An engineered Escherichia coli having a high intracellular level of ATP and enhanced recombinant protein production

Appl Microbiol Biotechnol. 2012 May;94(4):1079-86. doi: 10.1007/s00253-011-3779-0. Epub 2011 Dec 16.

Abstract

Artificial amplification of gluconeogenic phosphoenolpyruvate carboxykinase (PCK) under glycolytic conditions enables Escherichia coli to maintain a greater intracellular ATP concentration during its growth phase. To demonstrate the biotechnological benefit of E. coli harboring a high intracellular ATP concentration, we compared the recombinant protein synthesis of a soluble protein (enhanced green fluorescence protein, GFP) with that of a secretory protein (alkaline protease, AP), under control of the T7 promoter in E. coli BL21(DE3) overexpressing PCK. According to the batch fermentations, the strain overexpressing PCK produced more GFP and AP with a lower increase in biomass than the control strain. In a chemostat culture (D = 0.7 h(-1)), the GFP production in the PCK overexpressing strain was 99.0 ± 4.31 mg/g cell, with a biomass of 0.22 g/L, while that of the control strain was 53.5 ± 3.07 mg/g cell, with a biomass of 0.35 g/L. These results indicate that the PCK overexpressing E. coli strain harboring high intracellular levels of ATP can be useful as a protein-synthesizing host. The potential uses of the strain and associated rationale are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analysis*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cytoplasm / chemistry*
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Metabolic Engineering*
  • Phosphoenolpyruvate Carboxykinase (ATP) / genetics*
  • Phosphoenolpyruvate Carboxykinase (ATP) / metabolism*
  • Podoviridae
  • Recombinant Proteins / biosynthesis

Substances

  • Bacterial Proteins
  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Adenosine Triphosphate
  • Endopeptidases
  • alkaline protease
  • Phosphoenolpyruvate Carboxykinase (ATP)