Hiroyasu Onaka

The novel cyclic hexapeptides, piperapeptins A (1) and B (2) with rare N−OH-Leu and piperazic acid moieties, were discovered in the combined-culture broth of Amycolatopsis sp. 26-11 and Tsukamurella pulmonis TP-B0596. The planar structures were elucidated by HR-MS and 1D/2D NMR analyses. The stereochemistry of Ala and Phe was determined using our highly sensitive advanced Marfey’s method. The structures of N-OH-Leu, piperazic acids, and 4-OH-piperazic acids were confirmed by single-crystal X-ray diffraction analysis. Piperapeptins A (1) and B (2) exhibited weak antibacterial activity against Bacillus subtilis, and 1 showed an IC50 value of 14.9 ± 3.9 μM against HeLa S3 cells.

Microorganisms in nature form communities through diverse interactions, such as mutualism and competition, to adapt to their ecological environments. These interactions seem to be mediated by extracellular metabolites (exometabolites), yet the chemical and biological diversity underlying these processes remains largely unexplored. In this study, we examined the chemical basis of exometabolite-mediated interactions in the fission yeast Schizosaccharomyces pombe by a genome-wide screen employing 3,420 viable gene deletion mutants. We identified 37 strains that exhibited growth defects in monoculture on a minimal medium but exhibited growth recovery in the vicinity of wild-type colonies (co-culture), suggesting that exometabolites derived from wild-type cells compensated for the gene deletion. Both lipophilic and water-soluble fractions obtained by solvent partitioning of the wild-type culture supernatant promoted growth recovery. Among the 11 mutants rescued by the water-soluble fraction, 6 were cysteine auxotrophs, prompting analyses of thiol-containing metabolites by liquid chromatography-mass spectrometry (LC-MS), revealing the presence of glutathione (GSH) in the culture supernatant. GSH restored growth in most strains as a nutrient source. In contrast, GSH rescued cell morphology defects in the hob3 ∆ mutant, lacking the Bin/amphiphysin/Rvs (BAR) adaptor protein Hob3, through a mechanism independent of nutrition. This research advances understanding of exometabolite-mediated interactions in S. pombe by identifying GSH as an exometabolite that influences cellular processes and potentially shapes microbial communities. IMPORTANCE: Microorganisms secrete a wide range of metabolites that control microbial community behavior. These extracellular metabolites (exometabolites) include not only well-studied signaling molecules but also diverse primary and secondary metabolites, suggesting complex interactions among microbes. However, the molecular basis of these interactions remains poorly understood, partly due to challenges in detecting them experimentally. In this study, we surveyed exometabolites involved in cell–cell interactions in the model eukaryotic microorganism Schizosaccharomyces pombe . S. pombe releases a wide variety of metabolites outside the cells, including previously reported nitrogen signaling factors (NSFs) and glutathione (GSH) identified in this work. By analyzing gene deletion mutants whose growth is supported by extracellular GSH, we provide new insights into how secreted primary exometabolites compensate for specific genetic defects and influence cell physiology in microbial populations.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line enzymes that generate structurally diverse and biologically active natural products. Although adenylation (A) domain selectivity has been extensively characterized and engineered, the specificity of condensation (C) domains, which catalyze peptide bond formation and can constrain NRPS reprogramming, remains less well understood. Here, we report a systematic functional analysis of VibH, a stand-alone C domain VibH from the vibriobactin biosynthetic pathway. By integrating VibH with wild-type and engineered variants of the upstream aryl acid A domain EntE, we bypassed intrinsic A-domain constraints and independently interrogated donor- and acceptor-site substrate tolerance. The donor site of VibH exhibited stringent specificity, accepting only mono- and disubstituted benzoic acid derivatives closely resembling 2,3-dihydroxybenzoic acid (DHB), whereas bulkier aryl substrates were not processed. In contrast, the acceptor site displayed broad tolerance toward structurally diverse amines. Monoamines showed clear chain-length dependence, with productive turnover restricted to medium-chain substrates, whereas diamines were more broadly accepted. Systematic analysis further revealed distance-dependent steric tolerance, in which bulky substituents were accommodated when positioned distal to the reactive amine but were restricted beyond an upper steric threshold. This engineered reconstitution strategy enabled the synthesis of numerous non-native amide conjugates and uncovered an asymmetric substrate-recognition architecture characterized by a stringent donor site and a permissive yet spatially constrained acceptor site. Molecular docking analysis provided a structural rationale for these trends by suggesting distinct binding modes for native and non-native donor substrates and distance-dependent accommodation of bulky amine acceptors. These findings provide mechanistic insights into C-domain specificity and establish a framework for rational NRPS reprogramming.

