Comparison of Different Strategies to Reduce Acetate Formation in Escherichia coli Marjan De Mey,* ,² Gaspard J. Lequeux, ‡ Joeri J. Beauprez, ² Jo Maertens, ‡ Ellen Van Horen, ² Wim K. Soetaert, ² Peter A. Vanrolleghem, ‡ and Erick J. Vandamme ² Ghent University, Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, and BIOMATH, Department of Applied Mathematics, Biometrics and Process Control, Faculty of Bioscience Engineering, Coupure Links 653, B-9000 Ghent, Belgium E. coli cells 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, poxB knock-out); (2) anticipation on the underlying metabolic and regulatory mechanisms that lead to acetate (constitutive ppc expression mutant); and (3) both (1) and (2). Initially, these mutants were compared to the wild-type E. coli via 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 ). Introduction E. coli cells produce acetate as an extracellular coproduct of aerobic cultivations under excess-glucose conditions. This phenomenon is referred as overflow metabolism. Acetate is undesirable because it retards growth even at concentrations as low as 0.5 g/L (1) and inhibits protein formation (2). Most process designs or genetic modifications to overcome acetate formation ultimately aim to balance growth rate and oxygen consumption (3). Process improvement approaches involve designing growth media or conditions that eliminate or reduce acetate for- mation. In contrast, genetic approaches involve altering the genetic profile of the strain itself, to restrict the biochemical synthesis of acetate. Process approaches generally focus on reducing the carbon flux entering in the cells and thus dimin- ish the specific growth rate and efficiency of the production process. For these reasons, our research focuses on genetic approaches to overcome acetate formation. Several interrelated methods have been used to reduce acetate formation genetically. The three partially overlapping strategies are (1) approaches that directly reduce glucose consumption, (2) approaches that directly reduce carbon flow to acetate, and (3) approaches that address underlying metabolic and regulatory mechanisms leading to acetate formation. Because the first strategy is a genetic equivalent of bioprocess approaches, this research focuses on the two other strategies based on metabolic modeling and engineering. Materials and Methods Bacterial Strains. Escherichia coli MG1655 [ λ - ,F - , rph- 1, rfb-50, ilVG - ] was obtained from The Netherlands Culture Collection of Bacteria (NCCB). Escherichia coli MG1655 ΔackA-pta, ΔpoxB [ λ - ,F - , rph-1, rfb-50, ilVG - , ΔackA-pta, ΔpoxB] was constructed in the Laboratory of Genetics and Microbiology (MICR) using the method as described by Datsenko and Wanner (2000) (4). Escherichia coli MG1655 Δpppc ppc-p37 [ λ - ,F - , rph-1, rfb-50, ilVG - , Δpppc ppc-p37], Escherichia coli MG1655 Δpppc ppc-p55 [ λ - ,F - , rph-1, rfb- 50, ilVG - , Δpppc ppc-p55], Escherichia coli MG1655 ΔackA- pta, ΔpoxB, Δpppc ppc-p37 [ λ - ,F - , rph-1, rfb-50, ilVG - ,ΔackA- pta, ΔpoxB, Δpppc ppc-p37], and Escherichia coli MG1655 ΔackA-pta, ΔpoxB, Δpppc ppc-p55 [ λ - ,F - , rph-1, rfb-50, ilVG - , ΔackA-pta, ΔpoxB, Δpppc ppc-p55] were constructed in the Laboratory of Genetics and Microbiology (MICR, VUB, Belgium). Media. The culture medium Luria Broth (LB) consisted of 1% tryptone-peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco, Erembodegem, Belgium), and 0.5% sodium chloride (VWR, Leuven, Belgium). The pH of the medium was 6.7. For flask cultures, minimal medium (MM-flask) consisted of 18 µM FeCl 2 ‚4H 2 O (Merck, Leuven, Belgium), 34 µM CaCl 2 ‚2H 2 O (Merck, Leuven, Belgium), 8.3 µM MnCl 2 ‚ 2H 2 O (Merck, Leuven, Belgium), 2.2 µM CuCl 2 ‚2H 2 O (Sigma, Bornem, Belgium), 2.1 µM CoCl 2 ‚6H 2 O (Merck, Leuven, Belgium), 6.9 µM ZnCl 2 (Merck, Leuven, Belgium), * To whom correspondence should be addressed. Tel.: +32-9-264-60- 28. Fax: +32-9-264-62-31. E-mail: marjan.demey@ugent.be. Website: http://www.limab.UGent.be. ² Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology. ‡ BIOMATH, Department of Applied Mathematics, Biometrics and Process Control. 1053 Biotechnol. Prog. 2007, 23, 1053-1063 10.1021/bp070170g CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical Engineers Published on Web 08/23/2007