Fungal Secondary Metabolism

Fungal secondary metabolites (SMs) comprise a vast collection of compounds expendable for these organisms under laboratory conditions. They exhibit enormous chemical diversity, and usually belong to four major families: terpenoids, polyketides, non-ribosomal peptides, or a combination of the last two. Their functions are very diverse and are normally associated with a greater fitness of the producing fungi in their environment, which often compete with other microorganisms or interact with host plants. Many SMs have beneficial applications, e.g., as antibiotics or medical drugs, but others, known as mycotoxins, are harmful to health.

Screening of tenuazonic acid production-inducing compounds and identification of NPD938 as a regulator of fungal secondary metabolism

The control of secondary metabolism in fungi is essential for the regulation of various cellular functions. In this study, we searched the RIKEN Natural Products Depository (NPDepo) chemical library for inducers of tenuazonic acid (TeA) production in the rice blast fungus Pyricularia oryzae and identified NPD938. NPD938 transcriptionally induced TeA production. We explored the mode of action of NPD938 and observed that this compound enhanced TeA production via LAE1, a global regulator of fungal secondary metabolism. NPD938 could also induce production of terpendoles and pyridoxatins in Tolypocladium album RK99-F33. Terpendole production was induced transcriptionally. We identified the pyridoxatin biosynthetic gene cluster among transcriptionally induced secondary metabolite biosynthetic gene clusters. Therefore, NPD938 is useful for the control of fungal secondary metabolism.

Microevolution in the pansecondary metabolome of Aspergillus flavus and its potential macroevolutionary implications for filamentous fungi

Fungi produce a wealth of pharmacologically bioactive secondary metabolites (SMs) from biosynthetic gene clusters (BGCs). It is common practice for drug discovery efforts to treat species’ secondary metabolomes as being well represented by a single or a small number of representative genomes. However, this approach misses the possibility that intraspecific population dynamics, such as adaptation to environmental conditions or local microbiomes, may harbor novel BGCs that contribute to the overall niche breadth of species. Using 94 isolates of Aspergillus flavus, a cosmopolitan model fungus, sampled from seven states in the United States, we dereplicate 7,821 BGCs into 92 unique BGCs. We find that more than 25% of pangenomic BGCs show population-specific patterns of presence/absence or protein divergence. Population-specific BGCs make up most of the accessory-genome BGCs, suggesting that different ecological forces that maintain accessory genomes may be partially mediated by population-specific differences in secondary metabolism. We use ultra-high-performance high-resolution mass spectrometry to confirm that these genetic differences in BGCs also result in chemotypic differences in SM production in different populations, which could mediate ecological interactions and be acted on by selection. Thus, our results suggest a paradigm shift that previously unrealized population-level reservoirs of SM diversity may be of significant evolutionary, ecological, and pharmacological importance. Last, we find that several population-specific BGCs from A. flavus are present in Aspergillus parasiticus and Aspergillus minisclerotigenes and discuss how the microevolutionary patterns we uncover inform macroevolutionary inferences and help to align fungal secondary metabolism with existing evolutionary theory.

Interspecies Genomic Variation and Transcriptional Activeness of Secondary Metabolism-Related Genes in Aspergillus Section Fumigati

Filamentous fungi produce various bioactive compounds that are biosynthesized by sets of proteins encoded in biosynthesis gene clusters (BGCs). For an unknown reason, many BGCs are transcriptionally silent in laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional reactiveness of fungal secondary metabolism is not fully understood. To gain the comprehensive view, we conducted comparative genomic and transcriptomic analyses of nine closely-related species of Aspergillus section Fumigati (A. fumigatus, A. fumigatiaffinis, A. novofumigatus, A. thermomutatus, A. viridinutans, A. pseudoviridinutans, A. lentulus, A. udagawae, and Neosartorya fischeri). For expanding our knowledge, we newly sequenced genomes of A. viridinutans and A. pseudoviridinutans, and reassembled and reannotated the previously released genomes of A. lentulus and A. udagawae. Between 34 and 84 secondary metabolite (SM) backbone genes were identified in the genomes of these nine respective species, with 8.7–51.2% being unique to the species. A total of 247 SM backbone gene types were identified in the nine fungi. Ten BGCs are shared by all nine species. Transcriptomic analysis using A. fumigatus, A. lentulus, A. udagawae, A. viridinutans, and N. fischeri was conducted to compare expression levels of all SM backbone genes in four different culture conditions; 32–83% of SM backbone genes in these species were not expressed in the tested conditions, which reconfirmed that large part of fungal SM genes are hard to be expressed. The species-unique SM genes of the five species were expressed with lower frequency (18.8% in total) than the SM genes that are conserved in all five species (56%). These results suggest that the expression tendency of BGCs is correlated with their interspecies distribution pattern. Our findings increase understanding of the evolutionary processes associated with the regulation of fungal secondary metabolism.

Overexpression of Global Regulator Talae1 Leads to the Discovery of New Antifungal Polyketides From Endophytic Fungus Trichoderma afroharzianum

Transcription regulation caused by global regulators exerts important effects on fungal secondary metabolism. By overexpression of the global regulator Talae1 in a Ficus elastica-associated fungus Trichoderma afroharzianum, two structurally new polyketides (1 and 2) that were newly produced in the transformant were isolated and identified. Their structures, including the absolute configurations, were elucidated through a combination of high resolution mass spectrometer (HRMS), NMR, and electronic circular dichroism (ECD) calculations. The growth inhibitory activities of compounds 1 and 2 were evaluated against four bacteria and six plant-pathogenic fungi. Compound 1 showed the highest antifungal activity against Botrytis cinerea and Fusarium oxysporum f. sp. nicotianae with MIC of 8 μg/ml. To the best of our knowledge, this is the first study to report on the application of the global regulator in T. afroharzianum to activate the biosynthesis of bioactive secondary metabolites.

