Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

Abernathy, Mary H.; Zhang, Yuchen; Hollinshead, Whitney D.; Wang, George; Baidoo, Edward E. K.; Liu, Tiangang; Tang, Yinjie J.

doi:10.1002/aic.16367

Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

Journal Article · Fri Sep 21 00:00:00 EDT 2018 · AIChE Journal

DOI:https://doi.org/10.1002/aic.16367· OSTI ID:1472164

Abernathy, Mary H. ^[1]; Zhang, Yuchen ^[2]; Hollinshead, Whitney D. ^[1]; Wang, George ^[3]; Baidoo, Edward E. K. ^[3]; ^[2]; ^[1]

Dept. of Energy, Environmental and Chemical Engineering Washington University St. Louis MO 63130
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education and Wuhan University School of Pharmaceutical Sciences Wuhan 430071 China
Lawrence Berkeley National Laboratory Emeryville CA 64608

This study constructed cell‐free glycolytic enzyme systems and compared them to their in vivo functions in Escherichia coli . Under in vitro conditions, flux regulation followed enzyme concentrations and kinetics. In E. coli, only one of the isozymes of phosphofructokinase (PfkA) and fructose‐bisphosphate aldolase (FbaA) facilitate Embden‐Meyerhof‐Parnas (EMP) flux, but under in vitro assays, these isozymes were interchangeable. Additionally, in vitro introduction of the Entner–Doudoroff (ED) pathway improved glycolysis rates, while in vivo overexpression of the ED pathway could not capture significant flux unless its phosphotransferase system (PTS) was knocked out. Lastly, in vivo dynamic ¹³ C‐experiments revealed that the labeling order of EMP pathway intermediates was not strictly cascade, indicating intracellular metabolites were not well mixed. These enigmatic observations cannot be fully explained by thermodynamics or substrate level regulations. This article supports the long‐time conjecture that EMP enzymes are channeled, and the PTS may be an anchor point to initiate enzyme assemblies. © 2018 American Institute of Chemical Engineers AIChE J , 65: 483–490, 2019

View Accepted Manuscript (Publisher)

Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0018324; AC02-05CH11231

OSTI ID:: 1472164

Journal Information:: AIChE Journal, Journal Name: AIChE Journal Journal Issue: 2 Vol. 65; ISSN 0001-1541

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United States

Language:: English

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