Fungal Biology and Biotechnology最新文献

Background: Production of biofuels and bioproducts from lignocellulosic material is limited due to the complexity of the cell wall structure. This necessitates the use of physical, chemical, and/or physico-chemical pretreatment technologies, which adds significant capital, operational, and environmental costs. Biological pretreatment strategies have the potential to mitigate these expenses by harnessing the innate ability of specialized bacteria and fungi to deconstruct lignocellulose. White-rot fungi (e.g. Trametes versicolor) have been shown to be effective at biological pretreatment of lignocellulose, yet it was uncertain if these fungi are feedstock agnostic or are able to sense subtle changes in cell wall chemistry.

Results: The present study examined the transcriptome response by Trametes versicolor to transgenic hybrid poplar (Populus tremula × alba) lines with altered syringyl (S) and guaiacyl (G) lignin. Specifically, the transcriptional response of the fungus to wild-type wood was compared to that from the wood of six transgenic lines within three lignin phenotypes, LSX (low S with hydroxy-G), LSHG (low S with high G), and HS (high S), with 350 transcripts showing significant differences among the samples. The transcriptome of T. versicolor varied according to the lignin phenotype of the wood, with the LSX wood resulting in the most substantial changes in T. versicolor transcript abundance. Specifically, the LSX wood led to 50 upregulated and 48 downregulated transcripts from WT at the twofold or greater threshold. For example, transcripts for the lignin peroxidases LiP3 and LiP10 were downregulated (approximately 12X and 31X lower, respectively) by the fungus on LSX wood compared to wild-type wood. LSX wood also resulted in approximately 11X lower transcript numbers of endo-β-1,4-glucanase yet led to an increase in expression of certain hemicellulases, further highlighting the altered deconstruction strategy by the fungus on this wood type.

Conclusions: Overall, the results of this study demonstrated that T. versicolor was able to respond to transgenic poplar wood with the same genetic background, which has important implications for biological pretreatment strategies involving feedstocks that are genetically modified or have considerable natural variations in cell wall chemistry.

Background: Fungal-based composites have emerged as renewable, high-performance biomaterials that are produced on lignocellulosic residual streams from forestry and agriculture. Production at an industrial scale promises to revolutionize the world humans inhabit by generating sustainable, low emission, non-toxic and biodegradable construction, packaging, textile, and other materials. The polypore Fomes fomentarius is one of the basidiomycete species used for biomaterial production, yet nothing is known about the transcriptional basis of substrate decomposition, nutrient uptake, or fungal growth during composite formation. Co-expression network analysis based on RNA-Seq profiling has enabled remarkable insights into a range of fungi, and we thus aimed to develop such resources for F. fomentarius.

Results: We analysed gene expression from a wide range of laboratory cultures (n = 9) or biomaterial formation (n = 18) to determine the transcriptional landscape of F. fomentarius during substrate decomposition and to identify genes important for (i) the enzymatic degradation of lignocellulose and other plant-based substrates, (ii) the uptake of their carbon monomers, and (iii) genes guiding mycelium formation through hyphal growth and cell wall biosynthesis. Simple scripts for co-expression network construction were generated and tested, and harnessed to identify a fungal-specific transcription factor named CacA strongly co-expressed with multiple chitin and glucan biosynthetic genes or Rho GTPase encoding genes, suggesting this protein is a high-priority target for engineering adhesion and branching during composite growth. We then updated carbohydrate activated enzymes (CAZymes) encoding gene annotation, used phylogenetics to assign putative uptake systems, and applied network analysis to predict repressing/activating transcription factors for lignocellulose degradation. Finally, we identified entirely new types of co-expressed contiguous clusters not previously described in fungi, including genes predicted to encode CAZymes, hydrophobins, kinases, lipases, F-box domains, chitin synthases, amongst others.

Conclusion: The systems biology data generated in this study will enable us to understand the genetic basis of F. fomentarius biomaterial formation in unprecedented detail. We provided proof-of-principle for accurate network-derived predictions of gene function in F. fomentarius and generated the necessary data and scripts for analysis by any end user. Entirely new classes of contiguous co-expressed gene clusters were discovered, and multiple transcription factor encoding genes which are high-priority targets for genetic engineering were identified.

Mycelial materials are an emerging, natural material made from filamentous fungi that have the potential to replace unsustainable materials used in numerous commercial applications (e.g., packaging, textiles, construction). Efforts to change the mechanical properties of mycelial-materials have typically involved altering growth medium, processing approaches, or fungal species. Although these efforts have shown varying levels of success, all approaches have shown there is a strong correlation between phenotype (of both fungal mycelia and mycelial material's assembly) and resultant mechanical properties. We hypothesize that genetic means can be used to generate specific fungal phenotypes, leading to mycelial materials with specific mechanical properties. To begin to test this hypothesis, we used a mutant of the model filamentous fungus, Aspergillus nidulans, with a deletion in the gene encoding the last kinase in the cell wall integrity (CWI) signaling pathway, mpkA. We generated one set of mycelial materials from the ΔmpkA deletion mutant (A1404), and another from its isogenic parent (A1405; control). When subjected to tensile testing, and compared to material generated from the control, ΔmpkA material has similar elastic modulus, but significantly increased ultimate tensile strength, and strain at failure. When subjected to a fragmentation assay (i.e., resistance to shear-stress), the ΔmpkA material also had higher relative mechanical strength. To determine possible causes for this behavior, we carried out a comprehensive set of phenotype assessments focused on: three-dimensional structure, hyphal morphology, hyphal growth behaviors, and conidial development. We found, compared to the control, material generated from the ΔmpkA mutant manifests significantly less development, a modified cell wall composition, larger diameter hyphae, more total biomass, higher water capacity and more densely packed material, which all appear to impact the altered mechanical properties.

