Genomics and systematics of the white-rot fungus Phlebia radiata : special emphasis on wood-promoted transcriptome and proteome

The wood-decaying white-rot fungi have the profound ability to completely degrade lignocelluloses and all wood components. These fungi and their enzymes have evolved to modify the various lignocellulose feedstocks in nature, and thereby, they are important organisms for bioconversions as well as in fundamental research on fungal biology. The enzymes have many potential applications in biotechnology and industrial purposes including bioenergy production. Evolutionary background of the fungal species and their organelles thus requires deeper understanding to aid in elucidating the relationship of the species to their lifestyles. This PhD study concentrated on the white-rot fungal species Phlebia radiata, Finnish isolate number 79 (FBCC0043). The phylogenetic studies confirmed positioning of P. radiata species in the systematic class Agaricomycetes of Basidiomycota, and in the phlebioid clade of the order Polyporales. The sequenced and annotated mitochondrial genome of P. radiata was discovered to have features that indicate evolutionary pressure and structural diversity in fungal mitogenomes, not being as stable and compact entities than was previously believed. In this study, P. radiata together with species like Phlebia acerina and Phlebia brevispora was demonstrated to form a Phlebia sensu stricto group which consists of efficient producers of lignin-modifying enzymes. The results pinpointed that there is a species-level connection of fungal molecular systematics to the efficiency in the production of wood-decaying enzymes and activities. Norway spruce (Picea abies) is a common tree species in the boreal forests providing an important source of biomass for forest-based industry. Therefore, P. radiata was cultivated on Norway spruce wood under conditions mimicking natural solid-wood colonization, up to six weeks of growth, and the dynamics of fungal enzyme production and gene expression was studied. The lignin-modifying class-II peroxidases (LiPs and various MnPs) were produced, especially in the beginning of fungal growth and colonization of wood, thus indicating the essence of class-II peroxidase as the primary enzymes to function against coniferous wood lignin. Moreover, these extracellular oxidoreductases enhance the accessibility of lignocellulose carbohydrates and thereby, they promote fungal growth in wood. Simultaneously, lytic polysaccharide monooxygenases and several CAZyme glycoside hydrolases attacking cellulose, hemicellulose and pectin were produced, which demonstrates ongoing depolymerization of the polysaccharides to monomers and oligomers. Electron microscopic examination of fungal-colonized wood after six weeks of growth indicated that the decay of wood cell walls was initiated at the tracheid lumen side apparently proceeding towards the middle lamellae. Furthermore, degradation of spruce wood lignin was detected by pyrolysis-GC/MS as decrease in the amount of phenylpropane units with concomitant increase in the number of smaller fragmented products from these lignin units. Thus, the previously observed unique and strong ability of P. radiata to degrade wood lignin and lignin-like aromatic compounds was confirmed. According to the results of this PhD study, P. radiata produces the white-rot type of decay of wood components when growing on Norway spruce. This is due to the efficient ability of the fungus to express and produce a versatile enzyme repertoire for degradation of wood lignocellulose, and in consequence, to generate diverse