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Synthetic NAD(P)(H) Cycle for ATP Regeneration
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    Synthetic NAD(P)(H) Cycle for ATP Regeneration
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2023, 12, 7, 2118–2126
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    https://doi.org/10.1021/acssynbio.3c00172
    Published June 27, 2023
    Copyright © 2023 American Chemical Society

    Abstract

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    ATP is the energy currency of the cell and new methods for ATP regeneration will benefit a range of emerging biotechnology applications including synthetic cells. We designed and assembled a membraneless ATP-regenerating enzymatic cascade by exploiting the substrate specificities of selected NAD(P)(H)-dependent oxidoreductases combined with substrate-specific kinases. The enzymes in the NAD(P)(H) cycle were selected to avoid cross-reactions, and the cascade was driven by irreversible fuel oxidation. As a proof-of-concept, formate oxidation was chosen as the fueling reaction. ATP regeneration was accomplished via the phosphorylation of NADH to NADPH and the subsequent transfer of the phosphate to ADP by a reversible NAD+ kinase. The cascade was able to regenerate ATP at a high rate (up to 0.74 mmol/L/h) for hours, and >90% conversion of ADP to ATP using monophosphate was also demonstrated. The cascade was used to regenerate ATP for use in cell free protein synthesis reactions, and the ATP production rate was further enhanced when powered by the multistep oxidation of methanol. The NAD(P)(H) cycle provides a simple cascade for the in vitro regeneration of ATP without the need for a pH-gradient or costly phosphate donors.

    Copyright © 2023 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.3c00172.

    • Kinetic parameters for the PPNK enzyme with NAD+, NADH, ATP, and popy(P); relative activity of PPNK with different phosphate substrates; initial rate kinetic data for the PPNK enzyme; calibration curve for the luciferase assays used to determine ATP concentrations; native protein gel showing sfGFP produced with the CFPS reactions; calibration curves used to determine sfGFP concentrations and HRP concentrations; and protein and DNA sequences for the recombinant proteins used in the NAD(P)(H) cascade (PDF)

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    Cited By

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    This article is cited by 1 publications.

    1. Jun Liang, Kemeng Xiao, Xinyu Wang, Tianfeng Hou, Cuiping Zeng, Xiang Gao, Bo Wang, Chao Zhong. Revisiting Solar Energy Flow in Nanomaterial-Microorganism Hybrid Systems. Chemical Reviews 2024, 124 (15) , 9081-9112. https://doi.org/10.1021/acs.chemrev.3c00831

    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2023, 12, 7, 2118–2126
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.3c00172
    Published June 27, 2023
    Copyright © 2023 American Chemical Society

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