阅读说明：本技术 一种用于代谢稳态同位素非稳态下获取胞内中心碳代谢途径代谢通量的方法 (Method for acquiring metabolic flux of intracellular central carbon metabolic pathway under metabolic steady isotope unsteady state ) 是由 夏建业 李欢 陈敏 郑世媛 汪佳琪 于 2020-05-14 设计创作，主要内容包括：本发明公开了一种用于代谢稳态同位素非稳态下获取胞内中心碳代谢途径代谢通量的方法,包括批培养模块、恒化培养模块、取样模块、处理与分析样品模块；当所述批培养模块中的OUR和CER同时出现下降时转入所述恒化培养模块；所述恒化培养模块中具体包括：流加不含同位素标记底物的营养液,同时开启排废,进行恒化培养,直至使微生物细胞处于代谢稳态时,切换成流加含同位素标记底物的营养液,保持培养条件不变；当所述同位素标记底物流入反应器内时开始快速、连续取样,同时进入所述取样模块。本发明的方法在短时间内获得一系列发酵液样品,进而处理、分析样品得到精确的胞内代谢物同位素丰度数据,对计算代谢通量起着至关重要的作用。(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.)

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。

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.