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Filamentous fungi represent an incredibly rich and rather overlooked reservoir of natural products, which often show potent bioactivity and find applications in different fields. Increasing the naturally low yields of bioactive metabolites within their host producers can be problematic, and yield improvement is further hampered by such fungi often being genetic intractable or having demanding culturing conditions. Additionally, total synthesis does not always represent a cost-effective approach for producing bioactive fungal-inspired metabolites, especially when pursuing assembly of compounds with complex chemistry. This review aims at providing insights into heterologous production of secondary metabolites from filamentous fungi, which has been established as a potent system for the biosynthesis of bioactive compounds. Numerous advantages are associated with this technique, such as the availability of tools that allow enhanced production yields and directing biosynthesis towards analogues of the naturally occurring metabolite. Furthermore, a choice of hosts is available for heterologous expression, going from model unicellular organisms to well-characterised filamentous fungi, which has also been shown to allow the study of biosynthesis of complex secondary metabolites. Looking to the future, fungi are likely to continue to play a substantial role as sources of new pharmaceuticals and agrochemicals—either as producers of novel natural products or indeed as platforms to generate new compounds through synthetic biology.

Background Fungi are talented producers of secondary metabolites with applications in the pharmaceutical and agrochemical sectors. Aspergillus wentii CBS 141173 has gathered research interest due to its ability to produce high-value norditerpenoid compounds, including anticancer molecules. In this study, we aimed to expand the genomic information available for A. wentii to facilitate the identification of terpenoid biosynthetic genes that may be involved in the production of bioactive molecules. Results Long-read genome sequencing of Aspergillus wentii CBS 141173 was conducted using Oxford Nanopore Technologies (ONT) MinION MK1C. In addition, paired-end stranded RNA-seq data from two time points, 7 days and 30 days, was used for functional annotation of the assembled genome. Overall, we assembled a genome of approximately 31.2 Mb and identified 66 biosynthetic gene clusters from the annotated genome. Metabolic extracts of A. wentii were analysed and the production of the bioactive terpenoid asperolide A was confirmed. We further mined the assembled and annotated genome for BGCs involved in terpenoid pathways using a combination of antiSMASH and local BlastP and identified 16 terpene synthases. Phylogenetic analysis was conducted and allowed us to establish relationships with other characterised terpene synthases. We identified two terpene clusters potentially involved in pimarane-like diterpenoid biosynthesis. Finally, the analysis of the 16 terpene synthases in our 7-day and 30-day transcriptomic data suggested that only four of them were constitutively expressed under laboratory conditions. Conclusion These results provide a scaffold for the future exploration of terpenoid biosynthetic pathways for bioactive molecules in A. wentii . The terpenoid clusters identified in this study are candidates for heterologous gene expression and/or gene disruption experiments. The description and availability of the long-read genome assembly of A. wentii CBS 141173 further provides the basis for downstream genome analysis and biotechnological exploitation of this species.

Background Tetracladium spp. represent a group of fungi that inhabit various ecological niches, including soil and aquatic environments, where they are considered to have a saprotrophic lifestyle and within plant roots as endophytes. To date, a lack of sequenced Tetracladium spp. genomes has inhibited our understanding of their metabolic potential and ecological interactions. In this study, we aimed to elucidate the genetic differences between aquatic saprotrophic and endophytic strains of Tetracladium spp. by sequencing and analysing the genomes of T. maxilliforme (isolated from Brassica napus roots) and T. marchalianum (isolated from freshwater), alongside 41 publicly available saprotrophic and endophytic Ascomycetes. Results Genomic sequencing revealed that T. maxilliforme possesses a genome size of 35.5 Mbp with 9657 predicted genes, while T. marchalianum has a genome size of 33.2 Mbp with 15,230 predicted genes. Our analyses primarily focused on carbohydrate-active enzymes (CAZymes). Both genomes possessed the full range of enzymatic machinery for cellulose degradation, as well as the complete repertoire of genes necessary to degrade plant cell walls. Notably, the genomes lacked essential enzymes for lignin degradation or modification. Furthermore, we observed a complete repertoire of known fungal chitin-degrading enzymes in both genomes, which might be related to potential interactions with other fungi. Enzyme composition profiles revealed distinct groupings, with T. maxilliforme primarily clustering with endophytic or ecologically versatile species, while T. marchalianum was predominantly associated with saprotrophic species. We also identified secondary metabolite biosynthetic gene clusters in both genomes, including several that showed high homology to those of known bioactive compounds. Conclusions In summary, our findings offer valuable insights into the genomic adaptations of Tetracladium spp. to various ecological niches, highlighting their enzymatic capabilities for carbohydrate degradation and potential interactions within fungal communities.

