Expression of Syo_1.56 SARP Regulator Unveils Potent Elasnin Derivatives with Antibacterial Activity.

Actinomycetes are prolific producers of natural products, particularly antibiotics. However, a significant proportion of its biosynthetic gene clusters (BGCs) remain silent under typical laboratory conditions. This limits the effectiveness of conventional isolation methods for the discovery of novel natural products. Genetic interventions targeting the activation of silent gene clusters are necessary to address this challenge. Streptomyces antibiotic regulatory proteins (SARPs) act as cluster-specific activators and can be used to target silent BGCs for the discovery of new antibiotics. In this study, the expression of a previously uncharacterized SARP protein, Syo_1.56, in Streptomyces sp. RK18-A0406 significantly enhanced the production of known antimycins and led to the discovery of 12 elasnins (1-12), 10 of which were novel. The absolute stereochemistry of elasnin A1 was assigned for the first time to be 6S. Unexpectedly, Syo_1.56 seems to function as a pleiotropic rather than cluster-specific SARP regulator, with the capability of co-regulating two distinct biosynthetic pathways, simultaneously. All isolated elasnins were active against wild-type and methicillin-resistant Staphylococcus aureus with IC50 values of 0.5-20 µg/mL, some of which (elasnins A1, B2, and C1 and proelasnins A1, and C1) demonstrated moderate to strong antimalarial activities against Plasmodium falciparum 3D7. Elasnins A1, B3, and C1 also showed in vitro inhibition of the metallo-ß-lactamase responsible for the development of highly antibiotic-resistant bacterial strains.

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Production of Kinanthraquinone D with Antimalarial Activity by Heterologous Gene Expression and Biotransformation in Streptomyces lividans TK23.

Two new compounds, kinanthraquinone C (1) and kinanthraquinone D (2), were isolated along with two known compounds, kinanthraquinone (3) and kinanthraquinone B (4), produced by the heterologous expression of the kiq biosynthetic gene cluster and its pathway-specific regulator, kiqA, in Streptomyces lividans TK23. The chemical structures of compounds 1 and 2 were determined using mass spectrometry and nuclear magnetic resonance analyses. To examine a biosynthetic pathway of compounds 1 and 2, incubation experiments were conducted using S. lividans TK23 to supply the compounds 3 and 4. These experiments indicated that compounds 3 and 4 were converted to compounds 2 and 1, respectively, by the endogenous enzymes of S. lividans TK23. Compounds 2, 3, and 4 had antimalarial activities at half-maximal inhibitory concentration values of 0.91, 1.2, and 15 µM, respectively, without cytotoxicity up to 30 µM.

Linkage-Editing Pseudo-Glycans: A Reductive α-Fluorovinyl-C-Glycosylation Strategy to Create Glycan Analogs with Altered Biological Activities.

The acetal (O-glycoside) bonds of glycans and glycoconjugates are chemically and biologically vulnerable, and therefore C-glycosides are of interest as more stable analogs. We hypothesized that, if the O-glycoside linkage plays a vital role in glycan function, the biological activities of C-glycoside analogs would vary depending on their substituents. Based on this idea, we adopted a "linkage-editing strategy" for the creation of glycan analogs (pseudo-glycans). We designed three types of pseudo-glycans with CH2 and CHF linkages, which resemble the O-glycoside linkage in terms of bond lengths, angles, and bulkiness, and synthesized them efficiently by means of fluorovinyl C-glycosylation and selective hydrogenation reactions. Application of this strategy to isomaltose (IM), an inducer of amylase expression, and α-GalCer, which activates iNKT cells, resulted in the discovery of CH2-IM, which shows increased amylase production ability, and CHF-α-GalCer, which shows activity opposite that of native α-GalCer, serving as an antagonist of iNKT cells.

Total Synthesis of Clostrienose.

This paper considers the total synthesis of a cellular differentiation regulator of Clostridium acetobutylicum, clostrienose, which is a unique fatty-acid glycosyl ester consisting of clostrienoic acid, (3R,5E,8E,10E)-3-hydroxy-tetradeca-5,8,10-trienoic acid and α-d-galactofuranosyl-(1 → 2)-α-l-rhamnose. The key features of our synthesis include stereoselective construction of a skipped-triene system in clostrienoic acid and its esterification with a disaccharide residue. The partially protected clostrienoic acid employed for the coupling also served for the preparation of l-rhamnosyl clostrienoate, thus leading to confirmation of the proposed structure unambiguously.

Identification of a New Pyrrolyl Pyridoindole Alkaloid, Melpyrrole, and Flazin from Honey and Their Cough-Suppressing Effect in Guinea Pigs.

