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Estimation of phosphoenolpyruvate carboxylation mediated by phosphoenolpyruvate carboxykinase (PCK) in engineered Escherichia coli having high ATP.
MedLine Citation:
PMID:  23683699     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
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:
Hyo Jung Lee; Hye-Jung Kim; Jiyoon Seo; Yoon Ah Na; Jiyeon Lee; Joo-Young Lee; Pil Kim
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Publication Detail:
Type:  Journal Article     Date:  2013-04-06
Journal Detail:
Title:  Enzyme and microbial technology     Volume:  53     ISSN:  1879-0909     ISO Abbreviation:  Enzyme Microb. Technol.     Publication Date:  2013 Jun 
Date Detail:
Created Date:  2013-05-20     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8003761     Medline TA:  Enzyme Microb Technol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  13-7     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 Elsevier Inc. All rights reserved.
Affiliation:
Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi 420-743, Republic of Korea.
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