The Establishment Of ¹³C-Assisted Quantitative Metabolomics And Its Application In The Study Of Metabolism Of Pichia Pastoris

Metabolomics has already been one of the most important tools in metabolic engineering, and it is a reliable method to study the effects of endogenous and exogenous factors on organism. At present, the research of metabolomics focus on semi-quantitative, in this way only the multiples of increasing or reducing of metabolites concertration can be reached, it's impossible to achieve the absolutely quantitative, also bring much trouble for the study of cells metabolic regulation mechanism. In this study, we integrated the analysis method of LC-MS/MS and the advantage of 13C isotope labeling techniques and established a reliable and accurate method to abosultly quantitative metabolites, fanally this method was used to the research of the production of beta galactose glucoside enzyme in P. Pastor is.In this study,64 metabolites including organic acids, sugar phosphate, nucleoside substance, amino acid were successfully separated by UPLC in three kands of chromatographi column and the effects of mobile phase pH and ionic reagents on retention time was examined. Based on that, mass spectrometry condition was optimized, the approprate ion pairs and collision voltage were getting and a complete LC-MS/MS method was set up.Combined the detection method of LC-MS/MS with 13C isotope labeling techniques, the stand curve of isotope dilution mass spectrometry (IDMS) were built. Using these stand curve,53 metabolites can be absolutely quantitative and the limites of detection were under 1?mol/L. The evalution reasults show that the stand curve with high accuracy, the correlation coefficient are above 0.99, and with good reproducibility, the influence of experimental and equipment operating condition is very small.In the end of this study, the method of 13C quantitative metabolomics established in this work was used to study the metabolic defferences in beta galactose glucoside enzyme low yield becteria GHL, high yield becteria G1HL and G1HL adding with glutamate. Data of macroscopic metabolic shows that G1HL experienced significantly decreased specific growth rate and specific glucose uptake rate, affter adding glutamate its specific growth rate and specific glucose uptake rate can rise to the levels of GHL, and the yield was further improved. Quantitative metabolomics and intationary 13C metabolic flux analysis illustrates the reasion for these difference. The result shows that, in batch culture using glucose assole carbon source the over production of recombinant protein along with an increasing demand of redox, energy and precursor amino acids which will result in decreasing of metabolites concentration. When we adding glutamic acid in medium, the flux of downstream of TCA basically remaid unchanged, the upstream decrease about 50 percent and the flux of downstream of EMP increased significantly, due to these changes the metabolic burden can be released and the production increases. Tow aspects of macroscopic and microscopic reseach deepens our understanding of metabolic characteristics in, and providing the thoretical basis for further strain development or process optimizations.