Respirometric 13C flux analysis—Part II: In vivo flux estimation of lysine-producing Corynebacterium glutamicum

A novel method for metabolic flux studies of central metabolism which is based on respirometric 13C flux analysis, i.e., parallel 13C tracer studies with online CO2 labeling measurements is applied to flux quantification of a lysine-producing mutant of Corynebacterium glutamicum. For this purpose, 3 respirometric 13C labeling experiments with [1-13C1], [6-13C1] and [1,6-13C2] glucose were carried out in parallel. uxes comprising the reactions of glycolysis, of TCA cycle, of C3- and C4-metabolite interconversion and of lysine biosynthesis as well as the net reactions in the pentose phosphate pathway could be quantified solely using experimental data obtained from CO2 labeling and extracellular rate measurements. At key branch points, 68±5% of glucose 6-phosphate were observed to be metabolized into pentose phosphate pathway and 48±1% of pyruvate into TCA cycle via pyruvate dehydrogenase. The results showed a good agreement with the previous studies using 13C tracer cultivation and GC/MS analysis of proteinogenic amino acids. Also, respiratory quotient calculated from flux estimates using redox balance showed a high accordance with the value determined directly from the measured specific rates of O2 consumption and CO2 production. The results strongly support that the respirometric 13C metabolic flux analysis is suited as an alternative to the conventional methods to study functional and regulatory activities of cells. veloped method is applicable to study growing or non-growing cells, primary and secondary metabolism and immobilized cells. Due to the non-accumulating nature of CO2 labeling and instantaneous nature of the resulting fluxes, the method can also be used for dynamic profiling of metabolic activities. Therefore, it is complementary to conventional methods for metabolic flux analysis.