Isolation and Structure Elucidation of JBIR-157, a Skeletally Novel Aromatic Polyketide Produced by the Heterologous Expression of a Cryptic Gene Cluster.

Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.

Capability of a large bacterial artificial chromosome clone harboring multiple biosynthetic gene clusters for the production of diverse compounds.

The biosynthetic gene clusters (BGCs) for the macrocyclic lactone-based polyketide compounds are extremely large-sized because the polyketide synthases that generate the polyketide chains of the basic backbone are of very high molecular weight. In developing a heterologous expression system for the large BGCs amenable to the production of such natural products, we selected concanamycin as an appropriate target. We obtained a bacterial artificial chromosome (BAC) clone with a 211-kb insert harboring the entire BGC responsible for the biosynthesis of concanamycin. Heterologous expression of this clone in a host strain, Streptomyces avermitilis SUKA32, permitted the production of concanamycin, as well as that of two additional aromatic polyketides. Structural elucidation identified these additional products as ent-gephyromycin and a novel compound that was designated JBIR-157. We describe herein sequencing and expression studies performed on these BGCs, demonstrating the utility of large BAC clones for the heterologous expression of cryptic or near-silent loci.

New phenylspirodrimane metabolites MBJ-0030, MBJ-0031, and MBJ-0032 isolated from the soil fungal strain Stachybotrys sp. f23793.

Chemical screening of culture medium from the soil fungus Stachybotrys sp. resulted in the isolation of the three new phenylspirodrimanes MBJ-0030 (1), MBJ-0031 (2) and MBJ-0032 (3). Their structures were determined by detailed analysis of spectroscopic data. The absolute configurations of 1-3 were determined by modified Mosher's and Marfey's methods. In addition, cytotoxic and antimicrobial evaluations of the compounds were conducted.

Synthesis and biological evaluation of thielocin B1 analogues as protein-protein interaction inhibitors of PAC3 homodimer.

The synthesis and biological evaluation of thielocin B1 analogues have been demonstrated. Fourteen analogues modified in the central core and terminal carboxylic acid moiety were concisely synthesized by simple esterification or etherification reaction. The evaluation of synthetic analogues as inhibitors of proteasome assembling chaperone (PAC) complexes (the PAC3 homodimer and PAC1/PAC2) revealed that the natural product-like bending structure and terminal carboxylic acid groups were crucial for its biological activity. Moreover, SAR and in silico docking studies indicated that all methyl groups on the diphenyl ether moiety of thielocin B1 contribute to the potent and selective inhibition of the PAC3 homodimer via hydrophobic interactions.

Unprecedented Cyclization Catalyzed by a Cytochrome P450 in Benzastatin Biosynthesis.

Benzastatins have unique structures probably derived from geranylated p-aminobenzoic acids. The indoline and tetrahydroquinoline scaffolds are presumably formed by cyclization of the geranyl moiety, but the cyclization mechanism was unknown. We studied the benzastatin biosynthetic gene cluster of Streptomyces sp. RI18; functions of the six enzymes encoded by it were analyzed by gene disruption in a heterologous host and in vitro enzyme assays. We propose the biosynthetic pathway for benzastatins in which a cytochrome P450 (BezE) is responsible for the cyclization of geranylated p-acetoxyaminobenzoic acids; BezE catalyzes elimination of acetic acid to form an iron nitrenoid, nitrene transfer to form an aziridine ring, and nucleophilic addition of hydroxide ion to C-10 and chloride ion to C-9 to generate the indoline and tetrahydroquinoline scaffolds, respectively. Discovery of this enzyme, which should be termed cytochrome P450 nitrene transferase, provides an important insight into the functional diversity of cytochrome P450.

Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster.

During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP (ribosomally synthesized and post-translationally modified peptides), we discovered a novel cryptic biosynthetic gene cluster. During efforts to express this biosynthetic gene using heterologous expression of this biosynthetic gene cluster, a novel compound designated as neothioviridamide was produced. We report herein the cloning and heterologous expression of the neothioviridamide biosynthetic gene cluster and the isolation, structure determination, and cytotoxic activity of neothioviridamide.

Identification of a gene cluster for telomestatin biosynthesis and heterologous expression using a specific promoter in a clean host.

