Complete oxidation of hydroxymethylfurfural to furandicarboxylic acid by aryl-alcohol oxidase

Abstract Background 5-Hydroxymethylfurfural (HMF) is a highly valuable platform chemical that can be obtained from plant biomass carbohydrates. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a renewable substitute for the petroleum-based terephthalic acid in polymer production. Results Aryl-alcohol oxidase (AAO) from the white-rot fungus Pleurotus eryngii is able to oxidize HMF and its derivative 2,5-diformylfuran (DFF) producing formylfurancarboxylic acid (FFCA) thanks to its activity on benzylic alcohols and hydrated aldehydes. Here, we report the ability of AAO to produce FDCA from FFCA, opening up the possibility of full oxidation of HMF by this model enzyme. During HMF reactions, an inhibitory effect of the H2O2 produced in the first two oxidation steps was found to be the cause of the lack of AAO activity on FFCA. In situ monitoring of the whole reaction by 1H-NMR confirmed the absence of any unstable dead-end products, undetected in the HPLC analyses, that could be responsible for the incomplete conversion. The deleterious effect of H2O2 was confirmed by successful HMF conversion into FDCA when the AAO reaction was carried out in the presence of catalase. On the other hand, no H2O2 formation was detected during the slow FFCA conversion by AAO in the absence of catalase, in contrast to typical oxidase reaction with HMF and DFF, suggesting an alternative mechanism as reported in some reactions of related flavo-oxidases. Moreover, several active-site AAO variants that yield nearly complete conversion in shorter reaction times than the wild-type enzyme have been identified. Conclusions The use of catalase to remove H2O2 from the reaction mixture leads to 99% conversion of HMF into FDCA by AAO and several improved variants, although the mechanism of peroxide inhibition of the AAO action on the aldehyde group of FFCA is not fully understood.

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

Biomolecules ◽

10.3390/biom11101526 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1526

Author(s):

Joanna E. Kowalczyk ◽

Shreya Saha ◽

Miia R. Mäkelä

Keyword(s):

Genome Editing ◽

Plant Biomass ◽

Genetic Manipulation ◽

White Rot ◽

White Rot Fungus ◽

Detailed Knowledge ◽

Wild Type ◽

Low Frequencies ◽

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.

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Biodegradability of Extractives in Sapwood and Heartwood from Scots Pine by Sapstain and White-Rot Fungi

Holzforschung ◽

10.1515/hf.1999.042 ◽

1999 ◽

Vol 53 (3) ◽

pp. 247-252 ◽

Cited By ~ 53

Author(s):

M.J. Martínez-Inigo ◽

P. Immerzeel ◽

A. Gutierrez ◽

J.C. del Río ◽

R. Sierra-Alvarez

Keyword(s):

Scots Pine ◽

White Rot Fungi ◽

Inhibitory Effect ◽

Resin Acids ◽

White Rot ◽

White Rot Fungus ◽

Fungal Degradation ◽

Steryl Esters ◽

Funalia Trogii ◽

Total Extractives

SummaryThe fungal degradation of lipophilic extractives in sapwood and heartwood from Scots pine (Pinus sylvestris) was studied. In sapwood, the white rot fungi,Bjerkanderasp. andFunalia trogii, removed higher amounts of extractives than the sapstain strains,Ophiostoma ainoaeandCeratocystis allantospora. Triglycerides, long chain fatty acids, steryl esters and waxes in pine sapwood were almost completely degraded by all the fungi. Sterols and resin acids were also extensively degraded by the white rot strains; however, these components were not or only poorly removed by the sapstain fungi. The removal of total extractives by all the fungal strains was higher in sapwood as compared to heartwood. The highly concentrated extractive fraction in pine heartwood mainly consists of resin acids. As observed in sapwood, sapstain were also poorly effective in the degradation of the resin acids present in heartwood. The fungal degradation of heartwood extractives was not only limited by the degradative ability of the various test microorganisms, but also by the inhibitory effect exerted by the extractive fraction. The white rot fungusF. trogiiwas particularly inhibited on heartwood.Bjerkanderasp. showed a higher tolerance to toxic extractives and was the most efficient fungus in degrading extractive constituents in both Scots pine heartwood and sapwood. Therefore,Bjerkanderasp. strain BOS55 should be considered as a potential agent for pitch control in pulp and paper manufacture.

