Achieving practical biotechnol. processes for the prodn. of fine chems. requires optimization of metabolic fluxes. Attempts to alter the fluxes through a given pathway may be frustrated by compensatory changes in coupled pathways or by unexpected effects seemingly unrelated to the modification performed. Therefore, precise monitoring of metabolic responses to modifications is essential for successful and accelerated metabolic engineering. Commonly employed approaches are based on stoichiometric mass balancing and rely on assumptions which are subject to significant uncertainties. By combining the powers of stoichiometric mass balancing and biosynthetically-directed fractional 13C-labeling of amino acids, we have developed a new methodol. for the detn. of metabolic fluxes. Labeling of proteinogenic amino acids is achieved by growing the cells in a medium contg. a mixt. of 10% uniformly 13C-labeled and 90% unlabeled glucose. Metabolic fluxes in central carbon metab. of riboflavin-producing and wild-type Bacillus subtilis have been detd. using this methodol. [on SciFinder (R)]