Thiopeptides are a family of macrocycle-containing ribosomally synthesized and post-translationally modified peptides. Their elaborate scaffolds offer considerable potential for bioengineering toward thiopeptide-based pharmaceuticals and other practical applications. Among thiopeptides, lactazoles possess uniquely promiscuous biosynthetic machinery that enables the creation of diverse macrocyclic peptides. Previous bioengineering efforts have exploited this machinery to achieve the de novo design of bioactive lactazole-based thiopeptides in vitro. However, it remains unclear whether microbial systems can produce lactazole-based thiopeptides with dramatically engineered macrocycles, particularly those that are expanded and contain more than 50% amino acid divergence relative to native lactazole macrocycles. Here, we established a framework for the in vivo production of lactazole-based thiopeptides by tuning expression cassettes and selecting suitable heterologous hosts and culture conditions. Initially, we focused on transcriptional terminators in the 3′-untranslated region of the precursor gene and identified the lazA terminator as a critical determinant for lactazole production. We then evaluated several heterologous Streptomyces hosts and selected Streptomyces sp. TP-A0584 ΔgodA for the production of lactazole-based thiopeptides. Under optimized conditions, including low-temperature cultivation, more than 90% of the tested lactazole-based thiopeptides were successfully produced, and a large part of them reached mg-scale production, with a maximum titer of 46.4 mg/L. Notably, although their macrocycles showed up to 73% amino acid divergence from those of native lactazoles, most of these lactazole-based thiopeptides were successfully produced in vivo. Our framework represents an initial step toward enabling the large-scale supply of lactazole-based thiopeptides and should facilitate the development of thiopeptide-based bioactive molecules.

A series of novel sulfur-containing cyclic lipopeptides named thioamycolamides F-I (1-4), with thiazoline, sulfoxide/sulfide, and fatty acid moieties, was discovered by applying GNPS molecular networking technology against combined-culture broth of Amycolatopsis sp. 26-4 and Tsukamurella pulmonis TP-B0596. Planar structural elucidation was accomplished by HRMS and 1D/2D NMR spectroscopic data analyses. The absolute configuration was determined by the highly sensitive advanced Marfey's method developed by our group, electronic circular dichroism (ECD) spectroscopy, chemical synthesis, and Gaussian calculation. Moreover, compound 2 exhibited cytotoxicity against human cervix adenocarcinoma HeLa S3 cells with an IC₅₀ of 24.0 μM.

Microorganisms in nature form communities through diverse interactions, such as mutualism and competition, to adapt to their ecological environments. These interactions seem to be mediated by extracellular metabolites (exometabolites), yet the chemical and biological diversity underlying these processes remains largely unexplored. In this study, we examined the chemical basis of cell-cell communication in the fission yeast Schizosaccharomyces pombe by a genome-wide screen employing 3,420 viable gene deletion mutants. We identified 37 strains that exhibited growth defects in monoculture on a minimal medium but exhibited growth recovery in the vicinity of wild-type colonies (co-culture), suggesting that exometabolites secreted by wild-type cells compensated for the gene deletion. Both lipophilic and water-soluble fractions obtained by solvent partitioning of the wild-type culture supernatant promoted growth recovery. Among the 11 mutants rescued by the water-soluble fraction, six were cysteine auxotrophs, prompting analyses of thiol-containing metabolites by liquid chromatography-mass spectrometry (LC-MS), revealing the presence of glutathione (GSH) in the culture supernatant. GSH restored growth in most strains as a nutrient source. In contrast, GSH rescued cell morphology defects in the hob3 Δ mutant, lacking the Bin/Amphiphysin/Rvs (BAR) adaptor protein Hob3, through a mechanism independent of nutrition. This research advances understanding of exometabolite-mediated interactions in S. pombe by highlighting the role of GSH as one of the communication molecules that influence cellular processes and shape microbial communities. Author summary: Microorganisms secrete a wide range of metabolites that control microbial community behavior. These extracellular metabolites (exometabolites) include not only well-studied signaling molecules but also diverse primary and secondary metabolites, suggesting complex interactions among microbes. However, the molecular basis of these interactions remains poorly understood, partly due to challenges in detecting them experimentally. In this study, we surveyed exometabolites involved in cell-cell interactions in the model eukaryotic microorganism Schizosaccharomyces pombe . S. pombe secretes a wide variety of metabolites, including previously reported nitrogen signaling factors (NSFs) and glutathione identified in this work. By analyzing gene deletion mutants that depend on GSH for growth, we provide new insights into how microbes regulate collective behavior by sharing exometabolites.

Bisenarsan is an organoarsenic natural product identified from actinomycetes and a derivative of (2-hydroxyethyl)arsonic acid (2-HEA) esterified with 2,4,6-trimethyl-2-nonenoic acid (2,4,6-TMNA). Our previous study suggested that bisenarsan is biosynthesized from arsenate [As(V)] via arsonoacetaldehyde (AnAA). In contrast, the late-stage biosynthetic steps from AnAA to bisenarsan and the roles of transporter genes within the biosynthetic gene clusters (BGCs) of bisenarsan remain unclear. In this study, through in-frame deletions and heterologous expression targeting the bisenarsan BGC in Streptomyces lividans 1326 (bsn cluster), we identified bsnF (nicotinamide adenine dinucleotide phosphate-dependent oxidoreductase), bsnPKS (iterative type I polyketide synthase), and bsnFB (3-ketoacyl-acyl carrier protein synthase III family protein) as genes encoding enzymes likely responsible for the late-stage biosynthesis of bisenarsan. BsnF, BsnPKS, and BsnFB are presumed to catalyze the reduction of AnAA to 2-HEA, the formation of the 2,4,6-TMNA moiety, and the ester bond formation, respectively. Furthermore, based on the functional analysis of the transporter genes in the bsn cluster, BsnT2 (major facilitator superfamily transporter) appears to be involved in the efflux of bisenarsan. Although the roles of other transporters in bisenarsan biosynthesis remain unclear, they may contribute to the uptake and efflux of inorganic arsenic, presumably to ensure a consistent substrate supply and mitigate toxicity caused by its overaccumulation. Our study provides valuable insights into the biosynthesis of a rare class of organoarsenic natural products, with arsonopyruvate as an intermediate.