reactions and bioconversions important for carbon cycling in the forest ecosystems.Valkolahoa tuottavat sienet hajottavat tehokkaasti lignoselluloosaa ja puuaineksen biopolymeerejä, kuten selluloosaa, hemiselluloosaa ja ligniiniä. Valkolahottavat sienet ovat evoluution myötä kehittyneet tuottamaan erilaisia entsyymejä, jotka kykenevät muokkaamaan ja pilkkomaan monimutkaisia raaka-aineita. Tämän vuoksi valkolahottajat ovat kiinnostavia tutkimuskohteita niin teollisesti kuin perustutkimuksenkin näkökulmasta. Sienten tuottamia entsyymejä voidaan hyödyntää bioteknisissä ja teollisissa sovelluksissa, kuten biopolttoaineiden ja uusien biomateriaalien tuotannossa. Puunlahottajien ja niiden mitokondrioiden pitkän evoluutiohistorian selvittäminen on tärkeää, jotta sienilajien ja niiden elintapojen välisiä suhteita voidaan ymmärtää. Tässä väitöskirjatutkimuksessa oli keskeisessä roolissa Suomesta eristetty rusorypykän (Phlebia radiata) sienikanta 79 (FBCC0043). Fylogeneettinen monigeenitarkastelu vahvisti rusorypykän ja lajin muiden edustajien kuuluvan kantasienten Agaricomycetes-luokan Polyporales-lahkoon, ja siinä edelleen niin kutsuttuun phlebioid-sukuryhmään. Rusorypykän mitokondrion genomin sekvensointi osoitti, että tumallisten eliöiden mitogenomit eivät ole niin vakaita ja yhteneviä yksikköjä kuin aikaisemmin on uskottu. Sienten mitogenomit ovat monimuotoisia ja jatkuvan evolutiivisen paineen alla. Laajempi Phlebia-suvun lajien ja isolaattien fylogeneettinen tutkimus osoitti, että rusorypykkä muodostaa yhdessä lajien Phlebia acerina ja P. brevispora kanssa Phlebia sensu stricto -ryhmän, jonka jäsenet tuottavat tehokkaasti ligniiniä hajottavia hapetus-pelkistysentsyymeitä. Tässä tutkimuksessa huomattiin myös, että molekyylisystematiikan perusteella arvioidut lajien väliset geneettiset suhteet ovat yhteydessä puuta sisältävällä alustalla tuotettaviin entsyymiaktiivisuuksiin ja lajien entsyymiprofiileihin. Kuusi (metsäkuusi, Picea abies) on yksi pohjoisen havumetsävyöhykkeen tärkeimmistä raaka-aineista. Tästä syystä rusorypykän isolaattia 79 kasvatettiin kuusipuuaineksella sienirihmaston luonnollisia kasvuolosuhteita jäljittelevissä oloissa kuuden viikon ajan lahoentsyymien tuoton ja niitä koodaavien geenien ilmentymisen muutosten seuraamiseksi. Rusorypykkä tuotti ligniiniä muokkaavia hapetus-pelkistysentsyymeitä, kuten ligniini- ja mangaaniperoksidaaseja, runsaasti jo ensimmäisellä kasvuviikolla. Havainnot osoittivat näiden peroksidaasientsyymien tärkeyden havupuuligniinin hajotuksen alkuvaiheessa sekä merkityksen hiilihydraattipolymeerien saatavuuden ja sienirihmaston kasvun edistämisessä. Samanaikaisesti kun ligniiniä hajotettiin, rusorypykkä tuotti tasaisemmin lyyttisiä polysakkaridi-mono-oksygenaaseja sekä useita selluloosaa, hemiselluloosaa ja pektiiniä hajottavia glykosidihydrolaaseja, jotka pilkkovat hiilihydraattipolymeerejä helpommin hyödynnettäviksi mono- ja oligomeereiksi. Elektronimikroskopia osoitti, että kuusipuuaineksen hajotus alkoi puusolujen soluonteloista ja ilmeisesti eteni paksuihin puusoluseiniin ja kohti välilamelleja. Lisäksi massaspektrometrinen analyysi (pyrolyysi-GC/MS) osoitti, että ligniinin hajotusta oli tapahtunut kuuden viikon seuranta-aikana. Nämä havainnot vahvistavat aikaisempia tuloksia rusorypykän erinomaisesta kyvystä hajottaa puuainesta sekä muokata ja pilkkoa ligniiniä sekä ligniinin kaltaisia aromaattisia yhdisteitä. Tulosten perusteella voidaan todeta, että rusorypykkä P. radiata tuottaa valkolahottajille tyypillisen entsyymikirjon ja hyödyntää monipuolisia biokemiallisia reaktioita kierrättääkseen lignoselluloosan hiiltä kasvuympäristössään

