The 'emodin family' of fungal natural products-amalgamating a century of research with recent genomics-based advances.

Covering: up to 2022A very large group of biosynthetically linked fungal secondary metabolites are formed via the key intermediate emodin and its corresponding anthrone. The group includes anthraquinones such as chrysophanol and cladofulvin, the grisandienes geodin and trypacidin, the diphenyl ether pestheic acid, benzophenones such as monodictyphenone and various xanthones including the prenylated shamixanthones, the agnestins and dimeric xanthones such as the ergochromes, cryptosporioptides and neosartorin. Such compounds exhibit a wide range of bioactivities and as such have been utilised in traditional medicine for centuries, as well as garnering more recent interest from the pharmaceutical sector. Additional interest comes from industries such as textiles and cosmetics due to their use as natural colourants. A variety of biosynthetic routes and mechanisms have been proposed for this family of compounds, being altered and updated as new biosynthetic methods develop and new results emerge. After nearly 100 years of such research, this review aims to provide a comprehensive overview of what is currently known about the biosynthesis of this important family, amalgamating the early chemical and biosynthetic studies with the more recent genetics-based advances and comparative bioinformatics.

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

Covering: up to early 2022Maleidrides 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.

Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus oryzae as a Heterologous Host.

A suite of molecular techniques have been developed in recent decades, which allow gene clusters coding for the biosynthesis of fungal natural products to be investigated and characterized in great detail. Many of these involve the manipulation of the native producer, for example, to increase yields of natural products or investigate the biosynthetic pathway through gene disruptions. However, an alternative and powerful means of investigating biosynthetic pathways, which does not rely on a cooperative native host, is the refactoring and heterologous expression of pathways in a suitable host strain. This protocol aims to walk the reader through the various steps required for the heterologous expression of a fungal biosynthetic gene cluster, specifically using Aspergillus oryzae strain NSAR1 and the pTYGS series of expression vectors. Briefly, this process involves the design and construction of up to four multigene expression vectors using yeast recombination, PEG-mediation transformation of A. oryzae protoplasts, and chemical extraction of the resulting transformants to screen for the presence of metabolites.

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.

Cladobotric Acids: Metabolites from Cultures of Cladobotryum sp., Semisynthetic Analogues and Antibacterial Activity.

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.

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.

Cprp-An Unusual, Repetitive Protein Which Impacts Pleuromutilin Biosynthesis in the Basidiomycete Clitopilus passeckerianus.

Interrogation of an EST database for Clitopilus passeckerianus identified a putative homolog to the unusual stress response gene from yeast; ddr48, as being upregulated under pleuromutilin production conditions. Silencing of this gene, named cprp, produced a population of transformants which demonstrated significantly reduced pleuromutilin production. Attempts to complement a Saccharomyces cerevisiae ddr48 mutant strain (strain Y16748) with cprp were hampered by the lack of a clearly identifiable mutant phenotype, but interestingly, overexpression of either ddr48 or cprp in S. cerevisiae Y16748 led to a conspicuous and comparable reduction in growth rate. This observation, combined with the known role of DDR48 proteins from a range of fungal species in nutrient starvation and stress responses, raises the possibility that this family of proteins plays a role in triggering oligotrophic growth. Localization studies via the production of a Cprp:GFP fusion protein in C. passeckerianus showed clear localization adjacent to the hyphal septa and, to a lesser extent, cell walls, which is consistent with the identification of DDR48 as a cell wall-associated protein in various yeast species. To our knowledge this is the first study demonstrating that a DDR48-like protein plays a role in the regulation of a secondary metabolite, and represents the first DDR48-like protein from a basidiomycete. Potential homologs can be identified across much of the Dikarya, suggesting that this unusual protein may play a central role in regulating both primary and secondary metabolism in fungi.

Fusarochromene, a novel tryptophan-derived metabolite from Fusarium sacchari.

Fusarochromene isolated from the plant pathogenic fungus, Fusarium sacchari is closely related to a group of mycotoxins including fusarochromanone previously isolated from various Fusaria spp. Despite their assumed polyketide biogenesis, incorporation studies with 13C-labelled acetate, glycerol and tryptophans show that fusarochromene is unexpectedly derived via oxidative cleavage of the aromatic amino acid tryptophan. A putative biosynthetic gene cluster has been identified.

Uncovering biosynthetic relationships between antifungal nonadrides and octadrides.

Maleidrides are a class of bioactive secondary metabolites unique to filamentous fungi, which contain one or more maleic anhydrides fused to a 7-, 8- or 9- membered carbocycle (named heptadrides, octadrides and nonadrides respectively). Herein structural and biosynthetic studies on the antifungal octadride, zopfiellin, and nonadrides scytalidin, deoxyscytalidin and castaneiolide are described. A combination of genome sequencing, bioinformatic analyses, gene disruptions, biotransformations, isotopic feeding studies, NMR and X-ray crystallography revealed that they share a common biosynthetic pathway, diverging only after the nonadride deoxyscytalidin. 5-Hydroxylation of deoxyscytalidin occurs prior to ring contraction in the zopfiellin pathway of Diffractella curvata. In Scytalidium album, 6-hydroxylation - confirmed as being catalysed by the α-ketoglutarate dependent oxidoreductase ScyL2 - converts deoxyscytalidin to scytalidin, in the final step in the scytalidin pathway. Feeding scytalidin to a zopfiellin PKS knockout strain led to the production of the nonadride castaneiolide and two novel ring-open maleidrides.

The cycloaspeptides: uncovering a new model for methylated nonribosomal peptide biosynthesis.

The cycloaspeptides are bioactive pentapeptides produced by various filamentous fungi, which have garnered interest from the agricultural industry due to the reported insecticidal activity of the minor metabolite, cycloaspeptide E. Genome sequencing, bioinformatics and heterologous expression confirmed that the cycloaspeptide gene cluster contains a minimal 5-module nonribosomal peptide synthetase (NRPS) and a new type of trans-acting N-methyltransferase (N-MeT). Deletion of the N-MeT encoding gene and subsequent feeding studies determined that two modules of the NRPS preferentially accept and incorporate N-methylated amino acids. This discovery allowed the development of a system with unprecedented control over substrate supply and thus output, both increasing yields of specific metabolites and allowing the production of novel fluorinated analogues. Furthermore, the biosynthetic pathway to ditryptophenaline, another fungal nonribosomal peptide, was shown to be similar, in that methylated phenylalanine is accepted by the ditryptophenaline NRPS. Again, this allowed the directed biosynthesis of a fluorinated analogue, through the feeding of a mutant strain. These discoveries represent a new paradigm for the production of N-methylated cyclic peptides via the selective incorporation of N-methylated free amino acids.

Insights into the Classical Genetics of Clitopilus passeckerianus - the Pleuromutilin 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.