Shotaro Hoshino

Abstract Actinomycetes have produced a variety of bioactive secondary metabolites; however, discovering new actinobacterial natural products using conventional approaches has become increasingly challenging. Meanwhile, genomic studies of actinomycetes have revealed that numerous secondary metabolite biosynthetic gene clusters (SM-BGCs) remain untapped. Thus, utilizing these secondary metabolic pathways is expected to facilitate the discovery of new actinomycetes-derived natural products. In this review, I primarily describe our research on the utilization of these untapped actinobacterial SM-BGCs and the discovery of new secondary metabolites. First, I introduce our studies on the activation of silent SM-BGCs through the co-cultivation of various actinomycetes with mycolic acid-containing bacteria (MACB), which led to the identification of 20 actinobacterial secondary metabolites, including 16 new compounds. In the latter part, I describe our recent findings on arsenic-related secondary metabolism, which has been overlooked in model actinomycetes, including the identification of a novel organoarsenic natural product, and the elucidation of its unique biosynthetic strategy, which is independent of S-adenosylmethionine (SAM)-dependent enzymes. Graphical abstract

We summarize recent research in the discovery and biosynthesis of bacterial organoarsenic natural products, providing unique chemical architecture and enzymologies.

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.

Structure-based engineering of an Fe(ii)/2-oxoglutarate-dependent oxygenase AndA altered the catalytic function of the enzyme to catalyze spiro-ring formation reaction from isomerization reaction.

We report the first structures of bacterial HAQs bound to their target DHODH and provide insights into mechanism of inhibition.

C-S bond formation reactions are widely distributed in the biosynthesis of biologically active molecules, and thus have received much attention over the past decades. Herein, we report intramolecular C-S bond formation by a P450 monooxygenase, TleB, which normally catalyzes a C-N bond formation in teleocidin biosynthesis. Based on the proposed reaction mechanism of TleB, a thiol-substituted substrate analogue was synthesized and tested in the enzyme reaction, which afforded the unprecedented sulfur-containing thio-indolactam V, in addition to an unusual indole-fused 6/5/8-tricyclic product whose structure was determined by the crystalline sponge method. Interestingly, conformational analysis revealed that the SOFA conformation is stable in thio-indolactam V, in sharp contrast to the major TWIST form in indolactam V, resulting in differences in their biological activities.

Tenebrathin (1), a new C-5-substituted γ-pyrone with a nitroaryl side chain, was isolated from the rare actinomycete Streptoalloteichus tenebrarius NBRC 16177. The chemical structure of 1 was elucidated by a spectroscopic analysis using the crystalline sponge method of crystallization-free X-ray crystallography. The biosynthetic origin of the unusual C-5-substituted γ-pyrone in 1 was revealed by a 13C-labeling experiment. Compound 1 exhibited moderate cytotoxicity against several cancer cell lines and likely targets some protein kinases.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Abstract Bacterial secondary metabolites (SM) are rich sources of drug leads, and in particular, numerous metabolites have been isolated from actinomycetes. It was revealed by recent genome sequence projects that actinomycetes harbor much more secondary metabolite-biosynthetic gene clusters (SM-BGCs) than previously expected. Nevertheless, large parts of SM-BGCs in actinomycetes are dormant and cryptic under the standard culture conditions. Therefore, a widely applicable methodology for cryptic SM-BGC activation is required to obtain novel SM. Recently, it was discovered that co-culturing with mycolic-acid-containing bacteria (MACB) widely activated cryptic SM-BGCs in actinomycetes. This “combined-culture” methodology (co-culture methodology using MACB as the partner of actinomycetes) is easily applicable for a broad range of actinomycetes, and indeed, 33 novel SM have been successfully obtained from 12 actinomycetes so far. In this review, the development, application, and mechanistic analysis of the combined-culture method were summarized.

