Retrobisabolane A, a Novel Bisabolane-Derived Sesquiterpenoid Isolated from Deep-Sea-Derived Fungus Retroconis fusiformis MCCC 3A00792.

One novel bisabolane-derived sesquiterpenoid retrobisabolane A (1), featuring a methyl group location at the C-4 position instead of C-3 in the bisabolanes, and a known ester-substituted eremophilane-type sesquiterpenoid cryptosphaerolide (2), along with three known indole alkaloids (3-5) were discovered from the fermented cultures of a deep-sea-derived fungus Retroconis fusiformis MCCC 3A00792. The planar structure of new compound 1 was determined by extensive analysis of the NMR and HRESIMS spectra. The relative and absolute configurations of 1 were resolved by the coupling constant (J), calculation of ECD and NMR spectra, and the DP4+ probability analysis of the 1H and 13C NMR data. Interestingly, retrobisabolane A was the new subclass of bisabolanes bearing a methyl group linkage at C-4 instead of C-3 position. Three human cancer cell lines (Hela, AGS, and BIU-87) were subjected to evaluate the cytotoxic activities of compounds 1-5. As a result, compound 2 exhibited significant inhibitory activities against three cell lines with IC50 values ranging from 9.95 to 18.77 µM.

Malfilanol C, a new sesquiterpenoid isolated from the deep-sea-derived Aspergillus puniceus A2 fungus.

The chemical examination of the rice solid fermentation products of the deep-sea-derived fungus Aspergillus puniceus A2 resulted in the isolation of one new sesquiterpenoid malfilanol C (1), together with a rare analogue malfilanol B (2). The planar structure of 1 was resolved on the basis of the extensive analyses of the spectroscopic data (HRESIMS and NMR spectra), and its absolute configuration was assigned by quantum chemical calculation of the ECD data. Compound 1, featuring a bicyclo[5.4.0]-undecane nucleus skeleton, was the third example of this subclass sesquiterpenoids found from nature. Additionally, the subclass sesquiterpenoids 1 and 2 were discovered from marine-derived-fungi for the first time. All the isolated metabolites were evaluated the antibacterial activities against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. Compound 1 exhibited weak antibacterial activity against S. aureus ATCC 29213.

Phaeosphamides A and B, Cytotoxic Cyclodecadepsipeptides from the Mangrove-Derived Fungus Phaeosphaeriopsis sp. S296.

Chemical examination of the fermented broth of the mangrove-derived fungus Phaeosphaeriopsis sp. S296 resulted in the isolation of two new cyclodecadepsipeptides, namely phaeosphamides A (1) and B (2), as well as one known congener Sch 217048 (3). The structures of new metabolites, including absolute configurations, were established on the basis of extensive spectroscopic data analyses, chemical conversion, and Marfey's method. The 2-hydroxy-3-methylpentanoic acid (Hmp) moiety and pipecolic acid (Pip) unit in structures were rarely discovered in nature. Interestingly, compounds 1-3 are examples of peptides discovered from the fungal genus Phaeosphaeriopsis for the first time. All identified compounds were evaluated for their cytotoxicity against five tumor cell lines of AGS, BEL-7402, HepG2, B16, and BIU87. Among them, compound 1 showed inhibitory activities against these tumor cell lines with IC50 values ranging from 5.14 to 66.38 µM. A further mechanistic investigation found that 1 arrested AGS cells in the G2 phase and induced their apoptosis in a dose-dependent manner.

Ion Mobility-Derived Collision Cross-Sections Add Extra Capability in Distinguishing Isomers and Compounds with Similar Retention Times: The Case of Aphidicolanes.

The hyphenation of ion mobility spectrometry with high-resolution mass spectrometry has been widely used in the characterization of various metabolites. Nevertheless, such a powerful tool remains largely unexplored in natural products research, possibly mainly due to the lack of available compounds. To evaluate the ability of collision cross-sections (CCSs) in characterizing compounds, especially isomeric natural products, here we measured and compared the traveling-wave IMS-derived nitrogen CCS values for 75 marine-derived aphidicolanes. We established a CCS database for these compounds which contained 227 CCS values of different adducts. When comparing the CCS differences, 36 of 57 pairs (over 60%) of chromatographically neighboring compounds showed a ΔCCS over 2%. What is more, 64 of 104 isomeric pairs (over 60%) of aphidicolanes can be distinguished by their CCS values, and 13 of 18 pairs (over 70%) of chromatographically indistinguishable isomers can be differentiated from the mobility dimension. Our results strongly supported CCS as an important parameter with good orthogonality and complementarity with retention time. CCS is expected to play an important role in distinguishing complex and diverse marine natural products.

