The white-rot fungi Phlebia radiata and Dichomitus squalens in wood-based cultures : expression of laccases, lignin peroxidases, and oxalate decarboxylase

Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.Kantasieniin kuuluvat valkolahosienet ovat ainoita organismeja, jotka hajottavat tehokkaasti puun kaikkia komponentteja. Tämän lisäksi ne pystyvät mineralisoimaan vaikeasti hajoavaa ligniinipolymeeriä solujensa ulkopuolelle erittyvien, hapettavien ligniiniä muokkaavia entsyymien avulla. Näitä entsyymejä, joista valkolahosienet tuottavat tyypillisesti useita eri geenien koodaamia isoentsyymejä, ovat lakkaasi, ligniiniperoksidaasi (LiP), mangaaniperoksidaasi (MnP) ja versatiiliperoksidaasi (VP). -- Tässä tutkimuksessa keskityttiin kahteen ligniiniä tehokkaasti hajottavaan valkolahosieneen: rusorypykkään (Phlebia radiata) ja salokääpään (Dichomitus squalens). Molekyylitason tietoa Suomesta eristetyn P. radiata FBCC43 (79, ATCC 64658) sienen ligniiniä muokkaavista entsyymeistä täydennettiin kloonaamalla ja karakterisoimalla yksi lakkaasi (Pr-lac2) ja kolme lip-geeniä (Pr-lip1, Pr-lip3, Prlip4), joista vain Pr-lip3 on aiemmin kloonattu cDNA:sta. D. squalensista kloonattiin ja karakterisoitiin ensimmäisen kerran kaksi lakkaasigeeniä (Ds-lac3, Ds-lac4). Fylogeneettinen analyysi osoitti P. radiatan LiP-isoentsyymien olevan evolutiivisesti hyvin lähellä toisiaan. P. radiatan ja D. squalensin lakkaasit ovat puolestaan kaukaista sukua toisilleen, joten ne saattavat osallistua erilaisiin fysiologisiin toimintoihin. Lakkaasiaktiivisuus nousi huomattavasti, kun P. radiatan liemiviljelmiin lisättiin ylimäärin Cu2+-ioneja. Sieni kasvoi hyvin ravinteikkaissa, runsaasti orgaanista typpeä sisältävissä liemiviljelmissä. Puu on valkolahosienten luonnollinen kasvualusta, jolla sienet tuottavat kasvuunsa ja lignoselluloosan hajottamiseen tarvittavia entsyymejä ja aineenvaihduntatuotteita. Tässä työssä valkolahosieniä kasvatettiin puulla ja puuta sisältävillä, luonnollisia kasvuolosuhteita jäljittelevillä alustoilla. Transkriptianalyysit osoittivat kaikkien P. radiatan tunnettujen ligniiniä muokkaavia entsyymien ja D. squalensin lakkaasien ilmentyvän sienten kasvaessa puulla. Yksittäisten P. radiatan lip- ja lakkaasigeenien sekä D. squalensin lakkaasigeenien transkriptimäärissä oli eroja kasvun aikana. Näiden tulosten perusteella useilla ligniiniä muokkaavilla isoentsyymeillä on tärkeä merkitys valkolahosienten kasvaessa puulla. Entsyymien lisäksi valkolahosienet erittävät puun ja ligniinin hajotukseen osallistuvia yhdisteitä, kuten oksaalihappoa, joka on puuta lahottavien sienten yleinen aineenvaihduntatuote. Sienet tuottavat myös oksaalihappoa hajottavia entsyymejä, joista yleisin on oksalaattidekarboksylaasi (ODC). ODC:n merkitystä sienissä ei toistaiseksi tunneta tarkasti. Entsyymin on ehdotettu säätelevän solunsisäistä ja -ulkoista oksaalihappopitoisuutta sekä osallistuvan ATP:n tuottoon. Tässä tutkimuksessa solunsisäistä ODC-aktiivisuutta havaittiin neljällä uudella valkolahosienikannalla. Korkein ODC-aktiivisuus mitattiin D. squalensilla kasvualustoihin tehdyn oksaalihappolisäyksen jälkeen. Oksaalihappo oli ODC:a tuottavien sienten yleisimmin erittämä ja ainoa puualustalla havaittu orgaaninen happo. Työssä kahdesta D. squalens kannasta kloonattu odc-geeni on ensimmäinen valkolahottajakäävästä kuvattu odc-geeni. D. squalensin ODC oli biokemiallisilta ominaisuuksiltaan samankaltainen muista kantasienistä kuvattujen ODC-proteiinien kanssa. Ds-odc -geenistä käännetyn aminohapposekvenssin mukaan sen koodaaman proteiinin N-terminaalissa on kuitenkin uudenlainen, noin 60 aminohapon pituinen paljon alaniinia ja seriiniä sisältävä toistojakso. Ds-odc geenin ilmentymisen perusteella sen koodaamaa entsyymiä tuotetaan konstitutiivisesti. ODC saattaakin olla tärkeä entsyymi puuta lahottavien sienten kasvulle ja perusaineenvaihdunnalle

