Publications by authors named "Miia R Mäkelä"

71 Publications

Revisiting a 'simple' fungal metabolic pathway reveals redundancy, complexity and diversity.

Microb Biotechnol 2021 Mar 5. Epub 2021 Mar 5.

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

Next to d-glucose, the pentoses l-arabinose and d-xylose are the main monosaccharide components of plant cell wall polysaccharides and are therefore of major importance in biotechnological applications that use plant biomass as a substrate. Pentose catabolism is one of the best-studied pathways of primary metabolism of Aspergillus niger, and an initial outline of this pathway with individual enzymes covering each step of the pathway has been previously established. However, although growth on l-arabinose and/or d-xylose of most pentose catabolic pathway (PCP) single deletion mutants of A. niger has been shown to be negatively affected, it was not abolished, suggesting the involvement of additional enzymes. Detailed analysis of the single deletion mutants of the known A. niger PCP genes led to the identification of additional genes involved in the pathway. These results reveal a high level of complexity and redundancy in this pathway, emphasizing the need for a comprehensive understanding of metabolic pathways before entering metabolic engineering of such pathways for the generation of more efficient fungal cell factories.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1751-7915.13790DOI Listing
March 2021

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.02701-20DOI Listing
February 2021

Applicability of Recombinant Laccases From the White-Rot Fungus for Mediator-Promoted Oxidation of Biorefinery Lignin at Low pH.

Front Bioeng Biotechnol 2020 17;8:604497. Epub 2020 Dec 17.

Department of Chemistry, Faculty of Science, Chemicum, University of Helsinki, Helsinki, Finland.

Utilization of lignin-rich side streams has been a focus of intensive studies recently. Combining biocatalytic methods with chemical treatments is a promising approach for sustainable modification of lignocellulosic waste streams. Laccases are catalysts in lignin biodegradation with proven applicability in industrial scale. Laccases directly oxidize lignin phenolic components, and their functional range can be expanded using low-molecular-weight compounds as mediators to include non-phenolic lignin structures. In this work, we studied in detail recombinant laccases from the selectively lignin-degrading white-rot fungus for their properties and evaluated their potential as industrial biocatalysts for the modification of wood lignin and lignin-like compounds. We screened and optimized various laccase mediator systems (LMSs) using lignin model compounds and applied the optimized reaction conditions to biorefinery-sourced technical lignin. In the presence of both N-OH-type and phenolic mediators, the laccases were shown to selectively oxidize lignin in acidic reaction conditions, where a cosolvent is needed to enhance lignin solubility. In comparison to catalytic iron(III)-(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation systems, the syringyl-type lignin units were preferred in mediated biocatalytic oxidation systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2020.604497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773891PMC
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/dnares/dsaa011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406137PMC
April 2020

Engineering of primary carbon metabolism in filamentous fungi.

Biotechnol Adv 2020 11 11;43:107551. Epub 2020 May 11.

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

Filamentous fungi are important industrial cell factories used for the production of a wide range of enzymes and metabolites. Their primary metabolism is a significant source of industrially important compounds, as well as of monomeric building blocks for the production of secondary metabolites and extracellular enzymes. Therefore, large efforts have been made towards the development of suitable strains for the industrial scale production of primary metabolites. Over the last decades, metabolic engineering of primary metabolism has become a powerful tool to enhance production of both primary and secondary metabolites. This review summarises the different metabolic engineering methods that have been applied to rationally improve the production of industrially relevant primary metabolites in filamentous fungi, and discusses related challenges and future perspectives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biotechadv.2020.107551DOI Listing
November 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biortech.2020.123477DOI Listing
September 2020

Genomic and Postgenomic Diversity of Fungal Plant Biomass Degradation Approaches.

Trends Microbiol 2020 06 10;28(6):487-499. Epub 2020 Feb 10.

Department of Microbiology, University of Helsinki, Helsinki, Finland.