The cough-suppressing effect of honey was demonstrated for the first time using a guinea pig model whereby cough was induced by citric acid and capsaicin, and a new pyrrolyl pyridoindole, 1-(5-(hydroxymethyl)-1H-pyrrol-2-yl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1), named melpyrrole, and flazin (2) were identified as the active principle components. The structures of 1 and 2 were estimated using a combination approach of an activity-guided survey and LC-MS/MS multivariate analysis and were finally established by total synthesis of 1 and comparison with an authentic standard for 2. Both compounds showed antitussive activity comparable to that of dextromethorphan in guinea pigs. Their antitussive effects were unaffected by an opioid antagonist and reversed by a nitric oxide (NO) synthase inhibitor, indicating that these natural products do not act directly on opiate receptors but through the NO signaling pathway.

Wakodecaline C, new tetrahydrofuran-fused decalin metabolite isolated from fungus Pyrenochaetopsis sp. RK10-F058.

A new decalin-containing secondary metabolite, wakodecaline C, was isolated from a fungus Pyrenochaetopsis sp. RK10-F058 by screening structurally interesting metabolites based on LC/MS profiling. The structure including the absolute configuration was determined by a combination of spectroscopic methods including NMR and mass spectrometry, chemical reaction, and calculation of ECD spectra. Wakodecaline C has unique structural features containing a tetrahydrofuran-fused decalin skeleton and tetramic acid moiety, which are connected through a double bond. The compound showed moderate cytotoxicity against HL-60 cells and antimalarial activity against the Plasmodium falciparum 3D7 strain.

Ganglioside GM3 Analogues Containing Monofluoromethylene-Linked Sialoside: Synthesis, Stereochemical Effects, Conformational Behavior, and Biological Activities.

Glycoconjugates are an important class of biomolecules that regulate numerous biological events in cells. However, these complex, medium-size molecules are metabolically unstable, which hampers detailed investigations of their functions as well as their potential application as pharmaceuticals. Here we report sialidase-resistant analogues of ganglioside GM3 containing a monofluoromethylene linkage instead of the native O-sialoside linkage. Stereoselective synthesis of CHF-linked disaccharides and kinetically controlled Au(I)-catalyzed glycosylation efficiently furnished both stereoisomers of CHF-linked as well as CF 2 - and CH 2 -linked GM3 analogues. Like native GM3, the C-linked GM3 analogues inhibited the autophosphorylation of epidermal growth factor (EGF) receptor induced by EGF in vitro. Assay of the proliferation-enhancing activity toward Had-1 cells together with NMR-based conformational analysis showed that the (S)-CHF-linked GM3 analogue with exo-gauche conformation is the most potent of the synthesized compounds. Our findings suggest that exo-anomeric conformation is important for the biological functions of GM3.

6,9-Dihydroxytetrangulol, a novel angucyclinone antibiotic accumulated in kiqO gene disruptant in the biosynthesis of kinanthraquinone.

A novel angucyclinone, 6,9-dihydroxytetrangulol, was isolated from Streptomyces lividans TK23 transformed with a kinanthraquinone biosynthetic gene cluster in which the kiqO gene was disrupted. The chemical structure was elucidated by spectroscopic analyses. It showed significant antibacterial activities with an IC50 value of 1.9 µM against Staphylococcus aureus and moderate anticancer activities against HL-60 cells.

Identification and structural characterisation of a catecholate-type siderophore produced by Stenotrophomonas maltophilia K279a.

Siderophores are produced by several bacteria that utilise iron in various environments. Elucidating the structure of a specific siderophore may have valuable applications in drug development. Stenotrophomonas maltophilia, a Gram-negative bacterium that inhabits a wide range of environments and can cause pneumonia, produces siderophores. However, the structure was unknown, and therefore, in this study, we aimed to elucidate it. We purified siderophores from cultures of S. maltophilia K279a using preparative reversed-phase HPLC. The structure was analysed through LC-MS and 1H and 13C NMR. The results demonstrated that S. maltophilia K279a produces 2,3-dihydroxybenzoylserine (DHBS), a monomer unit of enterobactin. We suggested the uptake of Iron(III) by the DHBS complex. DHBS production by S. maltophilia K279a could be attributed to an incomplete enterobactin pathway. Drugs targeting DHBS synthesis could prevent S. maltophilia infection.

Dynamics in Catalytic Asymmetric Diastereoconvergent (3 + 2) Cycloadditions with Isomerizable Nitrones and α-Keto Ester Enolates.

Reaction design in asymmetric catalysis has traditionally been predicated on a structurally robust scaffold in both substrates and catalysts, to reduce the number of possible diastereomeric transition states. Herein, we present the stereochemical dynamics in the Ni(II)-catalyzed diastereoconvergent (3 + 2) cycloadditions of isomerizable nitrile-conjugated nitrones with α-keto ester enolates. Even in the presence of multiple equilibrating species, the catalytic protocol displays a wide substrate scope to access a range of CN-containing building blocks bearing adjacent stereocenters with high enantio- and diastereoselectivities. Our computational investigations suggest that the enantioselectivity is governed in the deprotonation process to form (Z)-Ni-enolates, while the unique syn addition is mainly controlled by weak noncovalent bonding interactions between the nitrone and ligand.

Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator.

Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

Total Synthesis of the Proposed Structure for Chaunopyran A and Its Absolute Configuration.

This paper describes a seven-step synthesis of the proposed structure for chaunopyran A produced by cocultivation of a Chaunopycnis sp. and Trichoderma hamatum. This synthesis included a coupling of a diene sulfone and a tetrahydropyranyl aldehyde as a key step. The sign of the specific rotation value of the synthetic sample was opposite that of the natural product, suggesting that the absolute configuration of the natural product should be revised.

Structural Studies on Stilbene Oligomers Isolated from the Seeds of Melinjo (Gnetum gnemon L.).

This paper describes the isolation and structural determination of a new stilbene dimer, named 7a-epi-gnetin C, from melinjo (Gnetum gnemon L.) seed extract. The relative structure was elucidated based on NMR spectroscopic evidence, while the absolute configuration was assigned by a combination of NMR and electronic circular dichroism spectroscopic analysis and chemical conversion. 7a-epi-Gnetin C was evaluated as an antioxidant and was shown to have a comparable activity to the known stilbene oligomers. In addition, the structural revision of gnetin L, a known stilbene dimer, was also discussed.

Kolavenic acid analog restores growth in HSET-overproducing fission yeast cells and multipolar mitosis in MDA-MB-231 human cells.

Although cancer cells often harbor supernumerary centrosomes, they form pseudo-bipolar spindles via centrosome clustering, instead of lethal multipolar spindles, and thus avoid cell death. Kinesin-14 HSET/KIFC1 is a crucial protein involved in centrosome clustering. Accordingly, a compound that targets HSET could potentially inhibit cancer cell proliferation in a targeted manner. Here, we report three natural compounds derived from Solidago altissima that restored the growth of fission yeast cells exhibiting lethal HSET overproduction (positive screening), namely solidagonic acid (SA) (1), kolavenic acid analog (KAA: a stereo isomer at C-9 and C-10 of 6ß-tigloyloxykolavenic acid) (2), and kolavenic acid (KA) (3). All three compounds suppressed fission yeast cell death and enabled reversion of the mitotic spindles from a monopolar to bipolar morphology. Compound 2, which exerted the strongest activity against HSET-overproducing yeast cells, also inhibited centrosome clustering in MDA-MB-231 human breast adenocarcinoma cells, which contained large numbers of supernumerary centrosomes. These natural compounds may be useful as bioprobes in studies of HSET function. Moreover, compound 2 is a prime contender in the development of novel agents for cancer treatment.

Isolation, Identification, and Synthesis of a New Prenylated Cinnamic Acid Derivative from Brazilian Green Propolis and Simultaneous Quantification of Bioactive Components by LC-MS/MS.

A new cinnamic acid derivative, (E)-3-[4-hydroxy-3-((E)-3-formyl-2-butenyl)phenyl]-2- propenoic acid (20) has been isolated from the ethanol extract of Brazilian green propolis along with three known cinnamic acid derivatives, 3,4-dihydroxy-5-prenyl-(E)-cinnamic acid (4), capillartemisin A (6), and 2,2-dimethylchromene-6-(E)-propenoic acid (8), and a flavonoid, dihydrokaempferide (16) by liquid-liquid participation, a series of column chromatography and preparative HPLC. Their structures have been determined by spectroscopic analyses and chemical synthesis of compound 20. The simultaneous quantification of 20 constituents, including 10 cinnamic acid derivatives, 7 flavonoids, and 3 caffeoylquinic acid derivatives, has also been developed and validated using LC-MS/MS. The new compound 20 was shown to activate PPAR α but not PPAR ß or γ.

Synthesis of Belt- and Möbius-Shaped Cycloparaphenylenes by Rhodium-Catalyzed Alkyne Cyclotrimerization.

A belt-shaped [8]cycloparaphenylene (CPP) and an enantioenriched Möbius-shaped [10]CPP have been synthesized by high-yielding rhodium-catalyzed intramolecular cyclotrimerizations of a cyclic dodecayne and a pentadecayne, respectively. This Möbius-shaped [10]CPP possesses stable chirality and isolated with high enantiomeric purity. It is evident from the reaction Gibbs energy calculation that the above irreversible cyclotrimerizations are highly exothermic; therefore establishing that the intramolecular alkyne cyclotrimerization is a powerful route to strained cyclic molecular strips.

Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire ß-Oxidation Cycle of the Cleaved B Ring.

Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by ß-oxidation. In this study, we revealed that 7ß,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of ß-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7ß,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters.IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire ß-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

A novel Ca2+-signal transduction inhibitor, kujigamberol C, isolated from Kuji amber.

A novel labdane type diterpenoid, 15-nor-8-labden-13-ol, named kujigamberol C, was isolated from Kuji amber using a modified isolation method to increase the yield of biologically active compounds. The structure was determined using HREIMS, 1D and 2D NMR. Kujigamberol C showed growth-restoring activity against mutant yeast via Ca2+-signal transduction.