The genetic basis for indole-diterpene chemical diversity in filamentous fungi

Indole-diterpenes are a structurally diverse group of secondary metabolites with a common cyclic diterpene backbone derived from geranylgeranyl diphosphate and an indole group derived from indole-3-glycerol phosphate. Different types and patterns of ring substitutions and ring stereochemistry generate this structural diversity. This group of compounds is best known for their neurotoxic effects in mammals, causing syndromes such as ‘ryegrass staggers’ in sheep and cattle. Because many of the fungi that synthesise these compounds form symbiotic relationships with plants, insects, and other fungi, the synthesis of these compounds may confer an ecological advantage to these associations. Considerable recent progress has been made on understanding indole-diterpene biosynthesis in filamentous fungi, principally through the cloning and characterisation of the genes and gene products for paxilline biosynthesis in Penicillium paxilli. Important insights into how the indole-diterpene backbone is synthesised and decorated have been obtained using P. paxilli mutants in this pathway. This review provides an overview of these recent developments.     

Regulation of secondary metabolite production in filamentous ascomycetes

Fungi are renowned for their ability to produce bioactive small molecules otherwise known as secondary metabolites. These molecules have attracted much attention due to both detrimental (e.g. toxins) and beneficial (e.g. pharmaceuticals) effects on human endeavors. Once the topic only of chemical and biochemical studies, secondary metabolism research has reached a sophisticated level in the realm of genetic regulation. This review covers the latest insights into the processes regulating secondary metabolite production in filamentous fungi.     

A new species of Phaeohelotium from Nothofagus forests in Argentina and Chile, with a key to the Southern Hemisphere species

Phaeohelotium nothofagi, sp.nov., found on fallen wood and leaves of Nothofagus dombeyi, is described and illustrated. The fungus is characterized by pale yellow discs, and fumose ascospores with asperulate walls. A key with distributional data for the five species of the genus now known in the Southern Hemisphere is provided.     

Hyperinduction of nitrilases in filamentous fungi

2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.     

Elephant dung decomposition and coprophilous fungi in two habitats of semi-arid Botswana

In order to understand the impact of habitat changes on ecosystem processes caused by increased populations of elephants, elephant dung decomposition was studied in semi-arid Botswana. Dung decomposition rates were studied with and without the presence of arthropods, using pairs of exposed dung and dung enclosed in nylon-mesh bags, respectively. Dung decomposition rates were lower in the absence of arthropods. The rates in the late wet season were higher in the scrubland than in the woodland. In the early dry season, immediately after the wet season, the rates were higher in the woodland than in the scrubland. The difference in decomposition rates between habitats was attributed to microclimatic conditions created by vegetation cover. With regard to fungal succession, Cladosporium cladosporioides and Eurotium brefeldianum occurred only in the late stages of dung decomposition whereas Talaromyces helicus, Cercophora coprophila and Sporormiella minima occurred in all the stages. Although there was no significant difference in Shannon–Weiner fungal species diversity index between habitats, seasons, dung ages and laboratory incubation periods, there were significant differences in fungal community composition between these parameters. Species richness was higher in the late wet season than in the early dry season, indicating the importance of moist conditions for a large diversity of fungal species.     

The sterile microfilamentous lichenized fungi Cystocoleus ebeneus and Racodium rupestre are relatives of plant pathogens and clinically important dothidealean fungi

The phylogenetic positions of the always-sterile microfilamentous lichens Cystocoleus ebeneus and Racodium rupestre were studied in a phylogenetic framework using sequence data of 5′ nuSSU, nuLSU, and mtSSU rDNA. The analysis reveals that both genera are ascomycetes and belong to Dothideomycetidae: they are not close to lichenized members within the subclass, but rather belong to Capnodiales. The macroscopically scarcely distinguishable C. ebeneus and R. rupestre do not form a monophyletic group. The well-supported clade of R. rupestre is basal to the one in which C. ebeneus is close to Mycosphaerellaceae. This study provides another example of ascomycetes with very different life-styles and ecologies being closely related.     

The significance of peroxisomes in secondary metabolite biosynthesis in filamentous fungi

Peroxisomes are ubiquitous organelles characterized by a protein-rich matrix surrounded by a single membrane. In filamentous fungi, peroxisomes are crucial for the primary metabolism of several unusual carbon sources used for growth (e.g. fatty acids), but increasing evidence is presented that emphasize the crucial role of these organelles in the formation of a variety of secondary metabolites. In filamentous fungi, peroxisomes also play a role in development and differentiation whereas specialized peroxisomes, the Woronin bodies, play a structural role in plugging septal pores. The biogenesis of peroxisomes in filamentous fungi involves the function of conserved PEX genes, as well as genes that are unique for these organisms. Peroxisomes are also subject to autophagic degradation, a process that involves ATG genes. The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells.     

Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.     

丝状真菌杀松材线虫代谢产物研究进展

松材线虫病是破坏我国森林生态系统最为严重的病害,具有极强的传播性和破坏性,防治此种病害迫在眉睫。基于对物理和化学方式防治松材线虫的研究,对环境友好度最高的生物防治具有更广的研究前景。丝状真菌及其次级代谢产物,来源于自然,与传统的化学杀线虫药剂相比,对环境影响较小,针对松材线虫的致死作用更为专一,因此,从丝状真菌的次级代谢产物中分离获得杀松材线虫活性产物并测定其结构和活性,对于松材线虫病的防治具有重要意义。本文对丝状真菌产生的具有杀松材线虫活性产物的结构、活性展开综述,发现近二十年共有57个活性产物被发现,且结构多种多样,活性差别较大,为了更好地开展此领域的研究,本文对所有产物的结构和活性进行了系统总结,最后又对该领域的研究进行了总结和展望,以期对松材线虫病的生物防治和丝状真菌杀松材线虫次级代谢产物的深入研究提供参考。     

Corylomyces: a new genus of Sordariales from plant debris in France

The new genus Corylomyces, isolated from the surface of a hazelnut (Corylus avellana) in the French Pyrenees, is described, illustrated and compared with morphologically similar taxa. It is characterised by tomentose, ostiolate ascomata possessing long necks composed of erect to sinuose hairs, and one- or two-celled, opaque, lunate to reniform ascospores. Analyses of the SSU and LSU fragments rDNA gene sequences support its placement in the Lasiosphaeriaceae (Sordariales).     

