Fungal Biology and Biotechnology最新文献

Asexuality was considered to be a common feature of a large part of fungi, including those of the genus Aspergillus. However, recent advances and the available genomic and genetic engineering technologies allowed to gather more and more indications of a hidden sexuality in fungi previously considered asexual. In parallel, the acquired knowledge of the most suitable conditions for crossings was shown to be crucial to effectively promote sexual reproduction in the laboratory. These discoveries not only have consequences on our knowledge of the biological processes ongoing in nature, questioning if truly asexual fungal species exist, but they also have important implications on other research areas. For instance, the presence of sexuality in certain fungi can have effects on their pathogenicity or on shaping the ecosystem that they normally colonize. For these reasons, further investigations of the sexual potential of Aspergillus species, such as the industrially important A. niger, will be carried on.

For centuries, filamentous fungi have been used in the making of food and beverages, and for decades for the production of enzymes and pharmaceuticals. In the last decades, the intellectual property (IP) landscape for fungal technology has seen an ever increasing upward trend, introducing new and promising applications utilising fungi. In this review, we highlight fungi-related patent applications published during the last 5 years (2015-2020), identify the key players in each field, and analyse future trends. New developments in the field of fungal technology include the increased use of filamentous fungi as a food source (mycoprotein), using fungi as biodegradable materials, in wastewater treatment, in integrated biorefineries and as biological pest agents. Biotechnology companies in Europe and the US are currently leading when it comes to the number of patents in these areas, but Asian companies and research institutes, in particular in China, are becoming increasingly important players, for example in pesticide formulation and agricultural practices.

Background: Two reference strains have been sequenced from the mushroom Coprinopsis cinerea, monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 (ade8-1) and a para-aminobenzoic acid (PABA)-auxotrophy in AmutBmut (pab1-1) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph, respectively.

Results: Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8 ⁺ vector pCcAde8 complements the auxotrophy of OK130 in transformations. Transformation rates with pCcAde8 in single-vector and co-transformations with ade8 ⁺-selection were similarly high, unlike for trp1 ⁺ plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected pCcAde8 helped in co-transformations of trp1 strains with a trp1 ⁺-selection vector to overcome suicidal effects by transferred trp1 ⁺. Co-transformation rates of pCcAde8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26-42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase.

Conclusions: ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. pCcAde8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8 ⁺ successive rounds of transformation possible.

Background: The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs)-secreted, membrane-bound nanoparticles containing molecular cargo-being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes.

Results: Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 and 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs.

Conclusion: We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

Background: Synthetic gene expression systems offer a possibility for controllable and targeted induction of the expression of genes of interest, which is a fundamental technique necessary for basic research and industrial applications. The human estrogen receptor α contains a ligand binding domain that enforces dimerization and nuclear import upon binding of the inducer 17β-estradiol. In this study, we tested the potential of this ligand binding domain to be used in filamentous fungi as an auto-regulatory domain in a synthetic transcription factor.

Results: We constructed the synthetic transcription factor SynX by fusing the DNA-binding domain of Xyr1 (Xylanase Regulator 1), the transactivation domain of Ypr1 (Yellow Pigment Regulator 1), and the ligand binding domain of the human estrogen receptor α. SynX is able to strongly induce the gene expression of xylanases and an aldose reductase by addition of 17β-estradiol, but SynX does not induce gene expression of cellulases. Importantly, the induction of xylanase activities is mostly carbon source independent and can be fine-tuned by controlling the concentration of 17β-estradiol.

Conclusion: The ability of SynX to induce gene expression of xylanase encoding genes by addition of 17β-estradiol demonstrates that the ligand binding domain of the human estrogen receptor α works in filamentous fungi, and that it can be combined with a transactivation domain other than the commonly used transactivation domain of herpes simplex virion protein VP16.

Background: Recently, a gene cluster responsible for biosynthesis of ustiloxin in Aspergillus flavus was identified as the first case of a ribosomally synthesized and post-translationally modified peptide (RiPP) synthetic pathway in Ascomycota. RiPPs are biosynthesized from precursor peptides, which are processed to produce the RiPP backbone (core peptides) for further modifications such as methylation and cyclization. Ustiloxin precursor peptide has two distinctive features: a signal peptide for translocation into the endoplasmic reticulum and highly repeated core sequences cleaved by Kex2 protease in the Golgi apparatus. On the basis of these characteristics, the ustiloxin-type RiPP precursor peptides or Kex2-processed repeat proteins (KEPs) in strains belonging to the Fungi kingdom were computationally surveyed, in order to investigate the distribution and putative functions of KEPs in fungal ecology.

