Publications by authors named "Kristiina S Hildén"

25 Publications

  • Page 1 of 1

Discovery and Functional Analysis of a Salicylic Acid Hydroxylase from Aspergillus niger.

Appl Environ Microbiol 2021 02 26;87(6). Epub 2021 Feb 26.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands

Salicylic acid plays an important role in the plant immune response, and its degradation is therefore important for plant-pathogenic fungi. However, many nonpathogenic microorganisms can also degrade salicylic acid. In the filamentous fungus , two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid, followed by decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). cleaves the aromatic ring of catechol catalyzed by catechol 1,2-dioxygenase (CrcA) to form ,-muconic acid. However, the identification and role of the genes and characterization of the enzymes involved in these pathways are lacking. In this study, we used transcriptome data of grown on salicylic acid to identify genes ( and ) involved in salicylic acid metabolism. Heterologous production in followed by biochemical characterization confirmed the function of ShyA and CrcA. The combination of ShyA and CrcA demonstrated that -muconic acid can be produced from salicylic acid. In addition, the roles of , , and were studied by creating deletion mutants which revealed the role of these genes in the fungal metabolism of salicylic acid. Nonrenewable petroleum sources are being depleted, and therefore, alternative sources are needed. Plant biomass is one of the most abundant renewable sources on Earth and is efficiently degraded by fungi. In order to utilize plant biomass efficiently, knowledge about the fungal metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable compounds such as ,-muconic acid. ,-Muconic acid is an important platform chemical that is used to synthesize nylon, polyethylene terephthalate (PET), polyurethane, resins, and lubricants. Currently, ,-muconic acid is mainly produced through chemical synthesis from petroleum-based chemicals. Here, we show that two enzymes from fungi can be used to produce ,-muconic acid from salicylic acid and contributes in creating alternative methods for the production of platform chemicals.
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http://dx.doi.org/10.1128/AEM.02701-20DOI Listing
February 2021

Impacts of holmium and lithium to the growth of selected basidiomycetous fungi and their ability to degrade textile dyes.

3 Biotech 2020 Aug 28;10(8):357. Epub 2020 Jul 28.

Department of Microbiology, University of Helsinki, Biocenter 1, PO Box 56, 00014 Helsinki, Finland.

The impacts of Ho and Li (0, 10, 50, 200 mg/L) were tested towards the growth of four basidiomycetous fungal species, their ability to decolorise synthetic dyes (Reactive Green 19, Reactive Orange 16, Reactive Black 5), and produce oxidative enzymes. All species; , , and grew with and without supplemented Ho or Li. The growth of was the most tolerant species towards Ho or Li (200 mg/L), whereas the growth of was the most sensitive of the studied species to both 200 mg Ho or Li/L. All fungi oxidized ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] forming colour zone on plate tests indicating production of lignin modifying laccase enzyme. and formed black MnO zone in Mn plates, which indicates the production of manganese peroxidase (MnP). and decolorised Reactive Black 5 indicating the production of versatile peroxide (VP) enzyme. Our study presents two new candidate species able to produce VP. was capable of decolorising all tested synthetic dyes in the presence of Ho or Li (0-200 mg/L) suggesting that this fungus is a promising species for bioremediation of multi dye-containing wastes.
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http://dx.doi.org/10.1007/s13205-020-02355-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387387PMC
August 2020

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus.

DNA Res 2020 Apr;27(2)

INRAE, UMR1163, Biodiversity and Biotechnology of Fungi, Aix Marseille University, 13009 Marseille, France.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.
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http://dx.doi.org/10.1093/dnares/dsaa011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406137PMC
April 2020

Penicillium subrubescens adapts its enzyme production to the composition of plant biomass.

