchers can analyze how bacteria adjust their MPs and energy distribution under changing conditions, such as lack of nutrients, temperature changes or the existence of harmful substances. For example, bacteria may start an anaerobic respiratory path when facing oxygen deficiency, or may survive by decomposing the resources stored in cells when nutrients are scarce. MFA can also help reveal how bacteria escape the host's defense mechanism by changing MPs under the pressure of host immune response or antibiotic treatment.
- Optimize the application of biotechnology: by identifying metabolic bottlenecks and underutilized MPs, MFA can provide guidance for genetic engineering, so that bacteria can use substrates more efficiently and maximize the production of products. When bacteria are used in large-scale production in bioreactor, MFA can help optimize culture conditions, such as temperature, pH and nutrient concentration, ensure efficient operation of Mps and maximize production efficiency. MFA can also help improve the tolerance of bacteria to specific production environment or product toxicity.
- Study on antibiotic resistance: MFA can reveal how bacteria can resist the interference of antibiotics by changing MPs, such as enhancing metabolic repair mechanism or synthesizing substances related to metabolic resistance. The drug resistance of some antibiotics is not caused by the traditional drug resistance genes, but by the metabolic changes of bacteria. MFA can help identify these metabolic reactions, thus providing a theoretical basis for new strategies of antibiotics and drug resistance monitoring. By analyzing the metabolic response of bacteria under antibiotic pressure, MFA can also help identify potential antibiotic targets.
Metabolic flux distribution based on 13C flux analysis using the extended metabolic model (Jordà J et al., 2013).
Working principle of MFA in bacterial research
- Introduction of Isotope Labels: The first step of MFA is to introduce substrates with stable isotope labels (such as ^13C, ^15N, etc.) into the bacterial culture system, so that we can track the flow of metabolites in the Mps through isotope tracing.
- Bacterial sample collection: Under strictly controlled culture conditions, we need to take samples from the culture solution at different time points, usually at the metabolic steady state or at a specific time point, and then extract the metabolites inside and outside the cell for subsequent analysis. The selection of sampling time is very important for the determination of metabolic flux, which can ensure the study of the dynamic process of Mps.
- Quantitative analysis of metabolites: analyze the samples by GC-MS or LC-MS, accurately determine the concentration of labeled metabolites, reveal the spectra and isotopic distribution of various metabolites inside and outside the cell, and then quantify the key intermediates in the Mps.
- Calculation of metabolic flux: after obtaining the data of metabolite concentration, use mathematical models (such as metabolic flux model based on Mps) to calculate the flux of metabolic reaction. Based on the stoichiometric relationship between metabolites and substrates, the process was deduced by using the metabolic flux balance equations (FBA), and combined with the topological structure of the Mps, the flow value of each reaction in each Mps was calculated.
- Data analysis and metabolic network reconstruction: the calculated metabolic flux was further analyzed and analyzed to evaluate the role of different MPs in the overall metabolism of bacteria. By comparing the distribution of metabolic flux under different physiological conditions, researchers can deeply understand how bacteria allocate energy, use carbon sources and optimize the allocation of other resources, and then analyze their metabolic adaptation mechanism under environmental changes or stress conditions.
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Application of MFA in bacterial research
A. Industrial biotechnology
Bacteria are often used to produce biofuels, chemicals and drugs. MFA can be used to optimize MPs related to the production of these products. By identifying bottlenecks and underutilized pathways, researchers can design more productive bacterial strains.
Due to the global oil crisis and the increase of greenhouse gases, alternative fuels have attracted attention, and biodiesel (produced by the reaction of vegetable oil or animal fat with lower alcohols) is one of the important alternatives. It has obvious advantages to use oil-bearing microorganisms (such as Yarrowia lipolytica) as the production source of biodiesel. They can grow on a variety of carbon sources and accumulate higher lipids in cells. Lipid accumulation is usually induced by limiting nitrogen sources. When the nitrogen source is exhausted, the cells will stop synthesizing protein and nucleotides, and turn the excess carbon source into lipids. In this study, the potential of Yarrowia lipolytica in producing single cell oil (which can be converted into biodiesel) was discussed, and the molecular mechanism of lipid accumulation under the conditions of sufficient nitrogen and insufficient nitrogen was studied by mfa. In the medium with insufficient nitrogen, the cell growth of Yarrowia lipolytica was inhibited, but the lipid accumulation increased significantly, and the proportion of lipid in biomass increased from 8.7% to 14.3%.
