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.
This article reviews CRISPR/Cas9-based toolkits available to investigate natural product biosynthesis and regulation in streptomycete bacteria.
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.
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 are compounds that are produced by living organisms. They have numerous applications in our everyday life, from antibiotics to herbicides. They possess great chemical and structural ...diversity, which gives them a leading position as a source of new drugs. Many institutions worldwide are focusing more and more on natural product research, with microorganisms and plants being the most common source for discovery of new compounds. On the 30th June and 1st July 2016, early-career scientists working in the field of natural products gathered at the University of Warwick for the 10th edition of the Chemistry and Biology of Natural Products Symposium (CBNP10). This critical reflection reviews, in the context of the current research in the field, the major considerations that arose from this meeting.
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.
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.
, 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
have been reported, but the availability of antagonistic fungi remains unknown. In this study, mycelial suspensions from five rhizospheric
isolates of Malaysian origin were found to exhibit notable antagonisms against
during co-cultivation. We further characterised three isolates,
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
and the genomic data opens possibilities to further explore the underlying molecular mechanisms behind their antimicrobial activity, with potential synthetic biology applications.
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,β-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.
WS9326A biosynthesis contains a dehydrotyrosine residue that we show is directly installed by the cytochrome P450 enzyme Sas16 during peptide assembly on the non-ribosomal peptide synthetase assembly line. Display omitted
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