Bioresour Technol 2020 Sep 5;311:123477. Epub 2020 May 5.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Electronic address:

Penicillium subrubescens is able to degrade a broad range of plant biomass and it has an expanded set of Carbohydrate Active enzyme (CAZyme)-encoding genes in comparison to other Penicillium species. Here we used exoproteome and transcriptome analysis to demonstrate the versatile plant biomass degradation mechanism by P. subrubescens during growth on wheat bran and sugar beet pulp. On wheat bran P. subrubescens degraded xylan main chain and side residues from Day 2 of cultivation, whereas it started to degrade side chains of pectin in sugar beet pulp prior to attacking the main chain on Day 3. In addition, on Day 3 the cellulolytic enzymes were highly increased. Our results confirm that P. subrubescens adapts its enzyme production to the available plant biomass and is a promising new fungal cell factory for the production of CAZymes.
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http://dx.doi.org/10.1016/j.biortech.2020.123477DOI Listing
September 2020

Colonies of the fungus Aspergillus niger are highly differentiated to adapt to local carbon source variation.

Environ Microbiol 2020 03 6;22(3):1154-1166. Epub 2020 Jan 6.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

Saprobic fungi, such as Aspergillus niger, grow as colonies consisting of a network of branching and fusing hyphae that are often considered to be relatively uniform entities in which nutrients can freely move through the hyphae. In nature, different parts of a colony are often exposed to different nutrients. We have investigated, using a multi-omics approach, adaptation of A. niger colonies to spatially separated and compositionally different plant biomass substrates. This demonstrated a high level of intra-colony differentiation, which closely matched the locally available substrate. The part of the colony exposed to pectin-rich sugar beet pulp and to xylan-rich wheat bran showed high pectinolytic and high xylanolytic transcript and protein levels respectively. This study therefore exemplifies the high ability of fungal colonies to differentiate and adapt to local conditions, ensuring efficient use of the available nutrients, rather than maintaining a uniform physiology throughout the colony.
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http://dx.doi.org/10.1111/1462-2920.14907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065180PMC
March 2020

Cinnamic Acid and Sorbic acid Conversion Are Mediated by the Same Transcriptional Regulator in .

Front Bioeng Biotechnol 2019 27;7:249. Epub 2019 Sep 27.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Utrecht, Netherlands.

Cinnamic acid is an aromatic compound commonly found in plants and functions as a central intermediate in lignin synthesis. Filamentous fungi are able to degrade cinnamic acid through multiple metabolic pathways. One of the best studied pathways is the non-oxidative decarboxylation of cinnamic acid to styrene. In , the enzymes cinnamic acid decarboxylase (CdcA, formally ferulic acid decarboxylase) and the flavin prenyltransferase (PadA) catalyze together the non-oxidative decarboxylation of cinnamic acid and sorbic acid. The corresponding genes, and , are clustered in the genome together with a putative transcription factor previously named sorbic acid decarboxylase regulator (SdrA). While SdrA was predicted to be involved in the regulation of the non-oxidative decarboxylation of cinnamic acid and sorbic acid, this was never functionally analyzed. In this study, deletion mutants of , and were made to further investigate the role of SdrA in cinnamic acid metabolism. Phenotypic analysis revealed that and are exclusively involved in the degradation of cinnamic acid and sorbic acid and not required for other related aromatic compounds. Whole genome transcriptome analysis of Δ grown on different cinnamic acid related compounds, revealed additional target genes, which were also clustered with , and in the genome. Synteny analysis using 30 genomes demonstrated a conserved cinnamic acid decarboxylation gene cluster in most Aspergilli of the Nigri clade. Aspergilli lacking certain genes in the cluster were unable to grow on cinnamic acid, but could still grow on related aromatic compounds, confirming the specific role of these three genes for cinnamic acid metabolism of .
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http://dx.doi.org/10.3389/fbioe.2019.00249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776626PMC
September 2019

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi.

Biotechnol Adv 2019 11 7;37(7):107396. Epub 2019 May 7.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Department of Microbiology, University of Helsinki, Viikinkaari 9, Helsinki, Finland. Electronic address:

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.
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http://dx.doi.org/10.1016/j.biotechadv.2019.05.002DOI Listing
November 2019

Draft Genome Sequences of Three Monokaryotic Isolates of the White-Rot Basidiomycete Fungus Dichomitus squalens.

Microbiol Resour Announc 2019 May 2;8(18). Epub 2019 May 2.

