title: An engineered Escherichia coli having a high intracellular level of ATP and enhanced recombinant protein production.
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.
authors.affiliation: Department of Biotechnology, Catholic University of Korea, Bucheon, Gyeonggi, Korea.
authors.forename: Hye-Jung
authors.initials: HJ
authors.lastname: Kim
authors.forename: Yeong Deok
authors.initials: YD
authors.lastname: Kwon
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
authors.forename: Pil
authors.initials: P
authors.lastname: Kim
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics* / Q000378:metabolism*; D017902:Podoviridae; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D010450:Endopeptidases; C087822:alkaline protease; D019764:Phosphoenolpyruvate Carboxykinase (ATP)