Results: In total, 7878 KEPs were detected in 1345 of 1461 strains belonging to 8 phyla. The average number of KEPs per strain was 5.25 in Ascomycota and 5.30 in Basidiomycota, but only 1.35 in the class Saccharomycetes (Ascomycota) and 1.00 in the class Tremellomycetes (Basidiomycota). The KEPs were classified into 838 types and 2560 stand-alone ones, which had no homologs. Nearly 200 types were distributed in more than one genus, and 14 types in more than one phylum. These types included yeast α-mating factors and fungal pheromones. Genes for 22% KEPs were accompanied by genes for DUF3328-domain-containing proteins, which are indispensable for cyclization of the core peptides. DUF3328-domain-containing protein genes were located at an average distance of 3.09 genes from KEP genes. Genes for almost all (with three exceptions) KEPs annotated as yeast α-mating factors or fungal pheromones were not accompanied by DUF3328-domain-containing protein genes.

Conclusion: KEPs are widely distributed in the Fungi kingdom, but their repeated sequences are highly diverse. From these results and some examples, a hypothesis was raised that KEPs initially evolved as unmodified linear peptides (e.g., mating factors), and then those that adopted a modified cyclic form emerged (e.g., toxins) to utilize their strong bioactivity against predators and competitive microorganisms.

Filamentous fungi are widely used for production of enzymes and chemicals, and are industrially cultivated both in liquid and solid cultures. Submerged culture is often used as liquid culture for filamentous fungi. In submerged culture, filamentous fungi show diverse macromorphology such as hyphal pellets and dispersed hyphae depending on culture conditions and genetic backgrounds of fungal strains. Although the macromorphology greatly affects the productivity of submerged cultures, the specific cellular components needed for hyphal aggregation after conidial germination have not been characterized. Recently we reported that the primary cell wall polysaccharide α-1,3-glucan and the extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae, and that a strain deficient in both α-1,3-glucan and GAG shows dispersed hyphae in liquid culture. In this review, we summarize our current understanding of the contribution of chemical properties of α-1,3-glucan and GAG to hyphal aggregation. Various ascomycetes and basidiomycetes have α-1,3-glucan synthase gene(s). In addition, some Pezizomycotina fungi, including species used in the fermentation industry, also have GAG biosynthetic genes. We also review here the known mechanisms of biosynthesis of α-1,3-glucan and GAG. Regulation of the biosynthesis of the two polysaccharides could be a potential way of controlling formation of hyphal pellets.

Truffles, the symbiotic hypogeous edible fungi, have been worldwide regarded as a great delicacy because of their unique flavor and high nutritional value. By identifying their bioactive components such as phenolics, terpenoids, polysaccharides, anandamide, fatty acids, and ergosterols, researchers have paid attention to their biological activities including antitumor, antioxidant, antibacterial, anti-inflammatory, and hepatoprotective activities. In addition, numerous factors have been investigating that can affect the quality and productivity of truffles to overcome their difficulty in culturing and preserving. To provide the information for their potential applications in medicine as well as in functional food, this review summarizes the relevant literature about the biochemical composition, aromatic and nutritional benefits, and biological properties of truffles. Besides, various factors affecting their productivity and quality as well as the preservation methods are also highlighted.

Background: 'Rice koji' is a solid culture of Aspergillus oryzae on steamed rice grains. Multiple parallel fermentation, wherein saccharification of rice by A. oryzae and alcohol fermentation by the budding yeast occur simultaneously, leads to the formation of a variety of ingredients of Japanese sake. In sake brewing, the degree of mycelial invasive growth into the steamed rice, called 'haze-komi', highly correlates with the digestibility and quality of rice koji, since the hyphae growing into the rice secrete amylases and digest starch.

Results: In this study, we investigated mycelial distribution of GFP-tagged A. oryzae in rice koji made with different types of rice, such as sake rice and eating rice, with 50 or 90% polishing rate to remove abundant proteins and lipids near the surface. In addition, we compared transcriptomes of A. oryzae in the different types of rice koji. Finally, we found that A. oryzae increases the nuclear number and hyphal width in the course of 1-3 days cultivation.

Conclusions: Our imaging analyses indicate that A. oryzae hyphae grew more deeply into 50% polished rice than 90% polished rice. The increases of nuclear number may be a selectively acquired characteristic for the high secretory capacity during the long history of cultivation of this species.