Method for obtaining metabolic flux of intracellular central carbon metabolic pathway in unsteady state of metabolic steady-state isotopes

Abstract

The invention discloses a method for obtaining metabolic flux of an intracellular central carbon metabolic pathway in an unsteady state of metabolic steady-state isotopes. The method adopts a batch culture module, a constant culture module, a sampling module and a sample processing and analyzing module. When the OUR and the CER in the batch culture module decline at the same time, culture is performed in the constant culture module. The constant culture module specifically adopts a process of feeding a nutrient solution containing no isotope labeling substrate, starting waste discharge at thesame time, carrying out constant culture, until the microbial cells are in a metabolic steady state, feeding a nutrient solution containing the isotope labeling substrate, and keeping culture conditions unchanged; and when the isotope labeling substrate flows into a reactor, starting rapid and continuous sampling, and entering the sampling module at the same time. According to the method, a seriesof fermentation liquor samples are obtained in a short time, and then the samples are processed and analyzed to obtain accurate intracellular metabolite isotope abundance data; and the method plays acrucial role in calculating the metabolic flux.

Description

technical field [0001] The invention relates to the technical field of calculating metabolic flow, in particular to a method for obtaining the metabolic flux of intracellular central carbon metabolic pathway under the unstable state of metabolic stable isotope. Background technique [0002] The rapid development of "multi-omics" technology has made it possible to obtain the abundance of various biomolecules with high throughput, so that the changes and subtle connections of each omics under different physiological states can be determined. Various omics have been applied in microbial systems biology research, including transcriptomics, which measures mRNA transcription levels; proteomics, which quantifies the abundance of proteins; metabolomics, which measures the abundance of cellular metabolites; metabonomics, which Establishment of flux distribution in intracellular metabolic networks. At the same time, metabolic flux is the result of quantifying the comprehensive networ...

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Problems solved by technology

[0003] At present, the theoretical method for calculating metabolic flux is very mature, but the relevant experimental method is not perfect, and there are the following problems: the large-capacity bioreactor will waste unnecessary energy during the isotope labeling experiment. 13 C glucose, which leads to high experimental costs; in the experimental operation of switching substrates, complex pipelines are likely to cause a window period for feeding substrates, which affects the physiological and metabolic state of microorganisms; cumbersome sampling devices will affect sampling efficiency, and sampling at the same time If the amount is too large, it will interfere with the microorganisms under metabolic homeostasis; in the process of extracting intracellular metabolites, it is easy to leak, which will affect the accuracy of the experimental results;

It can be seen that the current calculation of metabolic flux experiments is facing the problems of high cost, cumbersome operation, and poor stability.

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