C-13 TRACER EXPERIMENTS AND METABOLITE BALANCING FOR METABOLIC FLUX ANALYSIS - COMPARING 2 APPROACHES

Conventional metabolic flux analysis uses the information gained from determination of measurable fluxes and a steady-state assumption for i ntracellular metabolites to calculate the metabolic fluxes in a given metabolic network. The determination of intracellular fluxes depends h eavily on the correctness of the assumed stoichiometry including the p resence of all reactions with a noticeable impact on the model metabol ite balances. Determination of fluxes in complex metabolic networks of ten requires the inclusion of NADH and NADPH balances, which are subje ct: to controversial debate. Transhydrogenation reactions that transfe r reduction equivalents From NADH to NADPH or vice versa can usually n ot be included in the stoichiometric model, because they result in sin gularities in the stoichiometric matrix. However, it is the NADPH bala nce that, to a large extent, determines the calculated flux through th e pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estim ations of the intracellular flux distribution. Using C-13 tracer exper iments and NMR analysis, flux analysis can be performed on the basis o f only well established stoichiometric equations and measurements of t he labeling state of intracellular metabolites. Neither NADH/NADPH bal ancing nor assumptions on energy yields need to be included to determi ne the intracellular fluxes. Because metabolite balancing methods and the use of C-13 labeling measurements are two different approaches to the determination of intracellular fluxes, both methods can be used to verify each other or to discuss the origin and significance of deviat ions in the results. Flux analysis based entirely on metabolite balanc ing and flux analysis, including labeling information, have been perfo rmed independently for a wild-type strain of Aspergillus oryzae produc ing alpha-amylase. Two different nitrogen sources, NH4+ and NO3-, have been used to investigate the influence of the NADPH requirements on t he intracellular flux distribution. The two different approaches to th e calculation of fluxes are compared and deviations in the results are discussed. (C) 1998 John Wiley & Sons, Inc.