尾仲 宏康

Actinomycetes, particularly Streptomyces, are soil microorganisms that produce diverse secondary metabolites with pharmaceutical applications, such as antibiotics and anticancer drugs. These metabolites play important roles in microbial competition and survival. This review highlights three major aspects of actinomycete secondary metabolism: (1) the biosynthesis of indolocarbazoles, (2) the biosynthesis of RiPPs (ribosomally synthesized and post-translationally modified peptides), and (3) the activation of secondary metabolism through microbial interactions. Indolocarbazoles, including staurosporine and rebeccamycin, are potent inhibitors of kinases and DNA topoisomerase I, with potential as anticancer agents. Their biosynthetic pathways involve multiple enzymatic steps, notably carbon-carbon bond formation catalyzed by cytochrome P450 enzymes. RiPPs such as goadsporin and lactazole are highly modular peptide natural products; structural gene modification enables the generation of diverse analogs. A cell-free one-pot synthesis platform has been developed for efficient analog production. To activate cryptic biosynthetic pathways, we employed a combined-culture strategy using actinomycetes and mycolic acid-containing bacteria, resulting in the discovery of 42 novel compounds. Genetic and physiological data indicate that physical contact, rather than diffusible signaling, is essential for induction. These insights emphasize the importance of microbial interactions in natural product biosynthesis and offer new directions for drug discovery through synthetic biology and microbial ecology.

The combined-culture of actinomycetes with mycolic acid-containing bacteria (MACB) Tsukamurella pulmonis TP-B0596 is a promising strategy to produce cryptic metabolites in actinomycetes. In this study, Streptomyces sp. 23-50 was identified as an appropriate strain for co-culturing with T. pulmonis TP-B0596 using on-gel combined-culture screening of 160 strains of actinomycetes. A new pyranonaphthoquinone, actinoquinonal A (1), along with two known congeners, compound 2 and mevashuntin (3), were isolated from the combined-culture of Streptomyces sp. 23-50 with T. pulmonis TP-B0596 based on global natural product social (GNPS) molecular networking. The planar structures of 1-3 were elucidated by analyzing 2D nuclear magnetic resonance (NMR) and LC-MS/MS spectral data, and the absolute configurations of 1 and 3 were unambiguously determined by comparing experimental and calculated ECD spectra. Moreover, the combined-culture characteristic metabolites, including 3, were enhanced when Streptomyces sp. 23-50 was cultured in the presence of pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the mevalonate pathway, suggesting that T. pulmonis TP-B0596 triggered a shunt in the mevalonate pathway of Streptomyces sp. 23-50. Notably, compounds 1 and 3 exhibited cytotoxicity against human cervical epithelioid carcinoma HeLa S3 (IC50 = 60.5 μM for 1, 0.67 μM for 3) and human colorectal cancer HT29 cells (IC50 = 101.9 μM for 1, 0.45 μM for 3).

Covering: 1977 to presentArsenic is widely distributed throughout terrestrial and aquatic environments, mainly in highly toxic inorganic forms. To adapt to environmental inorganic arsenic, bacteria have evolved ubiquitous arsenic metabolic strategies by combining arsenite methylation and related redox reactions, which have been extensively studied. Recent reports have shown that some bacteria have specific metabolic pathways associated with structurally and biologically unique organoarsenic natural products. In this highlight, by exemplifying the cases of oxo-arsenosugars, arsinothricin, and bisenarsan, we summarize recent advances in the identification and biosynthesis of bacterial organoarsenic natural products. We also discuss the potential discoveries of novel arsenic-containing natural products of bacterial origins.

