CN111575336A - Method for acquiring metabolic flux of intracellular central carbon metabolic pathway under metabolic steady isotope unsteady state - Google Patents
Method for acquiring metabolic flux of intracellular central carbon metabolic pathway under metabolic steady isotope unsteady state Download PDFInfo
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Abstract
The invention discloses a method for acquiring metabolic flux of an intracellular central carbon metabolic pathway under a metabolic steady isotope non-steady state, which comprises a batch culture module, a chemostat culture module, a sampling module and a sample processing and analyzing module; transferring to the chemostat culture module when the OUR and the CER in the batch culture module are simultaneously reduced; the chemostat culture module specifically comprises: feeding nutrient solution without isotope labeled substrate, simultaneously starting waste discharge, performing chemostat culture, switching to feeding nutrient solution containing isotope labeled substrate when the microbial cells are in metabolic steady state, and keeping culture conditions unchanged; rapid, continuous sampling begins as the isotopically labeled substrate flows into the reactor, simultaneously entering the sampling module. The method of the invention obtains a series of fermentation liquor samples in a short time, and then processes and analyzes the samples to obtain accurate intracellular metabolite isotope abundance data, which plays an important role in calculating metabolic flux.
Description
Technical Field
The invention relates to the technical field of metabolic flux calculation, in particular to a method for acquiring metabolic flux of intracellular central carbon metabolic pathway under metabolic steady isotope unsteady state.
Background
The rapid development of the "multiomics" technology makes it possible to obtain the abundance of various biomolecules at high throughput, so that the variation and subtle relationship of the various omics under different physiological states can be determined. Various omics are applied in the biological research of a microbial system, including transcriptomics, and the transcription level of mRNA is measured; proteomics, quantifying the abundance of proteins; metabolomics, determining cellular metabolite abundance; and (4) metabolic flow group, namely establishing flow distribution in the intracellular metabolic network. Meanwhile, metabolic flux is a result of quantifying the comprehensive network reaction of the "gene-protein-metabolite" interaction, and is the most direct expression of the physiological metabolic state of cells. It can be seen that metabolic flux is very important for the study of the metabolic properties of microbial cells. Through decades of development, the analysis method of microbial cell metabolic flux is gradually mature. From the earliest metabolic flux analysis methods using classical metrology to the use of isotopic abundance data for constraints13C-MFA, and to the nearest isotopic unsteadiness, differential equations are used to estimate INST-MFA of metabolic flows. In the meantime, in order to simplify the complexity of calculation, researchers also propose an innovative EMU decomposition method, which promotes the development of metabonomics.
At present, the theoretical method for calculating metabolic flow is very mature, but the related experimental method is not perfect, and the following problems exist: the large-capacity bioreactor wastes unnecessary isotope labeling experiment process13C glucose, which results in high experimental costs; in the experimental operation of switching substrates, complicated pipelines are easy to cause flow additionA blank window of substrate, affecting the physiological metabolic state of the microorganism; the complicated sampling device can influence the sampling efficiency, and the excessive sampling quantity can interfere the microorganisms in the metabolic steady state; leakage is easy to occur in the process of extracting the intracellular metabolites, and the accuracy of an experimental result is influenced; in the concentration process, the inactivation of intracellular metabolites is easily caused by overlong time or overhigh temperature, and the like. Therefore, the problems of high cost, complex operation and poor stability are faced in the current metabolic flux calculation experiment.
Therefore, it is urgently needed to provide a method for acquiring metabolic flux of intracellular central carbon metabolic pathways under metabolic steady isotope unsteady state, a series of fermentation liquid samples are acquired in a short time through rapid sampling, and then the samples are processed and analyzed to obtain accurate intracellular metabolite isotope abundance data, and meanwhile, the accuracy of metabolic flux results obtained by calculating the experimental data acquired by the method can be ensured, and the method plays a crucial role in calculating metabolic flux.
Disclosure of Invention
The invention aims to provide a method for acquiring metabolic flux of an intracellular central carbon metabolic pathway under a metabolic steady isotope unsteady state, a series of fermentation liquid samples are acquired in a short time through a rapid sampling device, and then the samples are processed and analyzed to obtain accurate intracellular metabolite isotope abundance data and exchange reaction rate.
In order to achieve the purpose, the invention adopts the following technical scheme.
