Enzymatic plant cell wall degradation by the white rot fungus Dichomitus squalens

Basidiomycete white rot fungi are wood-rotting species and their impact to the global carbon cycle is significant. White rot fungi are capable of degrading all the polymeric cell wall components of the plant biomass from polysaccharides, cellulose, hemicelluloses and pectin, to the aromatic heteropolymer lignin. This is due to their ability to produce diverse set of extracellular enzymes that degrade or modify the plant cell wall concomitantly releasing carbon. Research on plant-biomass-degrading fungi has concentrated on isolation and characterization of enzymes especially from the ascomycete fungi for biotechnological applications, such as bioenergy, food processing and waste treatment. More recently genomic studies have opened the reservoir of the plant-biomass-degrading potential of basidiomycete fungi including wood-rotting, litter-decomposing, plant-pathogenic and ectomycorrhizal species. Dichomitus squalens is a white-rot fungus, which colonises softwood and is able to efficiently degrade lignin and cellulose. Previously, intensive studies on white rot fungi have been focused on lignin degradation by oxidative enzymes. The aim of this study was to analyse the potential of the plant-cell-wall-modifying enzymes of D. squalens. Plant biomass degradation by D. squalens was studied at different levels from gene expression to enzyme production. The focus was to dissect the overall degradation of plant biomass polymers, especially cellulose degrading enzymes of D. squalens. The cellulose degradation by D. squalens was studied at the transcript level during growth on spruce wood sticks and in microcrystalline cellulose-containing liquid medium. Selected cellulases and oxidoreductases, which putatively act on cellulose were expressed simultaneously on spruce, the natural substrate of the fungus, and microcrystalline cellulose in time- and substrate-dependent manner. To clarify the adaptation of D. squalens to different plant biomass, the transcriptome and secretome of the fungus were studied in different wood and non-woody substrates. The study confirmed that lignin degradation occurs at the initial stage of growth and D. squalens has retained the diverse enzyme set both for the degradation of wood and non-woody plant biomass. The cellobiohydrolases (CBHs) and cellobiose dehydrogenase of D. squalens were biochemically characterized. In hydrolysis of different plant-derived biomasses, CBHs released reducing sugars alone and in combination with oxidative laccase enzyme. The study shows that D. squalens encodes a complete enzymatic repertoire for plant biomass degradation. In addition, the data emphasise the role of oxidoreductases in the white rot fungal degradation of cellulose and other plant cell wall polymers. Results suggest that white rot fungal plant cell wall converting enzymes are promising candidates in the biotechnological applications using plant biomass.Valkolahosienet ovat puuta lahottavia kantasieniä, joilla on huomattava merkitys maapallon hiilen kierrossa. Ne hajottavat tehokkaasti kasvisoluseinää, joka koostuu pääosin hiilihydraattipolymeereistä eli selluloosasta, hemiselluloosista ja pektiinistä sekä aromaattisesta ligniinipolymeeristä. Tuottamiensa solunulkoisten entsyymien avulla valkolahosienet muokkaavat ja hajottavat näitä yhdisteitä ja siten vapauttavat hiiltä. Kasvibiomassaa hajottavilla entsyymeillä on lukuisia sovelluskohteita esimerkiksi polttoaine-, elintarvike- ja jätteenkäsittelyteollisuudessa. Tähänastinen tutkimus on kohdistunut lähinnä kotelosienten tuottamien entsyymien eristämiseen ja karakterisointiin sekä niiden bioteknologiseen hyödyntämiseen. Viimeisen kymmenen vuoden aikana genomitieto on osoittanut, että myös puuta lahottavien valkolahosienten sekä muiden kasvibiomassaa muokkaavien kantasienten lignoselluloosaa hajottavien entsyymien kirjo on laaja. Onkin todennäköistä, että näillä entsyymeillä on teollisesti kiinnostavia uusia katalyyttisiä ominaisuuksia. Valkolahosienten tutkimuksen pääkohteena ovat perinteisesti olleet ligniiniä hapettavat entsyymit. Tässä työssä täydennettiin kokonaiskuvaa valkolahosienten kasvibiomassan entsymaattisesta hajotuksesta selvittämällä myös kasvisoluseinän hiilihydraattipolymeerien, erityisesti selluloosan, hajotusta. Työn malliorganismiksi valittiin Dichomitus squalens -salokääpä, joka on havupuulla kasvava ja tehokkaasti selluloosaa ja ligniiniä hajottava valkolahosieni. Salokäävän selluloosan hajotuskykyä selvitettiin seuraamalla valikoitujen, solunulkoisia entsyymejä koodaavien geenien ilmentymistä sienen kasvaessa luonnollisella kasvualustallaan, kuusella, ja mikrokiteisellä selluloosalla. Tutkimuksessa havaittiin, että sieni ilmensi kasvunsa aikana yhtäaikaisesti geenejä, jotka koodaavat sellulaaseja ja hiilihydraattipolymeerejä hapetus-pelkistysreaktioilla hajottavia entsyymejä. Lisäksi sienen kasvun vaihe ja kasvualustan hiilen lähde vaikuttivat näiden geenien ilmentymiseen. D. squalens -sienen sopeutumista puu- ja ruohovartisten kasvibiomassojen hajotukseen selvitettiin tutkimalla sienen transkriptomeja ja eksoproteomeja erilaisilta lignoselluloosaa sisältäviltä kasvualustoilta. Nämä analyysit osoittivat, että salokääpä tuotti ligniinin hajottamiseen tarvittavia entsyymejä kasvun alkuvaiheessa, jonka jälkeen sieni eritti hiilihydraattipolymeerejä pilkkovia entsyymejä. Lisäksi työssä tuotettiin ja karakterisoitiin salokäävän mielenkiintoisimmat sellobiohydrolaasi- (CBH) ja sellobioosidehydrogenaasi-entsyymit. Kasviperäisten biomassojen hydrolyysikokeissa salokäävän CBH:t hajottivat selluloosaa pelkistäviksi sokereiksi sekä yksin että yhdessä hapettavan lakkaasientsyymin kanssa. Tässä työssä havaittiin, että D. squalens -sienellä on kattavat entsymaattiset mekanismit erilaisten kasvibiomassojen hajotukseen. Lisäksi tulokset osoittivat, että valkolahosienten tuottamilla hapettavilla entsyymeillä on olennainen rooli selluloosan ja muiden kasvisoluseinän polymeerien pilkkomisessa. Tutkimuksen mukaan valkolahosienten kasvisoluseinää hajottavat entsyymit ovat lupaavia katalyyttejä bioteknologisiin sovelluksiin

