Determination Of Isotopic Abundance Of Intracellular Metabolites By GC-MS With Selected Ion Monitoring Mode And Its Application In Coenzyme Q₁₀ Fermentation Process

Among different types of omics data, fluxomics can best reflect the characteristics of cell metabolism, and13C labeling experiments is the most reliable method to accurately obtain the intracellular metabolic flux. Currently,13C metabolic flux analysis based on analysis of protein-bound amino acids is only applicable in the study of biological processes closely associated with the cell growth. Compared to protein-bound amino acids, intracellular metabolites have advantages of fast response to environmental change and direct association with products. Accordingly, in order to expand the application of13C metabolic flux analysis to the biological processes of secondary metabolites, recombinant proteins and other products not directly associating with cell growth, it is necessary to develop a novel analysis method for determination of isotopic abundance of intracellular metabolites.Immediate ceasing of metabolic activities is a prerequesite in metabolic study because of the rapid turnover of intracellular metabolites. A rapid sampling device was developed consisting of piston pump, electric rotating disk, refrigeration systems and control systems. The device was tested in40experiments for sampling10ml broth on different conditions. The resulting average sampling time of28.14±0.13s, with0.14s deviation from theoretical value, indicated that the device has the ability to get sample in the second order.Full Scan mode is widely used in the determination of isotopic abundance of protein-bound amino acids by GC-MS. The low concentration of intracellular metabolites requires more sensitive analysis method. Selected ion monitoring mode has shown it’s potentials of high sensitivity in the quantification of metabolites concentration, but its application has seldom been reported yet in the field of13C MFA. In this paper, selected ion monitoring mode was introduced to the GC-MS determination of isotopic abundance of intracellular amino acids and organic acids. The analysis program was designed and operating conditions optimized. The analysis of naturally-labeled standard sample indicated the superiority of selected ion monitoring mode over full Scan mode, with10times improvement in signal-to-noise ratio. The isotopic abundance from selected ion monitoring mode was also closer to the theoretical value of the natural labeled compounds. Based on above method,13C-MFA methods for coenzyme Q10fermentation process was estabished. The metabolic model of central metabolism of the coenzyme Q10producing strain was constructed. The low acitivity of EMP and relatively high activities of HMP, TCA and ED pathway was discovered by isotopic labeling experiments. Using this metabolic model, the effect of potassium ion on central carbon metabolism was investigated. The results showed that the influences of potassium ion is associated with the Na-K pump activity. The lack of potassium ions results in the reduction in the fluxes of glucose absorption, HMP pathway and TCA cycle, thus insufficient supply of anabolic precursors may cause the decrease of specific growth rate.The device and metohd present in the paper will find it’s potential not only in academic research, but also in the wide application of13C metabolic flux analysis to the industrial production of secondary metabolites and recombinant proteins.