title: Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering.
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 L(-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(CDW)(-1) vs. 30±5 mg g(CDW)(-1), respectively.
authors: Centre of Expertise-Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Hendrik.Waegeman@UGent.be
authors: Hendrik
authors: H
authors: Waegeman
authors: Stijn
authors: S
authors: De Lausnay
authors: Joeri
authors: J
authors: Beauprez
authors: Jo
authors: J
authors: Maertens
authors: Marjan
authors: M
authors: De Mey
authors: Wim
authors: W
authors: Soetaert
mesh_terms: D061249:Batch Cell Culture Techniques; D019149:Bioreactors / Q000382:microbiology; D048168:Escherichia coli K12 / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D010447:Peptide Hydrolases / Q000378:metabolism; D011489:Protein Denaturation; D020673:Protein Renaturation; D059748:Proteolysis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D010447:Peptide Hydrolases