Nonribosomal peptides (NRPs), one of the most widespread secondary metabolites in nature, with therapeutically significant activities, are biosynthesized by modular nonribosomal peptide synthetases (NRPSs). Aryl acids contribute to the structural diversity of NRPs as well as nonproteinogenic amino acids and keto acids. We previously confirmed that a single Asn-to-Gly substitution in the 2,3-dihydroxybenzoic acid-activating adenylation (A) domain EntE involved in enterobactin biosynthesis accepts monosubstituted benzoic acid derivatives with nitro, cyano, bromo, and iodo functionalities at the 2 or 3 positions. Here, we showed that the mutant EntE (N235G) accommodates various disubstituted benzoic acid derivatives with halogen, methyl, methoxy, nitro, and cyano functionalities at the 2 and 3 positions and monosubstituted benzoic acid with an alkyne at the 3 position. Structural analysis of the mutant EntE (N235G) with nonhydrolyzable aryl-AMP analogues using 3-chloro-2-methylbenzoic acid and 3-prop-2-ynoxybenzoic acid revealed how bulky 3-chloro-2-methylbenzoic acid and clickable 3-prop-2-ynoxybenzoic acid are recognized by enlarging the substrate-binding pocket of the enzyme. When engineered EntE mutants were coupled with enterobactin and vibriobactin biosynthetic enzymes, 3-hydroxybenzoic acid-, salicylic acid-, and 3-bromo-2-fluorobenzoic acid-containing peptides were produced as early stage intermediates, highlighting the potential of NRP biosynthetic pathway engineering for constructing diverse aryl acid-containing metabolites.

A phytochemical investigation of Kaempferia champasakensis rhizomes led to the isolation of five new pimarane diterpenes, kaempferiols E-I (1-5). The structures of 1-5 were elucidated by extensive spectroscopic techniques, including HR-ESI-MS, UV, IR, and 1D and 2D NMR. The absolute configurations of 1-3 were determined by the modified Mosher method, and those of 4 and 5 were established by ECD calculations. Further cytotoxic assay for all isolated compounds against three human cancer cell lines, lung cancer (A549), cervical cancer (HeLa), and breast cancer (MCF-7) indicated that 5 showed moderate cytotoxic activities against the three tested cell lines, with IC50 values of 44.78, 25.97, and 41.39 Mμ for A549, HeLa, and MCF-7 cell lines, respectively.

A phytochemical investigation of Kaempferia champasakensis rhizomes led to the isolation of a new 3,4-seco-isopimarane diterpene, kaempferiol A (1), and three new isopimarane diterpenes, kaempferiols B-D (2-4), together with six known isopimarane diterpenes (5-10). The structures of 1-4 were elucidated by extensive spectroscopic analyses, including HR-ESI-MS, UV, IR, and 1D and 2D NMR. The absolute configurations of 1, 3, and 4 were determined by ECD calculations, while that of 2 was established using the modified Mosher method. All isolated compounds were tested for cytotoxicity against three human cancer cell lines, lung cancer (A549), cervical cancer (HeLa), and breast cancer (MCF-7). Among them, 6 and 7 showed moderate cytotoxic activities against the three tested cell lines, with IC50 values ranging from 38.04 to 27.77 μM, respectively.

Three neo-clerodane diterpenoids, including two new tinocordifoliols A (1) and B (2) and one known tinopanoid R (3), were isolated from the ethyl acetate-soluble fraction of the 70% ethanol extract of Tinospora cordifolia stems. The structures were elucidated by various spectroscopic methods, including one dimensional (1D) and 2D-NMR, high resolution-electrospray ionization (HR-ESI)-MS, and electronic circular dichroism (ECD) data. The T. cordifolia extract and all isolated compounds 1-3 possessed arginase I inhibitory activities. Among them, 3 exhibited moderate competitive inhibition of human arginase I (IC50 = 61.9 µM). Furthermore, docking studies revealed that the presence of a β-substituted furan in 3 may play a key role in the arginase I inhibitory activities.