Phenolic Metabolites from a Deep-Sea-Derived Fungus Aspergillus puniceus A2 and Their Nrf2-Dependent Anti-Inflammatory Effects.

Four undescribed phenolic compounds, namely asperpropanols A-D (1-4), along with two known congeners 5 and 6, were isolated from Aspergillus puniceus A2, a deep-sea-derived fungus. The gross structures of the compounds were established by detailed analyses of the HRESIMS and NMR data, and their absolute configurations were resolved by modified Mosher's method and calculations of ECD data. Compounds 1-6 were found to have excellent anti-inflammatory effect on lipopolysaccharide (LPS)-induced RAW264.7 cells at 20 µM, evidenced by the reduced nitric oxide (NO), tumor necrosis factor α, and interleukin 6 production. Among them, 5 and 6 showed inhibitory effects on NO production comparable with the positive control (BAY11-7083 at 10 µM). Additionally, the LPS-induced mRNA expressions of inducible nitric oxide synthase and cyclooxygenase-2 were also decreased. Interestingly, mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was downregulated by LPS and recovered by 1-6, suggesting a vital role of Nrf2 in their effect. We further found that pharmacological inhibition of Nrf2 by ML385 largely abrogated the effects of 1-6 on RAW264.7 cells. Therefore, 1-6 may share a common anti-inflammatory mechanism via Nrf2 upregulation and activation.

Antiviral Cyclopropane Acids from Deep-Sea-Derived Fungus Aspergillus sydowii.

Four novel monocyclic cyclopropane acids, namely, sydocyclopropanes A-D (1-4), along with one known congener hamavellone B (5), were isolated from the Aspergillus sydowii MCCC 3A00324 fungus, which was isolated from the deep-sea sediment. The gross structures of novel compounds were established by detailed analyses of the spectroscopic data (HRESIMS and NMR spectra), and their absolute configurations were resolved on the basis of the quantum chemical calculations of ECD and NMR data, in association with DP4+ probability analyses. Sydocyclopropanes A-D, featuring the 1,1,2,3-tetrasubstituted cyclopropane nucleus with different lengthy alkyl side chains, were discovered in nature for the first time. All compounds exhibited antiviral activities against A/WSN/33 (H1N1), with IC50 values ranging from 26.7 to 77.2 µM, of which compound 1 exhibited a moderate inhibitory effect (IC50 = 26.7 µM).

Generation of a live attenuated influenza A vaccine by proteolysis targeting.

The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin-proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

Bisabolane Sesquiterpenes with Anti-Inflammatory Activities from the Endophytic Fungus Penicillium citrinum DF47.

Seven new bisabolane-type sesquiterpenes (1-7), namely penicibisabolanes A-G, together with eight known analogs (8-15) were obtained from the AcOEt extract of the millet fermentation broth of the endophytic fungus Penicillium citrinum DF47, which was isolated from the fresh root of Codonopsis pilosula (Franch.) Nannf. The gross structures of new metabolites were determined on the basis of the spectroscopic data (HR-ESI-MS, 1D and 2D NMR spectra), while their absolute configurations were resolved by comparison of the experimental and calculated ECD spectra, in association with specific rotation data. Compound 1 is a rare seco-trinor-bisabolane sesquiterpene found in nature, while 3 is the first example of phenolic bisabolanes bearing a methoxy group at C-1. All the isolates were evaluated their inhibitory effects against NO production in lipopolysaccharides (LPS) stimulated RAW264.7 cells. Among them, compounds 7 and 13 showed moderately anti-inflammatory effects with the inhibitory rate more than 50 % at the concentration of 20 µM.

Acremolin D, a new acremolin alkaloid from the deep-sea sediment derived Aspergillus sydowii fungus.

Chemical investigation of the deep-sea-derived fungus Aspergillus sydowii MCCC 3A00324 led to the isolation of one new acremolin type alkaloid (acremolin D, 1) and five known alkaloids (2‒6). The planar structure of 1 was established by the extensive analyses of the NMR and HRESIMS data, while its absolute configuration was assigned by the comparison of the experimental and calculated ECD data. Acremolin D (1) represented the second analogue of acremolin found in nature. All compounds were evaluated for their cytotoxic activities against six human cancer cell lines (A549, Hela-S3, MCF-7, HepG2, K562, and SF-268). As a result, compounds 1 and 2 exhibited a certain inhibitory effects against the proliferation of the A549, Hela-S3, HepG2, and K562 cell lines at the concentration of 20 µM.[Formula: see text].