Functional diversity in Dichomitus squalens monokaryons

Abstract Dichomitus squalens is a white-rot fungus that colonizes and grows mainly on softwood and is commonly found in the northern parts of Europe, North America, and Asia. We analyzed the genetic and physiological diversity of eight D. squalens monokaryons derived from a single dikaryon. In addition, an unrelated dikaryon and a newly established dikaryon from two of the studied monokaryons were included. Both growth and lignocellulose acting enzyme profiles were highly variable between the studied monokaryotic and dikaryotic strains, demonstrating a high level of diversity within the species

Saccharification of lignocelluloses by carbohydrate active enzymes of the white rot fungus Dichomitus squalens

White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.Peer reviewe

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

Gel electrophoretic (SDS-PAGE) separation and isoelectric focusing (IEF) of <i>D</i>. <i>squalens</i> enzymes.

<p>(A) SDS-PAGE of chromatographically purified CBHI fraction, heterologously produced rCel6A, and purified CDH of <i>D</i>. <i>squalens</i>. Lane 1, CBHI fraction; lane 2, rCel6A; lane 3, <i>T</i>. <i>reesei</i> culture filtrate without <i>cel6a</i> insert; lane 4 CDH. (B) IEF analysis of CBHI fraction of <i>D</i>. <i>squalens</i>.</p

Plant-polysaccharide-degrading enzymes from basidiomycetes

SUMMARY: Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation

Hydrolysis of 1% (w/v) Avicel, sugar beet pulp (SBP) and wheat bran (WB).

<p>Reactions with CBHI fraction (5 μg/mg, red with horizontal stripes); rCel6A (5 μg/mg green with white horizontal stripes; 10 μg/mg, green with white dots); rCel6A and CDH (10 μg/mg and 1 μg/mg, respectively, green with white grid); rCel6A, CDH and laccase (10, 0.5 and 0.5 μg/mg, respectively, blue with grey grid); rCel6A and laccase (10 and 1 μg/mg, respectively, blue with grey vertical stripes); CBHI fraction and rCel6A (5 and 5 μg/mg, respectively, grey with diagonal stripes) or CBHI fraction, rCel6A and laccase (5, 5 and 1 μg/mg, respectively, grey with white vertical stripes), for 4 h at 50°C. Significant differences (p<0.05) in the amount of released reducing sugars between the compared reactions are indicated by asterisk.</p

Characterization of CBHI fraction of <i>D</i>. <i>squalens</i>

<p><b>(</b>A) Temperature (10 min reaction, ■; 1 h reaction, ━) and (B) pH optimum, (C) thermostability at 40 (■), 50 (○), 60 (▲) and 70°C (◇), and (D) pH stability at pH 3.0 (■), pH 4.0 (●), pH 5.0 (△), pH 6.0 (◆), pH 6.5 (□) and in distilled water (○) of the purified CBHI fraction. Reactions were conducted in 50 mM sodium citrate buffer at pH 5.0 with MULac as substrate, except pH stability, which was studied in various buffer pH values and in distilled water. Standard deviations of the activities of three technical replicates are shown as error bars.</p