Metabolic Flux Analysis ofEscherichia coliDeficient in the Acetate Production Pathway and Expressing theBacillus subtilisAcetolactate Synthase

Abstract Several approaches to reduce acetate accumulation in Escherichia coli cultures have recently been reported. This reduction subsequently led to a significant enhancement in recombinant protein production. In those studies, metabolically engineered E. coli strains with reduced acetate synthesis rates were constructed through the modification of glucose uptake rate, the elimination of critical enzymes that are involved in the acetate formation pathways, and the redirection of carbon flux toward less inhibitory byproducts. In particular, it has been shown that strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene not only produce less acetate but also have a higher ATP yield. Metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point revealed that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin. The main focus of this study is the systematic analysis of the effects of small perturbations in the host's existing pathways on the redistribution of carbon fluxes. Specifically, a mutant with deleted acetate kinase (ACK) and acetyl phosphotransferase (PTA) was constructed and studied. Results from the metabolic analysis of carbon redistribution show the ackA-pta mutation will reduce acetate level at the expense of the growth rate. In addition, in the ackA-pta deficient strain a much higher lactate formation rate with simultaneously lower formate and ethanol synthesis rates was found. Expression of the B. subtilis ALS in ackA-pta mutants further reduces acetate levels while cell density similar to that of the parent strain is attained.