Genomic Insights and Synthetic Biology Applications of Marine Actinomycete Streptomyces griseoincarnatus HNS054.

The marine bacterium Streptomyces sp. HNS054 shows promise as a platform for producing natural products. Isolated from a marine sponge, HNS054 possesses several desirable traits for bioengineering: rapid growth, salt tolerance, and compatibility with genetic tools. Its genome contains 21 potential biosynthetic gene clusters, offering a rich source of natural products. We successfully engineered HNS054 to increase the production of aborycin and actinorhodin by 4.5-fold and 1.2-fold, respectively, compared to S. coelicolor M1346 counterparts. With its unique features and amenability to genetic manipulation, HNS054 emerges as a promising candidate for developing novel marine-derived drugs and other valuable compounds.

Jejucarbosides B-E, Chlorinated Cycloaromatized Enediynes, from a Marine Streptomyces sp.

Four new chlorinated cycloaromatized enediyne compounds, jejucarbosides B-E (1-4), were discovered together with previously-identified jejucarboside A from a marine actinomycete strain. Compounds 1-4 were identified as new chlorinated cyclopenta[a]indene glycosides based on 1D and 2D nuclear magnetic resonance, high-resolution mass spectrometry, and circular dichroism (CD) spectra. Jejucarbosides B and E bear a carbonate functional group whereas jejucarbosides C and D are variants possessing 1,2-diol by losing the carbonate functionality. It is proposed that the production of 1-4 occurs via Bergman cycloaromatization capturing Cl- and H+ in the alternative positions of a p-benzyne intermediate derived from a 9-membered enediyne core. Jejucarboside E (4) displayed significant cytotoxicity against human cancer cell lines including SNU-638, SK-HEP-1, A549, HCT116, and MDA-MB-231, with IC50 values of 0.31, 0.40, 0.25, 0.29, and 0.48 µM, respectively, while jejucarbosides B-D (1-3) showed moderate or no cytotoxic effects.

Spiractinospora alimapuensis gen. nov., sp. nov., isolated from marine sediment of Valparaíso Bay (Chile) and proposal for reclassification of two species of the genus Nocardiopsis.

An alkaliphilic actinobacterium, designated VN6-2T, was isolated from marine sediment collected from Valparaíso Bay, Chile. Strain VN6-2T formed yellowish-white branched substrate mycelium without fragmentation. Aerial mycelium was well developed, forming wavy or spiral spore chains. Strain VN6-2T exhibited a 16S rRNA gene sequence similarity of 93.9 % to Salinactinospora qingdaonensis CXB832T, 93.7 % to Murinocardiopsis flavida 14-Be-013T, and 93.7 % to Lipingzhangella halophila 14-Be-013T. Genome sequencing revealed a genome size of 5.9 Mb and an in silico G+C content of 69.3 mol%. Both of the phylogenetic analyses based on 16S rRNA gene sequences and the up-to-date bacterial core gene sequences revealed that strain VN6-2T formed a distinct monophyletic clade within the family Nocardiopsaceae. Chemotaxonomic assessment of strain VN6-2T showed that the major fatty acids were iso-C16 : 0, anteiso-C17 : 0 and 10-methyl-C18 : 0, and the predominant respiratory quinones were MK-9, MK-9(H2) and MK-9(H4). Whole-cell hydrolysates contained meso-diaminopimelic acid as the cell-wall diamino acid, and ribose and xylose as the diagnostic sugars. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, aminophospholipids, glycolipid and phospholipid. Based on the results of this polyphasic study, a novel genus, Spiractinospora gen. nov., is proposed within the family Nocardiopsaceae and the type species Spiractinospora alimapuensis gen. nov., sp. nov. The type strain is VN6-2T (CECT 30026T, CCUG 66258T). On the basis of the phylogenetic results herein, we also propose that Nocardiopsis arvandica and Nocardiopsis litoralis are later heterotypic synonyms of Nocardiopsis sinuspersici and Nocardiopsis kunsanensis, respectively, for which emended descriptions are given.

Anti-Tyrosinase Compounds from the Deep-Sea-Derived Actinomycete Georgenia sp. 40DY180.

With the aim of finding new marine-derived skincare promoters, an assay-guided approach was employed to discover tyrosinase-modulating compounds from marine actinomycete. Here we describe a new 2,5-piperazinedione, named georgenione A (1), together with two previously described compounds, 5-(4'-hydroxybenzyl)hydantoin (2) and cyclo(Trp-Gly) (3), produced by actinomycete Georgenia sp. 40DY180, isolated from deep-sea sediments collected in the Pacific Ocean. Their structures were elucidated by a combination of spectroscopic analyses including 1D and 2D NMR and high-resolution mass spectrometric data. 5-(4'-hydroxybenzyl)hydantoin (2) displayed in vitro potent anti-tyrosinase activity with IC50 value of 36 µM, comparable to the commercially used positive control kojic acid (IC50 =46 µM) and arbutin (IC50 =100 µM). Compounds 1-3 were firstly reported from marine actinomycete Georgenia sp.

Antibacterial Activity of Chromomycins from a Marine-Derived Streptomyces microflavus.