Plant biomass degradation by fungi is a widely studied and applied field of science, due to its relevance for the global carbon cycle and many biotechnological applications. Before the genome era, many of the in-depth studies focused on a relatively small number of species, whereas now, many species can be addressed in detail, revealing the large variety in the approach used by fungi to degrade plant biomass. This variation is found at many levels and includes genomic adaptation to the preferred biomass component, but also different approaches to degrade this component by diverse sets of activities encoded in the genome. Even larger differences have been observed using transcriptome and proteome studies, even between closely related species, suggesting a high level of adaptation in individual species. A better understanding of the drivers of this diversity could be highly valuable in developing more efficient biotechnology approaches for the enzymatic conversion of plant biomass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tim.2020.01.004DOI Listing
June 2020

CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass.

Enzyme Microb Technol 2020 May 24;136:109508. Epub 2020 Jan 24.

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

The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of d-xylose and l-arabinose from wheat bran, and d-galacturonic acid from sugar beet pulp.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.enzmictec.2020.109508DOI Listing
May 2020

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

ACS Omega 2020 Mar 10;5(11):6130-6140. Epub 2020 Mar 10.

Department of Microbiology, University of Helsinki, Viikinkaari 9, Helsinki FI-00014 Helsinki, Finland.

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

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsomega.0c00142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098016PMC
March 2020

A comparative genomics study of 23 Aspergillus species from section Flavi.

Nat Commun 2020 02 27;11(1):1106. Epub 2020 Feb 27.

Department of Biotechnology and Bioengineering, Technical University of Denmark, Søltoft Plads 223, 2800, Kongens Lyngby, Denmark.

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-14051-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046712PMC
February 2020

Identification of a gene encoding the last step of the L-rhamnose catabolic pathway in Aspergillus niger revealed the inducer of the pathway regulator.

Microbiol Res 2020 Feb 7;234:126426. Epub 2020 Feb 7.

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

In fungi, L-rhamnose (Rha) is converted via four enzymatic steps into pyruvate and L-lactaldehyde, which enter central carbon metabolism. In Aspergillus niger, only the genes involved in the first three steps of the Rha catabolic pathway have been identified and characterized, and the inducer of the pathway regulator RhaR remained unknown. In this study, we identified the gene (lkaA) involved in the conversion of L-2-keto-3-deoxyrhamnonate (L-KDR) into pyruvate and L-lactaldehyde, which is the last step of the Rha pathway. Deletion of lkaA resulted in impaired growth on L-rhamnose, and potentially in accumulation of L-KDR. Contrary to ΔlraA, ΔlrlA and ΔlrdA, the expression of the Rha-responsive genes that are under control of RhaR, were at the same levels in ΔlkaA and the reference strain, indicating the role of L-KDR as the inducer of the Rha pathway regulator.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micres.2020.126426DOI Listing
February 2020

Corrigendum to "Mixtures of aromatic compounds induce ligninolytic gene expression in the wood-rotting fungus Dichomitus squalens" [J. Biotechnol. 380 (2020) 35-39].

J Biotechnol 2020 Feb 14;309:142. Epub 2020 Jan 14.

Department of Microbiology, University of Helsinki, P.O. Box 56 (Viikinkaari 9), Biocenter 1, Fl-00014, Helsinki, Finland. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiotec.2020.01.006DOI Listing
February 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.14907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065180PMC
March 2020

Mixtures of aromatic compounds induce ligninolytic gene expression in the wood-rotting fungus Dichomitus squalens.

J Biotechnol 2020 Jan 25;308:35-39. Epub 2019 Nov 25.

