The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants ...has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species.
Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1+5.8S+ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes.
Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates.
Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales.
Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to ...the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation.
The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a ...wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds.
The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far.
Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.
Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied ...a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-alpha (ERalpha), as validated by western blotting and quantitative real time-PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERalpha-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERalpha in 3'-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERalpha, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERalpha-negative as compared with ERalpha-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERalpha signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.
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.
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.
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.
Plant biomass degradation differs strongly among fungi.The diversity in plant biomass degradation approaches from fungi appears to be largely due to postgenomic/regulatory differences.Despite conservation of the enzymes involved in plant biomass degradation in basidiomycete and ascomycete fungi, these two phyla have distinct regulatory systems that control plant biomass degradation.Fungi are able to adapt the enzymes they produce to the prevailing substrate, even when this is not a substrate they naturally colonize.The lack of sufficient biochemically characterized enzymes in many CAZy families prevents reliable genome annotation of fungi.
•A. niger metabolic engineering improved xylitol production from lignocellulose.•The triple mutant ΔladAΔxdhAΔsdhA showed the best performance in xylitol production.•Wheat bran and cotton seed hulls ...were the best substrates for xylitol bioproduction.•Choice of lignocellulosic biomass plays an important role in xylitol bioproduction.
Xylitol is widely used in the food and pharmaceutical industries as a valuable commodity product. Biotechnological production of xylitol from lignocellulosic biomass by microorganisms is a promising alternative option to chemical synthesis or bioconversion from D-xylose. In this study, four metabolic mutants of Aspergillus niger were constructed and evaluated for xylitol accumulation from D-xylose and lignocellulosic biomass. All mutants had strongly increased xylitol production from pure D-xylose, beechwood xylan, wheat bran and cotton seed hulls compared to the reference strain, but not from several other feed stocks. The triple mutant ΔladAΔxdhAΔsdhA showed the best performance in xylitol production from wheat bran and cotton seed hulls. This study demonstrated the large potential of A. niger for xylitol production directly from lignocellulosic biomass by metabolic engineering.
Summary
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.
There are no records of established plant pathogenic Phytophthora species in Finnish forests, but they are likely in the future. Therefore, the effects of Phytophthora inoculations on young, ca. ...2‐month‐old silver birch (Betula pendula) seedling roots and shoots were investigated.
Visual inspection of dark discoloration, direct PCR and re‐isolation, and detailed root morphology analyses were used to evaluate the effects of Phytophthora inoculation on roots. Symptoms in leaves and stems were also recorded.
Phytophthora was successfully re‐isolated from 67% of the surface‐sterilized roots of inoculated seedlings, but not from the non‐inoculated control seedlings. Dark discolorations were found more often in the root segments of inoculated seedlings than in control seedlings. In the Phytophthora‐treated seedlings, discoloured root segments were usually linked and found primarily in the main root or lateral roots attached to it, whereas in the control seedlings a few single discoloured root segments were scattered throughout the root systems. The number of root segments was lower in the inoculated than in the control seedlings, indicating root loss after Phytophthora inoculation. In the shoots of inoculated birches, leaf and shoot wilting was observed.
The appearance of wilting in shoots without visible dark discoloration in the base of stems indicated that symptoms originated from roots inoculated with Phytophthora.
Phytophthora cactorum infection causes dark discolorations both in roots and shoots, decreases the number of root tips and the height of shoots, and finally induces wilting in leaves.
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