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Fungi are a rich source of secondary metabolites with potent biological activities. Co-culturing a fungus with another microorganism has drawn much attention as a practical method for stimulating fungal secondary metabolism. However, in most cases, the molecular mechanisms underlying the activation of secondary metabolite production in co-culture are poorly understood. To elucidate such a mechanism, in this study, we established a model fungal-fungal co-culture system, composed of Aspergillus nidulans and Aspergillus fumigatus. In the co-culture of A. nidulans and A. fumigatus, production of antibacterial diphenyl ethers was enhanced. Transcriptome analysis by RNA-sequencing showed that the co-culture activated expression of siderophore biosynthesis genes in A. fumigatus and two polyketide biosynthetic gene clusters (the ors and cic clusters) in A. nidulans. Gene disruption experiments revealed that the ors cluster is responsible for diphenyl ether production in the co-culture. Interestingly, the ors cluster was previously reported to be upregulated by co-culture of A. nidulans with the bacterium Streptomyces rapamycinicus; orsellinic acid was the main product of the cluster in that co-culture. In other words, the main product of the ors cluster was different in fungal-fungal and bacterial-fungal co-culture. The genes responsible for biosynthesis of the bacterial- and fungal-induced polyketides were deduced using a heterologous expression system in Aspergillus oryzae. The molecular genetic mechanisms that trigger biosynthesis of two different types of compounds in A. nidulans in response to the fungus and the bacterium were demonstrated, which provides an insight into complex secondary metabolic response of fungi to microorganisms. KEY POINTS: • Co-culture of two fungal species triggered antibiotic diphenyl ether production. • The co-culture affected expression levels of several genes for secondary metabolism. • Gene cluster essential for induction of the antibiotics production was determined.

Surugamides are a group of non-ribosomal peptides isolated from marine-derived Streptomyces. Surugamide A (1) and its closely related derivatives, surugamides B-E (2-5), are D-amino acid containing cyclic octapeptides with cathepsin B inhibitory activity. The D-isoleucine (Ile), the nonproteinogenic amino acid residue embedded in 1, is less common in natural peptides because a rare Cβ-epimerization is required for its biosynthesis. Taking advantage of the synthetic route of 2 previously established by our group, we synthesized the cyclic octapeptide 1 containing D-Ile by solid phase peptide synthesis. The structure of 1 actually contains D-allo-Ile in place of D-Ile, which was corroborated by chemical syntheses and chromatographic comparisons.

A revised structure of natural 14-mer cyclic depsipeptide MA026, isolated from Pseudomonas sp. RtlB026 in 2002 was established by physicochemical analysis with HPLC, MS/MS, and NMR and confirmed by total solid-phase synthesis. The revised structure differs from that previously reported in that two amino acid residues, assigned in error, have been replaced. Synthesized MA026 with the revised structure showed a tight junction (TJ) opening activity like that of the natural one in a cell-based TJ opening assay. Bioinformatic analysis of the putative MA026 biosynthetic gene cluster (BGC) of RtIB026 demonstrated that the stereochemistry of each amino acid residue in the revised structure can be reasonably explained. Phylogenetic analysis with xantholysin BGC indicates an exceptionally high homology (ca. 90 %) between xantholysin and MA026. The TJ opening activity of MA026 when binding to claudin-1 is a key to new avenues for transdermal administration of large hydrophilic biologics.

Bacterial cells travel along fungal highway and pay thiamine as a toll to the fungus. Simultaneous spatial and metabolic interactions in communicating bacterial and fungal species establish a mutualism that facilitates them to obtain an environmental niche and nutrient., Exclusivity in physical spaces and nutrients is a prerequisite for survival of organisms, but a few species have been able to develop mutually beneficial strategies that allow them to co-habit. Here, we discovered a mutualistic mechanism between filamentous fungus, Aspergillus nidulans, and bacterium, Bacillus subtilis. The bacterial cells co-cultured with the fungus traveled along mycelia using their flagella and dispersed farther with the expansion of fungal colony, indicating that the fungal mycelia supply space for bacteria to migrate, disperse, and proliferate. Transcriptomic, genetic, molecular mass, and imaging analyses demonstrated that the bacteria reached the mycelial edge and supplied thiamine to the growing hyphae, which led to a promotion of hyphal growth. The thiamine transfer from bacteria to the thiamine non-auxotrophic fungus was directly demonstrated by stable isotope labeling. The simultaneous spatial and metabolic interactions demonstrated in this study reveal a mutualism that facilitates the communicating fungal and bacterial species to obtain an environmental niche and nutrient, respectively.

