Conserved White-Rot Enzymatic Mechanism for Wood Decay In
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DNA Research, 2020, 0(0), 1–14 doi: 10.1093/dnares/dsaa011 Advance Access Publication Date: 12 June 2020 Full Paper Downloaded from https://academic.oup.com/dnaresearch/article/27/2/dsaa011/5856740 by University of Wisconsin-Madison Libraries user on 28 August 2020 Full Paper Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus 1,2,† 1 1 Shingo Miyauchi , Hayat Hage , Elodie Drula , 1,3 1 1,3 Laurence Lesage-Meessen , Jean-Guy Berrin , David Navarro , Anne 1,3 1,3 1 1 Favel , Delphine Chaduli , Sacha Grisel , Mireille Haon , Franc¸ois 1,‡ ‡ § 1 Piumi , Anthony Levasseur , , Anne Lomascolo , 4 4 4 5 Steven Ahrendt , Kerrie Barry , Kurt M LaButti , Didier Chevret , Chris 4 6 6 7 Daum ,Je´roˆ me Mariette , Christophe Klopp , Daniel Cullen , 8,9 10 11,12 Ronald P de Vries , Allen C Gathman , Matthieu Hainaut , 11,12 9 13,14 10 Bernard Henrissat , Kristiina S Hilde´n , Ursula Ku¨es , Walt Lilly , 4 9 15 Anna Lipzen , Miia R Ma¨ kela¨ , Angel T Martinez , 2 2 4 Me´ lanie Morel-Rouhier , Emmanuelle Morin , Jasmyn Pangilinan , Arthur 16 17 15 4 F J Ram , Han A B Wo¨ sten , Francisco J Ruiz-Duenas~ , Robert Riley , 1 4,18 1, Eric Record , Igor V Grigoriev , and Marie-Noe¨ lle Rosso * 1INRAE, UMR1163, Biodiversity and Biotechnology of Fungi, Aix Marseille University, 13009 Marseille, France, 2INRAE, UMR1136, Interactions Arbres/Microorganismes, Universite´ de Lorraine, Nancy, France, 3INRAE, CIRM-CF, UMR1163, Aix Marseille University, Marseille, France, 4US Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA, 5INRAE, UMR1319, Micalis, Plateforme d’Analyse Prote´ omique de Paris Sud-Ouest, Jouy-en- Josas, France, 6INRAE, Genotoul Bioinfo, UR875, Mathe´ matiques et Informatique Applique´ es de Toulouse, Castanet-Tolosan, France, 7USDA Forest Products Laboratory, Madison, WI, USA, 8Fungal Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands, 9Department of Microbiology, University of Helsinki, Helsinki, Finland, 10Department of Biology, Southeast Missouri State University, Cape Girardeau, MI, USA, 11CNRS, UMR7257, AFMB, Aix Marseille University, Marseille, France, 12INRAE, USC1408, AFMB, Marseille, France, 13Department of Molecular Wood Biotechnology and Technical Mycology, Bu¨ sgen-Institute, Georg-August-University Go¨ ttingen, Go¨ ttingen, Germany, 14Center for Molecular Biosciences (GZMB), Georg-August-University Go¨ ttingen, Go¨ ttingen, Germany, 15Centro de Investigaciones Biolo´ gicas, CSIC, Madrid, Spain, 16Molecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands, 17Microbiology, Utrecht University, Utrecht, The Netherlands, and 18Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA † Present address: Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, Ko¨ ln, Germany. ‡ Present address: UMR INRAE, BDR, ENVA, Universite´ Paris Saclay, 78350 Jouy en Josas, France. ¶Present address: IRD, Microbes, Evolution, Phylogeny and Infection, APHM, IHU Me´ diterrane´ e Infection, Aix Marseille University, Marseille, France. *To whom correspondence should be addressed. Tel. þ33 491828607. Fax þ33 491828601. Email: marie- [email protected] Received 18 March 2020; Accepted 5 June 2020 VC The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 2 Cross-species multi-omics and conserved gene regulations Abstract White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. Downloaded from https://academic.oup.com/dnaresearch/article/27/2/dsaa011/5856740 by University of Wisconsin-Madison Libraries user on 28 August 2020 However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early re- sponse of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellu- lose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysac- charide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process. Key words: wood decay, lignocellulose, CAZyme, lytic polysaccharide monooxygenase, Class II Peroxidase 1. Introduction mechanics in