Effect of variation of Klebsiella pneumoniae acetolactate synthase expression on metabolic flux redistribution in Escherichia coli

Escherichia coli strains carrying the Bacillus subtilis acetolactate syntha se (ALS) gene were previously shown to produce less acetate with higher ATP yields. Metabolic flux analysis was used to show that excess pyruvate was channeled into the less inhibitory product, acetoin. To further understand the role of intrinsic enzymatic properties and the effect of variations in enzyme levels in the alternation of metabolic fluxes, we constructed a chro mosomal integrant of the Klebsiella pneumoniae ALS gene. The reported in vi tro Michaelis-Menten constants (K-m) for the Bacillus and the Klebsiella AL S are 13.0 mM and 8.0 mM respectively. Furthermore, expression of the Klebs iella ALS is under the control of an inducible trp promoter system. Shake-f lask experiments showed a linear induction response (the ALS activity chang es from about 9 to 223 U/mg of protein when the inducer concentration [IAA] varied from 0 to 40 mg/L). Chemostat experiments showed a similar inductio n response. Interactions between the branched reactions catalyzed by the PF L, LDH, and the ALS enzymes at the pyruvate node were examined. The results indicate the importance of in vivo enzyme activities in the redistribution of metabolic fluxes. (C) 2000 John Wiley & Sons, Inc.