Application of capillary electrophoresis-mass spectrometry to synthetic in vitro glycolysis studies.
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.
Itoh
Aya
A
Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
Ohashi
Yoshiaki
Y
Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
Soga
Tomoyoshi
T
Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
Mori
Hirotada
H
Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.|Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Takayama-cho, Ikoma, Japan.
Nishioka
Takaaki
T
Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.|Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kita-shirakawa, Sakyo-ku, Kyoto, Japan.
Tomita
Masaru
M
Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
D002384:Catalysis; D003001:Cloning, Molecular; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D013058:Mass Spectrometry / Q000379:methods*; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013997:Time Factors
D011994:Recombinant Proteins; D019289:Pyruvic Acid; D005947:Glucose