The pentose phosphate pathway and the pyruvate shunt were identified as major pathways of glucose catabolism in a recombinant, riboflavin-producing Bacillus subtilis strain. Reactions connecting the tricarboxylic acid cycle and glycolysis, catalyzed by the malic enzyme and phosphoenolpyruvate carboxykinase, consume up to 23% of the metabolized glucose. These are examples of important fluxes that can be accessed explicitly using a novel anal. based on synergistic application of flux balancing and recently introduced techniques of fraction 13C-labeling and two-dimensional NMR spectroscopy. The overall flux distribution also suggests that B. subtilis metab. has an unusually high capacity for the reoxidn. of NADPH. Under the conditions investigated, riboflavin formation in B. subtilis is limited by the fluxes through the biosynthetic rather than the central carbon pathways, which suggests a focus for future metabolic engineering of this system. [on SciFinder (R)]