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Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering

Hendrik Waegeman (UGent) , Stijn De Lausnay (UGent) , Joeri Beauprez (UGent) , Jo Maertens (UGent) , Marjan De Mey (UGent) and Wim Soetaert (UGent)
(2013) NEW BIOTECHNOLOGY. 30(2). p.255-261
Author
Organization
Project
Biotechnology for a sustainable economy (Bio-Economy)
Project
Biotechnology for a sustainable economy (Bio-Economy)
Abstract
Escherichia coli strains are widely used as host for the production of recombinant proteins. Compared to E. coli K12, E. coli BL21 (DE3) has several biotechnological advantages, such as a lower acetate yield and a higher biomass yield, which have a beneficial effect on protein production. In a previous study (BMC Microbiol. 2011, 11:70) we have altered the metabolic fluxes of a K12 strain (i.e. E. coli MG1655) by deleting the regulators ArcA and IclR in such a way that the biomass yield is remarkably increased, while the acetate production is decreased to a similar value as for BL21 (DE3). In this study we show that the increased biomass yield beneficially influences recombinant protein production as a higher GFP yield was observed for the double knockout strain compared to its wild type. However, at higher cell densities (>2 g LÀ1 CDW), the GFP concentration decreases again, due to the activity of proteases which obstructs the application of the strain in high cell density cultivations. By further deleting the genes lon and ompT, which encode for proteases, this degradation could be reduced. Consequently, higher GFP yields were observed in the quadruple knockout strain as opposed to the double knockout strain and the MG1655 wild type and its yield approximates the GFP yield of E. coli BL21 (DE3), that is, 27 +- 5 mg g/g vs. CDW 30 +- 5 mg g/g , respectively.
Keywords
COLI BL21, HIGH CELL-DENSITY, EXPRESSION SYSTEMS, ACETATE FORMATION, GROWTH, GLUCOSE, ARCA, PROTEOLYSIS, REPRESSION, PROTEASES

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Chicago
Waegeman, Hendrik, Stijn De Lausnay, Joeri Beauprez, Jo Maertens, Marjan De Mey, and Wim Soetaert. 2013. “Increasing Recombinant Protein Production in Escherichia Coli K12 Through Metabolic Engineering.” New Biotechnology 30 (2): 255–261.
APA
Waegeman, H., De Lausnay, S., Beauprez, J., Maertens, J., De Mey, M., & Soetaert, W. (2013). Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering. NEW BIOTECHNOLOGY, 30(2), 255–261.
Vancouver
1.
Waegeman H, De Lausnay S, Beauprez J, Maertens J, De Mey M, Soetaert W. Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering. NEW BIOTECHNOLOGY. 2013;30(2):255–61.
MLA
Waegeman, Hendrik, Stijn De Lausnay, Joeri Beauprez, et al. “Increasing Recombinant Protein Production in Escherichia Coli K12 Through Metabolic Engineering.” NEW BIOTECHNOLOGY 30.2 (2013): 255–261. Print.
@article{1997615,
  abstract     = {Escherichia coli strains are widely used as host for the production of recombinant proteins. Compared to E. coli K12, E. coli BL21 (DE3) has several biotechnological advantages, such as a lower acetate yield and a higher biomass yield, which have a beneficial effect on protein production. In a previous study (BMC Microbiol. 2011, 11:70) we have altered the metabolic fluxes of a K12 strain (i.e. E. coli MG1655) by deleting the regulators ArcA and IclR in such a way that the biomass yield is remarkably increased, while the acetate production is decreased to a similar value as for BL21 (DE3). In this study we show that the increased biomass yield beneficially influences recombinant protein production as a higher GFP yield was observed for the double knockout strain compared to its wild type. However, at higher cell densities ({\textrangle}2 g L{\`A}1 CDW), the GFP concentration decreases again, due to the activity of proteases which obstructs the application of the strain in high cell density cultivations. By further deleting the genes lon and ompT, which encode for proteases, this degradation could be reduced. Consequently, higher GFP yields were observed in the quadruple knockout strain as opposed to the double knockout strain and the MG1655 wild type and its yield approximates the GFP yield of E. coli BL21 (DE3), that is, 27 +- 5 mg g/g vs. CDW 30 +- 5 mg g/g , respectively.},
  author       = {Waegeman, Hendrik and De Lausnay, Stijn and Beauprez, Joeri and Maertens, Jo and De Mey, Marjan and Soetaert, Wim},
  issn         = {1871-6784},
  journal      = {NEW BIOTECHNOLOGY},
  keyword      = {COLI BL21,HIGH CELL-DENSITY,EXPRESSION SYSTEMS,ACETATE FORMATION,GROWTH,GLUCOSE,ARCA,PROTEOLYSIS,REPRESSION,PROTEASES},
  language     = {eng},
  number       = {2},
  pages        = {255--261},
  title        = {Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering},
  url          = {http://dx.doi.org/10.1016/j.nbt.2011.11.008},
  volume       = {30},
  year         = {2013},
}
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