Interactions affect hyphal growth and enzyme profiles in combinations of coniferous wood-decaying fungi of Agaricomycetes

Fomitopsis pinicola is a species of Polyporales frequently encountered in Nordic temperate and boreal forests. In nature, the fungus causes destructive brown rot in wood, colonizing tree trunks often occupied by other Basidiomycota species. We mimicked these species-species interactions by introducing F. pinicola to five white rot species, all common saprotrophs of Norway spruce. Hyphal interactions and mycelial growth in various combinations were recorded, while activities of lignocellulose-acting CAZymes and oxidoreductases were followed in co-cultures on two different carbon-source media. Of the species, Phlebia radiata and Trichaptum abietinum were the strongest producers of lignin-modifying oxidoreductases (laccase, manganese peroxidase) when evaluated alone, as well as in co-cultures, on the two different growth media (low-nitrogen liquid medium containing ground coniferous wood, and malt extract broth). F. pinicola was an outstanding producer of oxalic acid (up to 61 mM), whereas presence of P. radiata prevented acidification of the growth environment in the liquid malt-extract cultures. When enzyme profiles of the species combinations were clustered, time-dependent changes were observed on wood-supplemented medium during the eight weeks of growth. End-point acidity and production of mycelium, oxalic acid and oxidoreductase activities, in turn clustered the fungal combinations into three distinct functional groups, determined by the presence of F. pinicola and P. radiata, by principal component analysis. Our findings indicate that combinations of wood-decay fungi have dramatic dynamic effects on the production of lignocellulose-active enzymes, which may lead to divergent degradative processes of dead wood and forest litter.Peer reviewe

Single-step, single-organism bioethanol production and bioconversion of lignocellulose waste materials by phlebioid fungal species

Ethanol production from non-pretreated lignocellulose was carried out in a consolidated bioprocess with wood-decay fungi of phlebioid Polyporales. Ethanol production was attempted on glucose, spruce wood sawdust and waste core board. Substantial quantities of ethanol were achieved, and isolate Phlebia radiata 0043 produced 5.9 g/L of ethanol reaching the yield of 10.4% ethanol from core board lignocellulose substrate. Acidic initial culture conditions (pH 3) induced ethanol fermentation compared to the more neutral environment. Together with bioethanol, the fungi were able to produce organic acids such as oxalate and fumarate, thus broadening their capacity and applicability as efficient organisms to be utilized for bioconversion of various lignocelluloses. In conclusion, fungi of Phlebia grow on, convert and saccharify solid lignocellulose waste materials without pre-treatments resulting in accumulation of ethanol and organic acids. These findings will aid in applying fungal biotechnology for production of biofuels and biocompounds.Peer reviewe

Genome description of Phlebia radiata 79 with comparative genomics analysis on lignocellulose decomposition machinery of phlebioid fungi

Background The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata. Results The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins. Conclusions The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems.Peer reviewe

Genome description of Phlebia radiata 79 with comparative genomics analysis on lignocellulose decomposition machinery of phlebioid fungi

Background The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata. Results The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins. Conclusions The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems.Peer reviewe

Lignocellulose-converting enzyme activity profiles correlate with molecular systematics and phylogeny grouping in the incoherent genus Phlebia (Polyporales, Basidiomycota)

Background. The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species. Methods. Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1 + 5.8S + ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes. Results. Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates. Conclusions. Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales.Peer reviewe

Depolymerization of biorefinery lignin by improved laccases of the white-rot fungus Obba rivulosa

Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic alpha-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.Peer reviewe

Fungal Treatment Modifies Kraft Lignin for Lignin- and Cellulose-Based Carbon Fiber Precursors

The kraft lignin's low molecular weight and too high hydroxyl content hinder its application in bio-based carbon fibers. In this study, we were able to polymerize kraft lignin and reduce the amount of hydroxyl groups by incubating it with the white-rot fungus Obba rivulosa. Enzymatic radical oxidation reactions were hypothesized to induce condensation of lignin, which increased the amount of aromatic rings connected by carbon-carbon bonds. This modification is assumed to be beneficial when aiming for graphite materials such as carbon fibers. Furthermore, the ratio of remaining aliphatic hydroxyls to phenolic hydroxyls was increased, making the structure more favorable for carbon fiber production. When the modified lignin was mixed together with cellulose, the mixture could be spun into intact precursor fibers by using dry-jet wet spinning. The modified lignin leaked less to the spin bath compared with the unmodified lignin starting material, making the recycling of spin-bath solvents easier. The stronger incorporation of modified lignin in the precursor fibers was confirmed by composition analysis, thermogravimetry, and mechanical testing. This work shows how white-rot fungal treatment can be used to modify the structure of lignin to be more favorable for the production of bio-based fiber materials.Peer reviewe

Time-scale dynamics of proteome and transcriptome of the white-rot fungus Phlebia radiata : growth on spruce wood and decay effect on lignocellulose

