Insights into the Classical Genetics of <i>Clitopilus passeckerianus</i> – the <i>Pleuromutilin </i>Producing Mushroom

Clitopilus passeckerianus is the fungal species responsible for the production of pleuromutilin, a diterpene antibiotic that is gaining in commercial interest. Production of the antibiotic is constrained by the low titers typically obtained from isolates. We therefore set out to investigate the possibility of using classical breeding techniques coupled with genetic manipulation as a means to develop such fungi. We show that the original production strain of C. passeckerianus is able to fruit under laboratory conditions, giving viable haploid meiotic basidiospores. The derived progeny displayed the typical physiological and genetic characteristics of a tetrapolar mating system. The monokaryon haploids produced pleuromutilin and haploid lines were amenable to genetic manipulation. Together this shows that the basic requirements for a classical breeding approach are present and the tools required to undertake directed genetic engineering on haploid strains are available, demonstrating that strain improvement may be feasible in this fungus

Insights into the Classical Genetics of <i>Clitopilus passeckerianus</i> – the <i>Pleuromutilin </i>Producing Mushroom

Clitopilus passeckerianus is the fungal species responsible for the production of pleuromutilin, a diterpene antibiotic that is gaining in commercial interest. Production of the antibiotic is constrained by the low titers typically obtained from isolates. We therefore set out to investigate the possibility of using classical breeding techniques coupled with genetic manipulation as a means to develop such fungi. We show that the original production strain of C. passeckerianus is able to fruit under laboratory conditions, giving viable haploid meiotic basidiospores. The derived progeny displayed the typical physiological and genetic characteristics of a tetrapolar mating system. The monokaryon haploids produced pleuromutilin and haploid lines were amenable to genetic manipulation. Together this shows that the basic requirements for a classical breeding approach are present and the tools required to undertake directed genetic engineering on haploid strains are available, demonstrating that strain improvement may be feasible in this fungus

In silico analyses of maleidride biosynthetic gene clusters

Maleidrides are a family of structurally related fungal natural products, many of which possess diverse, potent bioactivities. Previous identification of several maleidride biosynthetic gene clusters, and subsequent experimental work, has determined the ‘core’ set of genes required to construct the characteristic medium-sized alicyclic ring with maleic anhydride moieties. Through genome mining, this work has used these core genes to discover ten entirely novel putative maleidride biosynthetic gene clusters, amongst both publicly available genomes, and encoded within the genome of the previously un-sequenced epiheveadride producer Wicklowia aquatica CBS 125634. We have undertaken phylogenetic analyses and comparative bioinformatics on all known and putative maleidride biosynthetic gene clusters to gain further insights regarding these unique biosynthetic pathways. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40694-022-00132-z

Maleidride biosynthesis – construction of dimeric anhydrides – more than just heads or tails

Covering: up to early 2022 Maleidrides are a family of polyketide-based dimeric natural products isolated from fungi. Many maleidrides possess significant bioactivities, making them attractive pharmaceutical or agrochemical lead compounds. Their unusual biosynthetic pathways have fascinated scientists for decades, with recent advances in our bioinformatic and enzymatic understanding providing further insights into their construction. However, many intriguing questions remain, including exactly how the enzymatic dimerisation, which creates the diverse core structure of the maleidrides, is controlled. This review will explore the literature from the initial isolation of maleidride compounds in the 1930s, through the first full structural elucidation in the 1960s, to the most recent in vivo, in vitro, and in silico analyses

Cleaning the Cellular Factory:Deletion of McrA in Aspergillus oryzae NSAR1 and the generation of a novel kojic acid deficient strain for cleaner heterologous production of secondary metabolites

The use of filamentous fungi as cellular factories, where natural product pathways can be refactored and expressed in a host strain, continues to aid the field of natural product discovery. Much work has been done to develop host strains which are genetically tractable, and for which there are multiple selectable markers and controllable expression systems. To fully exploit these strains, it is beneficial to understand their natural metabolic capabilities, as such knowledge can rule out host metabolites from analysis of transgenic lines and highlight any potential interplay between endogenous and exogenous pathways. Additionally, once identified, the deletion of secondary metabolite pathways from host strains can simplify the detection and purification of heterologous compounds. To this end, secondary metabolite production in Aspergillus oryzae strain NSAR1 has been investigated via the deletion of the newly discovered negative regulator of secondary metabolism, mcrA (multicluster regulator A). In all ascomycetes previously studied mcrA deletion led to an increase in secondary metabolite production. Surprisingly, the only detectable phenotypic change in NSAR1 was a doubling in the yields of kojic acid, with no novel secondary metabolites produced. This supports the previous claim that secondary metabolite production has been repressed in A. oryzae and demonstrates that such repression is not McrA-mediated. Strain NSAR1 was then modified by employing CRISPR-Cas9 technology to disrupt the production of kojic acid, generating the novel strain NSARΔK, which combines the various beneficial traits of NSAR1 with a uniquely clean secondary metabolite background

Cladobotric acids: Metabolites from cultures of Cladobotryum sp., semi-synthetic analogues and antibacterial activity

[Image: see text] Three new polyketide-derived natural products, cladobotric acids G–I (1–3), and six known metabolites (4, 5, 8–11) were isolated from fermentation of the fungus Cladobotryum sp. grown on rice. Their structures were elucidated by extensive spectroscopic methods. Two metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of new products (12–20) by semisynthesis. The antibacterial activities of all these compounds were investigated against the Gram-positive pathogen Staphylococcus aureus including methicillin-susceptible (MSSA), methicillin-resistant and vancomycin-intermediate (MRSA/VISA), and heterogeneous vancomycin-intermediate (hVISA) strains. Results of these antibacterial assays revealed structural features of the unsaturated decalins important for biological activity

Structure revision of cryptosporioptides and determination of the genetic basis for dimeric xanthone biosynthesis in fungi

Three novel dimeric xanthones, cryptosporioptides A–C were isolated from Cryptosporiopsis sp. 8999 and their structures elucidated. Methylation of cryptosporioptide A gave a methyl ester with identical NMR data to cryptosporioptide, a compound previously reported to have been isolated from the same fungus. However, HRMS analysis revealed that cryptosporioptide is a symmetrical dimer, not a monomer as previously proposed, and the revised structure was elucidated by extensive NMR analysis. The genome of Cryptosporiopsis sp. 8999 was sequenced and the dimeric xanthone (dmx) biosynthetic gene cluster responsible for the production of the cryptosporioptides was identified. Gene disruption experiments identified a gene (dmxR5) encoding a cytochrome P450 oxygenase as being responsible for the dimerisation step late in the biosynthetic pathway. Disruption of dmxR5 led to the isolation of novel monomeric xanthones. Cryptosporioptide B and C feature an unusual ethylmalonate subunit: a hrPKS and acyl CoA carboxylase are responsible for its formation. Bioinformatic analysis of the genomes of several fungi producing related xanthones, e.g. the widely occurring ergochromes, and related metabolites allows detailed annotation of the biosynthetic genes, and a rational overall biosynthetic scheme for the production of fungal dimeric xanthones to be proposed

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

Strobilurin biosynthesis in Basidiomycete fungi

Strobilurins are fungal metabolites that inspired the creation of β-methoxyacrylate agricultural fungicides. Here, Nofiani et al. identify the strobilurin biosynthesis gene cluster, encoding a polyketide synthase as well as an FAD-dependent oxygenase for an oxidative rearrangement leading to β-methoxyacrylate formation