Dataset Information

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling.

ABSTRACT: Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications so far mainly in biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from the thermophilic and lignocellulose-degrading white rot fungus Phanerochaete chrysosporium. By employing a comparative ABPP-based functional screen, including a direct profiling of wood substrate-bound enzymes, we identify those lignocellulose-degrading carbohydrate esterase (CE1 and CE15) and glycoside hydrolase (GH3, GH5, GH16, GH17, GH18, GH25, GH30, GH74 and GH79) enzymes specifically active in presence of the substrate. As expression of fungal enzymes remains challenging, our ABPP-mediated approach represents a preselection procedure for focusing experimental efforts on the most promising biocatalysts. Furthermore, this approach may also allow the functional annotation of domains-of-unknown functions (DUFs). The ABPP-based biocatalyst screening described here may thus allow the identification of active enzymes in a process of interest and the elucidation of novel biocatalysts that share no sequence similarity to known counterparts.

SUBMITTER: Schmerling C

PROVIDER: S-EPMC9668830 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling.

Schmerling Christian C Sewald Leonard L Heilmann Geronimo G Witfeld Frederick F Begerow Dominik D Jensen Kenneth K Bräsen Christopher C Kaschani Farnusch F Overkleeft Herman S HS Siebers Bettina B Kaiser Markus M

Communications biology 20221116 1

Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications so far mainly in biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from the thermophilic and lignocellulose-degrading white rot fungus Phanerochaete chrysosporium. By employing a comparative ABPP-based functional screen, including a direct profiling of wood substrate-bound enzymes, we identify ...[more]

PMID: 36385496

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Project description:White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Project description:BACKGROUND:Wood-feeding insects often work in collaboration with microbial symbionts to degrade lignin biopolymers and release glucose and other fermentable sugars from recalcitrant plant cell wall carbohydrates, including cellulose and hemicellulose. Here, we present the midgut transcriptome of larval Anoplophora glabripennis, a wood-boring beetle with documented lignin-, cellulose-, and hemicellulose- degrading capabilities, which provides valuable insights into how this insect overcomes challenges associated with feeding in woody tissue. RESULTS:Transcripts from putative protein coding regions of over 9,000 insect-derived genes were identified in the A. glabripennis midgut transcriptome using a combination of 454 shotgun and Illumina paired-end reads. The most highly-expressed genes predicted to encode digestive-related enzymes were trypsins, carboxylesterases, ?-glucosidases, and cytochrome P450s. Furthermore, 180 unigenes predicted to encode glycoside hydrolases (GHs) were identified and included several GH 5, 45, and 48 cellulases, GH 1 xylanases, and GH 1 ?-glucosidases. In addition, transcripts predicted to encode enzymes involved in detoxification were detected, including a substantial number of unigenes classified as cytochrome P450s (CYP6B) and carboxylesterases, which are hypothesized to play pivotal roles in detoxifying host tree defensive chemicals and could make important contributions to A. glabripennis' expansive host range. While a large diversity of insect-derived transcripts predicted to encode digestive and detoxification enzymes were detected, few transcripts predicted to encode enzymes required for lignin degradation or synthesis of essential nutrients were identified, suggesting that collaboration with microbial enzymes may be required for survival in woody tissue. CONCLUSIONS:A. glabripennis produces a number of enzymes with putative roles in cell wall digestion, detoxification, and nutrient extraction, which likely contribute to its ability to thrive in a broad range of host trees. This system is quite different from the previously characterized termite fermentation system and provides new opportunities to discover enzymes that could be exploited for cellulosic ethanol biofuel production or the development of novel methods to control wood-boring pests.

Dataset Information

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling.

Publications

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling.

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