A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes

Yuka Kojima, Aniko Várnai, Takuya Ishida, Naoki Sunagawa, Dejan M. Petrovic, Kiyohiko Igarashi, Jody Jellison, Barry Goodell, Gry Alfredsen, Bjørge Westereng, Vincent G.H. Eijsink (Corresponding Author), Makoto Yoshida (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

32 Citations (Scopus)

Abstract

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the ß-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD andMSand that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolyticLPMOactivity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.
Original languageEnglish
Pages (from-to)6557-6572
JournalApplied and Environmental Microbiology
Volume82
Issue number22
DOIs
Publication statusPublished - 2016
MoE publication typeA1 Journal article-refereed

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Gloeophyllum trabeum
brown-rot fungi
xyloglucans
Mixed Function Oxygenases
polysaccharide
Cellulose
Polysaccharides
cellulose
Fungi
polysaccharides
fungus
Viscosity
chromatography
Anions
Chromatography
ion exchange
viscosity
Neurospora crassa
Carboxymethylcellulose Sodium
Pichia pastoris

Cite this

Kojima, Yuka ; Várnai, Aniko ; Ishida, Takuya ; Sunagawa, Naoki ; Petrovic, Dejan M. ; Igarashi, Kiyohiko ; Jellison, Jody ; Goodell, Barry ; Alfredsen, Gry ; Westereng, Bjørge ; Eijsink, Vincent G.H. ; Yoshida, Makoto. / A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes. In: Applied and Environmental Microbiology. 2016 ; Vol. 82, No. 22. pp. 6557-6572.
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abstract = "Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the {\ss}-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD andMSand that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolyticLPMOactivity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.",
author = "Yuka Kojima and Aniko V{\'a}rnai and Takuya Ishida and Naoki Sunagawa and Petrovic, {Dejan M.} and Kiyohiko Igarashi and Jody Jellison and Barry Goodell and Gry Alfredsen and Bj{\o}rge Westereng and Eijsink, {Vincent G.H.} and Makoto Yoshida",
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language = "English",
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Kojima, Y, Várnai, A, Ishida, T, Sunagawa, N, Petrovic, DM, Igarashi, K, Jellison, J, Goodell, B, Alfredsen, G, Westereng, B, Eijsink, VGH & Yoshida, M 2016, 'A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes', Applied and Environmental Microbiology, vol. 82, no. 22, pp. 6557-6572. https://doi.org/10.1128/AEM.01768-16

A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes. / Kojima, Yuka; Várnai, Aniko; Ishida, Takuya; Sunagawa, Naoki; Petrovic, Dejan M.; Igarashi, Kiyohiko; Jellison, Jody; Goodell, Barry; Alfredsen, Gry; Westereng, Bjørge; Eijsink, Vincent G.H. (Corresponding Author); Yoshida, Makoto (Corresponding Author).

In: Applied and Environmental Microbiology, Vol. 82, No. 22, 2016, p. 6557-6572.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes

AU - Kojima, Yuka

AU - Várnai, Aniko

AU - Ishida, Takuya

AU - Sunagawa, Naoki

AU - Petrovic, Dejan M.

AU - Igarashi, Kiyohiko

AU - Jellison, Jody

AU - Goodell, Barry

AU - Alfredsen, Gry

AU - Westereng, Bjørge

AU - Eijsink, Vincent G.H.

AU - Yoshida, Makoto

PY - 2016

Y1 - 2016

N2 - Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the ß-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD andMSand that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolyticLPMOactivity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.

AB - Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the ß-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD andMSand that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolyticLPMOactivity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.

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DO - 10.1128/AEM.01768-16

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