A comparative characterization of fungal melanin and the humin-like substances synthesized by <Emphasis Type="Italic">Cerrena maxima</Emphasis> 0275

Comparative studies of fungal melanin and two preparations of the high-molecular-weight humin-like substances formed during a solid-phase cultivation of the basidiomycete Cerrena maxima 0275 for 45 and 70 days were performed. The fungal melanin from Aspergillus niger and the humin-like substances synthesized by the basidiomycete C. maxima 0275 are similar in their physicochemical properties (elemental composition and behavior in acids and alkalis) and auxin-like activities. However, these biopolymers differ, essentially, at the structural level. According to IR spectroscopy data, the obtained humin-like substances display a higher similarity to natural humic acids and are more diverse in their functional groups compared with fungal melanins. Presumably, this is connected with the fact that laccase is involved in formation of humin-like substances; moreover, this enzyme is involved not only in the synthesis of these polymers, but also in their modification and degradation.     

Isolation and characterization of humin-like substances produced by wood-degrading fungi causing white rot

Three samples of high-molecular-weight humin-like substances were obtained by solid-phase cultivation of Coriolus hirsutus and/or Cerrena maxima on oat straw. The yield of humin-like substances amounted to 1.38-2.26% of the weight of the plant substrate consumed. These substances, produced both by individual and mixed cultures of the basidiomycetes, were shown to be similar in their structure and physicochemical properties. According to the data of IR and 13C-NMR spectroscopy, the substances contained aromatic fragments and were close to soil humic acids. Studies of the dynamics of laccase production suggested that the humin-like substances were produced bia direct degradation of lignin macromolecules with direct involvement of extracellular laccase.     

Isolation and Characterization of Humin-Like Substances Produced by Wood-Degrading White Rot Fungi

Three samples of high-molecular-weight humin-like substances were obtained by solid-phase cultivation of Coriolus hirsutus and/or Cerrena maxima on oat straw. The yield of humin-like substances amounted to 1.38–2.26% of the weight of the plant substrate consumed. These substances, produced both by individual and mixed cultures of the basidiomycetes, were shown to be similar in their structure and physicochemical properties. According to the data of IR and ¹³C-NMR spectroscopy, the substances contained aromatic fragments and were close to soil humic acids. Studies of the dynamics of laccase production suggested that the humin-like substances were produced via direct degradation of lignin macromolecules with direct involvement of extracellular laccase.     

Isolation of fungal cellobiohydrolase I genes from sporocarps and forest soils by PCR

Cellulose is the major component of plant biomass, and microbial cellulose utilization is a key step in the decomposition of plant detritus. Despite this, little is known about the diversity of cellulolytic microbial communities in soil. Fungi are well known for their cellulolytic activity and mediate key functions during the decomposition of plant detritus in terrestrial ecosystems. We developed new oligonucleotide primers for fungal exocellulase genes (cellobiohydrolase, cbhI) and used these to isolate distinct cbhI homologues from four species of litter-decomposing basidiomycete fungi (Clitocybe nuda, Clitocybe gibba, Clitopilus prunulus, and Chlorophyllum molybdites) and two species of ascomycete fungi (Xylaria polymorpha and Sarcoscypha occidentalis). Evidence for cbhI gene families was found in three of the four basidiomycete species. Additionally, we isolated and cloned cbhI genes from the forest floor and mineral soil of two upland forests in northern lower Michigan, one dominated by oak (Quercus velutina, Q. alba) and the other dominated by sugar maple (Acer saccharum) and American basswood (Tilia americana). Phylogenetic analysis demonstrated that cellobiohydrolase genes recovered from the floor of both forests tended to cluster with Xylaria or in one of two unidentified groups, whereas cellobiohydrolase genes recovered from soil tended to cluster with Trichoderma, Alternaria, Eurotiales, and basidiomycete sequences. The ability to amplify a key fungal gene involved in plant litter decomposition has the potential to unlock the identity and dynamics of the cellulolytic fungal community in situ.     

基于国际专利数据的真菌黑色素创新发展趋势分析

【背景】黑色素具有抗肿瘤、抗辐射等多种生物活性,在生产和生活中具有巨大的应用潜力,通过真菌生产是获取黑色素的一条重要途径,它与动植物相比具有更短的生产周期和更高的产量,并且易于实现商业应用。【目的】揭示真菌黑色素的生产及应用发展情况和创新趋势,为致力于真菌黑色素产业的科研人员和企业提供参考。【方法】基于Inco Pat科技创新情报平台,通过对全球真菌黑色素相关专利进行检索统计,从专利涉及的菌株、专利技术构成、申请人专利价值等多维度对真菌黑色素相关专利进行深入分析。【结果】真菌黑色素在生产制备领域申请专利数量最多(50.56%),作为化工染料和化妆品原料应用的专利申请数量最少(13.48%),涉及的真菌菌属主要有层孔菌属、短梗霉属、木耳属、纤孔菌属、粒毛盘菌属、灵芝属和曲霉属。目前真菌黑色素专利技术申请的热点领域主要在C12 (微生物发酵、培养基、突变或遗传工程)类和A61 (医学)类,并且在未来一段时间内,C12和A61将继续作为热点技术申请领域。中国申请的真菌黑色素专利数量最多,但拥有的高价值专利比例较低,较国外仍具有一定差距。【结论】我国科研人员需要加强在医药和化工领域核心专利技术的创新应用与海外布局,增强与企业的合作研发和技术转移,以抢占真菌黑色素在这些领域的应用市场,并推动真菌黑色素相关专利向高质量发展。     

Synthesis and assembly of fungal melanin

Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from l-3,4-dihydroxyphenylalanine (l-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur

Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.     

