Comparison of different strategies to reduce acetate formation in Escherichia coli.
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)).
De Mey
Marjan
M
Ghent University, Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Coupure Links 653, B-9000 Ghent, Belgium. marjan.demey@ugent.be
Lequeux
Gaspard J
GJ
Beauprez
Joeri J
JJ
Maertens
Jo
J
Van Horen
Ellen
E
Soetaert
Wim K
WK
Vanrolleghem
Peter A
PA
Vandamme
Erick J
EJ
D000085:Acetates / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation
D000085:Acetates; D029968:Escherichia coli Proteins; D002244:Carbon; D005947:Glucose