Telomestatin, a strong telomerase inhibitor with G-quadruplex stabilizing activity, is a potential therapeutic agent for treating cancers. Difficulties in isolating telomestatin from microbial cultures and in chemical synthesis are bottlenecks impeding the wider use. Therefore, improvement in telomestatin production and structural diversification are required for further utilization and application. Here, we discovered the gene cluster responsible for telomestatin biosynthesis, and achieved production of telomestatin by heterologous expression of this cluster in the engineered Streptomyces avermitilis SUKA strain. Utilization of an optimal promoter was essential for successful production. Gene disruption studies revealed that the tlsB, tlsC, and tlsO-T genes play key roles in telomestatin biosynthesis. Moreover, exchanging TlsC core peptide sequences resulted in the production of novel telomestatin derivatives. This study sheds light on the expansion of chemical diversity of natural peptide products for drug development.

Synthesis of Spiromamakone A Benzo Analogues via Double Oxa-Michael Addition of 1,8-Dihydroxynaphthalene.

Two benzo analogues of cytotoxic spiromamakone A, comprising carbon atoms with the same oxidation state and unsaturation degree as those of the natural products, are synthesized and biologically evaluated. Substitution of α,α'-dioxoketene dithioacetals, derived from 1,3-cyclopentanediones with protected (2-formylphenyl)magnesium bromide and 1,8-dihydroxynaphthalene, followed by deprotection, generated these analogues via an intramolecular aldol reaction. The cytotoxicity of benzo analogues and synthetic intermediates against cervical carcinoma HeLa cells shows the necessity of the 4-cyclopentene-1,3-dione moiety for biological activity.

Involvement of the Baeyer-Villiger Monooxygenase IfnQ in the Biosynthesis of Isofuranonaphthoquinone Scaffold of JBIR-76 and -77.

JBIR-76 and -77 are isofuranonaphthoquinones (IFNQs) isolated from Streptomyces sp. RI-77. Draft genome sequencing and gene disruption analysis of Streptomyces sp. RI-77 showed that a type II polyketide synthase (PKS) gene cluster (ifn cluster) was responsible for the biosynthesis of JBIR-76 and -77. It was envisaged that an octaketide intermediate (C16 ) could be synthesized by the minimal PKS (IfnANO) and that formation of the IFNQ scaffold (C13 ) would therefore require a C-C bond cleavage reaction. An ifnQ disruptant accumulated some shunt products (C15 ), which were presumably produced by spontaneous cyclization of the decarboxylated octaketide intermediate. Recombinant IfnQ catalyzed the Baeyer-Villiger oxidation of 1-(2-naphthyl)acetone, an analogue of the bicyclic octaketide intermediate. Based on these results, we propose a pathway for the biosynthesis of JBIR-76 and -77, involving IfnQ-catalyzed C-C bond cleavage as a key step in the formation of the IFNQ scaffold.

Unveiling the Biosynthetic Pathway of the Ribosomally Synthesized and Post-translationally Modified Peptide Ustiloxin B in Filamentous Fungi.

The biosynthetic machinery of the first fungal ribosomally synthesized and post-translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side-chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5'-phosphate (PLP)-dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.

tRNA-Dependent Aminoacylation of an Amino Sugar Intermediate in the Biosynthesis of a Streptothricin-Related Antibiotic.

UNLABELLED: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a ß-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics. IMPORTANCE: The antibiotic streptothricin (ST) possesses an amino sugar bound to an l-ß-lysine (ß-Lys) side chain via a peptide bond formed by a nonribosomal peptide synthetase (NRPS). BD-12, an analogue of ST, carries a glycine-derived side chain rather than ß-Lys, and here, we describe the BD-12-biosynthetic gene cluster from Streptomyces luteocolor NBRC13826, which contains the orf11 gene encoding a novel tRNA-dependent peptide bond-forming enzyme. The unique Fem-like enzyme (Orf11) accepts the amino sugar as a substrate and mediates the peptide formation between the amino sugar intermediate and glycine. Our studies demonstrate that the structural diversity of the side chains of ST-related compounds in nature is generated via two distinct peptide bond-forming mechanisms.

Class of cyclic ribosomal peptide synthetic genes in filamentous fungi.