The rise in antibiotic resistance is a major threat for human health. Basidiomycete fungi represent an untapped source of underexploited antimicrobials, with pleuromutilin—a diterpene produced by Clitopilus passeckerianus —being the only antibiotic from these fungi leading to commercial derivatives. Here we report genetic characterisation of the steps involved in pleuromutilin biosynthesis, through rational heterologous expression in Aspergillus oryzae coupled with isolation and detailed structural elucidation of the pathway intermediates by spectroscopic methods and comparison with synthetic standards. A. oryzae was further established as a platform for bio-conversion of chemically modified analogues of pleuromutilin intermediates, and was employed to generate a semi-synthetic pleuromutilin derivative with enhanced antibiotic activity. These studies pave the way for future characterisation of biosynthetic pathways of other basidiomycete natural products in ascomycete heterologous hosts, and open up new possibilities of further chemical modification for the growing class of potent pleuromutilin antibiotics. Pleuromutilin derivatives are potent antibacterial drugs obtained from Basidiomycete fungi. Here, the authors report the genetic characterisation of the steps involved in pleuromutilin biosynthesis through heterologous expression and generate a semi-synthetic pleuromutilin derivative with enhanced antibiotic activity.

Basidiomycota are a large and diverse phylum of fungi. They can make bioactive metabolites that are used or have inspired the synthesis of antibiotics and agrochemicals. Terpenoids are the most abundant class of natural products encountered in this taxon. Other natural product classes have been described, including polyketides, peptides, and indole alkaloids. The discovery and study of natural products made by basidiomycete fungi has so far been hampered by several factors, which include their slow growth and complex genome architecture. Recent developments of tools for genome and metabolome studies are allowing researchers to more easily tackle the secondary metabolome of basidiomycete fungi. Inexpensive long-read whole-genome sequencing enables the assembly of high-quality genomes, improving the scaffold upon which natural product gene clusters can be predicted. CRISPR/Cas9-based engineering of basidiomycete fungi has been described and will have an important role in linking natural products to their genetic determinants. Platforms for the heterologous expression of basidiomycete genes and gene clusters have been developed, enabling natural product biosynthesis studies. Molecular network analyses and publicly available natural product databases facilitate data dereplication and natural product characterisation. These technological advances combined are prompting a revived interest in natural product discovery from basidiomycete fungi. This article has an associated Future Leader to Watch interview with the first author of the paper.

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus , no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

Fungi represents a rich repository of taxonomically restricted, yet chemically diverse, secondary metabolites that are synthesised via specific metabolic pathways. An enzyme’s specificity and biosynthetic gene clustering are the bottleneck of secondary metabolite evolution. Trichoderma harzianum M10 v1.0 produces many pharmaceutically important molecules; however, their specific biosynthetic pathways remain uncharacterised. Our genomic-based analysis of this species reveals the biosynthetic diversity of its specialised secondary metabolites, where over 50 BGCs were predicted, most of which were listed as polyketide-like compounds associated clusters. Gene annotation of the biosynthetic candidate genes predicted the production of many medically/industrially important compounds including enterobactin, gramicidin, lovastatin, HC-toxin, tyrocidine, equisetin, erythronolide, strobilurin, asperfuranone, cirtinine, protoilludene, germacrene, and epi-isozizaene. Revealing the biogenetic background of these natural molecules is a step forward towards the expansion of their chemical diversification via engineering their biosynthetic genes heterologously, and the identification of their role in the interaction between this fungus and its biotic/abiotic conditions as well as its role as bio-fungicide.