Chemical epigenetic manipulation triggers the production of sesquiterpenes from the deep-sea derived Eutypella fungus.

Chemical epigenetic manipulation of a deep-sea-derived Eutypella sp. fungus by the co-treatment with a histonedeacetylase inhibitor (suberohydroxamic acid, SBHA) and a DNA methyltransferase inhibitor (5-azacytidine, 5-Aza), resulted in the activation of a sesquiterpene-related biosynthetic gene cluster. Chromatographic separation of the elicitor-treated cultures led the isolation of 21 sesquiterpenes, including 17 undescribed compounds, eutypeterpenes A-Q. Their structures were identified by the extensive analysis of the spectroscopic data, including the single-crystal X-ray diffraction, chemical conversion, and the calculated NMR and ECD data for configurational assignments. Eutypeterpene A is a first bergamotene-type sesquiterpene incorporated with a dioxolanone unit, and eutypeterpenes O-Q with a cyclopentane ring represent an undescribed subtype of sesquiterpenes. The bioassay results showed that most compounds exert inhibitory effects against the lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages, and eutypeterpene N is the most active. This study demonstrates that the epigenetic manipulation is an effective approach to trigger the production of cryptic metabolites from deep-sea derived fungus. The significant inhibition against LPS-induced NO production in vitro suggests eutypeterpenes to be potential for the development as anti-inflammatory agents.

Brevibacterium limosum sp. nov., Brevibacterium pigmenatum sp. nov., and Brevibacterium atlanticum sp. nov., three novel dye decolorizing actinobacteria isolated from ocean sediments.

During a study of the marine actinobacterial biodiversity, a large number of Brevibacterium strains were isolated. Of these, five that have relatively low 16S rRNA gene similarity (98.5-99.3%) with validly published Brevibacterium species, were chosen to determine taxonomic positions. On the basis of 16S rRNA gene sequence analysis and BOX-PCR fingerprinting, strains o2T, YB235T, and WO024T were selected as representative strains. Genomic analyses, including average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), clearly differentiated the three strains from each other and from their closest relatives, with values ranging from 82.8% to 91.5% for ANI and from 26.7% to 46.5% for dDDH that below the threshold for species delineation. Strains YB235T, WO024T, and o2T all exhibited strong and efficient decolorization activity in congo red (CR) dyes, moderate decolorization activity in toluidine blue (TB) dyes and poor decolorization in reactive blue (RB) dyes. Genes coding for peroxidases and laccases were identified and accounted for these strains' ability to effectively oxidize a variety of dyes with different chemical structures. Mining of the whole genome for secondary metabolite biosynthesis gene clusters revealed the presence of gene clusters encoding for bacteriocin, ectoine, NRPS, siderophore, T3PKS, terpene, and thiopeptide. Based on the phylogenetic, genotypic and phenotypic data, strains o2T, YB235T and WO024T clearly represent three novel taxa within the genus Brevibacterium, for which the names Brevibacterium limosum sp. nov. (type strain o2T = JCM 33844T = MCCC 1A09961T), Brevibacterium pigmenatum sp. nov. (type strain YB235T = JCM 33843T = MCCC 1A09842T) and Brevibacterium atlanticum sp. nov. (type strain WO024T = JCM 33846T = MCCC 1A16743T) are proposed.

Description of Microbacterium luteum sp. nov., Microbacterium cremeum sp. nov., and Microbacterium atlanticum sp. nov., three novel C50 carotenoid producing bacteria.

We have identified three Microbacterium strains, A18JL200T, NY27T, and WY121T, that produce C50 carotenoids. Taxonomy shows they represent three novel species. These strains shared < 98.5% 16S rRNA gene sequence identity with each other and were closely related to Microbacterium aquimaris JCM 15625T, Microbacterium yannicii JCM 18959T, Microbacterium ureisolvens CFH S00084T, and Microbacterium hibisci CCTCC AB 2016180T. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) showed differences among the three strains and from their closest relatives, with values ranging from 20.4% to 34.6% and 75.5% to 87.6%, respectively. These values are below the threshold for species discrimination. Both morphology and physiology also differed from those of phylogenetically related Microbacterium species, supporting that they are indeed novel species. These strains produce C50 carotenoids (mainly decaprenoxanthin). Among the three novel species, A18JL200T had the highest total yield in carotenoids (6.1 mg/L or 1.2 mg/g dry cell weight). Unusual dual isoprenoid biosynthetic pathways (methylerythritol phosphate and mevalonate pathways) were annotated for strain A18JL200T. In summary, we found strains of the genus Microbacterium that are potential producers of C50 carotenoids, but their genome has to be investigated further.

