Metabolic control sites in the aromatic group amino acid pathway (AAAP) of Bacillus subtilis for L-Phenylalanine (Phe) overproduction were determined; and aiming pathway flux amplification, by cloning the flux controlling gene aroH to a multi-copy plasmid, the impact of single gene cloning on pathway flux distributions were investigated. The branch-point metabolites E4P and PEP+E4P supplied in vitro, enhanced Phe production and well defined perturbations were achieved on the AAAP reactions. The intracellular reaction rate distributions calculated by a mass flux balance-based stoichiometric model for B. subtilis based on the metabolic reaction network that contains 184 metabolites and 232 reaction fluxes using time profiles of glucose, dry cell, organic and amino acids, revealed that induction by E4P influenced the pathway reactions via chorismate and prephanate towards Phe by increasing the flux values. The reaction catalysed by chorismate mutase (R96) has the lowest flux value among the preceding reactions of the pathway that reduces the proceeding reaction rates towards Phe. Thus, R96 is predicted to be the primary rate-limiting step among the Phe pathway reactions. On the basis of the strategy designed, the cloning of aroH gene encoding chorismate mutase was achieved first onto the E.coli plasmid pUC19, and its expression was demonstrated to compare its performance. Thereafter, aroH was sub-cloned onto the multi-copy plasmid pMK4 and transferred into host B.subtilis mutants. The performance of r-E.coli carrying pUC19::aroH was lower than the r-B.subtilis strains carrying the pMK4::aroH; and, the highest Phe production was obtained with r-B. subtilis 1A263. The comparison of the calculated intracellular fluxes of r-B. subtilis 1A263 with that of the wild type revealed that the flux of the first reaction of the AAAP was 1.2-fold, but the engineered R96 was 7.2-fold higher in r-B.subtilis 1A263. (C) 2004 Elsevier Ltd. All rights reserved.