Abstract Background The white-rot Agaricomycetes species Phlebia radiata is an efficient wood-decaying fungus degrading all wood components, including cellulose, hemicellulose, and lignin. We cultivated P. radiata in solid state cultures on spruce wood, and extended the experiment to 6 weeks to gain more knowledge on the time-scale dynamics of protein expression upon growth and wood decay. Total proteome and transcriptome of P. radiata were analyzed by peptide LC–MS/MS and RNA sequencing at specific time points to study the enzymatic machinery on the fungus’ natural growth substrate. Results According to proteomics analyses, several CAZy oxidoreductase class-II peroxidases with glyoxal and alcohol oxidases were the most abundant proteins produced on wood together with enzymes important for cellulose utilization, such as GH7 and GH6 cellobiohydrolases. Transcriptome additionally displayed expression of multiple AA9 lytic polysaccharide monooxygenases indicative of oxidative cleavage of wood carbohydrate polymers. Large differences were observed for individual protein quantities at specific time points, with a tendency of enhanced production of specific peroxidases on the first 2 weeks of growth on wood. Among the 10 class-II peroxidases, new MnP1-long, characterized MnP2-long and LiP3 were produced in high protein abundances, while LiP2 and LiP1 were upregulated at highest level as transcripts on wood together with the oxidases and one acetyl xylan esterase, implying their necessity as primary enzymes to function against coniferous wood lignin to gain carbohydrate accessibility and fungal growth. Majority of the CAZy encoding transcripts upregulated on spruce wood represented activities against plant cell wall and were identified in the proteome, comprising main activities of white-rot decay. Conclusions Our data indicate significant changes in carbohydrate-active enzyme expression during the six-week surveillance of P. radiata growing on wood. Response to wood substrate is seen already during the first weeks. The immediate oxidative enzyme action on lignin and wood cell walls is supported by detected lignin substructure sidechain cleavages, release of phenolic units, and visual changes in xylem cell wall ultrastructure. This study contributes to increasing knowledge on fungal genetics and lignocellulose bioconversion pathways, allowing us to head for systems biology, development of biofuel production, and industrial applications on plant biomass utilizing wood-decay fungi

Engineering towards catalytic use of fungal class-II peroxidases for dye-decolorizing and conversion of lignin model compounds

Background. Manganese peroxidases (MnP) and lignin peroxidases (LiP) are haem-including fungal secreted class-II peroxidases, which are interesting oxidoreductases in protein engineering aimed at design of biocatalysts for lignin and lignocellulose conversion, dye compound degradation, activation of aromatic compounds, and biofuel production. Objective. Recombinant short-type MnP (Pr-MnP3) of the white rot fungus Phlebia radiata, and its manganese-binding site (E40, E44, D186) directed variants were produced and characterized. To allow catalytic applications, enzymatic bleaching of Reactive Blue 5 and conversion of lignin-like compounds by engineered class-II peroxidases were explored. Method. Pr-MnP3 and its variants were expressed in Escherichia coli. The resultant body proteins were lysed, purified and refolded into haem-including enzymes in 6-7% protein recovery, and examined spectroscopically and kinetically. Results. Successful production of active enzymes was attained, with spectral characteristics of high-spin class-II peroxidases. Recombinant Pr-MnP3 demonstrated high affinity to Mn2+, which was noticeably affected by single (D186H/N) and double (E40H+E44H) mutations. Without addition of Mn2+, Pr-MnP3 was able to oxidize ABTS and decolorize Reactive Blue 5. Pc-LiPH8, its Trp-radical site variants, and engineered CiP-LiP demonstrated conversion of veratryl alcohol and dimeric non-phenolic lignin-model compounds (arylglycerol-β-aryl ethers) with production of veratraldehyde, which is evidence for cation radical formation with subsequent Cα-Cβ cleavage. Pc-LiPH8 and CiP variants were able to effectively oxidize and convert the phenolic dimer (guaiacylglycerol-β–guaiacyl ether). Conclusion. Our results demonstrate suitability of engineered MnP and LiP peroxidases for dye-decolorizing, and efficiency of LiP and its variants for activation and degradation of phenolic and non-phenolic lignin-like aryl ether-linked compounds.Peer reviewe