Epigenetic Manipulation to Trigger Production of Guaiane-Type Sesquiterpenes from a Marine-Derived Spiromastix sp. Fungus with Antineuroinflammatory Effects.

Epigenetic manipulation of a deep-sea sediment-derived Spiromastix sp. fungus using suberoylanilide hydroxamic acid (SAHA) induction resulted in the activation of a terpene-related biosynthetic gene cluster, and nine new guaiane-type sesquiterpenes, spiromaterpenes A-I (1-9), were isolated. Their structures were determined using various spectroscopic techniques, in association with the modified Mosher's method, computed electronic circular dichroism (ECD) spectra, and chemical conversion for configurational assignments. Compounds 4-6 exhibited significant effects against the NO production on lipopolysaccharide (LPS)-induced microglia cells BV2, and the preliminary SAR analyses demonstrated that a 2(R),11-diol unit is favorable. The most active 5 abolished LPS-induced NF-κB translocation from the cytosol to the nucleus in BV-2 microglial cells, accompanied by the marked reduction of the transcription levels of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α dose-dependently in both LPS-induced BV-2 and BV-2 cells, as well as the protein and mRNA levels of iNOS and COX-2. This study complements the gap in knowledge regarding the anti-neuroinflammatory activity of guaiane-type sesquiterpenoids at the cellular level and suggests that 5 is promising for further optimization as a multifunctional agent for antineuroinflammation.

Complete genome sequence of Streptomyces sp. HSG2 from rhizosphere soil of mangrove in Qingmei Gang, Sanya.

Streptomyces sp. HSG2 was isolated from rhizosphere soil of a mangrove forest sample at Qingmei Gang, Sanya. The complete genome sequence of the strain HSG2 was obtained using PacBio Sequel HGAP.4 and comprised of 5,282,528 base pairs with a 71.9 mol% G + C content, 4504 protein-coding genes, and 71 RNAs. An in-silico analysis confirmed that genes associated with polysaccharide hydrolyzation, hydrocarbon degradation, and aerobic denitrification were presented in the genome. We also identified 24 natural product biosynthetic gene clusters for secondary metabolites, including those for streptobactin and nystatin A1. The complete genome sequence indicated that Streptomyces sp. HSG2 will provide insight into the biosynthesis and regulatory mechanisms for its secondary metabolites, and propose a potential use in biotechnological and novel bioactive natural product biosynthetic applications.

Chlorinated metabolites with antibacterial activities from a deep-sea-derived Spiromastix fungus.

Chromatographic separation of the solid cultures of a deep-sea-derived Spiromastix fungus (MCCC 3A00308) resulted in the isolation of eight compounds. Their structures were identified on the basis of the spectroscopic data. Compounds 1-8 are classified as depsidone-type (1-4), isocoumarin-type (5 and 6), and benzothiazole-type (7 and 8), of which 1-7 are new compounds and 1-3 along with 5 and 6 are chlorinated. Compound 3 is characterized by trichlorination and shows potent activities against Gram-positive pathogenic bacteria including Staphylococcus aureus ATCC 25923, Bacillus thuringiensis ATCC 10792, and Bacillus subtilis CMCC 63501, with minimum inhibitory concentration (MIC) values ranging from 0.5 to 1.0 µg mL-1. This study extends the chemical diversity of chlorinated natural products from marine-derived fungi and provides a promising lead for the development of antibacterial agents.

Phenolic bisabolane and cuparene sesquiterpenoids with anti-inflammatory activities from the deep-sea-derived Aspergillus sydowii MCCC 3A00324 fungus.

