Dataset Information

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

ABSTRACT: Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies.

SUBMITTER: Hori C

PROVIDER: S-EPMC6182905 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

Hori Chiaki C Gaskell Jill J Cullen Dan D Sabat Grzegorz G Stewart Philip E PE Lail Kathleen K Peng Yi Y Barry Kerrie K Grigoriev Igor V IV Kohler Annegret A Fauchery Laure L Martin Francis F Zeiner Carolyn A CA Bhatnagar Jennifer M JM

Applied and environmental microbiology 20181001 20

Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following <i>de novo</i> metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entr ...[more]

PMID: 30097442

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Project description:Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: ?-glucosidase, ?-L-arabinofuranosidase, ?-xylosidase, and endo-1,4-?-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative ?-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl ?-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading ?-xylosidase and ?-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-?-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 ?M, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.

Dataset Information

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

Publications

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

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