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E. colicells produce acetate as an extracellular coproduct of aerobic cultures. Acetate is undesirable because it retards growth and inhibits protein formation. Most process designs or genetic modifications to minimize acetate formation aim at balancing growth rate and oxygen consumption. In this research, three genetic approaches to reduce acetate formation were investigated:  (1) direct reduction of the carbon flow to acetate (ackA-pta, poxBknock-out); (2) anticipation on the underlying metabolic and regulatory mechanisms that lead to acetate (constitutive ppcexpression mutant); and (3) both (1) and (2). Initially, these mutants were compared to the wild-type E. colivia batch cultures under aerobic conditions. Subsequently, these mutants were further characterized using metabolic flux analysis on continuous cultures. It is concluded that a combination of directly reducing the carbon flow to acetate and anticipating on the underlying metabolic and regulatory mechanism that lead to acetate, is the most promising approach to overcome acetate formation and improve recombinant protein production. These genetic modifications have no significant influence on the metabolism when growing the micro-organisms under steady state at relatively low dilution rates (less than 0.4 h-1). [ABSTRACT FROM AUTHOR]