Seventeen undescribed sesquiterpenoids including 14 phenolic bisabolanes, namely asperbisabolanes A-N (1-14), and 3 cuparenes (aspercuparenes A-C, 15-17), together with 10 known bisabolane analogues (18-27) were isolated from the EtOAc extract of fermented cultures of the deep sea sediment-derived fungus Aspergillus sydowii MCCC 3A00324. The new structures were established on the basis of extensive NMR and HRESIMS spectroscopic data analyses, while their absolute configurations were assigned by comparison of the experimental ECD spectra with those of the TDDFT-ECD calculated spectra or reported data in literature. Asperbisabolanes A (1) and B (2) are the first examples of bisabolane sesquiterpenoids featuring a 6/6/6 tricyclic nucleus. Compound 3 possessed a novel seco-bisabolane skeleton with a rare dioxolane ring moiety, while asperbisabolane K (11) represents the first case of bisabolanes bearing a rare methylsulfonyl group. All the isolated compounds (1-27) were evaluated their activities against NO secretion in LPS-activated BV-2 microglia cells. As a result, 6, 12, 16, and 25-27 exhibited the inhibition rate over 45% at a concentration of 10 µM. Moreover, 12 exerted the anti-inflammatory activity by inhibiting the NF-κB-activated pathway in dose-dependent manner.

New Monoterpenoids and Polyketides from the Deep-Sea Sediment-Derived Fungus Aspergillus sydowii MCCC 3A00324.

Chemical study of the secondary metabolites of a deep-sea-derived fungus Aspergillus sydowii MCCC 3A00324 led to the isolation of eleven compounds (1-11), including one novel (1) and one new (2) osmane-related monoterpenoids and two undescribed polyketides (3 and 4). The structures of the metabolites were determined by comprehensive analyses of the NMR and HRESIMS spectra, in association with quantum chemical calculations of the 13C NMR, ECD, and specific rotation data for the configurational assignment. Compound 1 possessed a novel monoterpenoid skeleton, biogenetically probably derived from the osmane-type monoperpenoid after the cyclopentane ring cleavage and oxidation reactions. Additionally, compound 3 was the first example of the α-pyrone derivatives bearing two phenyl units at C-3 and C-5, respectively. The anti-inflammatory activities of 1-11 were tested. As a result, compound 6 showed potent inhibitory nitric oxide production in lipopolysaccharide (LPS)-activated BV-2 microglia cells with an inhibition rate of 94.4% at the concentration of 10 µM. In addition, a plausible biosynthetic pathway for 1 and 2 was also proposed.

Synthesis and Biological Evaluation of Analogues of Butyrolactone I as PTP1B Inhibitors.

In recent years, a large number of pharmacologically active compounds containing a butenolide functional group have been isolated from secondary metabolites of marine microorganisms. Butyrolactone I was found to be produced by Aspergillus terreus isolated from several marine-derived samples. The hypoglycemic activity of butyrolactone I has aroused our great interest. In this study, we synthesized six racemic butenolide derivatives (namely BL-1-BL-6) by modifying the C-4 side chain of butyrolactone I. Among them, BL-3 and BL-5 improved the insulin resistance of HepG2 cells and did not affect the proliferation of RIN-m5f cell line, which indicated the efficacy and safety of BL-3 and BL-5. Furthermore, BL-3, BL-4, BL-5, and BL-6 displayed a significant protein tyrosine phosphatase 1B (PTP1B) inhibitory effect, while the enantiomers of BL-3 displayed different 50% percentage inhibition concentration (IC50) values against PTP1B. The results of molecular docking simulation of the BLs and PTP1B explained the differences of biological consequences observed between the enantiomers of BL-3, which supported BLs as PTP1B inhibitors, and also indicated that the chirality of C-4 might influence the inhibitory effect of the BLs. Our findings provide a novel strategy for the development of butyrolactone derivatives as potential PTP1B inhibitors for the treatment of type 2 diabetes mellitus.

Brevibacterium profundi sp. nov., isolated from deep-sea sediment of the Western Pacific Ocean.

A new Gram-stain-positive, aerobic, non-motile and rod-shaped actinobacterium, designated O1T, was isolated from a deep-sea sediment of the Western Pacific Ocean. Strain O1T showed optimal growth at 30 °C, between pH 6.0 and 8.0, and in the presence of 1-5 % (w/v) NaCl. The predominant menaquinone was MK-8 (H2), and anteiso-C15 : 0 and anteiso-C17 : 0 were the major fatty acids. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and one unknown glycolipid. The DNA G+C content of strain O1T was 64.9 mol% and the genome size was 4.17 Mb. Based on a similarity search and phylogenetic analysis of the 16S rRNA gene sequence, strain O1T belonged to the genus Brevibacterium. The values of average nucleotide identity and in silico DNA-DNA hybridization between strain O1T and its close relatives were well below the thresholds used for the delineation of a new species. On the basis of the morphological and chemotaxonomic characteristics, as well as the genotypic data, it is proposed that strain O1T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium profundi sp. nov. is proposed. The type strain is O1T (=JCM 33845T=MCCC 1A16744T).