Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.     

Fungal artificial chromosomes for mining of the fungal secondary metabolome

Background

With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30–80 kb in size, breakthrough techniques are needed to characterize this SM wealth.

Results

Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery.

Conclusion

The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1561-x) contains supplementary material, which is available to authorized users.     

Recent advances in genome mining of secondary metabolite biosynthetic gene clusters and the development of heterologous expression systems in Aspergillus nidulans

Fungi are prolific producers of secondary metabolites (SMs) that show a variety of biological activities. Recent advances in genome sequencing have shown that fungal genomes harbor far more SM gene clusters than are expressed under conventional laboratory conditions. Activation of these “silent” gene clusters is a major challenge, and many approaches have been taken to attempt to activate them and, thus, unlock the vast treasure chest of fungal SMs. This review will cover recent advances in genome mining of SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A. nidulans and recent developments in A. nidulans as a molecular genetic system, both of which are essential for rapid and efficient experimental verification of SM gene clusters on a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a heterologous expression system to aid in the analysis of SM gene clusters from other fungal species that do not have an established molecular genetic system.     

Genetic regulation of aflatoxin biosynthesis: From gene to genome

Aflatoxins are notorious toxic secondary metabolites known for their impacts on human and animal health, and their effects on the marketability of key grain and nut crops. Understanding aflatoxin biosynthesis is the focus of a large and diverse research community. Concerted efforts by this community have led not only to a well-characterized biosynthetic pathway, but also to the discovery of novel regulatory mechanisms. Common to secondary metabolism is the clustering of biosynthetic genes and their regulation by pathway specific as well as global regulators. Recent data show that arrangement of secondary metabolite genes in clusters may allow for an important global regulation of secondary metabolism based on physical location along the chromosome. Available genomic and proteomic tools are now allowing us to examine aflatoxin biosynthesis more broadly and to put its regulation in context with fungal development and fungal ecology. This review covers our current understanding of the biosynthesis and regulation of aflatoxin and highlights new and emerging information garnered from structural and functional genomics. The focus of this review will be on studies in Aspergillus flavus and Aspergillus parasiticus, the two agronomically important species that produce aflatoxin. Also covered will be the important contributions gained by studies on production of the aflatoxin precursor sterigmatocystin in Aspergillus nidulans.     

Post-translational modifications drive secondary metabolite biosynthesis in Aspergillus: a review

Post-translational modifications (PTMs) are important for protein function and regulate multiple cellular processes and secondary metabolites (SMs) in fungi. Aspergillus species belong to a genus renown for an abundance of bioactive secondary metabolites, many important as toxins, pharmaceuticals and in industrial production. The genes required for secondary metabolites are typically co-localized in biosynthetic gene clusters (BGCs), which often localize in heterochromatic regions of genome and are ‘turned off’ under laboratory condition. Efforts have been made to ‘turn on’ these BGCs by genetic manipulation of histone modifications, which could convert the heterochromatic structure to euchromatin. Additionally, non-histone PTMs also play critical roles in the regulation of secondary metabolism. In this review, we collate the known roles of epigenetic and PTMs on Aspergillus SM production. We also summarize the proteomics approaches and bioinformatics tools for PTM identification and prediction and provide future perspectives on the emerging roles of PTM on regulation of SM biosynthesis in Aspergillus and other fungi.     

Sporogenic Effect of Polyunsaturated Fatty Acids on Development of Aspergillus spp.

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.     

Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM‐gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de‐repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein‐1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM‐specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.     

Secondary Metabolism and Biotrophic Lifestyle in the Tomato Pathogen Cladosporium fulvum

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.     

New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.     

A viscometric study of the biodegradation of photographic gelatin by fungi isolated from cinematographic films

The biodegradation of photographic gelatin grade (Bloom 225) material was studied by viscometry in aqueous solution (at 37 °C, 6.67% w/w) using filamentous fungi isolated and identified from cinematographic film stored in different Spanish archives. From viscosity data, different variables such as molecular weight and chain scission were calculated. To ensure initial spore suspension concentration was standardized for all the biodegradation experiments, a correlation between transmittance at 530 nm of fungal spore suspensions and the corresponding cytometric determination of populations was established for all the fungal strains studied in this work. The bioassay experiments were carried out at 25 and 4 °C using an initial concentration of fungi of 4.5×10⁵ conidia/mL except in the case of the genus Alternaria, where the concentration was 10 times lower. The fungal strains were three species of Aspergillus, i.e., A .ustus, A. nidulans var. nidulans, A. versicolor, seven Penicillium chrysogenum strains, and Cladosporium cladosporioides, Alternaria alternata, Mucor racemosus, Phoma glomerata, and Trichoderma longibrachiatum. All were gelatinase positive. Through the viscosity decay profiles with bioassay-time and the corresponding calculated chain scission, the relative quantitative gelatinase efficiency of these fungi has been evaluated.     

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides.     

Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression

Mycosporines and mycosporine-like amino acids (MAAs), including shinorine (mycosporine-glycine-serine) and porphyra-334 (mycosporine-glycine-threonine), are UV-absorbing compounds produced by cyanobacteria, fungi, and marine micro- and macroalgae. These MAAs have the ability to protect these organisms from damage by environmental UV radiation. Although no reports have described the production of MAAs and the corresponding genes involved in MAA biosynthesis from Gram-positive bacteria to date, genome mining of the Gram-positive bacterial database revealed that two microorganisms belonging to the order Actinomycetales, Actinosynnema mirum DSM 43827 and Pseudonocardia sp. strain P1, possess a gene cluster homologous to the biosynthetic gene clusters identified from cyanobacteria. When the two strains were grown in liquid culture, Pseudonocardia sp. accumulated a very small amount of MAA-like compound in a medium-dependent manner, whereas A. mirum did not produce MAAs under any culture conditions, indicating that the biosynthetic gene cluster of A. mirum was in a cryptic state in this microorganism. In order to characterize these biosynthetic gene clusters, each biosynthetic gene cluster was heterologously expressed in an engineered host, Streptomyces avermitilis SUKA22. Since the resultant transformants carrying the entire biosynthetic gene cluster controlled by an alternative promoter produced mainly shinorine, this is the first confirmation of a biosynthetic gene cluster for MAA from Gram-positive bacteria. Furthermore, S. avermitilis SUKA22 transformants carrying the biosynthetic gene cluster for MAA of A. mirum accumulated not only shinorine and porphyra-334 but also a novel MAA. Structure elucidation revealed that the novel MAA is mycosporine-glycine-alanine, which substitutes l-alanine for the l-serine of shinorine.     

veA Is Required for Toxin and Sclerotial Production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.     

Bacterial Biosynthetic Gene Clusters Encoding the Anti-cancer Haterumalide Class of Molecules: BIOGENESIS OF THE BROAD SPECTRUM ANTIFUNGAL AND ANTI-OOMYCETE COMPOUND,OOCYDIN A

Haterumalides are halogenated macrolides with strong antitumor properties, making them attractive targets for chemical synthesis. Unfortunately, current synthetic routes to these molecules are inefficient. The potent haterumalide, oocydin A, was previously identified from two plant-associated bacteria through its high bioactivity against plant pathogenic fungi and oomycetes. In this study, we describe oocydin A (ooc) biosynthetic gene clusters identified by genome sequencing, comparative genomics, and chemical analysis in four plant-associated enterobacteria of the Serratia and Dickeya genera. Disruption of the ooc gene cluster abolished oocydin A production and bioactivity against fungi and oomycetes. The ooc gene clusters span between 77 and 80 kb and encode five multimodular polyketide synthase (PKS) proteins, a hydroxymethylglutaryl-CoA synthase cassette and three flavin-dependent tailoring enzymes. The presence of two free-standing acyltransferase proteins classifies the oocydin A gene cluster within the growing family of trans-AT PKSs. The amino acid sequences and organization of the PKS domains are consistent with the chemical predictions and functional peculiarities associated with trans-acyltransferase PKS. Based on extensive in silico analysis of the gene cluster, we propose a biosynthetic model for the production of oocydin A and, by extension, for other members of the haterumalide family of halogenated macrolides exhibiting anti-cancer, anti-fungal, and other interesting biological properties.     

Discovery of cyclohexadepsipeptides with anti-Zika virus activities and biosynthesis of the nonproteinogenic building block (3S)-methyl-l-proline

The fungal cyclohexadepsipeptides destruxins (DTXs), isaridins (ISDs), and isariins (ISRs) are nonribosomal peptides whose structures include a 19-membered ring composed of five amino acid residues and one α- or β-hydroxy acid residue. These cyclohexadepsipeptides contain unusual nonproteinogenic amino acid–building blocks and possess a range of antiviral, antibacterial, and other activities. The biosynthetic gene clusters for ISDs and ISRs have not been identified, and the biosynthesis of the nonproteinogenic (3S)-methyl-l-proline residue, which is found in DTXs, ISDs, and many other natural products, lacks full characterization. In an ongoing effort to identify compounds that can inhibit the Zika virus (ZIKV), we examined the extract of marine-derived fungus Beauveria felina SX-6-22 and discovered 30 DTXs, ISDs, and ISRs (1–30) including seven new compounds (1–7). The anti-ZIKV assays showed that 9–12 and 16–18 possess inhibitory activities against ZIKV RNA replication and NS5 (nonstructural protein 5) production in ZIKV-infected A549 cells. We sequenced the genome of B. felina SX-6-22 and identified three biosynthetic gene clusters detx, isd and isr, which are responsible for the biosynthesis of DTXs, ISDs, and ISRs, respectively. Comparative analyses of the three gene clusters clarified the biosynthetic relationships among these cyclohexadepsipeptides. Finally, we characterized the entire biosynthesis of nonproteinogenic building block (3S)-methyl-l-proline. The Δ¹-pyrroline-5-carboxylate reductases (P5CRs), also used in the biosynthesis of l-proline, were demonstrated to catalyze the final reduction step in (3S)-methyl-l-proline formation, suggesting potential cross talk between primary and secondary metabolisms. These results provide opportunities for biosynthetic pathway engineering to generate new anti-ZIKV cyclohexadepsipeptides.