Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

Yann Mathieu; Olanrewaju Raji; Annie Bellemare; Marcos Di Falco; Thi Truc Minh Nguyen; Alexander Holm Viborg; Adrian Tsang; Emma Master; Harry Brumer

doi:10.1186/s13068-023-02383-3

Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

Yann Mathieu
, Olanrewaju Raji
, Annie Bellemare
, Marcos Di Falco
, Thi Truc Minh Nguyen
, Alexander Holm Viborg
, Adrian Tsang
, Emma Master^*
, Harry Brumer^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Scientific › peer-review

12 Citations (Scopus)

65 Downloads (Pure)

Abstract

Background: Microbial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin.

Results: To further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern.

Conclusions: This work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.

Original language	English
Article number	132
Number of pages	14
Journal	Biotechnology for Biofuels and Bioproducts
Volume	16
Issue number	1
DOIs	https://doi.org/10.1186/s13068-023-02383-3
Publication status	Published - 7 Sept 2023
MoE publication type	A1 Journal article-refereed

Funding

This work was funded by grants awarded to Emma Master, Harry Brumer, and Adrian Tsang from Genome Canada, Ontario Genomics, Genome BC, and Génome Québec for Project Number 10405, “SYNBIOMICS-Functional genomics and techno-economic models for advanced biopolymer synthesis”).

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 7 Affordable and Clean Energy
SDG 12 Responsible Consumption and Production

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10.1186/s13068-023-02383-3Licence: CC BY

CHEM_Mathieu_et_al_Functional_characterization_2023_Biotechnol_BiofuelsFinal published version, 3.7 MBLicence: CC BY

Cite this

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title = "Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation",

abstract = "Background: Microbial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin. Results: To further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern. Conclusions: This work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.",

author = "Yann Mathieu and Olanrewaju Raji and Annie Bellemare and \{Di Falco\}, Marcos and Nguyen, \{Thi Truc Minh\} and Viborg, \{Alexander Holm\} and Adrian Tsang and Emma Master and Harry Brumer",

note = "Funding Information: This work was funded by grants awarded to Emma Master, Harry Brumer, and Adrian Tsang from Genome Canada, Ontario Genomics, Genome BC, and G{\'e}nome Qu{\'e}bec for Project Number 10405, “SYNBIOMICS-Functional genomics and techno-economic models for advanced biopolymer synthesis”). Publisher Copyright: {\textcopyright} 2023, BioMed Central Ltd., part of Springer Nature.",

year = "2023",

month = sep,

day = "7",

doi = "10.1186/s13068-023-02383-3",

language = "English",

volume = "16",

journal = "Biotechnology for Biofuels and Bioproducts",

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T1 - Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

AU - Mathieu, Yann

AU - Raji, Olanrewaju

AU - Bellemare, Annie

AU - Di Falco, Marcos

AU - Nguyen, Thi Truc Minh

AU - Viborg, Alexander Holm

AU - Tsang, Adrian

AU - Master, Emma

AU - Brumer, Harry

N1 - Funding Information: This work was funded by grants awarded to Emma Master, Harry Brumer, and Adrian Tsang from Genome Canada, Ontario Genomics, Genome BC, and Génome Québec for Project Number 10405, “SYNBIOMICS-Functional genomics and techno-economic models for advanced biopolymer synthesis”). Publisher Copyright: © 2023, BioMed Central Ltd., part of Springer Nature.

PY - 2023/9/7

Y1 - 2023/9/7

N2 - Background: Microbial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin. Results: To further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern. Conclusions: This work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.

AB - Background: Microbial lytic polysaccharide monooxygenases (LPMOs) cleave diverse biomass polysaccharides, including cellulose and hemicelluloses, by initial oxidation at C1 or C4 of glycan chains. Within the Carbohydrate-Active Enzymes (CAZy) classification, Auxiliary Activity Family 9 (AA9) comprises the first and largest group of fungal LPMOs, which are often also found in tandem with non-catalytic carbohydrate-binding modules (CBMs). LPMOs originally attracted attention for their ability to potentiate complete biomass deconstruction to monosaccharides. More recently, LPMOs have been applied for selective surface modification of insoluble cellulose and chitin. Results: To further explore the catalytic diversity of AA9 LPMOs, over 17,000 sequences were extracted from public databases, filtered, and used to construct a sequence similarity network (SSN) comprising 33 phylogenetically supported clusters. From these, 32 targets were produced successfully in the industrial filamentous fungus Aspergillus niger, 25 of which produced detectable LPMO activity. Detailed biochemical characterization of the eight most highly produced targets revealed individual C1, C4, and mixed C1/C4 regiospecificities of cellulose surface oxidation, different redox co-substrate preferences, and CBM targeting effects. Specifically, the presence of a CBM correlated with increased formation of soluble oxidized products and a more localized pattern of surface oxidation, as indicated by carbonyl-specific fluorescent labeling. On the other hand, LPMOs without native CBMs were associated with minimal release of soluble products and comparatively dispersed oxidation pattern. Conclusions: This work provides insight into the structural and functional diversity of LPMOs, and highlights the need for further detailed characterization of individual enzymes to identify those best suited for cellulose saccharification versus surface functionalization toward biomaterials applications.

UR - https://www.scopus.com/pages/publications/85170248124

U2 - 10.1186/s13068-023-02383-3

DO - 10.1186/s13068-023-02383-3

M3 - Article

AN - SCOPUS:85170248124

SN - 2731-3654

VL - 16

JO - Biotechnology for Biofuels and Bioproducts

JF - Biotechnology for Biofuels and Bioproducts

IS - 1

M1 - 132

ER -

Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

Abstract

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