Biocontrol agents play a pivotal role in managing pests and contribute to sustainable agriculture. Recent advancements in genetic engineering can facilitate the development of entomopathogenic fungi with desired traits to enhance biocontrol efficacy. In this study, a CRISPR-Cas9 ribonucleoprotein system was utilized to genetically improve the virulence of Beauveria bassiana, a broad-spectrum insect pathogen used in biocontrol of arthropod pests worldwide. CRISPR-Cas9-based disruption of the transcription factor-encoding gene Bbsmr1 led to derepression of the oosporein biosynthetic gene cluster resulting in overproduction of the red-pigmented dibenzoquinone oosporein involved in host immune evasion, thus increasing fungal virulence. Mutants defective for Bbsmr1 displayed a remarkable enhanced insecticidal activity by reducing lethal times and concentrations, while concomitantly presenting negligible or minor pleiotropic effects. In addition, these mutants displayed faster germination on the insect cuticle which correlated with higher density of free-floating blastospores in the hemolymph and accelerated mortality of the host. These findings emphasize the utility of genetic engineering in developing enhanced fungal biocontrol agents with customized phenotypic traits, and provide an efficient and versatile genetic transformation tool for application in other beneficial entomopathogenic fungi.

Mycelium composite materials are comprised of renewable organic substrates interconnected by fungal mycelium, allowing full biodegradability after use. Due to their promising material properties, adaptability, and sustainable nature, these biomaterials are investigated intensively. However, one crucial aspect that has hardly been covered so far is the proportion of fungal biomass in the composites, which would be necessary to assess its contribution to the material characteristics. Since a complete physical separation of mycelium and substrate is not feasible, we approached this issue by isolating the fungal DNA and relating it to the mass of mycelium with the help of quantitative PCR. Overall, 20 different combinations of fungi and biogenic side streams were evaluated for their handling stability, and growth observations were related to the quantification results. Ganoderma sessile was able to form stable composites with almost all substrates, and a positive correlation between mycelial biomass and composite stability could be found. However, the amount of mycelium required for fabricating firm materials strongly depends on the combination of substrate and fungal species used. Less than five mass percent of fungal biomass can suffice to achieve this, as for example when combining Trametes versicolor with sugar beet pulp, whereas a mass fraction of twenty percent leads to crumbly materials when using Pleurotus pulmonarius on green waste. These results indicate that the mycelial biomass is an important factor for the composite's stability but that the properties of the fungal hyphae, as well as those of the substrate, are also relevant. The presented quantification method not only allows to estimate fungal growth during composite production but can also improve our understanding of how the mycelium influences the material.

Microbial production of aromatic compounds from renewable feedstocks has gained increasing interest as a means towards sustainable production of chemicals. The potential of filamentous fungi for production of aromatic compounds has nonetheless not yet been widely exploited. Notably, many filamentous fungi can naturally break down lignin and metabolize lignin-derived aromatic compounds. A few examples where a fungal cell factory, often of Aspergillus spp., is used to produce an aromatic compound, typically through the conversion of one compound to another, have already been reported. In this review, we summarize fungal biosynthesis of biotechnologically interesting aromatic compounds. The focus is on compounds produced from the shikimate pathway. Biorefinery-relevant efforts for valorizing residual biomass or lignin derived compounds are also discussed. The advancement in engineering tools combined with the increasing amounts of data supporting the discovery of new enzymes and development of new bioprocesses has led to an increased range of potential production hosts and products. This is expected to translate into a wider utilization of fungal cell factories for production of aromatic compounds.

Background: Fomitopsis pinicola is one of the most common fungi found in temperate zone of Europe, widely distributed spread in Asia and North America. Fungus has a wide range of therapeutic activity: antitumor, antimicrobial, anti-inflammatory, antidiabetic, antifungal, hepatoprotective, hemostatic action. A number of studies have confirmed the significant antioxidant activity of F. pinicola fruiting bodies. However, the controlled cultivation conditions that influence fungal growth and metabolite production of F. pinicola, particularly the mycelial growth and biosynthesis of metabolites in its culture broth, as well as the antioxidant activity of its mycelium, remain poorly understood.