Combined-cultures involving mycolic acid-containing bacteria (MACB) can stimulate secondary metabolite (SM) production in actinomycetes. In a prior investigation, we screened Streptomyces coelicolor JCM4020 mutants with diminished production of SMs, specifically undecylprodigiosin (RED), which was enhanced by introducing the MACB Tsukamurella pulmonis TP-B0596. In this study, we conducted mutational analysis that pinpointed the sco1842 gene, which we assigned the gene name ccr1 (combined-culture related regulatory protein no. 1), as a crucial factor in the deficient phenotype observed in the production of various major SMs in S. coelicolor A3(2). Notably, the Ccr1 (SCO1842) homolog was found to be highly conserved throughout the Streptomyces genome. Although Ccr1 lacked conserved motifs, in-depth examination revealed the presence of a helix-turn-helix (HTH) motif in the N-terminal region and a helicase C-terminal domain (HCTD) motif in the C-terminal region in some of its homologs. Ccr1 was predicted to be a nucleoid-associated protein (NAP), and its impact on gene transcription was validated by RNA-seq analysis that revealed genome-wide variations. Furthermore, RT-qPCR demonstrated that ccr1 was transcriptionally activated in combined-culture with T. pulmonis, which indicated that Ccr1 is involved in the response to bacterial interaction. We then investigated Streptomyces nigrescens HEK616 in combined-culture, and the knockout mutant of the ccr1 homolog displayed reduced production of streptoaminals and 5aTHQs. This finding reveals that the Ccr1 homolog in Streptomyces species is associated with SM production. Our study elucidates the existence of a new family of NAP-like proteins that evolved in Streptomyces species and play a pivotal role in SM production.

Cancer cells including colorectal cancer cells are resistant to anoikis, an anchorage-independent programmed death, which enables metastasis and subsequent survival in a new tumor microenvironment. In this study, we identified a new anoikis inducer, amoxetamide A (1) with a β-lactone moiety, that was produced by combined-culture of Amycolatopsis sp. 26-4 and mycolic acid-containing bacteria (MACB) Tsukamurella pulmonis TP-B0596. The structure of 1 including the stereochemistry of C8 was determined by MS and NMR spectroscopy and modified Mosher's method, and the absolute configurations of C11 and C12 were suggested as 11R and 12S, respectively, by GIAO NMR calculations. Amoxetamide A (1) exhibited anoikis-inducing activity in human colorectal cancer HT-29 cells in anchorage-independent culture conditions.

The unique bioactivities of arsenic-containing secondary metabolites have been revealed recently, but studies on arsenic secondary metabolism in microorganisms have been extremely limited. Here, we focused on the organoarsenic metabolite with an unknown chemical structure, named bisenarsan, produced by well-studied model actinomycetes and elucidated its structure by combining feeding of the putative biosynthetic precursor (2-hydroxyethyl)arsonic acid to Streptomyces lividans 1326 and detailed NMR analyses. Bisenarsan is the first characterized actinomycete-derived arsenic secondary metabolite and may function as a prototoxin form of an antibacterial agent or be a detoxification product of inorganic arsenic species. We also verified the previously proposed genes responsible for bisenarsan biosynthesis, especially the (2-hydroxyethyl)arsonic acid moiety. Notably, we suggest that a C-As bond in bisenarsan is formed by the intramolecular rearrangement of a pentavalent arsenic species (arsenoenolpyruvate) by the cofactor-independent phosphoglycerate mutase homologue BsnN, that is entirely distinct from the conventional biological C-As bond formation through As-alkylation of trivalent arsenic species by S-adenosylmethionine-dependent enzymes. Our findings will speed up the development of arsenic natural product biosynthesis.

We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid (O-allyl-L-serine) was successfully incorporated into gramicidin S.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are newly found lipopeptide-class natural products. We recently employed a combined approach of genome mining and stable isotope labeling and discovered solabiomycins as one of the polar-functionalized fatty-acylated RiPPs (PFARs) from Streptomyces lydicus NBRC13058. The solabiomycins contained a characteristic sulfoxide group in the labionin moiety referred to as the 'solabionin' structure for the RiPP moiety. A previous gene knockout experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of an alkyl sulfide in the solabionin. In this study, we isolated deoxysolabiomycins A and B from ΔsolS mutant and fully determined the chemical structures using a series of NMR experiments. We also tested the bioactivity of deoxysolabiomycins against Gram-positive bacteria, including Mycolicibacterium smegmatis, and notably found that the sulfoxide is critical for the antibacterial activity. To characterize the catalytic activity of SolS, the recombinant protein was incubated with a putative substrate, deoxysolabiomycins, and the cofactors FAD and NADPH. In vitro reactions demonstrated that SolS catalyzes the sulfoxidation, converting deoxysolabiomycins to solabiomycins.