The invention provides a method for acquiring metabolic flux of an intracellular central carbon metabolic pathway under a metabolic steady isotope non-steady state, which comprises a batch culture module, a chemostat culture module, a sampling module and a sample processing and analyzing module; wherein the content of the first and second substances,
oxygen Uptake Rate (OUR) and CO in the batch culture module2Transferring to the chemostat culture module when the release rate (CER) is reduced simultaneously;
the chemostat culture module specifically comprises: feeding nutrient solution without isotope labeled substrate, simultaneously starting waste discharge, performing chemostat culture, switching to feeding nutrient solution containing isotope labeled substrate when the microbial cells are in metabolic steady state, and keeping culture conditions unchanged;
beginning rapid, continuous sampling when the isotopically-labeled substrate flows into the reactor, at which point it enters the sampling module;
the sample processing and analyzing module is used for processing, detecting and analyzing the sample so as to obtain intracellular metabolite isotope abundance data and exchange reaction rate.
Further, the intracellular metabolite isotope abundance was determined by LC-MS/MS method.
Further, the isotopically labeled substrate is13And C, labeling the substrate. For example, adopt13C-labeled glucose.
Further, the time consumed for each sampling does not exceed 1 s.
Further, after sampling, quenching the taken sample, filtering to obtain thalli, adding the thalli into a preheated 75% ethanol solution, carrying out water bath reaction at 95 ℃, extracting intracellular metabolites, cooling to normal temperature, carrying out suction filtration to obtain a filtrate containing the intracellular metabolites, concentrating, removing ethanol, and fixing the volume to obtain an intracellular metabolite sample.
Further, after sampling, putting the taken sample into cold methanol at the temperature of-40 ℃ for quenching, filtering to obtain thalli, adding the thalli into 75% ethanol solution preheated to the temperature of 70-75 ℃, carrying out water bath reaction at the temperature of 95 ℃ for 3min, then quickly cooling to the normal temperature, and carrying out suction filtration to obtain filtrate containing intracellular metabolites; concentrating at 30 deg.C and vacuum degree of 100, removing ethanol, adding ultrapure water to desired volume to obtain intracellular metabolite sample.
Further, the concentration is: and (3) concentrating the filtrate by using a rotary evaporator under the conditions of 30 ℃ and 100 vacuum degree, and removing ethanol to prevent interference with subsequent analysis samples.
Further, the intracellular metabolite samples were stored at-80 ℃.
Further, in the chemostat culture module, Oxygen Uptake Rate (OUR) and CO2The metabolic homeostasis is reached when the release rate (CER) tends to be constant.
Further, the intracellular metabolite sample is pretreated before being injected, and the method comprises the following steps: taking an intracellular metabolite sample, adjusting the temperature to room temperature, sucking 15 mu L, quickly filtering at 0.22 mu m, and removing bubbles to obtain a pretreated sample for sample introduction and analysis.
Further, in the sample processing and analyzing module, detecting and analyzing the isotope abundance of the intracellular metabolites of the pretreated sample by using an LC-MS/MS method so as to obtain the data of the isotope abundance of the intracellular metabolites; the mobile phase adopted for detection comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 5% acetonitrile containing 5mmol/L dibutylammonium acetate (DBAA), and the mobile phase B is 84% acetonitrile containing 5mmol/L dibutylammonium acetate (DBAA).
Further, the sample introduction conditions of the pretreated sample in LC-MS/MS detection are as follows:
0-20 minutes: only introducing mobile phase A;
20-22 minutes: introducing 80% of mobile phase A and 20% of mobile phase B;
22-32 minutes: only the mobile phase B is introduced until the detection is finished.
Further, the flow rate of the mobile phase is 0.2 mL/min; the amount of each sample was 2. mu.L.
Further, in the method, the determination of extracellular by-products is performed by HPLC method to obtain the concentration of extracellular metabolites for calculating the rate of exchange reaction; wherein the mobile phase is 3M H2SO4The flow rate was 0.4L/min.
In the invention, a tee joint and a water stop clamp are used for regulating and controlling the addition of different feeding materials (nutrient solution) into the reactor. For example, a high temperature resistant tee is used to connect the reactor and the vessel containing the feeding material, a first end of the tee is connected to the reactor, a second end of the tee is connected to the vessel not containing the isotope labeled substrate, and a third end of the tee is connected to the vessel containing the isotope labeled substrate.
In the invention, an LC/MS-MS instrument is used for analyzing a sample to obtain intracellular metabolite isotope abundance data.
In the present invention, the concentration of extracellular metabolites is measured for calculating the rate of the exchange reaction. By usingHPLC was used for the determination of extracellular by-products using an Agilent Technologies Hi-Plex H column (300X 7.7mm) equipped with a guard column (50X 7.7mm) and a mobile phase of 3M H2SO4The flow rate was 0.4L/min.
In the present invention, the method of the glucose kit measures the glucose concentration.
