Fungal endophytes and bioprospecting

Horizontally transmitted fungal endophytes are an ecological group of fungi, mostly belonging to the Ascomycota, that reside in the aerial tissues and roots of plants without inducing any visual symptoms of their presence. These fungi appear to have a capacity to produce an array of secondary metabolites exhibiting a variety of biological activity. Although the ability of fungi to produce unique bioactive metabolites is well known, endophytes have not been exploited, perhaps because we are only beginning to understand their distribution and biology. This review emphasizes the need to routinely include endophytic fungi in the screening of organisms for bioactive metabolites and novel drugs; it also underscores the need to use information obtained concerning fungal secondary metabolite production from other groups of fungi for a targeted screening approach.     

Secondary metabolism: regulation and role in fungal biology

Filamentous fungi produce a diverse array of secondary metabolites--small molecules that are not necessary for normal growth or development. Secondary metabolites have a tremendous impact on society; some are exploited for their antibiotic and pharmaceutical activities, others are involved in disease interactions with plants or animals. The availability of fungal genome sequences has led to an enhanced effort at identifying biosynthetic genes for these molecules. Genes that regulate production of secondary metabolites have been identified and a link between secondary metabolism, light and sexual/asexual reproduction established. However, the role of secondary metabolites in the fungi that produce them remains a mystery. Many of these fungi live saprophytically in the soil and such molecules may provide protection against other inhabitants in this ecological niche.     

Bioactive secondary metabolites produced by microorganisms associated with plants

In the past few decades groups of scientists have focused their study on relatively new microorganisms called endophytes. By definition these microorganisms, mostly fungi and bacteria, colonise the intercellular spaces of the plant tissues. The mutual relationship between endophytic microorganisms and their host plants, taxanomy and ecology of endophytes are being studied. Some of these microorganisms produce bioactive secondary metabolites that may be involved in a host-endophyte relationship. Recently, many endophytic bioactive metabolites, known as well as new substances, possesing a wide variety of biological activities as antibiotic, antitumor, antiinflammatory, antioxidant, etc. have been identified. The microorganisms such as endophytes may be very interesting for biotechnological production of bioactive substances as medicinally important agents. Therefore the aim of this review is to briefly characterize endophytes and summarize the structuraly different bioactive secondary metabolites produced by endophytic microorganisms as well as microbial sources of these metabolites and their host plants.     

How many fungi make sclerotia?

Most fungi produce some type of durable microscopic structure such as a spore that is important for dispersal and/or survival under adverse conditions, but many species also produce dense aggregations of tissue called sclerotia. These structures help fungi to survive challenging conditions such as freezing, desiccation, microbial attack, or the absence of a host. During studies of hypogeous fungi we encountered morphologically distinct sclerotia in nature that were not linked with a known fungus. These observations suggested that many unrelated fungi with diverse trophic modes may form sclerotia, but that these structures have been overlooked. To identify the phylogenetic affiliations and trophic modes of sclerotium-forming fungi, we conducted a literature review and sequenced DNA from fresh sclerotium collections. We found that sclerotium-forming fungi are ecologically diverse and phylogenetically dispersed among 85 genera in 20 orders of Dikarya, suggesting that the ability to form sclerotia probably evolved ≥14 different times in fungi.     

丝状真菌基因工程进展

丝状真菌被广泛应用于生物工程来生产化学药品、药物制剂及酶。为了提高其生产能力,基因工程战略是一种较好的途径。为了使丝状真菌更好的用于工业生产,近年来出现许多新的转化技术。然而,由于缺乏有效的基因工程战略,许多真菌在生产新的、有商业价值的代谢产物上都存在着不足。在此总结了几种最新介导和控制基因表达的方法,并讨论了各自的优、缺点。此外,对丝状真菌今后的发展进行了展望。     

Cryopreservation of Microorganisms

Abstract

A great deal of recent interest has been shown in the ability of some microbes to synthesize exopolysaccharides. Most attention has been directed toward the prokaryote producers, yet many filamentous fungi also produce exopolysaccharides that have chemical and physical properties of considerable commercial potential. Surprisingly little is known about how and why fungi overproduce these metabolites and how yields are affected by both the physical and chemical environments. This review attempts to critically appraise the current literature on fungal exopolysaccharides, considers their chemical diversity, and examines factors that seem to affect their production. Although much of the published work has been carried out with the α-glucan pullulan, there is considerable literature on the β-glucans and, hence, both of these are discussed.     

TNT biotransformation: when chemistry confronts mineralization

The recent increase and availability of whole genome sequences have revised our view of the metabolic capabilities of microorganisms. From these data, a large number of orphan biosynthesis pathways have been identified by bio-informatics. Orphan biosynthetic pathways are gene clusters for which the encoded natural product is unknown. It is worthy to note that the number of orphan pathways coding for putative natural products outnumbers by far the number of currently known metabolites for a given organism. Whilst Streptomyces coelicolor was known to produce only 4 secondary metabolites, the genome analysis revealed 18 additional orphan biosynthetic pathways. It is intriguing to note that this is not a particular case because analysis of other microbial genomes originating from myxobacteria, cyanobacteria and filamentous fungi showed the presence of a comparable or even larger number of orphan pathways. The discovery of these numerous pathways represents a treasure trove, which is likely to grow exponentially in the future, uncovering many novel and possibly bio-active compounds. The few natural products that have been correlated with their orphan pathway are merely the tip of the iceberg, whilst plenty of metabolites await discovery. The recent strategies and methods to access these promising hidden natural products are discussed in this review.     