This shows that the lack of nitrogen source will stimulate the synthesis of lipids. MFA showed that the flux of pentose phosphate pathway did not change significantly under different nitrogen concentrations. This shows that the production of NADPH is not the limiting factor of lipid accumulation in Yarrowia lipolytica. However, the metabolic flux of malic enzyme is almost undetectable, which proves that it does not play a regulatory role in the lipid accumulation of Yarrowia lipolytica. Nitrogen restriction significantly increased the metabolic flux through ATP: citrate lyase. This shows that ACL plays a key role in providing acetyl coenzyme A, which is the precursor molecule of lipid synthesis. These results reveal how Yarrowia lipolytica can promote lipid accumulation by regulating key MPs (such as providing acetyl coenzyme A through ACL) under nitrogen limitation, thus providing a new idea for biodiesel production (Zhang H et al., 2016).
B. Microbial physiology
MFA can provide a detailed understanding of the physiological state of bacteria under different conditions. For example, by studying how bacteria adjust their metabolism under malnutrition or environmental pressure, scientists can understand their adaptation mechanism and survival strategy.
By analyzing the effects of temperature and dilution rate on the metabolism of Saccharomyces cerevisiae (EC1118) in anaerobic continuous culture, the accurate production and consumption rates of metabolites under different environmental conditions were studied to understand the physiological characteristics of yeast. Based on mfa, glucose is used as the only carbon source to replace the combination of glucose and fructose with equal molar amount. In this study, the ratio of amino acid incorporation rate to biomass and uptake rate is used to determine whether it is necessary to include synthetic or catabolic reactions. In the model, 19 kinds of amino acids and ammonium nitrogen transport reactions were included to describe the role of EC1118 in amino acid metabolism. The research shows that in anaerobic environment, carbon flux is mainly used for energy production, such as the generation of ethanol and CO2. The carbon flux of TCA cycle remains below 1%, which is enough to maintain the intracellular level of key metabolic intermediates. At 28℃, the carbon flux of glycolysis is high, which leads to the low carbon flux of PPP and carbohydrate biosynthesis. Under different temperatures and sugar concentrations, the carbon flux of PPP also shows differences, suggesting that this branch plays a flexible role in regulating metabolism. Glycolysis is dominant under all conditions, but the flux is different at different temperatures. At low temperature (16℃), the flux of glycolysis is high, while at high temperature (28℃), the flux of glycerol production increases. High sugar concentration (such as 280 g/L glucose) promotes glycerol synthesis because osmotic pressure increases. At 16℃, the intake of amino acids increased, which led to the increase of NADH consumption, thus inhibiting glycerol production. Pyruvate is mainly converted into acetaldehyde and enters ethanol synthesis. At low temperature, the carbon flux of TCA cycle decreases, and acetic acid production is related to sugar concentration, and acetic acid production is higher at higher sugar concentration. Lactic acid is less produced under anaerobic conditions, and its concentration is lower than the detection limit at 16℃.
In addition, the consumption of amino acids does not show a uniform law. The consumption of some amino acids (such as lysine, alanine, tryptophan, etc.) is significantly lower than that of other amino acids, while the consumption of some amino acids is greatly affected by sugar concentration (Quirós M et al., 2013).
C. Synthetic biology
In synthetic biology, researchers engineer bacteria to perform specific functions, such as producing bio-based materials or sensing environmental changes. MFA helps to ensure that engineered bacteria can use resources efficiently and that the designed Mps operates as expected.
