Fungal Biology and Biotechnology最新文献

Background: Ribosomally synthesized and posttranslationally modified peptides (RiPPs) represent a relatively recent addition to the biosynthetic gene cluster (BGC) repertoire of fungi. These BGCs are primarily involved in toxins production and defense-related functions and resulting metabolites also have a significant therapeutic potential. While only a limited number of fungal RiPPs, primarily from a few model fungi, have been characterized, genome mining approaches show that RiPP BGCs are nearly ubiquitous across the fungal kingdom. However, the RiPP biosynthetic landscape of fungi involved in intricate relationship as symbiosis, such as lichen-forming fungi (LFF), remains unexplored.

Results: This study presents the first comprehensive survey of RiPP BGCs across 111 LFF genomes employing an integrative framework that combines genome mining, phylogenetic inference, and gene network reconstruction. We identified 987 RiPP BGCs, constituting approximately 17% of the total biosynthetic diversity in LFF, a proportion significantly higher than previously estimated. Most lichen RiPP BGCs are unique and do not cluster with any known RiPP gene cluster. We found two RiPP BGCs that were shared among the members of the family Parmeliaceae (Lecanoromycetes), with the signature gene homologous to ustiloxin signature enzyme, indicating a putative similarity to fungal mycotoxin-related BGCs. While one of these BGCs, members of Clan R1, contains the accessory genes for dikaritin synthesis (tyrosinase and methyltransferase), the accessory genes of other BGCs, members of Clan R2, have not yet been reported from any characterized fungal RiPP BGC but only from bacteria. Additionally, for lichen RiPP BGCs that do not cluster with any known BGCs in the RiPP network, we unraveled the presence of the conserved HXXHC motif in the signature gene and, based on this we report the widespread distribution of putative dikaritin homologs across Lecanoromycetes.

Conclusions: This study highlights the presence and distribution of RiPP BGCs in Lecanoromycetes and identifies two conserved RiPP clusters putatively homologous to dikaritins (involved in mycotoxin production) within the Lecanoromycete family Parmeliaceae and a general prevalence of putative signature dikaritin genes (not the cluster) in Lecanoromycetes. Our study highlights the widespread presence of putative mycotoxin-related BGCs in lichenized fungi.

Background: CRISPR technologies have revolutionized strain engineering of Aspergillus species, and drastically increased the ease and speed at which genomic modifications can be performed. One of the advantages of CRISPR technologies is the possibility of rapid strain engineering using multiplex experiments. This can be achieved by using a set of different guiding RNA molecules (gRNA) to target multiple loci in the same experiment. Two major challenges in such experiments are firstly, the delivery of multiple guides simultaneously, and secondly, ensuring that each target locus is cut efficiently by the CRISPR nuclease. The CRISPR nuclease Cas12a, also known as Cpf1, presents a unique advantage to bypass this challenge. Specifically, and unlike Cas9, Cpf1 is able to release several gRNAs from a common precursor RNA molecule through its own RNase activity, eliminating the need for elements such as ribozymes or tRNA machinery for gRNA maturation. This feature sets the stage for much more straightforward construction of vectors for the delivery of many gRNAs, which in turn allows each locus to be targeted by multiple gRNAs to increase the odds of successfully inducing a break in the DNA.

Results: Here we present a toolbox that can be used to assemble plasmids containing a gRNA multiplex expression cassette, which is able to express a multi gRNA precursor. The precursor can be processed via Cpf1 RNase activity to produce multiple functional gRNAs in vivo. Using our setup, we have constructed plasmids that are able to deliver up to ten gRNAs. In addition, we show that three simultaneous deletions can be introduced robustly in Aspergillus niger by targeting each gene with several gRNAs, without prior gRNA validation or the use of genomically integrated selection markers.

Conclusion: In this study we have established an efficient system for the construction of CRISPR-Cpf1 vectors that are able to deliver a large number of gRNAs for multiplex genome editing in Aspergillus species. Our strategy allows multiple specific genomic modifications to be performed in a time frame of less than two weeks, and we envision this will be able to speed up cell factory construction efforts significantly.

Background: Lion's mane (Hericium erinaceus) mycelium produces erinacines, a suite of cyathane diterpenoids with established neuroactivities. While H. erinaceus fruit body tissue has its own characteristic secondary metabolites, it generally does not produce detectable amounts of erinacines. Substrate composition influences the erinacine content of H. erinaceus mycelial cultures, similar to production of secondary metabolites in other fungi. This study explored the relationship between biosynthetic gene expression and erinacine content in H. erinaceus, comparing fruit body tissue to mycelial tissue cultured in two liquid media formulations.

Results: In this study, we compared erinacine production in H. erinaceus fruit body to mycelial tissue cultivated in two liquid media formulations (Complex and Minimal) by quantifying mRNA transcript levels of the erinacine biosynthetic genes eriE, eriG, eriI, eriC, eriJ, eriB, and eriM (collectively, eri genes) alongside high performance liquid chromatography (HPLC) evaluation of erinacines Q, P, A, and C. We also predicted coding sequences for these seven eri genes. The Complex media preparation yielded mycelium with significantly higher erinacine C content, while the Minimal media yielded mycelium with greater erinacine Q content, suggesting an alteration of the biosynthetic pathway related to differences in substrate composition. Despite evident differences in erinacine concentrations, mycelial eri gene transcript levels did not differ significantly between the two liquid media preparations. When evaluated by gene expression or compound concentration, erinacine biosynthesis was substantially greater in mycelia compared to fruit body tissue in H. erinaceus.

Conclusions: Alongside the absence of detectable erinacines within fruit body samples, eri gene transcripts were consistently downregulated in the fruit body compared to the mycelium, particularly at early stages of the biosynthetic pathway. Substrate composition is a critical factor in production of erinacines by H. erinaceus, and large differences in mycelial erinacine content can occur without significant differences in expression of eri genes. Our data support the hypothesis that production of fungal secondary metabolites can be influenced by tissue type and substrate components, and that the expression of eri genes is enriched in the mycelium when compared to the fruit body.

Background: Quorn mycoprotein, a protein-rich meat alternative, is produced through large-scale fermentation of the fungus Fusarium venenatum. However, a major challenge during F. venenatum fermentation is the consistent appearance of mutants called colonial variants (C-variants). These C-variants have a highly branched morphology, which ultimately lead to a less desirable final product and early termination of the fermentation process. This study aimed to identify the genetic mutations responsible for C-variant morphology.

Results: We first isolated both C-variant and wild-type strains from commercial fermentation samples and characterised radial growth rates on solid media. Whole genome sequencing facilitated the identification of mutations in a gene called jg4843 in 11 out of 12 C-variant isolates, which were not observed in the wild-type isolates. The jg4843 gene was identified as the ortholog of LRG1, a Rho-GTPase activating protein that regulates the Rho1 signalling pathway affecting fungal growth. Notably, the mutations in jg4843 were primarily located in the RhoGAP domain responsible for LRG1 activity. To confirm the role of these mutations, we used CRISPR/Cas9-mediated homology-directed recombination to introduce the C-variant mutations into the wild-type isolate, which successfully recapitulated the characteristic C-variant morphology.

Conclusions: This study identified mutations in the LRG1 ortholog jg4843 as the genetic cause of C-variant morphology in commercial fermentation F. venenatum isolates. Understanding this genetic basis paves the way for developing strategies to prevent C-variants arising, potentially leading to more efficient and sustainable production of Quorn mycoprotein.

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.