In the invention, after the isotope labeling experiment is started, the bioreactor needs to be continuously and rapidly sampled, and the time consumed for sampling once is about 1 s. The sample is quenched in time to ensure that no reaction occurs in the microbial cells.
In the invention, the metabolic stability is reached when the fed-batch nutrient solution is subjected to chemostat culture until the elution volume is more than five.
In the invention, a rapid sampling device is connected with the sampling port of the reactor for sampling, and the device has the characteristic of simple operation.
In the invention, the raw materials are all commercial products.
In the present invention, the steps in the process may, unless otherwise specified, be carried out using conventional process steps.
The invention has the beneficial effects that:
in the invention, under the physiological state that the metabolism of the cultured microorganisms is stable, a series of fermentation liquor samples are obtained in a short time by adopting an isotope labeling experimental method through a quick sampling device, and then the samples are processed and analyzed to obtain accurate intracellular metabolite isotope abundance data and exchange reaction rate. The data obtained by the method is applied to the calculation of the metabolic flux, and the metabolic flux can be accurately calculated.
The batch culture module is used for culturing microorganisms; the chemostat culture module is used for culturing microorganisms to reach a metabolic steady state and performing an isotope labeling experiment; the sampling module is used for taking a fermentation liquid sample from the bioreactor; the processing and analyzing sample module is used for obtaining the isotopic abundance and the exchange reaction rate data of the intracellular metabolites. The experimental data obtained by the method can be used for calculating the intracellular metabolic flux. The experimental method has the advantages of simple operation, cost saving, good stability and the like.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a table diagram of the correlation data in embodiment 2 of the present invention.
FIG. 2 is a schematic diagram of the steps for calculating metabolic flux in the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a method for acquiring metabolic flux of an intracellular central carbon metabolic pathway under a metabolic steady isotope non-steady state, which comprises a batch culture module, a chemostat culture module, a sampling module and a sample processing and analyzing module; wherein the content of the first and second substances,
oxygen Uptake Rate (OUR) and CO in the batch culture module2Transferring to the chemostat culture module when the release rate (CER) is reduced simultaneously;
the chemostat culture module specifically comprises: feeding nutrient solution without isotope labeled substrate, simultaneously starting waste discharge, performing chemostat culture, switching to feeding nutrient solution containing isotope labeled substrate when the microbial cells are in metabolic steady state, and keeping culture conditions unchanged;
beginning rapid, continuous sampling when the isotopically-labeled substrate flows into the reactor, at which point it enters the sampling module;
the sample processing and analyzing module comprises: and (3) processing, detecting and analyzing the sample to obtain intracellular metabolite isotope abundance data and exchange reaction rate.
In this embodiment, the isotopically labeled substrate is13And C, labeling the substrate. The time consumed for each sampling does not exceed 1 s.
In this example, after sampling, the taken sample was quenched, filtered to obtain a cell, added to a preheated 75% ethanol solution, reacted in a water bath at 95 ℃ to extract intracellular metabolites, cooled to room temperature, filtered to obtain a filtrate containing the intracellular metabolites, concentrated, ethanol removed, and the volume was fixed to obtain an intracellular metabolite sample.
In the chemostat culture module, Oxygen Uptake Rate (OUR) and CO2The metabolic homeostasis is reached when the release rate (CER) tends to be constant.
Example 2
The embodiment provides a method for acquiring metabolic flux of an intracellular central carbon metabolic pathway under a metabolic steady isotope non-steady state, which comprises a batch culture module, a chemostat culture module, a sampling module and a sample processing and analyzing module.
