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Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink

Background: Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink.

Results: We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs.

Conclusions: In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

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Work Title Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink
Access
Open Access
Creators
  1. Zachary K. Haviland
  2. Daguan Nong
  3. Nerya Zexer
  4. Ming Tien
  5. Charles T. Anderson
  6. William O. Hancock
License CC0 1.0 (Public Domain Dedication)
Work Type Article
Publisher
  1. Biotechnology for Biofuels and Bioproducts
Publication Date January 19, 2024
Publisher Identifier (DOI)
  1. https://doi.org/10.1186/s13068-023-02456-3
Related URLs
  1. https://doi.org/10.26207/8pe3-rz48
Deposited April 25, 2025

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Version 1
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  • Created

    April 25, 2025 09:43 by avs5190

  • Updated

    April 25, 2025 09:43 by [unknown user]

  • Added Creator Zachary K Haviland

    April 25, 2025 09:44 by avs5190

  • Added Creator D Nong

    April 25, 2025 09:44 by avs5190

  • Added Creator Nerya Zexer

    April 25, 2025 09:44 by avs5190

  • Added Creator M Tien

    April 25, 2025 09:44 by avs5190

  • Added Creator Charles Anderson

    April 25, 2025 09:44 by avs5190

  • Added Creator William O. Hancock

    April 25, 2025 09:44 by avs5190

  • Updated Keyword, Publisher, Publisher Identifier (DOI), and 3 more Show Changes

    April 25, 2025 09:44 by avs5190

    Keyword
    • Lignin, Cellulase, Bacterial Cellulose, Cellulose, Cel7a, Bioethanol, Biofuel, Pretreatment, Lignification, Lignocellulosic Biomass, Enzymatic Degradation, Plant Biomass, Sustainable Source, Nanoscale, Cell Wall, Electron Microscopy, Lignocellulose, Trichoderma Reesei, Fluorescence Microscopy, Processive, Inhibition Mechanism, Plant Cell Wall, Optical Microscopy, Sequester, Production Efficiency, Dominant Role, In Vitro System, Ethanol, Cellulose Degradation, Polyphenolic Polymers, Efficient Production, Biofuel Production, Nonspecific Adsorption, Single Molecule Fluorescence Microscopy, Lignin Removal, Coniferyl Alcohol, Cellulose Surface, Catalytic Degradation, Density, Surface (Surface Science)
    Publisher
    • Biotechnology for Biofuels and Bioproducts
    Publisher Identifier (DOI)
    • 10.1186/s13068-023-02456-3
    Related URLs
    • https://scholarsphere.psu.edu/resources/659fb752-e6da-48c6-8077-1bb5b50032c0
    Description
    • Background: Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. Results: We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. Conclusions: In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.
    Publication Date
    • 2024-01-01
  • Updated

    April 25, 2025 09:44 by avs5190

  • Updated Keyword, Publisher Identifier (DOI), Related URLs, and 1 more Show Changes

    April 25, 2025 09:46 by avs5190

    Keyword
    • Lignin, Cellulase, Bacterial Cellulose, Cellulose, Cel7a, Bioethanol, Biofuel, Pretreatment, Lignification, Lignocellulosic Biomass, Enzymatic Degradation, Plant Biomass, Sustainable Source, Nanoscale, Cell Wall, Electron Microscopy, Lignocellulose, Trichoderma Reesei, Fluorescence Microscopy, Processive, Inhibition Mechanism, Plant Cell Wall, Optical Microscopy, Sequester, Production Efficiency, Dominant Role, In Vitro System, Ethanol, Cellulose Degradation, Polyphenolic Polymers, Efficient Production, Biofuel Production, Nonspecific Adsorption, Single Molecule Fluorescence Microscopy, Lignin Removal, Coniferyl Alcohol, Cellulose Surface, Catalytic Degradation, Density, Surface (Surface Science)
    Publisher Identifier (DOI)
    • 10.1186/s13068-023-02456-3
    • https://doi.org/10.1186/s13068-023-02456-3
    Related URLs
    • https://scholarsphere.psu.edu/resources/659fb752-e6da-48c6-8077-1bb5b50032c0
    • https://doi.org/10.26207/8pe3-rz48
    Publication Date
    • 2024-01-01
    • 2024-01-19
  • Renamed Creator Zachary K. Haviland Show Changes

    April 25, 2025 09:47 by avs5190

    • Zachary K Haviland
    • Zachary K. Haviland
  • Renamed Creator Daguan Nong Show Changes

    April 25, 2025 09:47 by avs5190

    • D Nong
    • Daguan Nong
  • Updated Creator Nerya Zexer

    April 25, 2025 09:47 by avs5190

  • Renamed Creator Ming Tien Show Changes

    April 25, 2025 09:47 by avs5190

    • M Tien
    • Ming Tien
  • Renamed Creator Charles T. Anderson Show Changes

    April 25, 2025 09:47 by avs5190

    • Charles Anderson
    • Charles T. Anderson
  • Updated Creator William O. Hancock

    April 25, 2025 09:47 by avs5190

  • Added LigninPaperRevised_Final.pdf

    April 25, 2025 09:47 by avs5190

  • Updated License Show Changes

    April 25, 2025 09:48 by avs5190

    License
    • http://creativecommons.org/publicdomain/zero/1.0/
  • Published

    April 25, 2025 09:48 by avs5190

  • Updated Description Show Changes

    April 25, 2025 09:49 by avs5190

    Description
    • Background: Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. Results: We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. Conclusions: In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.
    • Background: Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink.
    • Results: We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from _Trichoderma reesei_, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs.
    • Conclusions: In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.
  • Updated

    April 25, 2025 22:05 by [unknown user]