Contractile injection systems (CISs) are a large group of phage tail-like nanostructures conserved among bacteria. Despite their wide distribution, the biological significance of CISs in bacteria remains largely unclear except for a few unicellular bacteria. Here, we show that Streptomyces lividans-a model organism of filamentous Gram-positive bacteria with highly conserved CIS-related gene clusters-produces intracellular CIS-like nanostructures (Streptomyces phage tail-like particles [SLPs]) that affect phenotypes of this bacterium under hyperosmotic conditions. In contrast to typical CISs released from the cells, SLPs are localized in the cytoplasm of S. lividans. In addition, loss of SLPs leads to (i) delayed erection of aerial mycelia on hyperosmotic solid medium and (ii) decreased growth during the transition from exponential growth phase to stationary phase in hyperosmotic liquid medium. Localization of fluorescent protein-tagged SLPs showed partial correlation with cell wall synthesis-related proteins, including MreB, an actin-like cytoskeleton protein. Our pulldown assay and subsequent quantitative proteome analysis also suggest that 30S ribosomal proteins and cell wall-related proteins, including MreB, are coeluted with SLPs. Furthermore, an interaction assay using the recombinant proteins revealed a direct interaction between a sheath protein of SLP and ribosomal protein S16. Results of cross-linking experiments show indirect interactions between SLPs and translation elongation factors. These findings collectively suggest that SLPs are directly or indirectly associated with a protein interaction network within the cytoplasm of S. lividans and that SLP loss ultimately affects the susceptibility of the bacterium to certain stress conditions. IMPORTANCE Recent bioinformatic analyses have revealed that CIS-related gene clusters are highly conserved in Gram-positive actinomycetes, especially members of the genus Streptomyces known for their ability to produce therapeutic antibiotics. While typical CISs are released from the cells and can act as protein translocation systems that inject effector proteins into the target cells, our results indicate the unique intracellular localization of SLPs, CIS-related nanostructures produced by S. lividans. In addition, the direct and indirect interactions of SLPs with cytoplasmic proteins and SLP localization within specific regions of mycelia suggest that the biological significance of SLPs is related to intracellular processes. Further, SLP loss leads to increased susceptibility of S. lividans to osmotic stress, suggesting that production of these phage tail-like nanostructures ultimately affects the fitness of the bacterium under certain stress conditions. This work will provide new insight into the phage tail-like nanostructures highly conserved in Streptomyces species.

Streptomyces spp. are well-known producers of bioactive secondary metabolites (SMs) that serve as pharmaceutical agents. In addition to their ability to produce SMs, Streptomyces spp. have evolved diverse membrane transport systems to protect cells against antibiotics produced by itself or other microorganisms. We previously screened mutants of Streptomyces coelicolor that show a phenotype of reduced undecylprodigiosin (RED) production in a combined-culture with Tsukamurella pulmonis. Here, we identified a point mutation, which reduced RED production, by performing genome resequencing and genetic complementation. We found that inactivation of the sco1718 gene encoding the TetR family transcriptional regulator (TFR) produced a deficient phenotype for several SMs in Streptomyces coelicolor A3(2). In the genome of S. coelicolor A3(2), two other sets of TFR and two-component ATP-binding cassette (ABC) transporter genes (sco4358-4360 and sco5384-5382) were found which had similar effects on the phenotype for both secondary metabolism and antibiotic resistance. An electrophoretic mobility shift assay and quantitative reverse transcription-PCR experiments demonstrated that TFRs repressed the expression of each adjacent two-component ABC transporter genes by binding to the operator sequence. Notably, the Δsco1718 mutant showed increased resistance to several antibiotics of other actinomycete origin. Our results imply the switching of cell metabolism to direct offense (antibiotic production) or defense (efflux pump activation) using costly and limited quantities of cell energy sources (e.g., ATP) in the soil ecosystem. IMPORTANCE The bacterial metabolic potential to synthesize diverse secondary metabolites in the environment has been revealed by recent (meta)genomics of both unculturable and culturable bacteria. These studies imply that bacteria are continuously exposed to harmful chemical compounds in the environment. Streptomyces spp. contain antibiotic efflux pumps and SM biosynthetic gene clusters. However, the mechanism by which soil bacteria, including Streptomyces, survive against toxic compounds in the environment remains unclear. Here, we identified three sets of TFR-ABC transporter genes in Streptomyces coelicolor A3(2). We found that each TFR controlled the expression of respective ABC transporter, and the expression of all ABC transporters negatively impacted SM production and increased antibiotic resistance. Notably, bioinformatic analysis indicated that these TFR-ABC transporter gene sets are highly conserved and widely distributed in the genome of Streptomyces species, indicating the importance of systematic regulation that directs antibiotic production and xenobiotic excretion.