In situ hybridisation in filamentous fungi using peptide nucleic acid probes

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.     

Stability of classification of filamentous fungi under changes in character coding strategy

The characteristics of a number of filamentous fungal cultures were obtained from two previously published numerical taxonomic studies on Penicillium and Phoma. The coding strategies for some of the physiological and morphological properties employed in the original studies were re-examined and the data was re-coded by combining sets of characters into single ordered multistate characters. The different coding procedures were compared by generating average linkage (UPGMA) dendrograms which were in turn compared by calculating correlation coefficients between the final similarity matrices implied by these dendrograms. The character conversions had no significant effect on the final outcome of the clustering.     

Liposome-mediated mycelial transformation of filamentous fungi

Liposome-mediated transformation is common for cells with no cell wall, but has very limited usage in cells with walls, such as bacteria, fungi, and plants. In this study, we developed a procedure to introduce DNA into mycelium of filamentous fungi, Rhizopus nigricans LH 21 and Pleurotus ostreatus TD 300, by liposome-mediation but with no protoplast preparation. The DNA was transformed into R. nigricans via plasmid pEGFP-C1 and into P. ostreatus via 7.2 kb linear DNA. The mycelia were ground in 0.6 M mannitol without any grinding aids or glass powder for 15 min to make mycelial fragments suspension; the suspension was mixed with a mixture of the DNA and Lipofectamine 2000, and placed on ice for 30 min; 100 μL of the transformation solution was plated on potato dextrose agar (PDA) plate and cultivated at 28 °C for transformant screening. The plasmid and the linear DNA were confirmed to be integrated into the host chromosome, proving the success of transformation. The transformation efficiencies were similar to those of electroporation-mediated protoplast transformation (EMPT) of R. nigricans or PEG/CaCl₂-mediated protoplast transformation (PMT) of P. ostreatus, respectively. The results showed that our procedure was effective, fast, and simple transformation method for filamentous fungi.     

Phylogenetic and populational study of the Tuber indicum complex

When examined using SEM, Chinese samples of Tuber indicum and T. sinense displayed the same ascospore ornamentation as that of T. pseudohimalayense, T. indicum, collected in India by Duthie in 1899, and samples renamed T. himalayense in 1988. The different authors who named the four taxa (T. indicum, T. himalayense, T. sinense, T. pseudohimalyense) described differences in the surface of the peridium which could be considered as usual variations within a single species. We consider T. indicum, T. himalayense, T. sinense and T. pseudohimalayense as one species, T. indicum. Within this T. indicum complex, according to ITS and β-tubulin sequences, there are two groups in China, which could be considered as geographical ecotypes. This study is the first to identify a genetic and phylogeographical structure within the Chinese Tuber species.     

Strategies for improving heterologous protein production from filamentous fungi

Despite the naturally high capacity for protein secretion by many species of filamentous fungi, secteted yields of many heterologous proteins have been comparatively low. The strategies for yield improvement have included the use of strong homologous promoters, increased gene copy number, gene fusions with a gene encoding a naturally well-secreted protein, protease-deficient host strains and screening for high yields following random mutagenesis. Such approaches have been effective with some target heterologous proteins but not others.Approaches used in heterologous protein production from filamentous fungi are discussed and a perspective on emerging strategies is presented.     

丝状真菌次级代谢产物生物合成的表观遗传调控

丝状真菌产生的次级代谢产物是新药的重要来源之一,其生物合成过程受到众多因素的调控。最近的研究表明,表观遗传对多种丝状真菌次级代谢产物的生物合成具有调控作用。DNA和组蛋白的甲基化与乙酰化修饰是目前所知的丝状真菌主要的表观遗传调控形式。通过过表达或缺失相关表观修饰基因和利用小分子表观遗传试剂改变丝状真菌染色体的修饰形式,不仅可以提高多种已知次级代谢产物产量,而且可以通过激活沉默的生物合成基因簇诱导丝状真菌产生新的未知代谢产物。丝状真菌表观遗传学正逐渐成为真菌菌株改良的新策略以及挖掘真菌次级代谢产物合成潜力的强有力手段。     

A quantitative bioassay for extracellular metabolites that antagonize growth of filamentous fungi,and its use with biocontrol fungi

A bioassay and an empirically derived formula were developed to quantify fungitoxic effects. This bioassay can be easily performed and objectively read, and it is suitable for low-volume samples originating from aqueous or organic solvents. The formula defines the Inhibition Index (I), a single value that incorporates both the response to concentrations of the inhibitory compound and the persistence of inhibition. Antagonistic efficacy of metabolites produced by biocontrol strains of Trichoderma spp. were measured based on inhibition of growth of Rhizoctonia solani. Although the bioassay itself was not influenced by pH or light conditions, these factors affected metabolite production or activity. Aqueous extracts from light-grown germlings of Trichoderma virens inhibited R. solani more than extracts from germlings grown in the dark. Low pH increased the inhibitory activity of extracts from T. virens. Tests of fungal strains developed for biocontrol demonstrated that the bioassay reflected their activity both in the field and in other in vitro tests. The bioassay and formula are readily adapted for use with other fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.