Summary Aphids, including the peach‐potato aphid, Myzus persicae, are major insect pests of agriculture and horticulture, and aphid control measures are limited. There is therefore an urgent need to develop alternative and more sustainable means of control. Recent studies have shown that environmental microbes have varying abilities to kill insects. We screened a range of environmental bacteria isolates for their abilities to kill target aphid species. Tests demonstrated the killing aptitude of these bacteria against six aphid genera (including Myzus persicae). No single bacterial strain was identified that was consistently toxic to insecticide‐resistant aphid clones than susceptible clones, suggesting resistance to chemicals is not strongly correlated with bacterial challenge. Pseudomonas fluorescens PpR24 proved the most toxic to almost all aphid clones whilst exhibiting the ability to survive for over three weeks on three plant species at populations of 5–6 log CFU cm−2 leaf. Application of PpR24 to plants immediately prior to introducing aphids onto the plants led to a 68%, 57% and 69% reduction in aphid populations, after 21 days, on Capsicum annuum, Arabidopsis thaliana and Beta vulgaris respectively. Together, these findings provide new insights into aphid susceptibility to bacterial infection with the aim of utilizing bacteria as effective biocontrol agents. An aphid‐feeding system was adapted to screen environmental bacteria for aphid‐killing properties and a selection of bacteria exhibited low‐to‐high killing effects in a range of aphid species including insecticide‐resistant clones. Pseudomonas fluorescens PpR24 demonstrated the best killing effect in vitro, whilst being able to survive on plant leaves over three weeks. Application of PpR24 to plants immediately prior to introducing aphids onto the plants led to a 50–60% reduction in aphid populations, after 21 days, on three different plant species.

Erwinia mallotivora, the causal agent of papaya dieback disease, is a devastating pathogen that has caused a tremendous decrease in Malaysian papaya export and affected papaya crops in neighbouring countries. A few studies on bacterial species capable of suppressing E. mallotivora have been reported, but the availability of antagonistic fungi remains unknown. In this study, mycelial suspensions from five rhizospheric Trichoderma isolates of Malaysian origin were found to exhibit notable antagonisms against E. mallotivora during co-cultivation. We further characterised three isolates, Trichoderma koningiopsis UKM-M-UW RA5, UKM-M-UW RA6, and UKM-M-UW RA3a, that showed significant growth inhibition zones on plate-based inhibition assays. A study of the genomes of the three strains through a combination of Oxford nanopore and Illumina sequencing technologies highlighted potential secondary metabolite pathways that might underpin their antimicrobial properties. Based on these findings, the fungal isolates are proven to be useful as potential biological control agents against E. mallotivora, and the genomic data opens possibilities to further explore the underlying molecular mechanisms behind their antimicrobial activity, with potential synthetic biology applications.

Since the olden times, infectious diseases have largely affected human existence. The newly emerged infections are excessively caused by viruses that are largely associated with mammal reservoirs. The casualties of these emergencies are significantly influenced by the way human beings interact with the reservoirs, especially the animal ones. In our review we will consider the evolutionary and the ecological scales of such infections and their consequences on the public health, with a focus on the pathogenic influenza A virus. The nutraceutical properties of fungal and plant terpene-like molecules will be linked to their ability to lessen the symptoms of viral infections and shed light on their potential use in the development of new drugs. New challenging methods in antiviral discovery will also be discussed in this review. The authors believe that pharmacognosy is the “wave of future pharmaceuticals”, as it can be continually produced and scaled up under eco-friendly requirements. Further diagnostic methods and strategies however are required to standardise those naturally occurring resources.