Fungal Physiology, Westerdijk Fungal Biodiversity Centre, Utrecht, The Netherlands

Here, we report the draft genome sequences of three isolates of the wood-decaying white-rot basidiomycete fungus The genomes of these monokaryons were sequenced to provide more information on the intraspecies genomic diversity of this fungus and were compared to the previously sequenced genome of LYAD-421 SS1.
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http://dx.doi.org/10.1128/MRA.00264-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498232PMC
May 2019

Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation.

Biotechnol Adv 2019 11 27;37(6):107361. Epub 2019 Feb 27.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Electronic address:

Fungal strain engineering is commonly used in many areas of biotechnology, including the production of plant biomass degrading enzymes. Its aim varies from the production of specific enzymes to overall increased enzyme production levels and modification of the composition of the enzyme set that is produced by the fungus. Strain engineering involves a diverse range of methodologies, including classical mutagenesis, genetic engineering and genome editing. In this review, the main approaches for strain engineering of filamentous fungi in the field of plant biomass degradation will be discussed, including recent and not yet implemented methods, such as CRISPR/Cas9 genome editing and adaptive evolution.
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http://dx.doi.org/10.1016/j.biotechadv.2019.02.017DOI Listing
November 2019

Dichomitus squalens partially tailors its molecular responses to the composition of solid wood.

Environ Microbiol 2018 11 18;20(11):4141-4156. Epub 2018 Oct 18.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.

White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.
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http://dx.doi.org/10.1111/1462-2920.14416DOI Listing
November 2018

Draft Genome Sequence of the Basidiomycete White-Rot Fungus Phlebia centrifuga.

Genome Announc 2018 Apr 5;6(14). Epub 2018 Apr 5.

Department of Microbiology, University of Helsinki, Helsinki, Finland

Here, we report the genome sequence of wood-decaying white-rot fungus strain FBCC195, isolated from Norway spruce () in Finnish Lapland. The 34.66-Mb genome containing 13,785 gene models is similar to the genome length reported for other saprobic white-rot species.
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http://dx.doi.org/10.1128/genomeA.01414-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887033PMC
April 2018

Fungal feruloyl esterases: Functional validation of genome mining based enzyme discovery including uncharacterized subfamilies.

N Biotechnol 2018 Mar 23;41:9-14. Epub 2017 Nov 23.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands. Electronic address:

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs.
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http://dx.doi.org/10.1016/j.nbt.2017.11.004DOI Listing
March 2018

Fungal glucuronoyl esterases: Genome mining based enzyme discovery and biochemical characterization.

N Biotechnol 2018 Jan 16;40(Pt B):282-287. Epub 2017 Oct 16.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Electronic address:

4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl d-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.
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http://dx.doi.org/10.1016/j.nbt.2017.10.003DOI Listing
January 2018

Genetic transformation of the white-rot fungus Dichomitus squalens using a new commercial protoplasting cocktail.

J Microbiol Methods 2017 12 4;143:38-43. Epub 2017 Oct 4.

Department of Food and Environmental Sciences, University of Helsinki, Viikinkaari 9, Helsinki, Finland. Electronic address:

D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.
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http://dx.doi.org/10.1016/j.mimet.2017.10.001DOI Listing
December 2017

Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC.

N Biotechnol 2017 Jul 8;37(Pt B):200-209. Epub 2017 Mar 8.

Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, Viikki Biocenter 1, University of Helsinki, Finland. Electronic address:

A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass.
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http://dx.doi.org/10.1016/j.nbt.2017.02.007DOI Listing
July 2017