The method comprises the following specific steps:
culturing microorganism (Saccharomyces cerevisiae CEN. PK113-7D strain) in 1L bioreactor, and performing batch culture, wherein Oxygen Uptake Rate (OUR) and CO are determined when batch culture is completed2When the release rate (CER) is reduced at the same time, feeding nutrient solution without isotope labeling substrate into the reactor, simultaneously starting waste discharge, and performing chemostat culture;
when the chemostat culture reaches more than five elution volumes, the microorganisms in the reactor are in a metabolic steady state (both OUR and CER tend to be a constant), and nutrient solution with isotope-labeled substrates is supplemented into the reactor;
when the substrate with the isotope label flows into the reactor, the rapid sampling is started; the rapid sampling device is used for controlling sampling, and the tank pressure is used as the driving force for sampling to finish rapid and continuous sampling;
taking out the sampled fermentation broth (volume about 1mL) from the fermentation tank, putting the fermentation broth into a centrifugal tube containing 20mL of cold methanol (-40 ℃) which is weighed in advance for quenching, quickly whirling and shaking, and fully mixing; rapidly weighing the centrifuge tube containing the quenched fermentation liquid; then, the mixture is rapidly filtered by a filtering device provided with a cellulose filter membrane (the size is 47mm, the aperture is 0.8 mu m), and 10mL of cold methanol (-40 ℃) is used for rinsing the centrifuge tube; at this time, the quenched cells were applied to the filter, and then the filter with the cells was quickly transferred to a centrifuge tube containing 20mL of a preheated (75 ℃ C.) ethanol solution (75% v/v);
screwing down a centrifuge tube containing a filter membrane, fully and uniformly mixing (dissolving), transferring into a water bath kettle at 95 ℃ for reaction for 3min, and extracting intracellular metabolites; after the reaction is finished, quickly transferring the centrifugal tube to an ice box, cooling to normal temperature, and removing the filter membrane through a suction filtration device to obtain filtrate containing intracellular metabolites; then, putting the centrifuge tube filled with the filtrate into a rotary evaporator to realize rapid evaporation of the ethanol-water mixed solution and finish the concentration of the intracellular metabolite extracting solution; finally, obtaining concentrated solution with volume less than 600 mu L, adding ultrapure water into a centrifuge tube to fix the volume to 600 mu L, fully mixing and dissolving, transferring the solution into a 1.5mL EP tube, wherein the sample is called intracellular metabolite sample hereinafter;
before sample injection detection, pretreatment is carried out on an intracellular metabolite sample: taking the intracellular metabolite sample out of a refrigerator at minus 80 ℃, after the intracellular metabolite sample is heated to room temperature and completely melted, sucking 15 mu L of the intracellular metabolite sample by using an injector, quickly filtering the intracellular metabolite sample to a clean liquid phase small bottle through a 0.22 mu m water system nylon filter head, covering a cover, and shaking the liquid phase small bottle for several times to ensure that no air bubbles exist in the small bottle, thus obtaining the liquid phase small bottle containing the pretreated sample;
the liquid phase vial containing the pretreated sample was used for injection, analysis: the intracellular metabolite isotopic abundance was determined using an LC-MS/MS instrument in which the mobile phase was divided into two portions, acetonitrile containing 5mmol/L DBAA (dibutylammonium acetate) and acetonitrile containing 5mmol/L DBAA 84%, respectively, and the flow rate of the mobile phase was 0.2 mL/min. When detecting substances, only acetonitrile containing 5mmol/L DBAA 5% is passed through the sample in the first 0-20 minutes, and then acetonitrile containing 5mmol/L DBAA 84% in 20% is introduced for 2 minutes; then acetonitrile containing 5mmol/L DBAA 5% of acetonitrile is introduced again for 10 minutes until the detection is finished. The amount of each sample was 2. mu.L.
In this example, the top of the 1L bioreactor has a sampling port for taking fermentation broth from the reactor. A quick sampling device is connected with a sampling port of the reactor for sampling, and the device has the characteristic of simple operation.
In this example, the results of the experiment are shown in the following table, in which the metabolic fluxes are all relative values. Intracellular metabolite isotope abundance data are obtained through experiments, and intracellular metabolic fluxes of the saccharomyces cerevisiae at three different dilution rates are calculated and are shown in table 1.
TABLE 1
In this embodiment, the metabolic flux of intracellular central carbon metabolism can be obtained by the experimental method for obtaining intracellular metabolic flux under the metabolic steady isotope non-steady state of the present invention (the experimental results are shown in table 1). In contrast, the experimental results obtained by this method were compared with published literature values, and the comparison results are shown in fig. 1.
In FIG. 1, B represents the metabolic flux obtained by the method of the present invention, C represents a literature value, and correlation analysis (Pearson correlation, Spearman) was performed on both values, and the p-value was 6.51 × 10, respectively-4、3.63×10-7And the value is far less than 0.05, which shows that B and C are significantly related, and the obtained metabolic flux of the method is very close to the value reported in the literature, and further proves that the method has quite high accuracy and scientificity in the aspect of obtaining the metabolic flux.
In the invention, the intracellular metabolite isotope abundance data and the exchange reaction rate can be obtained by utilizing the method, the obtained data is applied to the calculation of the metabolic flux, and the calculated metabolic flux has quite high accuracy.