Mutagens manufactured in fungal culture may affect DNA/RNA of producing fungi

Self-produced mutagens in culture by fungi may affect DNA analysis of the same fungi. This has not been considered previously. Many fungi produce numerous mutagenic secondary metabolites (SM) in culture. There is a paradox of growing fungi in media to produce representative DNA which also support mutagenic SM. This is a crucial issue in developing diagnostic and phylogenetic methods, especially for closely-related fungi. For example, idh gene analysis of the patulin metabolic pathway in fungi can be interpreted as producing some false negative and positive results in terms of possession, or nonpossession, of the gene from mutated strains. The most obvious mycotoxins and fungi to consider in this regard are aflatoxins and Aspergillus , as aflatoxins are the most mutagenic natural compounds. Many other fungi and SM are relevant. Conditions to grow fungi have not been selected to inhibit SM production although relevant data exist. In fact, fungi repair damaged nucleic acid (NA) and are capable of removing toxins by employing transporter proteins. These and NA repair mechanisms could be inhibited by secondary metabolites. Mutagenic effects may involve inhibition of DNA stabilizing enzymes. There may be an equivalent situation for bacteria. Researchers need to devise methods to reduce SM for valid protocols. More work on how mutagens affect the NA of producing fungus in vitro is required. The current review assesses the potential seriousness of the situation with selected papers.     

Genomics reveals traces of fungal phenylpropanoid-flavonoid metabolic pathway in the f ilamentous fungus Aspergillus oryzae

Fungal secondary metabolites constitute a wide variety of compounds which either play a vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to play a vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.     

Biochemical mutagens affect the preservation of fungi and biodiversity estimations

Many fungi have significant industrial applications or biosafety concerns and maintaining the original characteristics is essential. The preserved fungi have to represent the situation in nature for posterity, biodiversity estimations, and taxonomic research. However, spontaneous fungal mutations and secondary metabolites affecting producing fungi are well known. There is increasing interest in the preservation of microbes in Biological Resource Centers (BRC) to ensure that the organisms remain viable and stable genetically. It would be anathema if they contacted mutagens routinely. However, for the purpose of this discussion, there are three potential sources of biochemical mutagens when obtaining individual fungi from the environment: (a) mixtures of microorganisms are plated routinely onto growth media containing mutagenic antibiotics to control overgrowth by contaminants, (b) the microbial mixtures may contain microorganisms capable of producing mutagenic secondary metabolites, and (c) target fungi for isolation may produce “self” mutagens in pure culture. The probability that these compounds could interact with fungi undermines confidence in the preservation process and the potential effects of these biochemical mutagens are considered for the first time on strains held in BRC in this review.     

Endophytic fungi—The treasure chest of antibacterial substances

Over more than 20 years, the endophytic fungi have been explored as “biofactories” of novel bioactive substances, and they have not disappointed. Among the extracts and pure substances obtained from the culture broths or fungal biomass, some have exerted antibacterial activity ranging from moderate to powerful when tested on the bacterial strains resistant to the antibiotics currently in use. In this article we review the accumulated data on endophytic fungi isolated from plants that produce metabolites with antibacterial activity against human pathogenic bacteria.     

The Effect of Stereochemistry on the Biological Activity of Natural Phytotoxins,Fungicides, Insecticides and Herbicides

Phytotoxins are secondary microbial metabolites that play an essential role in the development of disease symptoms induced by fungi on host plants. Although phytotoxins can cause extensive—and in some cases devastating—damage to agricultural crops, they can also represent an important tool to develop natural herbicides when produced by fungi and plants to inhibit the growth and spread of weeds. An alternative strategy to biologically control parasitic plants is based on the use of plant and fungal metabolites, which stimulate seed germination in the absence of the host plant. Nontoxigenic fungi also produce bioactive metabolites with potential fungicide and insecticide activity, and could be applied for crop protection. All these metabolites represent important tools to develop eco‐friendly pesticides. This review deals with the relationships between the biological activity of some phytotoxins, seed germination stimulants, fungicides and insecticides, and their stereochemistry. Chirality 25:59–78, 2013. © 2012 Wiley Periodicals, Inc.     

