Unveiling novel Neocosmospora species from Thai mangroves as potent biocontrol agents against Colletotrichum species.

AIMS: Neocosmospora species are saprobes, endophytes, and pathogens belonging to the family Nectriaceae. This study aims to investigate the taxonomy, biosynthetic potential, and application of three newly isolated Neocosmospora species from mangrove habitats in the southern part of Thailand using phylogeny, bioactivity screening, genome sequencing, and bioinformatics analysis. METHODS AND RESULTS: Detailed descriptions, illustrations, and a multi-locus phylogenetic tree with large subunit ribosomal DNA (LSU), internal transcribed spacer (ITS), translation elongation factor 1-alpha (ef1-α), and RNA polymerase II second largest subunit (RPB2) regions showing the placement of three fungal strains, MFLUCC 17-0253, MFLUCC 17-0257, and MFLUCC 17-0259 clustered within the Neocosmospora clade with strong statistical support. Fungal crude extracts of the new species N. mangrovei MFLUCC 17-0253 exhibited strong antifungal activity to control Colletotrichum truncatum CG-0064, while N. ferruginea MFLUCC 17-0259 exhibited only moderate antifungal activity toward C. acutatum CC-0036. Thus, N. mangrovei MFLUCC 17-0253 was sequenced by Oxford nanopore technology. The bioinformatics analysis revealed that 49.17 Mb genome of this fungus harbors 41 potential biosynthetic gene clusters. CONCLUSION: Two fungal isolates of Neocosmospora and a new species of N. mangrovei were reported in this study. These fungal strains showed activity against pathogenic fungi causing anthracnose in chili. In addition, full genome sequencing and bioinformatics analysis of N. mangrovei MFLUCC 17-0253 were obtained.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,ß-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

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.

Biosynthesis of pleuromutilin congeners using an Aspergillus oryzae expression platform.

Pleuromutilin is an antibiotic diterpenoid made by Clitopilus passeckerianus and related fungi, and it is the progenitor of a growing class of semi-synthetic antibiotics used in veterinary and human medicine. To harness the biotechnological potential of this natural product class, a full understanding of its biosynthetic pathway is essential. Previously, a linear pathway for pleuromutilin biosynthesis was established. Here we report two shunt pathways involving Pl-sdr and Pl-atf that were identified through the rational heterologous expression of combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. Three novel pleuromutilin congeners were isolated, and their antimicrobial activity was investigated, alongside that of an additional derivative produced through a semi-synthetic approach. It was observed that the absence of various functional groups - 3 ketone, 11 hydroxyl group or 21 ketone - from the pleuromutilin framework affected the antibacterial activity of pleuromutilin congeners. This study expands our knowledge on the biosynthesis of pleuromutilin and provides avenues for the development of novel pleuromutilin analogues by combining synthetic biology and synthetic chemistry.

CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function.

The CRISPR/Cas9 technology allows fast and marker-less genome engineering that can be employed to study secondary metabolism in actinobacteria. Here, we report a standard experimental protocol for the deletion of a biosynthetic gene in a Streptomyces species, using the vector pCRISPomyces-2 developed by Huimin Zhao and collaborators. We also describe how carrying out metabolite analysis can reveal the putative biosynthetic function of the inactivated gene.

Genome Sequencing and Analysis of Trichoderma (Hypocreaceae) Isolates Exhibiting Antagonistic Activity against the Papaya Dieback Pathogen, Erwinia mallotivora.

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.

Identification of novel aphid-killing bacteria to protect plants.

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.

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.

Recent developments of tools for genome and metabolome studies in basidiomycete fungi and their application to natural product research.

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.

Engineering Isoprenoid Quinone Production in Yeast.

Isoprenoid quinones are bioactive molecules that include an isoprenoid chain and a quinone head. They are traditionally found to be involved in primary metabolism, where they act as electron transporters, but specialized isoprenoid quinones are also produced by all domains of life. Here, we report the engineering of a baker's yeast strain, Saccharomyces cerevisiae EPYFA3, for the production of isoprenoid quinones. Our yeast strain was developed through overexpression of the shikimate pathway in a well-established recipient strain (S. cerevisiae EPY300) where the mevalonate pathway is overexpressed. As a proof of concept, our new host strain was used to overproduce the endogenous isoprenoid quinone coenzyme Q6, resulting in a nearly 3-fold production increase. EPYFA3 represents a valuable platform for the heterologous production of high value isoprenoid quinones. EPYFA3 will also facilitate the elucidation of isoprenoid quinone biosynthetic pathways.

Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery.

In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-l-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Synthetic standards of scleric acid and a key biosynthetic intermediate were also prepared to confirm the chemical structures we proposed. The assembly of scleric acid involves two unique condensation reactions catalysed by a single NRPS module and an ATP-grasp enzyme that link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate, respectively. Scleric acid was shown to exhibit moderate inhibition activity against Mycobacterium tuberculosis, as well as inhibition of the cancer-associated metabolic enzyme nicotinamide N-methyltransferase (NNMT).

Editing streptomycete genomes in the CRISPR/Cas9 age.

Covering: up to December 2018 This article aims to highlight advantages, drawbacks and issues that users should consider when implementing the use of CRISPR/Cas9-tools for genome editing in streptomycetes, the most prolific source of antimicrobial natural products to date. Here, we examine four toolkits that have so far been made available for streptomycete in vivo-engineering and one for in vitro-editing, and review how they have been applied over the last three years. Our critical evaluation of these toolkits intends to support potential users in determining what they could achieve, what they should consider and what system they should select/optimise for their application.

Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives.

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.

Natural products from filamentous fungi and production by heterologous expression.

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.

Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production.

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.