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

Authors:
Ronald P de Vries Robert Riley Ad Wiebenga Guillermo Aguilar-Osorio Sotiris Amillis Cristiane Akemi Uchima Gregor Anderluh Mojtaba Asadollahi Marion Askin Kerrie Barry Evy Battaglia Özgür Bayram Tiziano Benocci Susanna A Braus-Stromeyer Camila Caldana David Cánovas Gustavo C Cerqueira Fusheng Chen Wanping Chen Cindy Choi Alicia Clum Renato Augusto Corrêa Dos Santos André Ricardo de Lima Damásio George Diallinas Tamás Emri Erzsébet Fekete Michel Flipphi Susanne Freyberg Antonia Gallo Christos Gournas Rob Habgood Matthieu Hainaut María Laura Harispe Bernard Henrissat Kristiina S Hildén Ryan Hope Abeer Hossain Eugenia Karabika Levente Karaffa Zsolt Karányi Nada Kraševec Alan Kuo Harald Kusch Kurt LaButti Ellen L Lagendijk Alla Lapidus Anthony Levasseur Erika Lindquist Anna Lipzen Antonio F Logrieco Andrew MacCabe Miia R Mäkelä Iran Malavazi Petter Melin Vera Meyer Natalia Mielnichuk Márton Miskei Ákos P Molnár Giuseppina Mulé Chew Yee Ngan Margarita Orejas Erzsébet Orosz Jean Paul Ouedraogo Karin M Overkamp Hee-Soo Park Giancarlo Perrone Francois Piumi Peter J Punt Arthur F J Ram Ana Ramón Stefan Rauscher Eric Record Diego Mauricio Riaño-Pachón Vincent Robert Julian Röhrig Roberto Ruller Asaf Salamov Nadhira S Salih Rob A Samson Erzsébet Sándor Manuel Sanguinetti Tabea Schütze Kristina Sepčić Ekaterina Shelest Gavin Sherlock Vicky Sophianopoulou Fabio M Squina Hui Sun Antonia Susca Richard B Todd Adrian Tsang Shiela E Unkles Nathalie van de Wiele Diana van Rossen-Uffink Juliana Velasco de Castro Oliveira Tammi C Vesth Jaap Visser Jae-Hyuk Yu Miaomiao Zhou Mikael R Andersen David B Archer Scott E Baker Isabelle Benoit Axel A Brakhage Gerhard H Braus Reinhard Fischer Jens C Frisvad Gustavo H Goldman Jos Houbraken Berl Oakley István Pócsi Claudio Scazzocchio Bernhard Seiboth Patricia A vanKuyk Jennifer Wortman Paul S Dyer Igor V Grigoriev

Genome Biol 2017 02 14;18(1):28. Epub 2017 Feb 14.

US Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, 94598, USA.

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.

Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli.

Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.
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http://dx.doi.org/10.1186/s13059-017-1151-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5307856PMC
February 2017

Diversity of fungal feruloyl esterases: updated phylogenetic classification, properties, and industrial applications.

Biotechnol Biofuels 2016 28;9:231. Epub 2016 Oct 28.

Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584CT Utrecht, The Netherlands ; Division of Microbiology and Biotechnology, Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland.

Feruloyl esterases (FAEs) represent a diverse group of carboxyl esterases that specifically catalyze the hydrolysis of ester bonds between ferulic (hydroxycinnamic) acid and plant cell wall polysaccharides. Therefore, FAEs act as accessory enzymes to assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass conversion. Their ability to release ferulic acid and other hydroxycinnamic acids from plant biomass makes FAEs potential biocatalysts in a wide variety of applications such as in biofuel, food and feed, pulp and paper, cosmetics, and pharmaceutical industries. This review provides an updated overview of the knowledge on fungal FAEs, in particular describing their role in plant biomass degradation, diversity of their biochemical properties and substrate specificities, their regulation and conditions needed for their induction. Furthermore, the discovery of new FAEs using genome mining and phylogenetic analysis of current publicly accessible fungal genomes will also be presented. This has led to a new subfamily classification of fungal FAEs that takes into account both phylogeny and substrate specificity.
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http://dx.doi.org/10.1186/s13068-016-0651-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5084320PMC
October 2016

Penicillium subrubescens is a promising alternative for Aspergillus niger in enzymatic plant biomass saccharification.

N Biotechnol 2016 Dec 25;33(6):834-841. Epub 2016 Jul 25.

Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, Viikki Biocenter 1, University of Helsinki, Finland.