A scytalone dehydratase gene from<Emphasis Type="Italic"> Ophiostoma floccosum</Emphasis> restores the melanization and pathogenicity phenotypes of a melanin-deficient<Emphasis Type="Italic"> Colletotrichum lagenarium</Emphasis> mutant

Scytalone dehydratase is involved in the production of fungal dihydroxynaphthalene (DHN) melanin. We have isolated and characterized OSD1, a gene encoding scytalone dehydratase from the sap-staining fungus Ophiostoma floccosum by PCR-based cloning. Sequence analysis suggests that the OSD1 gene encodes a protein of 216 amino acids with a molecular weight of 24.2 kDa that shows 51-70% sequence identity to other scytalone dehydratases. The cloned OSD1 contains two introns of 76 bp and 63 bp in length, and is the longest scytalone dehydratase gene sequence so far reported. Transformation of a DHN melanin-deficient, non-pathogenic, mutant of Colletotrichum lagenarium with the OSD1 gene restored melanin production and pathogenicity. The ability of the mutant to produce the OSD1 gene product was confirmed by RT-PCR analysis. These data show that the cloned OSD1 gene product can function in the DHN melanin biosynthetic pathway in C. lagenarium.     

Phospholipid acylhydrolases trigger membrane degradation during fungal sporogenesis

Armillaria ostoyae is a phytopathogen infecting coniferous trees. Fruiting bodies of this basidiomycete contain high phospholipase A(1) (PLA(1)) activity. In this paper, the role of phospholipid-deacylating activity, which was also detected in fruiting bodies of other basidiomycetes, in the fungal lipid metabolism is elucidated. For A. ostoyae the occurrence of PLA(1) activity is shown to be restricted to the late reproductive phase, correlating with the release of mature spores. Specific expression in the spore-producing tissue provides evidence for the involvement of PLA(1) in spore formation. Based on lipid analysis, the degradation of membrane phospholipids in this tissue can be ascribed mainly to PLA(1) activity because other enzymes such as phospholipases C and D, triglyceride lipase and phosphatidic acid phosphatase had only low activities. A concomitant increase in the concentration of fatty acids and their anabolites (di- and triglycerides), which are used as storage lipids in the developing fungal spore cells, was observed. Therefore, PLA(1) contributes to the formation of spores by providing membrane constituents as a source of fatty acids.     

Characterization of genes encoding two manganese peroxidases from the lignin-degrading fungus Dichomitus squalens(1).

Genes encoding two manganese peroxidases from the white-rot basidiomycete Dichomitus squalens were cloned and sequenced. The mnp1 and mnp2 genes encode mature proteins of 369 and 365 amino acids, respectively. The amino acids involved in peroxidase function, those forming the Mn(II) binding site, and those forming the five disulfide bonds in other Mn peroxidases are conserved in these sequences. Both predicted D. squalens proteins contain multiple acidic residues in their C-terminal sequences, which may be involved in additional metal binding. Both genes contain seven small introns, the locations of which align with each other. The promoters of both D. squalens genes contain putative AP-2 sites, which may be involved in their regulation by nutrient nitrogen. Southern blot analysis of genomic PCR fragments suggests that these sequences represent separate genes rather than allelic variants.     

Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

水生生物的紫外防护剂——类菌胞素氨基酸

类菌胞素氨基酸(mycosporine-like amino acids, MAAs)是一大类以环己烯酮为基本骨架, 与不同类型氨基酸通过缩合作用形成的水溶性物质。它能在真菌、海洋细菌、蓝藻和真核藻类细胞中合成, 并在海生无脊椎动物和鱼类等生物体内积累, 广泛存在于多种水生生物中。它具有紫外光防护、渗透、繁殖调节及发育保护等功能。本文主要介绍有关类菌胞素氨基酸的分布、结构、生物合成和积累以及生理功能的研究进展。     

水生生物的紫外光防护剂--类菌胞素氨基酸

类菌胞素氨基酸（mycosporine—like amino acids,MAAs）是一大类以环己烯酮为基本骨架,与不同类型氨基酸通过缩合作用形成的水溶性物质。它能在真菌、海洋细菌、蓝藻和真核藻类细胞中合成,并在海生无脊椎动物和鱼类等生物体内积累,广泛存在于多种水生生物中。它具有紫外光防护、渗透、繁殖调节及发育保护等功能。本文主要介绍有关类菌胞素氨基酸的分布、结构、生物合成和积累以及生理功能的研究进展。     

Genetic analysis of biosurfactant production in Ustilago maydis

The dimorphic basidiomycete Ustilago maydis produces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated beta-d-mannopyranosyl-d-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes, emt1 and cyp1, that are essential for the production of fungal extracellular glycolipids. emt1 is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-d-erythritol by transfer of GDP-mannose. Deletion of the gene cyp1 resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.