Velvet activated McrA plays a key role in cellular and metabolic development in Aspergillus nidulans

McrA is a key transcription factor that functions as a global repressor of fungal secondary metabolism in Aspergillus species. Here, we report that mcrA is one of the VosA-VelB target genes and McrA governs the cellular and metabolic development in Aspergillus nidulans. The deletion of mcrA resulted in a reduced number of conidia and decreased mRNA levels of brlA, the key asexual developmental activator. In addition, the absence of mcrA led to a loss of long-term viability of asexual spores (conidia), which is likely associated with the lack of conidial trehalose and increased β-(1,3)-glucan levels in conidia. In supporting its repressive role, the mcrA deletion mutant conidia contain more amounts of sterigmatocystin and an unknown metabolite than the wild type conidia. While overexpression of mcrA caused the fluffy-autolytic phenotype coupled with accelerated cell death, deletion of mcrA did not fully suppress the developmental defects caused by the lack of the regulator of G-protein signaling protein FlbA. On the contrary to the cellular development, sterigmatocystin production was restored in the ΔflbA ΔmcrA double mutant, and overexpression of mcrA completely blocked the production of sterigmatocystin. Overall, McrA plays a multiple role in governing growth, development, spore viability, and secondary metabolism in A. nidulans.

Host-dependent fungus-fungus competition suppresses fungal pathogenesis in Arabidopsis thaliana

Like animals, plants accommodate a rich diversity of microbes, typically without discernible disease symptoms. How their pathogenesis is prevented in the host remains obscure. Here, we show that the root-infecting fungus Colletotrichum fructicola of the C. gloeosporioides clade (CgE), isolated from field-grown healthy Brassicaceae plants, inhibits growth of pathogenic fungi in Arabidopsis thaliana, in a phosphate status-dependent manner. Loss of host ethylene signaling or phytoalexins, camalexin or indole glucosinolates, however, allows CgE to display pathogenesis, suggesting host contributions to endophytic CgE colonization and benefit. Compared to a closely-related C. gloeosporioides pathogen (CgP), CgE is characterized by genome expansion and >700 fungal genes (4.34%) specifically induced in the host roots when co-inoculated with CgP, including genes related to fungal secondary metabolism. This may underlie antimicrobial tolerance of CgE and its dominance over pathogenic fungi within the host, pointing to a role for fungus-fungus competition in asymptomatic fungal colonization in plants.

Comparative genome and transcriptome analyses revealing interspecies variations in the expression of fungal biosynthetic gene clusters

Filamentous fungi produce various bioactive compounds that are biosynthesized by a set of proteins encoded in biosynthetic gene clusters (BGCs). For an unknown reason, large parts of the BGCs are transcriptionally silent under laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional regulation of fungal secondary metabolism is not fully understood from an evolutionary viewpoint. To address this issue, we conducted comparative genomic and transcriptomic analyses using five closely related species of the Aspergillus section Fumigati: Aspergillus fumigatus, Aspergillus lentulus, Aspergillus udagawae, Aspergillus pseudoviridinutans, and Neosartorya fischeri. From their genomes, 298 secondary metabolite (SM) core genes were identified, with 27.4% to 41.5% being unique to a species. Compared with the species-specific genes, a set of section-conserved SM core genes was expressed at a higher rate and greater magnitude, suggesting that their expression tendency is correlated with the BGC distribution pattern. However, the section-conserved BGCs showed diverse expression patterns across the Fumigati species. Thus, not all common BGCs across species appear to be regulated in an identical manner. A consensus motif was sought in the promoter region of each gene in the 15 section-conserved BGCs among the Fumigati species. A conserved motif was detected in only two BGCs including the gli cluster. The comparative transcriptomic and in silico analyses provided insights into how the fungal SM gene cluster diversified at a transcriptional level, in addition to genomic rearrangements and cluster gains and losses. This information increases our understanding of the evolutionary processes associated with fungal secondary metabolism.Author summaryFilamentous fungi provide a wide variety of bioactive compounds that contribute to public health. The ability of filamentous fungi to produce bioactive compounds has been underestimated, and fungal resources can be developed into new drugs. However, most biosynthetic genes encoding bioactive compounds are not expressed under laboratory conditions, which hampers the use of fungi in drug discovery. The mechanisms underlying silent metabolite production are poorly understood. Here, we attempted to show the diversity in fungal transcriptional regulation from an evolutionary viewpoint. To meet this goal, the secondary metabolisms, at genomic and transcriptomic levels, of the most phylogenetically closely related species in Aspergillus section Fumigati were compared. The conserved biosynthetic gene clusters across five Aspergillus species were identified. The expression levels of the well-conserved gene clusters tended to be more active than the species-specific, which were not well-conserved, gene clusters. Despite highly conserved genetic properties across the species, the expression patterns of the well-conserved gene clusters were diverse. These findings suggest an evolutionary diversification at the transcriptional level, in addition to genomic rearrangements and gains and losses, of the biosynthetic gene clusters. This study provides a foundation for understanding fungal secondary metabolism and the potential to produce diverse fungal-based chemicals.