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Induction of Genes Encoding Plant Cell Wall-Degrading Carbohydrate-Active Enzymes by Lignocellulose-Derived Monosaccharides and Cellobiose in the White-Rot FungusDichomitus squalens

Applied and Environmental Microbiology ◽

10.1128/aem.00403-18 ◽

2018 ◽

Vol 84 (11) ◽

Cited By ~ 11

Author(s):

Sara Casado López ◽

Mao Peng ◽

Tedros Yonatan Issak ◽

Paul Daly ◽

Ronald P. de Vries ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Plant Biomass ◽

Expression Patterns ◽

Fine Tuning ◽

White Rot ◽

White Rot Fungus ◽

Content Type ◽

Dichomitus Squalens

ABSTRACTFungi can decompose plant biomass into small oligo- and monosaccharides to be used as carbon sources. Some of these small molecules may induce metabolic pathways and the production of extracellular enzymes targeted for degradation of plant cell wall polymers. Despite extensive studies in ascomycete fungi, little is known about the nature of inducers for the lignocellulolytic systems of basidiomycetes. In this study, we analyzed six sugars known to induce the expression of lignocellulolytic genes in ascomycetes for their role as inducers in the basidiomycete white-rot fungusDichomitus squalensusing a transcriptomic approach. This identified cellobiose andl-rhamnose as the main inducers of cellulolytic and pectinolytic genes, respectively, ofD. squalens. Our results also identified differences in gene expression patterns between dikaryotic and monokaryotic strains ofD. squalenscultivated on plant biomass-derived monosaccharides and the disaccharide cellobiose. This suggests that despite conservation of the induction between these two genetic forms ofD. squalens, the fine-tuning in the gene regulation of lignocellulose conversion is differently organized in these strains.IMPORTANCEWood-decomposing basidiomycete fungi have a major role in the global carbon cycle and are promising candidates for lignocellulosic biorefinery applications. However, information on which components trigger enzyme production is currently lacking, which is crucial for the efficient use of these fungi in biotechnology. In this study, transcriptomes of the white-rot fungusDichomitus squalensfrom plant biomass-derived monosaccharide and cellobiose cultures were studied to identify compounds that induce the expression of genes involved in plant biomass degradation.

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An extracellular aryl-alcohol oxidase from the white-rot fungus Bjerkandera adusta

Enzyme and Microbial Technology ◽

10.1016/0141-0229(90)90039-s ◽

1990 ◽

Vol 12 (3) ◽

pp. 204-209 ◽

Cited By ~ 75

Author(s):

Andreas Muheim ◽

Roland Waldner ◽

Matti S.A. Leisola ◽

Armin Fiechter

Keyword(s):

Alcohol Oxidase ◽

White Rot ◽

White Rot Fungus ◽

Bjerkandera Adusta

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Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass

Biotechnology for Biofuels ◽

10.1186/s13068-018-1198-5 ◽

2018 ◽

Vol 11 (1) ◽

Cited By ~ 7

Author(s):

Shingo Miyauchi ◽

Anaïs Rancon ◽

Elodie Drula ◽

Hayat Hage ◽

Delphine Chaduli ◽

...

Keyword(s):

Plant Biomass ◽

White Rot ◽

Oxidative Enzymes ◽

White Rot Fungus ◽

Biotechnological Potential ◽

Polyporus Brumalis

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Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass

10.1101/296152 ◽

2018 ◽

Author(s):

Shingo Miyauchi ◽

Anaïs Rancon ◽

Elodie Drula ◽

Delphine Chaduli ◽

Anne Favel ◽

...

Keyword(s):

Wheat Straw ◽

Lignin Degradation ◽

Plant Biomass ◽

White Rot ◽

Fungal Genome ◽

Oxidative Enzymes ◽

White Rot Fungus ◽

Large Set ◽

Combining Data ◽

Polyporus Brumalis

AbstractWhite-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found the fungus possessed an unexpectedly large set of genes coding for enzymes related to lignin degradation, and that these were highly expressed and massively secreted under solid-state fermentation conditions. The examination of interrelated multi-omics patterns revealed the coordinated regulation of lignin-active peroxidases and H2O2-generating enzymes along with the activation of cellular mechanisms for detoxification, which combined to result in the efficient lignin breakdown by the fungus.ImportancePlant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e. agriculture and forestry by-products) are major obstacles for biomass conversions. From a biotechnological aspect, these compounds could be a potential source of aromatic platform molecules for bio-based polymers. Here we describe the extraordinary ability of Polyporus brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. We observed unusual expansions of gene families coding for; 1) Class II peroxidases involved in lignin degradation; and 2) GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity. Our findings suggested the fungus massively mobilizes this oxidative machinery during growth on wheat straw. Overall, we identified sets of co-regulated enzymes, which could potentially augment the efficiency of biotechnological plant biomass conversions.