terms of transcriptome and exo-proteome responses to Saprotrophic fungi of Northern Hemisphere and tropical forests impact lignocellulosic substrates. We sequenced the genomes of P. coccineus, the carbon cycling through mineralization and alteration of C storage in P. sanguineus and P. puniceus monokaryotic strains. We overviewed wood and litter dead organic matter.1,2 White-rot (WR) fungi are wood the genomic features among the three species, in comparison to the decayers with the capacity to mineralize lignin with ultimate formation previously sequenced genome of P. cinnabarinus9 and to other evolu- 3 of CO2 and H2O. WR fungi deploy a wide arsenal of hydrolytic and tionarily related wood-decay fungi. Then, we captured the transcrip- oxidative enzymes to degrade wood and their genomes typically contain tomic and exoproteomic responses of three Pycnoporus species to a genes coding for glycoside hydrolases, carbohydrate esterases and poly- panel of cellulosic and lignocellulosic substrates representative of saccharide lyases that collectively cleave cellulose, hemicellulose and Gramineae and hardwoods. The focus was the early responses to the pectin backbones and lateral chains, and oxidative enzymes that target substrates in order to minimize inter-species differences influenced by the highly recalcitrant lignin, crystalline cellulose or cellulose-bound xy- varied growth abilities on the substrates. Our omics integrative ap- 4,5 lan. Beyond these shared features, several studies have highlighted sig- proach enabled to identify a common set of lignocellulose-degrading nificant polymorphism between WR fungi regarding their ability to enzymes mobilized by the fungi at the initial stage of lignocellulose 6–8 selectively degrade lignin over cellulose and in the gene portfolios in- degradation, leading to discoveries of genus-wide conserved expres- 9–11 volved in lignocellulose breakdown. Scarce studies at the intra- sion patterns indicative of conserved enzymatic synergies. genus level have shown that functional diversity between species may arise from diversity in gene content.12,13 Among WR fungi, the genus Pycnoporus (Basidiomycota, 2. Materials and methods Agaricomycetes) has been studied for the efficiency of lignin degrada- 2.1. Genome sequencing and assembly tion, the capacity to secrete laccases and biotechnological applica- tions related to aromatic compound functionalization, biopolymer The monokaryotic strains P. coccineus BRFM 310, P. sanguineus synthesis and biomass pre-treatment in the pulp and paper indus- BRFM 1264 and P. puniceus BRFM 1868 were generated after fruit- try.14 Four Pycnoporus species have been differentiated,15,16 which ing of the parental strains BRFM 66 (IMB WOO6-2), BRFM 902 14 form a monophyletic group within the Trametes clade.17 The four and BRFM 1856, respectively, as described previously species are found in different geo-climatic areas with limited geo- (Supplementary Information). All strains were maintained at the graphical overlap; P. cinnabarinus is widely distributed in the International Centre of Microbial Resources (CIRM; https://www6. Northern hemisphere, P. coccineus is found in countries bordering inra.fr/cirm/). The P. coccineus BRFM 310 genome was sequenced the Indian and Pacific Oceans, P. sanguineus is found in the tropics using the Illumina platform (99.4) and assembled with AllPathsLG 18 and subtropics of both hemispheres and P. puniceus is found in pale- version R46652 (GenBank accession number: NCSW00000000). otropical areas.15,16 The four species are found on stumps and either The P. sanguineus BRFM 1264 genome was sequenced using 454 standing or fallen trunks of deciduous trees. (16.8) and Solexa (87) technologies and assembled with Our aim was to investigate the Pycnoporus intra-genus genomic CABOG19 (GenBank accession number: VOCM00000000). The P. and functional diversity focusing on lignocellulose breakdown. We ex- puniceus BRFM 1868 genome was sequenced using