In the open source procedure for calculating metabolic flux (as shown in FIG. 2), the steps are as follows: through a metabolic network model and a matrix equation (AX ═ B), a series of reaction flux estimates can be obtained; substituting the estimated value of the flux into a program for calculating the isotopic abundance of each metabolite, and further obtaining a series of analog values of the isotopic abundance of the metabolite; through an isotope labeling experiment, an experimental value of isotope abundance changing along with time can be obtained, an analog value of the isotope abundance is compared with the experimental value and is fitted, and metabolic flow is optimized by utilizing a nonlinear optimization algorithm, so that the sum of squares of residual errors between the analog value of the abundance and the experimental value is minimum, a group with the best fitting effect is found, and the flux value of the group is considered as the optimal solution of the metabolic flow. Through the three steps, the metabolic flux of each reaction in the metabolic model can be solved by combining experimental measurement values.
In conclusion, the experimental method disclosed by the invention is simple to operate and high in accuracy, and can reduce the experimental cost. Through different tee joint connection modes and the application of the rapid sampling device, the experimental efficiency is greatly improved, and the experimental operation is simplified; meanwhile, the experimental method for processing the sample is optimized, and the accuracy of the experimental result is ensured. Provides a simple and effective experimental method for the subsequent research of the metabolic flux field.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for obtaining metabolic flux of a carbon metabolic pathway in a cell under a metabolic steady isotope non-steady state is characterized by comprising a batch culture module, a chemostat culture module, a sampling module and a sample processing and analyzing module;
when oxygen uptake and CO in the batch cultivation module2When the release rate is reduced at the same time, the cells are transferred to the chemostat culture module;
the chemostat culture module specifically comprises: feeding nutrient solution without isotope labeled substrate, simultaneously starting waste discharge, performing chemostat culture, switching to feeding nutrient solution containing isotope labeled substrate when the microbial cells are in metabolic steady state, and keeping culture conditions unchanged;
beginning rapid, continuous sampling when the isotopically-labeled substrate flows into the reactor, at which point it enters the sampling module;
the sample processing and analyzing module is used for processing, detecting and analyzing samples to obtain intracellular metabolite isotope abundance data and exchange reaction rate for calculating metabolic flux.
2. The method of claim 1, wherein the isotopically labeled substrate is13C labeling a substrate; the time consumed for each sampling does not exceed 1 s.
3. The method as claimed in claim 1, wherein after sampling, quenching the sample taken out, filtering to obtain thalli, adding the thalli into a preheated 75% ethanol solution, carrying out a water bath reaction at 95 ℃, extracting intracellular metabolites, cooling to normal temperature, carrying out suction filtration to obtain a filtrate containing the intracellular metabolites, concentrating, removing ethanol, and carrying out volume fixing to obtain an intracellular metabolite sample.
4. The method of claim 3, wherein after sampling, the sample is put into cold methanol at-40 ℃ for quenching, filtered to obtain thalli, added into 75% ethanol solution preheated to 70-75 ℃, reacted in water bath at 95 ℃ for 3min to extract intracellular metabolites, then rapidly cooled to normal temperature, and filtered to obtain filtrate containing the intracellular metabolites; concentrating the filtrate at 30 deg.C and vacuum degree of 100, removing ethanol, adding ultrapure water to desired volume to obtain intracellular metabolite sample.
5. The method of claim 4, wherein the intracellular metabolite sample is stored at-80 ℃.
6. The method of claim 1, wherein the chemostat culture module comprises oxygen uptake and CO2The metabolic steady state is reached when the release rate tends to be constant.
7. The method according to any one of claims 3 to 5, wherein the intracellular metabolite sample is pre-treated prior to sample introduction, comprising the steps of: taking an intracellular metabolite sample, adjusting the temperature to room temperature, sucking 15 mu L, quickly filtering at 0.22 mu m, and removing bubbles to obtain a pretreated sample for sample introduction and analysis.
8. The method of claim 7, wherein in the sample processing and analyzing module, the preprocessed sample is subjected to detection and analysis of intracellular metabolite isotope abundance by using an LC-MS/MS method to obtain data of the intracellular metabolite isotope abundance; the mobile phase adopted for detection comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 5% acetonitrile containing 5mmol/L dibutylammonium acetate, and the mobile phase B is 84% acetonitrile containing 5mmol/L dibutylammonium acetate.
9. The method according to claim 8, wherein the sample introduction conditions of the pretreated sample in the detection are as follows:
0-20 minutes: only introducing mobile phase A;
20-22 minutes: introducing 80% of mobile phase A and 20% of mobile phase B;
22-32 minutes: only introducing the mobile phase B until the detection is finished;
the flow rate of the mobile phase is 0.2 mL/min; the amount of each sample was 2. mu.L.
10. The method according to claim 1, wherein in the method, the determination of extracellular by-products is performed by HPLC method to obtain the concentration of extracellular metabolites for calculating the rate of exchange reaction; wherein the mobile phase is 3M H2SO4The flow rate is 0.4L/min。
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