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Glucose-Mediated Repression of Plant Biomass Utilization in the White-Rot Fungus Dichomitus squalens

Applied and Environmental Microbiology ◽

10.1128/aem.01828-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 2

Author(s):

Paul Daly ◽

Mao Peng ◽

Marcos Di Falco ◽

Anna Lipzen ◽

Mei Wang ◽

...

Keyword(s):

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Plant Biomass ◽

Carbon Sources ◽

White Rot Fungi ◽

White Rot ◽

White Rot Fungus ◽

Content Type ◽

Dichomitus Squalens ◽

Genes Encoding

ABSTRACT The extent of carbon catabolite repression (CCR) at a global level is unknown in wood-rotting fungi, which are critical to the carbon cycle and are a source of biotechnological enzymes. CCR occurs in the presence of sufficient concentrations of easily metabolizable carbon sources (e.g., glucose) and involves downregulation of the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. We investigated this phenomenon in the white-rot fungus Dichomitus squalens using transcriptomics and exoproteomics. In D. squalens cultures, approximately 7% of genes were repressed in the presence of glucose compared to Avicel or xylan alone. The glucose-repressed genes included the essential components for utilization of plant biomass—carbohydrate-active enzyme (CAZyme) and carbon catabolic genes. The majority of polysaccharide-degrading CAZyme genes were repressed and included activities toward all major carbohydrate polymers present in plant cell walls, while repression of ligninolytic genes also occurred. The transcriptome-level repression of the CAZyme genes observed on the Avicel cultures was strongly supported by exoproteomics. Protease-encoding genes were generally not glucose repressed, indicating their likely dominant role in scavenging for nitrogen rather than carbon. The extent of CCR is surprising, given that D. squalens rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of D. squalens for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains. IMPORTANCE White-rot fungi are critical to the carbon cycle because they can mineralize all wood components using enzymes that also have biotechnological potential. The occurrence of carbon catabolite repression (CCR) in white-rot fungi is poorly understood. Previously, CCR in wood-rotting fungi has only been demonstrated for a small number of genes. We demonstrated widespread glucose-mediated CCR of plant biomass utilization in the white-rot fungus Dichomitus squalens. This indicates that the CCR mechanism has been largely retained even though wood-rotting fungi rarely experience commonly considered CCR conditions in their woody environment. The general lack of repression of genes encoding proteases along with the reduction in secreted CAZymes during CCR suggested that the retention of CCR may be connected with the need to conserve nitrogen use during growth on nitrogen-scarce wood. The widespread repression indicates that derepressed strains could be beneficial for enzyme production.

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Aryl-alcohol oxidase and lignin peroxidase from the white-rot fungus Bjerkandera adusta

Journal of Biotechnology ◽

10.1016/0168-1656(90)90101-g ◽

1990 ◽

Vol 13 (2-3) ◽

pp. 159-167 ◽

Cited By ~ 40

Author(s):

Andreas Muheim ◽

Matti S.A. Leisola ◽

Hans E. Schoemaker

Keyword(s):

Lignin Peroxidase ◽

Alcohol Oxidase ◽

White Rot ◽

White Rot Fungus ◽

Bjerkandera Adusta

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Characterization of a novel laccase purified from the fungus Hohenbuehelia serotina and its decolourisation of dyes.

Acta Biochimica Polonica ◽

10.18388/abp.2015_1091 ◽

2015 ◽

Vol 63 (2) ◽

Cited By ~ 3

Author(s):

Yingyin Xu ◽

Yuxiao Lu ◽

Rui Zhang ◽

Hexiang Wang ◽

Qinghong Liu

Keyword(s):

Molecular Weight ◽

Enzyme Activity ◽

Enzymatic Activity ◽

Inhibitory Effect ◽

Single Band ◽

White Rot ◽

White Rot Fungus ◽

Sds Page

A novel laccase was purified from the white rot fungus, Hohenbuehelia serotina, to investigate the applications of this laccase in the decoloration of various dyes. SDS-PAGE revealed a single band of this laccase corresponding to a molecular weight of approximately 57.8 kDa. The enzyme showed activity towards several substrates, the most sensitive of which was 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). The highest enzymatic activity using ABTS as a substrate was observed at pH 6.8 and 30°C. The enzyme activity was found to be significantly enhanced in the presence of Zn(2+) ions and inhibited by Fe(2+) ions. Moreover, SDS and β-mercaptoethanol were inhibitory, and inhibition by L-cysteine was observed while EDTA and DMSO had almost no inhibitory effect. The laccase could effectively decolorize seven different dyes within 30 minutes at 40°C.

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