Secretion systems for secondary metabolites: how producer cells send out messages of intercellular communication

Many secondary metabolites (e.g. antibiotics and mycotoxins) are toxic to the microorganisms that produce them. The clusters of genes that are responsible for the biosynthesis of secondary metabolites frequently contain genes for resistance to these toxic metabolites, such as different types of multiple drug resistance systems, to avoid suicide of the producer strains. Recently there has been research into the efflux systems of secondary metabolites in bacteria and in filamentous fungi, such as the large number of ATP-binding cassette transporters found in antibiotic-producing Streptomyces species and that are involved in penicillin secretion in Penicillium chrysogenum. A different group of efflux systems, the major facilitator superfamily exporters, occur very frequently in a variety of bacteria that produce pigments or antibiotics (e.g. the cephamycin and thienamycin producers) and in filamentous fungi that produce mycotoxins. Such efflux systems include the CefT exporters that mediate cephalosporin secretion in Acremonium chrysogenum. The evolutionary origin of these efflux systems and their relationship with current resistance determinants in pathogenic bacteria has been analyzed. Genetic improvement of the secretion systems of secondary metabolites in the producer strain has important industrial applications.     

Endophyte research: going beyond isolation and metabolite documentation

Many fungi belonging to mostly Ascomycota inhabit living tissues of plants of all major lineages without causing any visible symptoms. Termed horizontally transmitted endophytes, they have been investigated mostly for their capacity to produce bioactive secondary metabolites. However, many questions regarding the interactions between endophytes and their plant hosts, phytophagous insects and other fungi remain unanswered. This review highlights some of these areas of endophyte biology about which very little or no knowledge exists. Information garnered' using modern methodologies' on these grey areas of ‘endophytism’ (endophytic mode of lifestyle) would help immensely in understanding the evolution of endophytes of aerial plant tissues and in exploiting endophytes in various fields of biotechnology.     

真菌天然产物异源生产研究进展

真菌天然产物是天然药物的重要来源之一,大规模真菌基因组序列测序的完成表明真菌具有产生丰富的次级代谢产物的潜能。然而,许多真菌或生长缓慢,或不适宜在实验室条件下培养,或难以进行遗传操作,或化合物产量极低等,这些因素导致大量有价值的真菌天然产物无法获得。利用异源表达系统对真菌天然产物进行生产是发现新天然产物及解析其生物合成途径的有效手段,并为定向的以合成生物学的手段去合成重要活性分子奠定基础。本文对目前用于真菌天然产物生产的各种异源表达系统进行了综述,并结合最新的DNA组装技术展望了异源表达系统在真菌天然产物研究中的应用价值和前景。     

The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi

A secondary metabolite is a chemical compound produced by a limited number of fungal species in a genus, an order, or even phylum. A profile of secondary metabolites consists of all the different compounds a fungus can produce on a given substratum and includes toxins, antibiotics and other outward-directed compounds. Chemotaxonomy is traditionally restricted to comprise fatty acids, proteins, carbohydrates, or secondary metabolites, but has sometimes been defined so broadly that it also includes DNA sequences. It is not yet possible to use secondary metabolites in phylogeny, because of the inconsistent distribution throughout the fungal kingdom. However, this is the very quality that makes secondary metabolites so useful in classification and identification. Four groups of organisms are particularly good producers of secondary metabolites: plants, fungi, lichen fungi, and actinomycetes, whereas yeasts, protozoa, and animals are less efficient producers. Therefore, secondary metabolites have mostly been used in plant and fungal taxonomy, whereas chemotaxonomy has been neglected in bacteriology. Lichen chemotaxonomy has been based on few biosynthetic families (chemosyndromes), whereas filamentous fungi have been analysed for a wide array of terpenes, polyketides, non-ribosomal peptides, and combinations of these. Fungal chemotaxonomy based on secondary metabolites has been used successfully in large ascomycete genera such as Alternaria, Aspergillus, Fusarium, Hypoxylon, Penicillium, Stachybotrys, Xylaria and in few basidiomycete genera, but not in Zygomycota and Chytridiomycota.     

Analysis of myxobacterial secondary metabolism goes molecular

During the last 20 years myxobacteria have made their way from highly exotic organisms to one of the major sources of microbial secondary metabolites besides actinomycetes and fungi. The pharmaceutical interest in these peculiar prokaryotes lies in their ability to produce a variety of structurally unique compounds and/or metabolites with rare biological activities. This review deals with the recent progress toward a better understanding of the biology, the genetics, the biochemistry and the regulation of secondary metabolite biosynthesis in myxobacteria. These research efforts paved the way to sophisticated in vitro studies and to the heterologous expression of complete biosynthetic pathways in conjunction with their targeted manipulation. The progress made is a prerequisite for using the vast resource of myxobacterial diversity regarding secondary metabolism more efficiently in the future.     

Natural product discovery through microbial genome mining

The advent of the genomic era has opened up enormous possibilities for the discovery of new natural products. Also known as specialized metabolites, these compounds produced by bacteria, fungi, and plants have long been sought for their bioactive properties. Innovations in both DNA sequencing technologies and bioinformatics now allow the wealth of sequence data to be mined at both the genome and metagenome levels for new specialized metabolites. However, a key problem that remains is rapidly and efficiently linking these identified genes to their corresponding compounds. Within this review, we provide specific examples of studies that have used the power of genomic or metagenomic data to overcome these problems and identify new small molecules and their biosynthetic pathways.     

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。