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Comparative Genomics Platform and Phylogenetic Analysis of Fungal Laccases and Multi-Copper Oxidases Mycobiology ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tmyb20 Comparative Genomics Platform and Phylogenetic Analysis of Fungal Laccases and Multi-Copper Oxidases Jiayao Wu , Jaeyoung Choi , Fred O. Asiegbu & Yong-Hwan Lee To cite this article: Jiayao Wu , Jaeyoung Choi , Fred O. Asiegbu & Yong-Hwan Lee (2020) Comparative Genomics Platform and Phylogenetic Analysis of Fungal Laccases and Multi-Copper Oxidases, Mycobiology, 48:5, 373-382, DOI: 10.1080/12298093.2020.1816151 To link to this article: https://doi.org/10.1080/12298093.2020.1816151 © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. View supplementary material Published online: 11 Sep 2020. Submit your article to this journal Article views: 509 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tmyb20 MYCOBIOLOGY 2020, VOL. 48, NO. 5, 373–382 https://doi.org/10.1080/12298093.2020.1816151 RESEARCH ARTICLE Comparative Genomics Platform and Phylogenetic Analysis of Fungal Laccases and Multi-Copper Oxidases aà bà a c Jiayao Wu , Jaeyoung Choi , Fred O. Asiegbu and Yong-Hwan Lee aDepartment of Forest Sciences, University of Helsinki, Helsinki, Finland; bSmart Farm Research Center, Korea Institute of Science and Technology, Gangneung, Republic of Korea; cDepartment of Agricultural Biotechnology, Center for Fungal Genetic Resources, Plant Immunity Research Center, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea ABSTRACT ARTICLE HISTORY Laccases (EC 1.10.3.2), a group of multi-copper oxidases (MCOs), play multiple biological Received 27 April 2020 functions and widely exist in many species. Fungal laccases have been extensively studied Revised 6 August 2020 for their industrial applications, however, there was no database specially focused on fungal Accepted 24 August 2020 laccases. To provide a comparative genomics platform for fungal laccases, we have devel- KEYWORDS oped a comparative genomics platform for laccases and MCOs (http://laccase.riceblast.snu.ac. Laccase; multicopper kr/). Based on protein domain profiles of characterized sequences, 3,571 laccases were pre- oxidase; database; dicted from 690 genomes including 253 fungi. The number of putative laccases and their phylogenetic analysis; properties exhibited dynamic distribution across the taxonomy. A total of 505 laccases from fungal genome 68 genomes were selected and subjected to phylogenetic analysis. As a result, four clades comprised of nine subclades were phylogenetically grouped by their putative functions and analyzed at the sequence level. Our work would provide a workbench for putative laccases mainly focused on the fungal kingdom as well as a new perspective in the identification and classification of putative laccases and MCOs. 1. Introduction bilirubin oxidase activity and in brown pigmentation in spore coat [9,10]. Laccases were also known to be Laccases (EC 1.10.3.2), the biggest subgroup of involved in ectopic lignin polymerization and seed multi-copper oxidases (MCOs), are known to cata- coat browning in Arabidopsis thaliana [11,12]. In lyze the oxidation of a broad range of substrates other plant species Liriodendron tulipifera, laccases such as phenolic compounds and aromatic amines were reported to have putative roles in iron uptake [1]. Laccases have been actively investigated for their or weak electron acceptor in cytokinin degrad- ability in the degradation of a variety of compounds, ation [13,14]. such as phenols, ascorbates, amines, lignin, and Within the fungal kingdom, laccases were known phosphates. Laccases incorporate at least two copper to be dynamically associated with regulatory func- centers, a mononuclear T1 copper center for the tions at diverse developmental stages. A laccase- oxidation of substrate and a trinuclear including encoding gene lac4 in edible straw mushroom, one T2 and two T3 copper ions for reduction of Volvariella volvacea, was induced and strongly oxygen into water [2]. Laccases could be classified expressed during fruit body formation [15]. “ ” by spectroscopy; blue laccases exhibit an absorp- Increased activity of a secretory laccase LCC1 in tion band at 610 nm, while “white/yellow” laccases Hypsizygus marmoreus was involved in mycelial do not show such significant absorbance [3–5]. growth and primordium initiation, whereas the Laccases are commonly found in organisms from decreased activity of secretory laccases was observed bacteria to higher eukaryotes [6,7]. Typical laccases in Pleurotus tuber-regium when mycelia gradually are known to be three-domain MCOs, however, became mature [16,17]. Transcription of a laccase two-domain laccase-like enzymes have been discov- gene in wild-type Neurospora crassa was initiated ered in archaeal species, suggesting the evolutionary during the sexual differentiation stage, while its origin of laccases [8]. CotA, a laccase in Bacillus activity in a recombined strain was only achievable subtilis, for example, played additional roles as when copper ions were added [18]. Fungal genes CONTACT Yong-Hwan Lee [email protected]; Fred O. Asiegbu [email protected] à These authors contributed equally. Supplemental data for this article can be accessed here. ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. 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 use, distribution, and reproduction in any medium, provided the original work is properly cited. 374 J. WU ET AL. encoding laccases were also inducible to deal with attain this end, we present the Fungal Laccase different conditions. Laccase genes in Aspergillus Database (http://laccase.riceblast.snu.ac.kr/) which nidulans were transcribed upon carbon starvation would serve as a database mainly focused on fungal and highly induced during sexual development [19]. species based on protein domain profiles. In-depth In addition, laccase genes in Lentinula edodes were phylogenetic analysis using the predicted laccases expressed to resist low temperature and osmotic and MCOs would also provide a new perspective on pressure [20]. the functional classification of fungal laccases. A number of fungal laccases were known to be involved in biosynthesis of pigments. For instance, 2. Materials and methods laccases were involved in green pigment synthesis in conidial spores of Trichoderma atroviride and 2.1. Establishment of protein domain profiles T. harzianum [21]. A laccase encoded by yA in A. and collection of proteomes and nidulans was reported to oxidize a yellow precursor characterized sequences into mature green pigment during asexual develop- A total of 93 protein sequences of laccases were ment, while another one encoded by TilA was obtained from UniProtKB/SwissProt Database to expressed at the growing fungal hyphal tip only after determine domain profiles of fungal laccases by spore germination and associated with melanin bio- using InterProScan (version 58.0) [39,40]. The com- synthesis [22,23]. In L. edodes, laccases were also monly shared domain profile was applied to prote- involved in melanin synthesis during developmental ome sequences for the prediction of putative stages in fruiting body formation [24]. In addition, laccases and MCOs, supporting the previously laccases were also involved in the biosynthesis of reported structural relationships (Figure 1 and different types of melanin in Sinorhizobium meli- Supplementary Table 1)[41]. In total, 3,571 laccases loti [25]. predicted from 690 genomes of 472 species were It has been reported that laccases of white-rot archived and freely available at Fungal Laccase fungi exhibited capability in lignin degradation Database (http://laccase.riceblast.snu.ac.kr/). Proteome aided by their high redox potential [1,26]. During sequences were obtained from the standardized the cultivation of two soft-rot ascomycetes, Xylaria genome data warehouse built in the Comparative polymorpha and X. hypoxylon, on the beech-wood, Fungal Genomics Platform (CFGP 2.0; http://cfgp. laccases were identified as secreted lignin-modifying snu.ac.kr/)[42]. enzymes [27]. Missing with lignin- or manganese- type peroxidase, a white-rot fungus Pycnoporus cin- nabarinus possibly contains alternative pathways of 2.2. Prediction of secretory laccases lignin degradation by secreting laccase during To achieve better accuracy, prediction of secretory lac- growth in synthetic liquid media [28]. The activity cases was carried out by the protocol using SignalP, of fungal extracellular laccase was also found to be TMHMM, Wolf PSORT, Phobius, and PS-Scan as changed across mycelia during their interactions described previously [43]. Signal peptides were between white- and brown-rot basidiomycetes from scanned by SignalP 4.1 and Phobius [44,45]. different wood decay stages as well as fungal inter- Subsequently, transmembrane helices were predicted actions [29]. by TMHMM, and then subcellular localization of each Many efforts were also made to express or engin- sequence was determined by WoLF PSORT [46,47]. eer laccases to improve industrial performance, for Proteins containing ER-targeting signals were removed example, separation and degradation of lignin in the by PS-Scan (PROSITE:
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