Results: This study investigated the impact of cultivation conditions on F. pinicola mycelium growth, phenols synthesis and antioxidant activity. Difference in the biosynthetic activity of F. pinicola under tested cultivation conditions was established. A highest value of 2,2-diphenyl-1-picryl-hydrazyl (DPPH•) inhibition (78.2 ± 0.9%) was found for a mycelium cultivated at 30 ºC, while cultivation at a lower temperature (20 ºC) was suitable for biomass growth (8.5 ± 0.3 g/L) and total phenolic content (TPC) 11.0 ± 0.6 mg GAE/g. Carbon and nitrogen sources in a cultivation broth significantly influenced the studied characteristics. Xylose supported the highest DPPH• inhibition (89.91 ± 0.5%) and TPC (16.55 ± 0.4 mg GAE/g), while galactose yielded the best biomass (4.0 ± 0.3 g/L). Peptone was the most effective nitrogen source for obtaining the mycelium with high potential of DPPH• radical inactivation (90.42 ± 0.5%) and TPC (17.41 ± 0.5 mg GAE/g), while the maximum biomass yield (7.8 ± 0.6 g/L) was found with yeast extract in cultivation medium. F. pinicola demonstrated the ability to grow and produce bioactive metabolites across a wide pH range from 2.5 to 7.5. Shaking cultivation resulted in the highest TPC (21.44 ± 0.10 mg GAE/g), though the same level of antioxidant activity (93%) was achieved under both shaking and static cultivation on the 7th and 28th days, respectively.

Conclusion: Controlling cultivation parameters makes it possible to regulate the metabolic and biochemical processes of F. pinicola, facilitating the balance needed to obtain optimal biomass, phenols and antioxidant activity. The findings show the potential to increase phenol production by 2.25 and 2.23 times under shaking and static conditions, respectively, while maintaining a high level of activity.

Background: Spores produced by the filamentous fungus Aspergillus niger are abundant in a variety of environments. The proliferation of this fungus in indoor environments has been associated to health risks and its conidia can cause allergic reaction and severe invasive disease in animals and humans. Therefore, the detection and monitoring of Aspergillus conidia is of utmost importance to prevent serious fungal infections and contaminations. Among others, aptamers could serve as biosensors for the specific detection of fungal spores.

Results: In this study, DNA aptamers specific to conidia of A. niger were developed by optimizing a whole-cell SELEX approach. Three whole-cells SELEX experiments were performed in parallel with similar conditions. Quantification of recovered ssDNA and melting curve analyses were applied to monitor the ongoing SELEX process. Next-generation sequencing was performed on selected recovered ssDNA pools, allowing the identification of DNA aptamers which bind with high affinity to the target cells. The developed aptamers were shown to be species-specific, being able to bind to A. niger but not to A. tubingensis or to A. nidulans. The binding affinity of two aptamers (AN01-R9-006 and AN02-R9-185) was measured to be 58.97 nM and 138.71 nM, respectively, which is in the range of previously developed aptamers.

Conclusions: This study demonstrates that species-specific aptamers can be successfully developed via whole-cell SELEX to distinguish different Aspergillus species and opens up new opportunities in the field of diagnostics of fungal infections.

Background: Ashbya gossypii is a filamentous fungus widely utilized for industrial riboflavin production and has a great potential as a microbial chassis for synthesizing other valuable metabolites such as folates, biolipids, and limonene. Engineered strains of A. gossypii can effectively use various waste streams, including xylose-rich feedstocks. Notably, A. gossypii has been identified as a proficient biocatalyst for producing limonene from xylose-rich sources. This study aims to investigate the capability of engineered A. gossypii strains to produce various plant monoterpenes using agro-industrial waste as carbon sources.

Results: We overexpressed heterologous terpene synthases to produce acyclic, monocyclic, and bicyclic monoterpenes in two genetic backgrounds of A. gossypii. These backgrounds included an NPP synthase orthogonal pathway and a mutant erg20^F95W allele with reduced FPP synthase activity. Our findings demonstrate that A. gossypii can synthesize linalool, limonene, pinene, and sabinene, with terpene synthases showing differential substrate selectivity for NPP or GPP precursors. Additionally, co-overexpression of endogenous HMG1 and ERG12 with heterologous NPP synthase and terpene synthases significantly increased sabinene yields from xylose-containing media. Using mixed formulations of corn-cob lignocellulosic hydrolysates and either sugarcane or beet molasses, we achieved limonene and sabinene productions of 383 mg/L and 684.5 mg/L, respectively, the latter representing a significant improvement compared to other organisms in flask culture mode.

Conclusions: Engineered A. gossypii strains serve as a suitable platform for assessing plant terpene synthase functionality and substrate selectivity in vivo, which are crucial to understand monoterpene bioproduction. The NPP synthase pathway markedly enhances limonene and sabinene production in A. gossypii, achieving levels comparable to those of other industrial microbial producers. Furthermore, these engineered strains offer a novel approach for producing monoterpenes through the valorization of agro-industrial wastes.

Background: Aspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.

Results: Here we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13-20% of the transformants while 80-87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35-61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85-90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.

Conclusion: The awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.