title: Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP.
abstract: We have previously reported that phosphoenolpyruvate carboxykinase (PCK) overexpression under glycolytic conditions enables Escherichia coli to harbor a high intracellular ATP pool resulting in enhanced recombinant protein synthesis. To estimate how much PCK-mediated phosphoenolpyruvate (PEP) carboxylation is contributed to the ATP increase under engineered conditions, the kinetics of PEP carboxylation by PCK and substrate competing phosphoenolpyruvate carboxylase (PPC) were measured using recombinant enzymes. The PEP carboxylation catalytic efficiency (kcat/Km) of the recombinant PCK was 660mM(-1)min(-1), whereas that of the recombinant PPC was 1500mM(-1)min(-1). Under the presence of known allosteric effectors (fructose 1,6-bisphosphate, acetyl-CoA, ATP, malate, and aspartate) close to in vivo conditions, the catalytic efficiency of PCK-mediated PEP carboxylation (84mM(-1)min(-1)) was 28-folds lower than that of PPC (2370mM(-1)min(-1)). To verify the above results, an E. coli strain expressing native PCK and PPC under control of identical promoter was constructed by replacing PCK promoter region with that of PPC in chromosome. The native PCK activity (33nmol/mg-proteinmin) was 5-folds lower than PPC activity (160nmol/mg-proteinmin) in the cell extract from the promoter-exchanged strain. Intracellular modifications of ATP concentration by PCK activity and the consequences for biotechnology are further discussed.
authors.affiliation: Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi 420-743, Republic of Korea.
authors.forename: Hyo Jung
authors.initials: HJ
authors.lastname: Lee
authors.forename: Hye-Jung
authors.initials: HJ
authors.lastname: Kim
authors.forename: Jiyoon
authors.initials: J
authors.lastname: Seo
authors.forename: Yoon Ah
authors.initials: YA
authors.lastname: Na
authors.forename: Jiyeon
authors.initials: J
authors.lastname: Lee
authors.forename: Joo-Young
authors.initials: JY
authors.lastname: Lee
authors.forename: Pil
authors.initials: P
authors.lastname: Kim
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D007700:Kinetics; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010730:Phosphoenolpyruvate Carboxylase; D019764:Phosphoenolpyruvate Carboxykinase (ATP)