Study On Metabolic Mechanism Of Saccharomyces Cerevisiae Under Different Physlological Conditions Using Non-stationary ¹³C Metabolic Flux Analysis

Saccharomyces cerevisiae,also known as beer yeast.S.cerevisiae plays an important role in alcoholic fermentation and various alcoholic brewing processes,and plays an important role in chemical,food and beverage industries.S.cerevisiae is one the well-studied eukaryotic cell,which has become a model organism for eukaryotic biological physiological characteristics study.Therefore,it makes sense to study the metabolic regulation of S.cerevisiae.In this paper,the metabolic fluxes of S.cerevisiae under different physiological conditions were obtained by isotope labeling experiments,and the metabolic regulation mechanism of S.cerevisiae was studied.It is generally believed that S.cerevisiae produces alcohol by fermentation under anaerobic conditions,while under aerobic conditions,the cells do respiration and do not produce alcohol.However,it was observed that S.cerevisiae could produce ethanol in medium with high glucose concentration,even supplying adequate oxygen,while no ethanol was produced as glucose concentration was limited.This phenomenon is called glucose effect or Crabtree effect.This indicates that during this glucose concentration change process,S.cerevisiae switches its central carbon metabolic flux due to metabolic regulation mechanism.Nowadays,reliable intracellular metabolic flux is always obtained by 13C isotope labeling experiment.Steady 13C-MFA is a mature technique for calculating metabolic flux based on isotope steady state.Due to high cost of isotopes to achieve isotope homeostasis,its application is limited.The newly developed metabolic flow analysis under unstable isotope(INST-13C-MFA)is being adopted by researchers.Three chemostat cultures of S.cerevisiae were conducted with dilution rates(D=0.12,0.22,0.32 h-1),and the corresponding intracellular metabolic flux distributions were obtained by using the INST-13C-MFA method.Combined with the metabolome and metabolic fluxome data,we found obvious metabolic flux shift under the three different physiological states.In this process,pyruvate decarboxylase,ethanol dehydrogenase and acetyl-CoA synthase catalyzed reactions were key points.Negative correlation between relative flux of EMP and TC A and biomass yield,while positive correlation for PPP were observed.Yield of acetate and glycerol did not change significantly,while that of ethanol increased sharply.In the central carbon metabolism(CCM),most of the carbon flux(70%)was directed to the EMP pathway.At the same time,the energy charge increased with dilution rate,and the cell's energy supply mode gradually shifted from oxidative respiration to substrate level phosphorylation mode.Through this analysis and study of S.cerevisiae in different physiological condition,it is more clear how does S.cerevisiae shift from aerobic respiration to fermentation.At the same time,further it updates our understanding of eukaryotic cell metabolism regulation,which will be of great importance for engineering yeast cell.