In recent years, metabolic flux analysis (MFA) has become an important tool in metabolic engineering. The MFA involves the quantification of intracellular metabolic fluxes in a microorganism. The result of MFA is a metabolic flux map that allows the systematic study of cellular responses to genetic and environmental perturbations. Carbon isotope labeling-based MFA, with analysis by NMR or GC/MS, has gained wide use in the metabolic engineering community for estimating metabolic fluxes in central carbon metabolism.

However, the ¹³C labeling based MFA has been used for aerobic system and has not done been for anaerobic system. The ¹³C MFA is challenging under anaerobic condition as there is not less rearrangement because TCA cycle is not complete. The information from the labeled substrate can be increased through the proper choice of labeled substrate. We have come up with the optimal choice of labeled substrate for the experimental condition by flux identifiability analysis. The metabolic network model for MFA has been constructed for E. coli and it consists of all principal pathway of primary metabolism (PTS transport of glucose, glycolysis, pentose phosphate pathway, TCA cycle, glyoxylate shunt, and fermentative reactions) and the biosynthetic pathways that convert  the primary metabolic precursors to sink metabolites.