Department of Microbiology, University of Helsinki, 56 (Viikinkaari 9) Biocenter 1, Fl-00014, Helsinki, Finland. Electronic address:

Heterologous production of fungal ligninolytic cocktails is challenging due to the low yields of catalytically active lignin modifying peroxidases. Production using a natural system, such as a wood-rotting fungus, is a promising alternative if specific or preferential induction of the ligninolytic activities could be achieved. Using transcriptomics, gene expression of the white-rot Dichomitus squalens during growth on mixtures of aromatic compounds, with ring structures representing the two major lignin sub-units, was compared to a wood substrate. Most of the genes encoding lignin modifying enzymes (laccases and peroxidases) categorised as highly or moderately expressed on wood were expressed similarly on aromatic compounds. Higher expression levels of a subset of manganese and versatile peroxidases was observed on di- compared to mono-methoxylated aromatics. The expression of polysaccharide degrading enzymes was lower on aromatic compounds compared to wood, demonstrating that the induction of lignin modifying enzymes became more specific. This study suggests potential for aromatic waste streams, e.g. from lignocellulose pretreatment, to produce a lignin-specific enzyme cocktail from D. squalens or other white-rot fungi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiotec.2019.11.014DOI Listing
January 2020

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

Bioresour Technol 2020 Jan 11;295:122258. Epub 2019 Oct 11.

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

α-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular α-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All α-galactosidases exhibited high affinity to pNPαGal, 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 α-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other α-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 α-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biortech.2019.122258DOI Listing
January 2020

The White-Rot Basidiomycete Shows Highly Specific Transcriptional Response to Lignocellulose-Related Aromatic Compounds.

Front Bioeng Biotechnol 2019 20;7:229. Epub 2019 Sep 20.

Department of Microbiology, University of Helsinki, Helsinki, Finland.

Lignocellulosic plant biomass is an important feedstock for bio-based economy. In particular, it is an abundant renewable source of aromatic compounds, which are present as part of lignin, as side-groups of xylan and pectin, and in other forms, such as tannins. As filamentous fungi are the main organisms that modify and degrade lignocellulose, they have developed a versatile metabolism to convert the aromatic compounds that are toxic at relatively low concentrations to less toxic ones. During this process, fungi form metabolites some of which represent high-value platform chemicals or important chemical building blocks, such as benzoic, vanillic, and protocatechuic acid. Especially basidiomycete white-rot fungi with unique ability to degrade the recalcitrant lignin polymer are expected to perform highly efficient enzymatic conversions of aromatic compounds, thus having huge potential for biotechnological exploitation. However, the aromatic metabolism of basidiomycete fungi is poorly studied and knowledge on them is based on the combined results of studies in variety of species, leaving the overall picture in each organism unclear. is an efficiently wood-degrading white-rot basidiomycete that produces a diverse set of extracellular enzymes targeted for lignocellulose degradation, including oxidative enzymes that act on lignin. Our recent study showed that several intra- and extracellular aromatic compounds were produced when was cultivated on spruce wood, indicating also versatile aromatic metabolic abilities for this species. In order to provide the first molecular level systematic insight into the conversion of plant biomass derived aromatic compounds by basidiomycete fungi, we analyzed the transcriptomes of when grown with 10 different lignocellulose-related aromatic monomers. Significant differences for example with respect to the expression of lignocellulose degradation related genes, but also putative genes encoding transporters and catabolic pathway genes were observed between the cultivations supplemented with the different aromatic compounds. The results demonstrate that the transcriptional response of is highly dependent on the specific aromatic compounds present suggesting that instead of a common regulatory system, fine-tuned regulation is needed for aromatic metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2019.00229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763618PMC
September 2019

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

Appl Environ Microbiol 2019 12 14;85(23). Epub 2019 Nov 14.

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

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 using transcriptomics and exoproteomics. In 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-arbohydrate-ctive en (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 rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains. 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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AEM.01828-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856337PMC
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biotechadv.2019.02.017DOI Listing
November 2019

The presence of trace components significantly broadens the molecular response of Aspergillus niger to guar gum.

