MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Genomic Based Analysis of the Biocontrol Species Trichoderma harzianum: A Model Resource of Structurally Diverse Pharmaceuticals and Biopesticides.

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.

Naturally Occurring Terpenes: A Promising Class of Organic Molecules to Address Influenza Pandemics.

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.

Further Biochemical Profiling of Hypholoma fasciculare Metabolome Reveals Its Chemogenetic Diversity.

Natural products with novel chemistry are urgently needed to battle the continued increase in microbial drug resistance. Mushroom-forming fungi are underutilized as a source of novel antibiotics in the literature due to their challenging culture preparation and genetic intractability. However, modern fungal molecular and synthetic biology tools have renewed interest in exploring mushroom fungi for novel therapeutic agents. The aims of this study were to investigate the secondary metabolites of nine basidiomycetes, screen their biological and chemical properties, and then investigate the genetic pathways associated with their production. Of the nine fungi selected, Hypholoma fasciculare was revealed to be a highly active antagonistic species, with antimicrobial activity against three different microorganisms: Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. Genomic comparisons and chromatographic studies were employed to characterize more than 15 biosynthetic gene clusters and resulted in the identification of 3,5-dichloromethoxy benzoic acid as a potential antibacterial compound. The biosynthetic gene cluster for this product is also predicted. This study reinforces the potential of mushroom-forming fungi as an underexplored reservoir of bioactive natural products. Access to genomic data, and chemical-based frameworks, will assist the development and application of novel molecules with applications in both the pharmaceutical and agrochemical industries.

The Biogenetic Origin of the Biologically Active Naematolin of Hypholoma Species Involves an Unusual Sesquiterpene Synthase.

Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5ß,6α,8ß-trihydroxycariolan, 5ß,8ß-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.

Improved vectors for Agrobacterium mediated genetic manipulation of Hypholoma spp. and other homobasidiomycetes.

The basidiomycete fungi Hypholoma fasciculare and H. sublateritium are both prolific producers of sesquiterpenes and triterpenes, some of which have relevant pharmaceutical properties. Although H. sublateritium has been transformed in the past, the low reported efficiencies highlighted the need for establishing an effective simple transformation system for these valuable species. We have optimized Agrobacterium tumefaciens-mediated transformation through testing various parameters in these two Hypholoma species, showing that a mixture of homogenized mycelia and Agrobacterium (strain LBA4404) co-cultivated for 84h at 25°C is optimal for efficient transformation in these basidiomycetes. This study also reveals the requirements for transgene expression, with the first report of GFP expression in these Hypholoma, the need for an intron for such transgene expression, and further demonstrates the functionality of the expression vector by its use in Clitopilus passeckerianus. This development of transformation system and expression constructs, can facilitate further genetic investigation such as gene functionality in these fungi.