In industrial applications, efficient mixtures of polysaccharide-degrading enzymes are needed to convert plant biomass into fermentable sugars. Most of the commercially produced lignocellulolytic enzymes are from a limited number of filamentous fungi, such as Trichoderma and Aspergillus species. In contrast, the plant biomass-degrading capacity of Penicillia has been less explored. We performed growth profiling of several Penicillia on diverse plant biomass-related substrates demonstrating the capacity particularly of Penicillium subrubescens to degrade crude lignocellulose feedstock, as well as polysaccharides, and metabolise their monomeric components. We focussed on the lignocellulolytic potential of P. subrubescens FBCC1632, which produced a variable set of (hemi-)cellulolytic activities on plant biomass substrates with activity levels comparable to those of Aspergillus niger. The good ability of the extracellular enzyme mixtures produced by P. subrubescens to saccharify complex plant biomasses, wheat bran and sugar beet pulp, indicated a high potential for this strain as a producer of industrial enzyme cocktails.
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http://dx.doi.org/10.1016/j.nbt.2016.07.014DOI Listing
December 2016

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

Biotechnol Biofuels 2015 1;8:107. Epub 2015 Aug 1.

Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre and Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

Background: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails.

Results: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition.

Conclusions: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.
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http://dx.doi.org/10.1186/s13068-015-0285-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522099PMC
August 2015

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

Environ Microbiol 2015 Aug 4;17(8):3098-109. Epub 2015 Aug 4.

Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre & Fungal Molecular Physiology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush.
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http://dx.doi.org/10.1111/1462-2920.12967DOI Listing
August 2015

Aromatic metabolism of filamentous fungi in relation to the presence of aromatic compounds in plant biomass.

Adv Appl Microbiol 2015 24;91:63-137. Epub 2015 Feb 24.

Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.

The biological conversion of plant lignocellulose plays an essential role not only in carbon cycling in terrestrial ecosystems but also is an important part of the production of second generation biofuels and biochemicals. The presence of the recalcitrant aromatic polymer lignin is one of the major obstacles in the biofuel/biochemical production process and therefore microbial degradation of lignin is receiving a great deal of attention. Fungi are the main degraders of plant biomass, and in particular the basidiomycete white rot fungi are of major importance in converting plant aromatics due to their ability to degrade lignin. However, the aromatic monomers that are released from lignin and other aromatic compounds of plant biomass are toxic for most fungi already at low levels, and therefore conversion of these compounds to less toxic metabolites is essential for fungi. Although the release of aromatic compounds from plant biomass by fungi has been studied extensively, relatively little attention has been given to the metabolic pathways that convert the resulting aromatic monomers. In this review we provide an overview of the aromatic components of plant biomass, and their release and conversion by fungi. Finally, we will summarize the applications of fungal systems related to plant aromatics.
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http://dx.doi.org/10.1016/bs.aambs.2014.12.001DOI Listing
August 2015

An improved and reproducible protocol for the extraction of high quality fungal RNA from plant biomass substrates.

Fungal Genet Biol 2014 Nov 18;72:201-206. Epub 2014 Jun 18.

Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland.

Isolation of high quantity and quality RNA is a crucial step in the detection of meaningful gene expression data. Obtaining intact fungal RNA from complex lignocellulosic substrates is often difficult, producing low integrity RNA which perform poorly in downstream applications. In this study we developed an RNA extraction method using CsCl centrifugation procedure, modified from previous reports and adapted for isolation of RNA from plant biomass. This method provided high level of integrity and good quantity of RNA which were suitable for reliable analyses of gene expression and produced consistent and reproducible results.
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http://dx.doi.org/10.1016/j.fgb.2014.06.001DOI Listing
November 2014

Molecular characterization of the basidiomycete isolate Nematoloma frowardii b19 and its manganese peroxidase places the fungus in the corticioid genus Phlebia.

Microbiology (Reading) 2008 Aug;154(Pt 8):2371-2379

Laboratory of Fungal Biotechnology, Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, FIN-00014 Helsinki, Finland.