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi

Identification of an l-Arabitol Transporter from Aspergillus niger

l-arabitol is an intermediate of the pentose catabolic pathway in fungi but can also be used as a carbon source by many fungi, suggesting the presence of transporters for this polyol. In this study, an l-arabitol transporter, LatA, was identified in Aspergillus niger. Growth and expression profiles as well as sugar consumption analysis indicated that LatA only imports l-arabitol and is regulated by the arabinanolytic transcriptional activator AraR. Moreover, l-arabitol production from wheat bran was increased in a metabolically engineered A. niger mutant by the deletion of latA, indicating its potential for improving l-arabitol-producing cell factories. Phylogenetic analysis showed that homologs of LatA are widely conserved in fungi

Prosessoitujen lietteiden ja lannan käytöstä fosforilannoitteena

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Sensing the Rhythm

Glassblowing is a craft in which both human and material movement are essential. However, what kind of movement is required in glassblowing, and why does that movement happen? This article presents a practice-led case study conducted in a glassblowing studio. During a glassblowing session, clear drinking glass blanks are blown in a wooden turn mould. The process of glassblowing is documented on video and analysed using the visual data. Additionally, diary notes and participant observation are used to understand and contextualise video data more profoundly. The analysis focuses on the movement of the two main actants of the process: the human movement of the glassblower and the non-human movement of the hot glass. Altogether, six categories were identified to represent the human and material activity. This article concludes that the movement under investigation is relational between the glassblower and the hot glass, and it happens as a consequence of the glassblowers’ situated embodied knowledge. Furthermore, we discovered that research in the processes of glassblowing offers a rich ground for practice-led research that adopts relational ontology and sociomateriality as its theoretical perspectives. The purpose of this study is to fortify the craft of glassblowing as a vital practice in the fields of art, design, and craft

Occurrence and function of enzymes for lignocellulose degradation in commercial Agaricus bisporus cultivation

The white button mushroom Agaricus bisporus is economically the most important commercially produced edible fungus. It is grown on carbon- and nitrogen-rich substrates, such as composted cereal straw and animal manure. The commercial mushroom production process is usually performed in buildings or tunnels under highly controlled environmental conditions. In nature, the basidiomycete A. bisporus has a significant impact on the carbon cycle in terrestrial ecosystems as a saprotrophic decayer of leaf litter. In this mini-review, the fate of the compost plant cell wall structures, xylan, cellulose and lignin, is discussed. A comparison is made from the structural changes observed to the occurrence and function of enzymes for lignocellulose degradation present, with a special focus on the extracellular enzymes produced by A. bisporus. In addition, recent advancements in whole genome level molecular studies in various growth stages of A. bisporus in compost are reviewed.Peer reviewe

Safety of tacrolimus 0.03% and 0.1% ointments in young children with atopic dermatitis: a 36-month follow-up study

Summary Background Topical tacrolimus is used off-label in young children, but data are limited on its use in children under 2?years of age and for long-term treatment. Aim To compare safety differences between topical tacrolimus (0.03% and 0.1% ointments) and topical corticosteroids (mild and moderate potency) in young children with atopic dermatitis (AD). Methods We conducted a 36-month follow-up study with 152 young children aged 1?3?years with moderate to severe AD. The children were followed up prospectively, and data were collected on infections, disease severity, growth parameters, vaccination responses and other relevant laboratory tests were gathered. Results There were no significant differences between the treatment groups for skin-related infections (SRIs) (P?=?0.20), non-SRIs (P?=?0.20), growth parameters height (P?=?0.60), body weight (P?=?0.81), Eczema Area and Severity Index (EASI) (P?=?0.19), vaccination responses (P?=?0.62), serum cortisone levels (P?=?0.23) or serum levels of interleukin (IL)-4, IL-10, IL-12, IL-31 and interferon-?. EASI decreased significantly in both groups (P?Peer reviewe

Recombinant production and characterization of six novel GH27 and GH36 alpha-galactosidases from Penicillium subrubescens and their synergism with a commercial mannanase during the hydrolysis of lignocellulosic biomass

alpha-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular alpha-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All alpha-galactosidases exhibited high affinity to pNP alpha Gal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining alpha-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other alpha-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal alpha-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses.Peer reviewe