Ustiloxins were found recently to be the first example of cyclic peptidyl secondary metabolites that are ribosomally synthesized in filamentous fungi. In this work, two function-unknown genes (ustYa/ustYb) in the gene cluster for ustiloxins from Aspergillus flavus were found experimentally to be involved in cyclization of the peptide. Their homologous genes are observed mainly in filamentous fungi and mushrooms. They have two "HXXHC" motifs that might form active sites. Computational genome analyses showed that these genes are frequently located near candidate genes for ribosomal peptide precursors, which have signal peptides at the N-termini and repeated sequences with core peptides for the cyclic portions, in the genomes of filamentous fungi, particularly Aspergilli, as observed in the ustiloxin gene cluster. Based on the combination of the ustYa/ustYb homologous genes and the nearby ribosomal peptide precursor candidate genes, 94 ribosomal peptide precursor candidates that were identified computationally from Aspergilli genome sequences were classified into more than 40 types including a wide variety of core peptide sequences. A set of the predicted ribosomal peptide biosynthetic genes was experimentally verified to synthesize a new cyclic peptide compound, designated as asperipin-2a, which comprises the amino acid sequence in the corresponding precursor gene, distinct from the ustiloxin precursors.

Foxo3a Inhibitors of Microbial Origin, JBIR-141 and JBIR-142.

JBIR-141 (1) and JBIR-142 (2) were discovered as potent Foxo3a inhibitors that consist of three quite unique substructures, a 1-((dimethylamino)ethyl)-5-methyl-4,5-dihydrooxazole-4-carboxylic acid that is originated from Ala-Thr amino acid residues, a 3-acetoxy-4-amino-7-(hydroxy(nitroso)amino)-2,2-dimethylheptanoic acid, and an α-acyl tetramic acid fused with a 2-methylpropan-1-ol moiety. Their structures involving absolute configurations were determined by spectroscopic data, chemical degradation, anisotropy methods, and LC-MS analyses of diastereomeric derivatives. Compounds 1 and 2 exhibited specific inhibition against Foxo3a transcriptional activity with IC50 values of 23.1 and 166.2 nM, respectively.

Identification and Characterization of the Streptazone†E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08.

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2-like superfamily are responsible for C-C bond formation coupled with epoxide ring opening to give the five-membered ring of streptazone E.

Asymmetric Total Synthesis of ent-Pyripyropene†A.

An asymmetric total synthesis of ent-pyripyropene A was achieved by a convergent synthetic route. We used our originally developed Ti(III) -catalyzed radical cyclization to construct an AB-ring portion that consisted of a trans-decalin skeleton with five contiguous stereogenic centers. The coupling between the AB-ring and the DE-ring portions, and a subsequent C-ring cyclization, led to the total synthesis of ent-pyripyropene A. An evaluation of the insecticidal activity of ent-pyripyropene A against two aphid species revealed that ent-pyripyropene A was 35-175 times less active than naturally occurring pyripyropene A. This result indicated that the biological target of pyripyropene A recognizes the absolute configuration of pyripyropene A.

Saccharothriolides A-C, novel phenyl-substituted 10-membered macrolides isolated from a rare actinomycete Saccharothrix sp.

Three new 10-membered macrolides, saccharothriolides A-C (1-3), were discovered from a rare actinomycete Saccharothrix sp. A1506. All of the sp(3) carbons in the 10-membered ring had chirality, which was determined by extensive spectroscopic analysis and TDDFT-calculation of ECD spectra. Saccharothriolide B (2) exhibited cytotoxicity against human tumor cell lines HeLa and HT1080.

Stereochemical assignment of the protein-protein interaction inhibitor JBIR-22 by total synthesis.

Recent reports have highlighted the biological activity associated with a subfamily of the tetramic acid class of natural products. Despite the fact that members of this subfamily act as protein-protein interaction inhibitors that are of relevance to proteasome assembly, no synthetic work has been reported. This may be due to the fact that this subfamily contains an unnatural 4,4-disubstitued glutamic acid, the synthesis of which provides a key challenge. A highly stereoselective route to a masked form of this unnatural amino acid now enabled the synthesis of two of the possible diastereomers of JBIR-22 and allowed the assignment of its relative and absolute stereochemistry.