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.

Surugamide F is a linear decapeptide (1) isolated along with the cyclic octapeptides surugamides A-E (2-6), from a marine-derived Streptomyces species. The linear peptide 1 is produced by two nonribosomal peptide synthetases (NRPSs) encoded in adjacent open reading frames, which are further flanked by an additional pair of NRPS genes responsible for the biosyntheses of the cyclic peptides 2-6. While the cyclic peptides 2-6 were identified to be cathepsin B inhibitors, the biological activity of the new metabolite 1 still remained unclear. In order to elucidate its unique biosynthetic pathway and biological activity in detail, we planned to develop an efficient synthetic route toward 1. Here we report the diastereoselective total synthesis of 1, utilizing 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. During this study, we found that the structural correction of 1 was required, due to the mislabeling of the commercially obtained 3-amino-2-methylpropionic acid, and the true structure of 1 was corroborated by the chemical synthesis and chromatographic comparison.

The rubbing conditions are researched in terms of the rubbing cloth materials for ferroelectric liquid crystal (FLC) display application. Although cotton is generally used as the rubbing cloth, a good FLC molecular alignment can be obtained even in (semi) synthetic fiber clothes such as acetate, cellulose, and polyester. In order to obtain the good alignment without rubbing scratches, it is important to use a soft cloth. On the other hand, for a hard cloth, there is a possibility to obtain the good alignment by increasing the rubbing number and thus, polishing the alignment film surface.

Peptidic Natural Products (PNPs) are widely used compounds that include many antibiotics and a variety of other bioactive peptides. While recent breakthroughs in PNP discovery raised the challenge of developing new algorithms for their analysis, identification of PNPs via database search of tandem mass spectra remains an open problem. To address this problem, natural product researchers utilize dereplication strategies that identify known PNPs and lead to the discovery of new ones even in cases when the reference spectra are not present in existing spectral libraries. DEREPLICATOR is a new dereplication algorithm that enabled high-throughput PNP identification and that is compatible with large-scale mass spectrometry-based screening platforms for natural product discovery. After searching nearly one hundred million tandem mass spectra in the Global Natural Products Social (GNPS) molecular networking infrastructure, DEREPLICATOR identified an order of magnitude more PNPs (and their new variants) than any previous dereplication efforts.

The cathepsin B inhibitor surugamide B (2), along with structurally related derivatives (A and C-E), has previously been isolated from the marine actinomycete Streptomyces sp. JAMM992. The biosynthetic genes are unexpectedly part of a cluster of four non-ribosomal peptide synthetase (NRPS) genes, two of which are responsible for the biosynthesis of the additional linear decapeptide surugamide F. However, the thioesterase domain required for the later stage of the biosynthesis of the cyclic peptides surugamides A-E is not present in any module architecture of the surugamide NRPSs. Herein, we report the first total synthesis of surugamide B (2) through the macrocyclization at the biomimetic position, which not only alleviated the Cα epimerization in the macrolactamization process, but also efficiently provided 2 in 34 % yield for 18 steps. Furthermore, both the chemical and enzymatic studies with the biosynthetic precursor mimics revealed that the stand-alone enzyme SurE, which belongs to the penicillin-binding protein family, is responsible for macrocyclization of the tethered octapeptidyl intermediate.

Surugamides A-E (1-5), cyclic octapeptides with four D-amino acid residues, were isolated from the broth of marine-derived Streptomyces sp. Their planar structures were determined by analyses of spectroscopic data, and the absolute configuration of constituent amino acid residues was determined by the Marfey's method. Differentiation of D-Ile and L-Ile in the sequence was established by chiral analysis of fragment peptides obtained from the partial hydrolysate, whose identification was conducted by LC-MS/MS.

Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A-E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non-ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A-E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3-amino-2-methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid-phase peptide synthesis.