Abstract Chemical cross talks betweenMycolicibacterium septicumHEK138M andBacillus subtilis168 affect the bacterial morphology ofStreptomyces variegatusHEK138A. We found thatS. variegatusexhibits unusual hyphae branching by the bacterial interaction. We aimed to elucidate the mechanism by performing activity guided purification of substances that induce the unusual cell morphology. We found that pyrogallol, a redox active aromatic small molecule induced significant hyphae branching inS. variegatusand the activity was also observed in some of otherStreptomycesspecies. Interestingly, the pyrogallol activity was diminished by adding catalase, which broke down H₂O₂. To further confirm the involvement, H₂O₂was tested and similar activity which induced hyphal branching was observed. This indicates that reactive oxygen species (ROS) generated by redox-active compound (RAC) is the inducing factor of hyphae branching. Further investigation revealed that pyrogallol was generated by NahG enzyme homolog ofM. septicumusing 2,3-dihydroxybenzoic acid as substrate by heterologous expression inE. coli. Moreover, co-culture with gene knock-out mutants revealed that 2,3-dihydroxybenzoic acid was supplied byB. subtilisproduced as intermediate of bacterial siderophore bacillibactin. Since the hyphae branching of vegetative mycelium can increase the density of filamentous network and consequently help secure the milieu in soil, our results suggested that those filamentous soil bacteria use ROS which can be supplied from plant derived RAC as a signal. As those RAC ubiquitously exist in soil environment, the system will be beneficial for sensing the nutrient sources in addition to the generally considered defensive response to oxidative stress. Importance The characterization of interactions between three or more bacteria are lacking as these interactions are visually imperceptible in general. Our current study revealed changes of morphological behavior by the bacterial interaction. This study showed that hydrogen peroxide generated by redox-active compound derived from a breakdown product of siderophore can significantly increase the number of hyphae tip extension in filamentous bacteria. Our result implies the existence of oxidative response system using a low amount of reactive oxygen species as an integrated signal to sense the plant-derived carbon source by the filamentous soil bacteria. As a result of sensing, filamentous soil bacteria may decide whether the hypha tip should be extended to further explore the area or increase the tips to densify filamentous network to monopolize the nutrients in the milieu.

Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥1012) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affinity selection against Traf2- and NCK-interacting kinase (TNIK), a protein involved in several cancers, which yielded a plethora of candidate thiopeptides. Of the 11 synthesized compounds, 9 had high affinities for the target kinase (best KD = 1.2 nM) and 10 inhibited its enzymatic activity (best Ki = 3 nM). X-ray structural analysis of the TNIK/thiopeptide interaction revealed the unique mode of substrate-competitive inhibition exhibited by two of the discovered compounds. The thiopeptides internalized to the cytosol of HEK293H cells as efficiently as the known cell-penetrating peptide Tat (4-6 μM). Accordingly, the most potent compound, TP15, inhibited TNIK in HCT116 cells. Altogether, our platform enables the exploration of pseudo-natural thiopeptides with favorable pharmacological properties in drug discovery applications.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are a rarely found untapped class of natural products. Although polar-functionalized fatty-acylated RiPPs (PFARs) have potential as antimicrobial agents, the repertoire is still limited. Therefore, expanding the chemical space is expected to contribute to the development of pharmaceutical agents. In this study, we performed genome mining and stable isotope-guided comparative metabolomics to discover new PFAR natural products. We focused on the feature that PFARs incorporate l-arginine or l-lysine as the starter unit of the fatty acyl group and fed 13C6,15N4-l-arginine or 13C6,15N2-l-lysine to bacterial cultures. Metabolites were extracted and compared with those extracted from nonlabeled l-arginine or l-lysine fed cultures. We identified putative PFARs and successfully isolated solabiomycin A and B from Streptomyces lydicus NBRC 13 058 and albopeptin B from Streptomyces nigrescens HEK616, which contained a sulfoxide group in the labionin moiety. The gene disruption experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of aryl sulfides. The solabiomycins showed antibacterial activity against Gram-positive bacteria, including Mycobacterium tuberculosis H37Rv with a minimum 95% inhibitory concentration (MIC95) of 3.125 μg/mL, suggesting their potential as antituberculosis agents.