Phytochemical investigation of the CHCl ₃ extract of Premna serratifolia (syn: P. integrifolia) wood collected in Myanmar led to the isolation of a new tetrahydrofuran type lignan, 7,9-dihydroxydolichanthin B (1), together with two known triterpenoids, oleanonic acid (2) and (2a, 3α)-dihydroxyolean-12-en-28-oic acid (3). The structure of the new compound was determined using various spectroscopic techniques, mainly 1D- and 2D-NMR, HRESIMS, IR, and optical rotation, and by comparisons with the reported literatures. Compounds 1-3 had anti-melanin deposition activities against IBMX and α-MSH induced B16-F10 mouse melanoma cell line with IC ₅₀ values of 18.4, 17.7 and 11.2 μM, respectively. However, 2 exhibited cytotoxicity at concentrations above 50 μM.

Reprogramming of the NRPS/PKS assembly line is an attractive method for the production of new bioactive molecules. However, it is usually hampered by the loss of intimate domain/module interactions required for the precise control of chain transfer and elongation reactions. In this study, we first establish heterologous expression systems of the unique antimycin-type cyclic depsipeptides: JBIR-06 (tri-lactone) and neoantimycin (tetra-lactone), and engineer their biosyntheses by taking advantage of bioinformatic analyses and evolutionary insights. As a result, we successfully accomplish three manipulations: (i) ring contraction of neoantimycin (from tetra-lactone to tri-lactone), (ii) ring expansion of JBIR-06 (from tri-lactone to tetra-lactone), and (iii) alkyl chain diversification of JBIR-06 by the incorporation of various alkylmalonyl-CoA extender units, to generate a set of unnatural derivatives in practical yields. This study presents a useful strategy for engineering NRPS-PKS module enzymes, based on nature's diversification of the domain and module organizations.

Abstract Non-heme iron and α-ketoglutarate (αKG) oxygenases catalyze remarkably diverse reactions using a single ferrous ion cofactor. A major challenge in studying this versatile family of enzymes is to understand their structure–function relationship. AusE from Aspergillus nidulans and PrhA from Penicillium brasilianum are two highly homologous Fe(II)/αKG oxygenases in fungal meroterpenoid biosynthetic pathways that use preaustinoid A1 as a common substrate to catalyze divergent rearrangement reactions to form the spiro-lactone in austinol and cycloheptadiene moiety in paraherquonin, respectively. Herein, we report the comparative structural study of AusE and PrhA, which led to the identification of three key active site residues that control their reactivity. Structure-guided mutagenesis of these residues results in successful interconversion of AusE and PrhA functions as well as generation of the PrhA double and triple mutants with expanded catalytic repertoire. Manipulation of the multifunctional Fe(II)/αKG oxygenases thus provides an excellent platform for the future development of biocatalysts.

Combined-culture is a fermentation method which efficiently induces secondary metabolite production in Streptomyces by co-culturing them with mycolic acid-containing bacteria. As a result of combined-culture screening of our terrestrial Streptomyces collection using UV-HPLC, one of the tested strains, Streptomyces sp. TAKO-2, produced two known aromatic polyketides, julichrome Q ₆ (1) and julichrome Q _8·8 (2), when co-cultured with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. The structures of 1 and 2 were confirmed by spectroscopic analysis and literature data.

Abstract Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin—lyngbyatoxin A—and its biosynthetic intermediates by heterologous expression of the Streptomyces‐derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk.

Abstract Prenylation reactions play crucial roles in controlling the activities of biomolecules. Bacterial prenyltransferases, TleC from Streptomyces blastmyceticus and MpnD from Marinactinospora thermotolerans, catalyse the ‘reverse’ prenylation of (−)-indolactam V at the C-7 position of the indole ring with geranyl pyrophosphate or dimethylallyl pyrophosphate, to produce lyngbyatoxin or pendolmycin, respectively. Using in vitro analyses, here we show that both TleC and MpnD exhibit relaxed substrate specificities and accept various chain lengths (C₅–C₂₅) of the prenyl donors. Comparisons of the crystal structures and their ternary complexes with (−)-indolactam V and dimethylallyl S-thiophosphate revealed the intimate structural details of the enzyme-catalysed ‘reverse’ prenylation reactions and identified the active-site residues governing the selection of the substrates. Furthermore, structure-based enzyme engineering successfully altered the preference for the prenyl chain length of the substrates, as well as the regio- and stereo-selectivities of the prenylation reactions, to produce a series of unnatural novel indolactams.