The basidiomycete isolate b19, originally identified by morphological characteristics of the fruiting body as Nematoloma frowardii, efficiently produces manganese peroxidase (MNP) and is used for degradation of natural, persistent aromatic polymers (lignin, humic acids and brown coal components). The N. frowardii MNP has shown good activity in conversion of xenobiotic compounds such as polycyclic hydrocarbons and trinitrotoluene. However, this biotechnologically promising fungus has not previously been studied at the molecular biology level. We show here that according to the molecular characterization of its main MNP isozyme, Nf b19 MNP2, and partial sequencing of its MNP3-, three lignin peroxidase- and two laccase-encoding genes, and the gene encoding the ribosomal SSU 18S RNA, that the fungus has a close phylogenetic relationship to the white-rot basidiomycete Phlebia radiata (Fr.). Ribosomal internal transcribed spacer (ITS) sequence (ITS1+5.8S+ITS2) phylogeny reclassifies Nf b19 as a possible representative of a new species of the genus Phlebia, nearest to the Phlebia acerina clade. The genus Phlebia belongs to a completely different family (Corticiaceae) and order (Aphyllophorales) within the phylum Basidiomycota than the genus Nematoloma, which is classified in the order Agaricales, family Strophariaceae. Our results thus indicate a need for systematic re-identification of the previously named N. frowardii isolate b19.
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http://dx.doi.org/10.1099/mic.0.2008/018747-0DOI Listing
August 2008

Expression and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown on wood.

Curr Genet 2006 Nov 23;50(5):323-33. Epub 2006 Aug 23.

Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, Helsinki, Finland.

Laccases are phenol-oxidizing, multicopper enzymes produced by fungi, plants, insects and bacteria. Fungal laccases are involved in ecologically important processes such as decomposition of lignocellulose (wood and plant material). In this work, in order to find out the role of fungal laccases upon wood colonisation and lignin decay, we describe expression of laccase-encoding genes in the white rot basidiomycete Phlebia radiata 79, when the fungus grows on its natural substrates, that is on softwood (Alnus incana) and hardwood (Picea abies). Clones for two laccase-encoding genes, the previously described Pr-lac1 and a new gene Pr-lac2 were characterized. Pr-lac2 coding region is interrupted by 12 introns and the deduced Lac2 protein displays a higher pI value (5.8) than Lac1 (pI 3.2-3.5). Phylogenetic analysis indicates differential evolution for the two laccases, and Lac2 demonstrates the highest sequence identity with Trametes laccases (66%). Transcripts of Pr-lac1 were the most abundant both in solid-state softwood and semi-solid hardwood cultures, as analyzed by competitive RT-PCR and Northern hybridization. On spruce wood chips, Pr-lac1 and Pr-lac2 were expressed within 2-3 weeks of growth together with manganese and lignin peroxidase-encoding genes. Our results indicate wood-promoted but time-dependent regulation of expression for the two, at protein and gene level distinct P. radiata laccases.
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http://dx.doi.org/10.1007/s00294-006-0090-1DOI Listing
November 2006

Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata.

Curr Genet 2006 Feb 7;49(2):97-105. Epub 2005 Dec 7.

Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, Viikki Biocenter 1, P.O.Box 56, 00014 Helsinki, Finland.

Lignin peroxidase (LiP) is the first enzyme connected to oxidative breakdown of the aromatic plant heteropolymer lignin and related xenobiotics. However, this extracellular enzyme has been described in only a few species of wood-decaying basidiomycetous fungi. The white rot basidiomycete Phlebia radiata 79 readily produces a versatile set of lignin-oxidizing enzymes including lignin and manganese peroxidases (LiPs and MnPs) and laccases. Here we describe genomic and primary structure of two new LiP-encoding genes, Pr-lip1 and Pr-lip4, and genomic characterization for isozyme LiP3/LIII of P. radiata, encoded by the gene depicted Pr-lip3. Pr-lip1 and Pr-lip4 code for 370- and 361-amino-acid long proteins beginning with 26- and 24-amino-acid secretion pre-propeptides, respectively. Translated LiP1 and LiP4 share the highest protein sequence identity (74 and 86%) with P. radiata LiP3, and 70% identity with the one deduced LiP from Bjerkandera adusta. The three P. radiata LiP sequences form a coherent phylogenetic cluster, which is further supported by similarities within gene organization interrupted by 11-introns. To find out the significance of LiP upon fungal growth on natural lignocellulose, such as wood, we studied ligninolytic gene expression on hardwood (milled alder) and softwood (spruce chips). All the LiP-encoding genes were expressed on wood with predominance of Pr-lip3 transcript abundance, in particular on spruce wood chips, where also time-dependent expression of the multiple lip genes was observed.
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http://dx.doi.org/10.1007/s00294-005-0045-yDOI Listing
February 2006