To investigate the potential for secondary metabolite biosynthesis by Streptomyces species, we employed a coculture method to discover natural bioactive products and identified specific antibacterial activity from a combined-culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596. Molecular networking using ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) data revealed a specific clade of metabolites in this combined-culture that were not detected in both monocultures. Using the chemical profiles, a previously unidentified conjugate between FabF inhibitor and catechol-type siderophore was successfully identified and named harundomycin A. Harundomycin A was a conjugate between the 2,4-dihydroxy-3-aminobenzoate moiety of platensimycin and N,N'-bis(2,3-dihydroxybenzoyl)-O-seryl-cysteine (bisDHBA-Ser-Cys) with a thioester linkage. Along with the production of harundomycin A, platensimycin, its thiocarboxylic acid form thioplatensimycin, enterobactin, and its degradation product N,N'-bis(2,3-dihydroxybenzoyl)-O-l-seryl-dehydroalanine (bisDHBA-Ser-Dha) were also induced in the combined-culture. Genomic data of S. hygroscopicus HOK021 and T. pulmonis TP-B0596 indicated that strain HOK021 possessed biosynthetic gene clusters for both platensimycin and enterobactin, and thereby revealed that T. pulmonis stimulates HOK021 and acts as an inducer of both of these metabolites. Although the harundomycin A was modified by bulky bisDHBA-Ser-Cys, responsible for the binding to the target molecule FabF, it showed a similar antibacterial spectrum to platensimycin, including against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, suggesting that the pharmacophore is platensimycin. Additionally, Chrome Azurol S assay showed that harundomycin A possesses ferric iron-chelating activity comparable to that of enterobactin. Our study demonstrated the transformation of existing natural products to bifunctional molecules driven by bacterial interaction.

Promiscuous post-translational modification (PTM) enzymes often display nonobvious substrate preferences by acting on diverse yet well-defined sets of peptides and/or proteins. Understanding of substrate fitness landscapes for PTM enzymes is important in many areas of contemporary science, including natural product biosynthesis, molecular biology, and biotechnology. Here, we report an integrated platform for accurate profiling of substrate preferences for PTM enzymes. The platform features (i) a combination of mRNA display with next-generation sequencing as an ultrahigh throughput technique for data acquisition and (ii) deep learning for data analysis. The high accuracy (>0.99 in each of two studies) of the resulting deep learning models enables comprehensive analysis of enzymatic substrate preferences. The models can quantify fitness across sequence space, map modification sites, and identify important amino acids in the substrate. To benchmark the platform, we performed profiling of a Ser dehydratase (LazBF) and a Cys/Ser cyclodehydratase (LazDEF), two enzymes from the lactazole biosynthesis pathway. In both studies, our results point to complex enzymatic preferences, which, particularly for LazBF, cannot be reduced to a set of simple rules. The ability of the constructed models to dissect such complexity suggests that the developed platform can facilitate a wider study of PTM enzymes.

The complete 1 H and 13 C NMR characterization of streptogramin B (1), the major component of a clinically important synergistic antibiotic complex, was presented for the first time, along with those of L-156,587 (2), a dehydrated congener of streptogramin A (3). Compounds 1 and 2 were not synergistic and produced by Streptomyces albogriseolus in co-culture with Tsukamurella pulmonis, which poses a question on the adaptive significance of the induced production of this antibiotic pair.

