Transient metabolic modeling of Escherichia coli MG1655 and MG1655 ΔackA-pta, ΔpoxB Δpppc ppc-p37 for recombinant β-galactosidase production |
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Authors: | Marjan De Mey Gaspard J Lequeux Joeri J Beauprez Jo Maertens Hendrik J Waegeman Inge N Van Bogaert Maria R Foulquié-Moreno Daniel Charlier Wim K Soetaert Peter A Vanrolleghem Erick J Vandamme |
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Institution: | 1. Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium 2. BIOMATH, Department of Applied Mathematics, Biometrics and Process Control, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000, Ghent, Belgium 3. Laboratory for Genetics and Microbiology, Department of Applied Biological Sciences, Faculty of Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Elsene, Belgium 4. Laboratory of Molecular Cell Biology, Department of Molecular Microbiology (VIB), Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, bus 2438, 3001, Heverlee, Belgium
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Abstract: | Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, β-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards β-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production. |
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