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Application of capillary electrophoresis‐mass spectrometry to synthetic in vitro glycolysis studies
Author(s) -
Itoh Aya,
Ohashi Yoshiaki,
Soga Tomoyoshi,
Mori Hirotada,
Nishioka Takaaki,
Tomita Masaru
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305905
Subject(s) - glycolysis , metabolite , capillary electrophoresis , escherichia coli , mass spectrometry , biochemistry , chemistry , metabolic pathway , enzyme , in vitro , chromatography , metabolic engineering , gene
We propose an approach designed to reconstitute a metabolic pathway composed of multistep biochemical reactions, rather than to dissect the individual reactions that make up the pathway. A synthetic in vitro glycolysis was reconstructed from ten purified Escherichia coli ( E. coli ) enzymes to obtain a better understanding of the regulation of sequential enzymatic reactions. The key to the success of this approach is the ability to perform direct and simultaneous determination of the diverse metabolic intermediates in the pathway by capillary electrophoresis‐mass spectrometry. We observed that the pathway is regulated by a delicate balance between the changing metabolite concentrations and behaves like a natural biological oscillating network that has hitherto not been reported for E. coli glycolysis. The end‐product, pyruvate, was periodically synthesized from glucose at an overall efficiency of 30%, corresponding to an average of 90% conversion efficiency for each of the ten steps involved. This approach is likely useful for the synthesis of natural products requiring complex sequential biocatalytic reactions.