N Biotechnol 2019 Jul 20;51:57-66. Epub 2019 Feb 20.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute, Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT, Utrecht, the Netherlands; Microbiology, Utrecht University, Padualaan 8, 3584, CH, Utrecht, the Netherlands. Electronic address:

Guar gum consists mainly of galactomannan and constitutes the endosperm of guar seeds that acts as a reserve polysaccharide for germination. Due to its molecular structure and physical properties, this biopolymer has been considered as one of the most important and widely used gums in industry. However, for many of these applications this (hemi-)cellulosic structure needs to be modified or (partially) depolymerized in order to customize and improve its physicochemical properties. In this study, transcriptome, exoproteome and enzyme activity analyses were employed to decipher the complete enzymatic arsenal for guar gum depolymerization by Aspergillus niger. This multi-omic analysis revealed a set of 46 genes encoding carbohydrate-active enzymes (CAZymes) responding to the presence of guar gum, including CAZymes not only with preferred activity towards galactomannan, but also towards (arabino-)xylan, cellulose, starch and pectin, likely due to trace components in guar gum. This demonstrates that the purity of substrates has a strong effect on the resulting enzyme mixture produced by A. niger and probably by other fungi as well, which has significant implications for the commercial production of fungal enzyme cocktails.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbt.2019.02.005DOI Listing
July 2019

Genomic and Genetic Insights Into a Cosmopolitan Fungus, (Eurotiales).

Front Microbiol 2018 13;9:3058. Epub 2018 Dec 13.

School of BioSciences, University of Melbourne, Melbourne, VIC, Australia.

Species in the genus , a member of the fungal order Eurotiales, are ubiquitous in nature and impact a variety of human endeavors. Here, the biology of one common species, , was explored using genomics and functional genetics. Sequencing the genome of two isolates revealed key genome and gene features in this species. A striking feature of the genome was the two-part nature, featuring large stretches of DNA with normal GC content separated by AT-rich regions, a hallmark of many plant-pathogenic fungal genomes. These AT-rich regions appeared to have been mutated by repeat-induced point (RIP) mutations. We developed methods for genetic transformation of , including forward and reverse genetics as well as crossing techniques. Using transformation and crossing, RIP activity was identified, demonstrating for the first time that RIP is an active process within the order Eurotiales. A consequence of RIP is likely reflected by a reduction in numbers of genes within gene families, such as in cell wall degradation, and reflected by growth limitations on on diverse carbon sources. Furthermore, using these transformation tools we characterized a conserved protein containing a domain of unknown function (DUF1212) and discovered it is involved in pigmentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2018.03058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300479PMC
December 2018

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri.

Nat Genet 2018 12 22;50(12):1688-1695. Epub 2018 Oct 22.

Department of Biotechnology and Bioengineering, Technical University of Denmark, Kongens Lyngby, Denmark.

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-018-0246-1DOI Listing
December 2018

Selective Cleavage of Lignin β--4 Aryl Ether Bond by β-Etherase of the White-Rot Fungus .

ACS Sustain Chem Eng 2018 Mar 25;6(3):2878-2882. Epub 2018 Jan 25.

Division of Microbiology and Biotechnology, Department of Food and Environmental Sciences, University of Helsinki, Viikinkaari 9, FI-00014 Helsinki, Finland.

Production of value-added compounds from a renewable aromatic polymer, lignin, has proven to be challenging. Chemical procedures, involving harsh reaction conditions, are costly and often result in nonselective degradation of lignin linkages. Therefore, enzymatic catalysis with selective cleavage of lignin bonds provides a sustainable option for lignin valorization. In this study, we describe the first functionally characterized fungal intracellular β-etherase from the wood-degrading white-rot basidiomycete . This enzyme, Ds-GST1, from the glutathione--transferase superfamily selectively cleaved the β-4 aryl ether bond of a dimeric lignin model compound in a glutathione-dependent reaction. Ds-GST1 also demonstrated activity on polymeric synthetic lignin fractions, shown by a decrease in molecular weight distribution of the laccase-oxidized guaiacyl dehydrogenation polymer. In addition to a possible role of Ds-GST1 in intracellular catabolism of lignin-derived aromatic compounds, the cleavage of the most abundant linkages in lignin under mild reaction conditions makes this biocatalyst an attractive green alternative in biotechnological applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acssuschemeng.7b03619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156110PMC
March 2018

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.14416DOI Listing
November 2018

Efficient Extraction Method for High Quality Fungal RNA from Complex Lignocellulosic Substrates.