We report a method for the high-throughput reactivity profiling of genetically encoded libraries as a tool to study substrate fitness landscapes for RiPP (ribosomally synthesized and post-translationally modified peptide) biosynthetic enzymes. This method allowed us to rapidly analyze the substrate preferences of the lactazole biosynthetic pathway using a saturation mutagenesis mRNA display library of lactazole precursor peptides. We demonstrate that the assay produces accurate and reproducible in vitro data, enabling the quantification of reaction yields with temporal resolution. Our results recapitulate the previously established knowledge on lactazole biosynthesis and expand it by identifying the extent of substrate promiscuity exhibited by the enzymes. This work lays a foundation for the construction and screening of mRNA display-based combinatorial thiopeptide libraries for the discovery of lactazole-inspired thiopeptides with de novo designed biological activities.

Fusions of fatty acids and peptides expand the structural diversity of natural products; however, polyketide/ribosomally synthesized and post-translationally modified peptides (PK/RiPPs) hybrid lipopeptides are relatively rare. Here we report a family of PK/RiPPs called goadvionins, which inhibit the growth of Gram-positive bacteria, and an acyltransferase, GdvG, which catalyses the condensation of the PK and RiPP moieties. Goadvionin comprises a trimethylammonio 32-carbon acyl chain and an eight-residue RiPP with an avionin structure. The positions of six hydroxyl groups and one double bond in the very-long acyl chain were determined by radical-induced dissociation tandem mass spectrometry, which collides radical ion species to generate C-C bond cleavage fragments. GdvG belongs to the Gcn5-related N-acetyltransferase superfamily. Unlike conventional acyltransferases, GdvG transfers a very long acyl chain that is tethered to an acyl carrier protein to the N-terminal amino group of the RiPP moiety. gdvG homologues flanked by PK/fatty acid and RiPP biosynthesis genes are widely distributed in microbial species, suggesting that acyltransferase-catalysed condensation of PKs and RiPPs is a general strategy in biosynthesis of similar lipopeptides.

Enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, yet usually the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate the biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level and supplement this study with substrate scope analysis for participating enzymes. Our results reveal an unusual but well-defined assembly process where cyclodehydration, dehydroalanine formation, and azoline dehydrogenation events are intertwined due to minimal substrate recognition requirements characteristic of every lactazole enzyme. Additionally, each enzyme plays a role in directing LazBF-mediated dehydroalanine formation, which emerges as the central theme of the assembly process. Cyclodehydratase LazDE discriminates a single serine residue for azoline formation, leaving the remaining five as potential dehydratase substrates. Pyridine synthase LazC exerts kinetic control over LazBF to prevent the formation of overdehydrated thiopeptides, whereas the coupling of dehydrogenation to dehydroalanine installation impedes generation of underdehydrated products. Altogether, our results indicate that substrate-level cooperation between the biosynthetic enzymes maintains the integrity of lactazole assembly. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.

富山県高岡市産大麦麦芽より醸造に適した「とやま産まれの酵母」を分離した。本菌株はITS領域解析の結果，Saccharomyces cerevsiae NBRC2114（Kotobukiya whisky No. 1）に最も近い配列を有することが明らかとなった。清酒小仕込み試験による醸造特性解析の結果，とやま産まれの酵母のもろみ日数はK701よりも3日長くなったものの，両者の到達アルコール濃度はほぼ同じ18%であった。K701と比較し，有機酸組成では酢酸，リンゴ酸，コハク酸の含有量が顕著に異なっており，揮発性物質では高級アルコール含量が高く，エステル類の含量が低かった。アミノ酸度はとやま酵母の方が顕著に高かった。これらの性質が官能特性評価にも現れており，K701に比較して，酸味の違いや，旨み，重い，雑味などの味の多さに特徴がある個性的な酒質であった。<br>とやま産まれの酵母による実地醸造は成政酒造にて，これまでに5回行われており，試行錯誤の末，現在では比較的濃醇でキレのある酒質となっている。また，本酵母は野生酵母で特定の酒類醸造に特化した育種もなされていないため，富山県地域のクラフトビール，ワイン，ウイスキーの醸造にも利用されるに至っている。