Lignocellulose biomass (mainly composed of cellulose and hemicellulose) can be converted into fuels and other chemicals, especially glucose and xylose. However, there are relatively few studies on xylose metabolism. In this paper, the metabolism of glucose and xylose in Escherichia coli under aerobic and anaerobic conditions was comprehensively quantified by combining parallel labeling experiment and 13C-MFA method, and the changes of biomass composition and macromolecular turnover rate were discussed. 13C-MFA of wild-type Escherichia coli under different growth conditions showed that the aerobic growth rate (0.70 and 0.50 h-1, respectively) was significantly higher than the anaerobic growth rate (0.33 and 0.13 h-1, respectively) when glucose or xylose was used as carbon source. Under aerobic conditions, the biomass yield is also significantly higher than that under anaerobic conditions, especially in aerobic culture of glucose and xylose, the biomass yield is 0.44 and 0.35 gDW/g, compared with 0.14 and 0.08 gDW/g under anaerobic conditions. Under aerobic conditions, the only metabolite is acetate. Under anaerobic conditions, acetate, ethanol, formate and succinate are produced, and the secretion ratio of acetate, ethanol and formate is about 1:1:2. Compared with aerobic growth, the carbon and electron recovery rates of glucose and xylose under anaerobic growth are higher, which are 87% and 81% respectively. The RNA content is related to the growth rate, and it is higher in aerobic growth than in anaerobic growth. In anaerobic and aerobic cultures, the mass isotope isomers of glucose and xylose behave differently under different conditions, especially the labeling of aspartic acid, glutamic acid and related amino acids. Gene knockout experiments showed that the growth of ΔfadD, ΔfadK and ΔfadDΔfadK strains on anaerobic xylose medium was significantly limited, which proved the necessity of β -oxidation for anaerobic xylose growth. Under aerobic conditions, classical central MPs (such as glycolysis, pentose phosphate pathway and TCA cycles) are active, while under anaerobic conditions, some MPs (such as oxPPP and TCA cycles) are significantly weakened, indicating that energy generation in anaerobic growth mainly depends on lipid turnover (Gonzalez JE et al., 2017).
D. Pathogenicity research
MFA is used to study how pathogenic bacteria change their metabolism in order to survive and reproduce in host organisms. Understanding how bacteria modify their Mps during infection can help to find potential therapeutic targets.
Bacterial pathogens, such as Salmonella (STm), can adapt and survive in host cells by evolving complex mechanisms. The environment in the host cell provides a new niche for bacteria, and bacteria must quickly adjust their MPs to cope with limited or lack of metabolites. Mannitol is an ideal carbon source, which can be used to specifically label STm growing in cells. Mannitol can enter the host cell, but it will not be metabolized by the host cell. At the same time, it is effectively utilized by STm and does not interfere with the metabolism of other carbon sources. By providing 13C-labeled mannitol to host cells, researchers can track the metabolic process of bacteria and then analyze the Mps of STm in host cells. In the process of STm infection, the synthesis of nucleotides in STm cells depends on ribose 5- phosphate (5P) and nucleobases. The sources of ribose 5P include carbon assimilation and de novo synthesis, in which about 60% of ribose 5P is synthesized by de novo, while the synthesis of nucleobases mainly depends on absorption from the host. Specifically, uracil is partially synthesized by STm ab initio, while adenine and guanine are almost completely dependent on the host. Through 13C- MFA, it is shown that the synthesis of ribose 5P is realized by forward oxidation of pentose phosphate pathway (PPP) and reduction of PPP (through the action of transketolase TktA2/B2 and Rpe). Specific marker patterns (M+2, M+3) in glutamic acid, aspartic acid and malic acid were observed, and the existence of these markers was considered to be related to the activity of phosphoenolpyruvate carboxylase (Ppc). Ppc is a key enzyme in anaerobic reaction, and it is an important way for STm cells to maintain growth under anoxic conditions by participating in the conversion of pyruvate into succinate. Through the experiment of ppc gene knockout, it is found that Ppc plays an important role in the growth of STm cells, especially in the environment lacking amino acids. The ppc mutant grew close to the wild-type (wt) in enriched medium, but showed growth defects in the absence of amino acids, which indicated that Ppc played a decisive role in the growth of STm under different environmental conditions (Mitosch K et al., 2023).
References
- Zhang H, Wu C, Wu Q, Dai J, Song Y. "Metabolic Flux Analysis of Lipid Biosynthesis in the Yeast Yarrowia lipolytica Using 13C-Labled Glucose and Gas Chromatography-Mass Spectrometry." PLoS One. 2016 ;11(7):e0159187. doi: 10.1371/journal.pone.0159187
- Quirós M, Martínez-Moreno R, Albiol J, Morales P, Vázquez-Lima F, Barreiro-Vázquez A, Ferrer P, Gonzalez R. "Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations." PLoS One. 2013;8(8):e71909. doi: 10.1371/journal.pone.0071909
- Gonzalez JE, Long CP, Antoniewicz MR. "Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis." Metab Eng. 2017 ;39:9-18. doi: 10.1016/j.ymben.2016.11.003
- Mitosch K, Beyß M, Phapale P, Drotleff B, Nöh K, Alexandrov T, Patil KR, Typas A. "A pathogen-specific isotope tracing approach reveals metabolic activities and fluxes of intracellular Salmonella." PLoS Biol. 2023 ;21(8):e3002198. doi: 10.1371/journal.pbio.3002198