Methods Mol Biol 2018 ;1775:69-73

Department of Microbiology, University of Helsinki, Helsinki, Finland.

Here we describe an efficient and reproducible method for the extraction of fungal RNA from complex lignocellulose containing materials. The fungal cells are snap-frozen and disrupted in chaotropic guanidinium thiocyanate buffer, after which the extracted RNA is isolated by using CsCl gradient ultracentrifugation. By lowering the pH of the extraction buffer, the procedure is also suitable for sample materials rich in humic acids. The method results in high quantity and quality RNA that is separated from endogenous contaminants (e.g., RNases) and substances derived from plant biomass (e.g., colored aromatic compounds). In addition, no further steps such as DNase treatment are needed. The extracted RNA is highly suitable for downstream gene expression analyses such as RNA sequencing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7804-5_6DOI Listing
February 2019

Analysis of Putative Sugar Transporter Genes in Using Phylogeny and Comparative Transcriptomics.

Front Microbiol 2018 18;9:1045. Epub 2018 May 18.

Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland.

is one of the most widely used fungi to study the conversion of the lignocellulosic feedstocks into fermentable sugars. Understanding the sugar uptake system of is essential to improve the efficiency of the process of fungal plant biomass degradation. In this study, we report a comprehensive characterization of the sugar transportome of by combining phylogenetic and comparative transcriptomic analyses. We identified 86 putative sugar transporter (ST) genes based on a conserved protein domain search. All these candidates were then classified into nine subfamilies and their functional motifs and possible sugar-specificity were annotated according to phylogenetic analysis and literature mining. Furthermore, we comparatively analyzed the ST gene expression on a large set of fungal growth conditions including mono-, di- and polysaccharides, and mutants of transcriptional regulators. This revealed that transporter genes from the same phylogenetic clade displayed very diverse expression patterns and were regulated by different transcriptional factors. The genome-wide study of STs of provides new insights into the mechanisms underlying an extremely flexible metabolism and high nutritional versatility of and will facilitate further biochemical characterization and industrial applications of these candidate STs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2018.01045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5968117PMC
May 2018

The fungus Aspergillus niger consumes sugars in a sequential manner that is not mediated by the carbon catabolite repressor CreA.

Sci Rep 2018 04 27;8(1):6655. Epub 2018 Apr 27.

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

In nature, the fungus Aspergillus niger degrades plant biomass polysaccharides to monomeric sugars, transports them into its cells, and uses catabolic pathways to convert them into biochemical building blocks and energy. We show that when grown in liquid cultures, A. niger takes up plant-biomass derived sugars in a largely sequential manner. Interestingly, this sequential uptake was not mediated by the fungal general carbon catabolite repressor protein CreA. Furthermore, transcriptome analysis strongly indicated that the preferential use of the monomeric sugars is arranged at the level of transport, but it is not reflected in transcriptional regulation of sugar catabolism. Therefore, the results indicate that the regulation of sugar transport and catabolism are separate processes in A. niger.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-25152-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923239PMC
April 2018

Characterization of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes from Carbohydrate Esterase family 1 of the carbohydrate-active enzymes (CAZy) database.

Microb Biotechnol 2018 09 26;11(5):869-880. Epub 2018 Apr 26.

Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Viikinkaari 9, Helsinki, Finland.

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1751-7915.13273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116738PMC
September 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/genomeA.01414-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887033PMC
April 2018