A marine-derived actinomycete (Streptomyces sp. MBTI36) exhibiting antibacterial activities was investigated in the present study. The strain was identified using genetic techniques. The 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces microflavus. Furthermore, a new chromomycin A9 (1), along with chromomycin Ap (2), chromomycin A2 (3), and chromomycin A3 (4), were isolated from the ethyl acetate extract. Their structures were determined using extensive spectroscopic methods including 1D and 2D NMR, and HRMS, as well as comparisons with previously reported data. Compounds 1-4 showed potent antibacterial activities against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). During a passage experiment, minimum inhibitory concentration (MIC) values for compounds 1-4 showed no more than a 4-fold increase from the starting MIC value, indicating that no resistance was detected over the 21 passages.

Actinomadura craniellae sp. nov., isolated from a marine sponge in the South China Sea.

A novel marine actinomycete, designated LHW63021T, was isolated from a marine sponge, genus Craniella, collected in the South China Sea. A polyphasic approach was applied to characterize the taxonomic position of this strain. The strain was found to have scarce aerial mycelia that differentiated into spore chains. The cell-wall hydrolysates contained meso-diaminopimelic acid as the diagnostic diamino acid. Glucose, galactose, mannose and madurose were found in the whole-cell hydrolysates. The dominant polar lipids were phosphatidylinositol and diphosphatidylglycerol. MK-9(H6) and MK-9(H8) were the predominant menaquinones. The major fatty acids were iso-C16 : 0, iso-C18 : 0, 10-methyl C17 : 0 and C18 : 1 ω9c. The DNA G+C content based on the draft genome sequence was 72.0 mol%. 16S rRNA gene sequence analysis indicated that strain LHW63021T was a member of the genus Actinomadura and had the highest similarity to Actinomadura echinospora DSM 43163T (97.3 %). Phylogenetic trees supported their close relationship. The average nucleotide identity and digital DNA-DNA hybridization values between the whole genome sequences of strain LHW63021T and A. echinospora DSM 43163T were 79.13 and 23.20 %, respectively. The evidence from the polyphasic study shows that strain LHW63021T represents a novel species of the genus Actinomadura, for which the name Actinomadura craniellae sp. nov. is proposed. The type strain is LHW63021T (=DSM 106125T=CCTCC AA 2018015T).

Corynebacterium alimapuense sp. nov., an obligate marine actinomycete isolated from sediment of Valparaíso bay, Chile.

A novel Gram-positive, non-motile, non-spore-forming and aerobic bacterium, designated strain VA37-3T, was isolated from a marine sediment sample collected at 19.2 m water depth from Valparaíso bay, Chile. Strain VA37-3T exhibits 97.6 % 16S rRNA gene sequence similarity to Corynebacterium marinum D7015T, 96.4 % to Corynebacterium humireducens MFC-5T and 96 % to Corynebacterium testudinoris M935/96/4T; and a rpoB gene sequence similarity of 85.1 % to Corynebacterium pollutisoli VMS11T, both analyses suggesting that strain VA37-3T represents a novel species of Corynebacterium. Physiological testing indicated that strain VA37-3T requires artificial sea water or sodium-supplemented media for growth, representing the first obligate marine actinomycete of the genus Corynebacterium. The genome of the proposed new species, along with the type strains of its most closely related species were sequenced and characterized. In silico genome-based similarity analyses revealed an ANIb of 72.8 % (C. marinum D7015T), ANIm of 85.0 % (Corynebacterium mustelae DSM 45274T), tetra of 0.90 (Corynebacterium callunae DSM 20147T) and ggdc of 24.7 % (Corynebacterium kutscheri DSM 20755T) when compared with the closest related strains. The genomic DNA G+C content of strain VA37-3T was 57.0 %. Chemotaxonomic assessment of strain VN6-2T showed the major fatty acids were C18 : 1ω9c and C16 : 0. Menaquinones predominantly consisted of MK-8(II-H2). Polar lipids consisted of diphosphatidylglycerol, glycolipids, phosphatidylglycerol, phosphoglycolipid and phosphatidylinositol. Mycolic acids also were present. Overall, the results from phylogenetic, phenotypic and genomic analyses confirmed that strain VA37-3T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium alimapuense sp. nov. is proposed, with VA37-3T as the type strain (=CCUG 69366T=NCIMB 15118T).

2-Alkyl-4-hydroxyquinolines from a Marine-Derived Streptomyces sp. Inhibit Hyphal Growth Induction in Candida albicans.

Four 2-alkyl-4-hydroxyquinoline derivatives (1⁻4) were isolated from a semisolid rice culture of the marine-derived actinomycete Streptomyces sp. MBTG13. The structures of these compounds were elucidated by a combination of spectroscopic methods, and their data were in good agreement with previous reports. Compounds 1 and 2 exhibited weak to moderate antibacterial activity against pathogenic bacteria. Unexpectedly, we found that compound 1 acted as a potent inhibitor of hyphal growth induction in the dimorphic fungus Candida albicans, with an IC50 value of 11.4 µg/mL. Growth experiments showed that this compound did not inhibit yeast cell growth, but inhibited hyphal growth induction. Semi-quantitative reverse transcription (RT)-PCR analysis of hyphal-inducing signaling pathway components indicated that compound 1 inhibited the expression of mRNAs related to the cAMP-Efg1 pathway. The expression of HWP1 and ALS3 mRNAs (hypha-specific genes positively regulated by Efg1, an important regulator of cell wall dynamics) was significantly inhibited by the addition of compound 1. These results indicate that compound 1 acts on the Efg1-mediated cAMP pathway and regulates hyphal growth in Candida albicans.

Inhibitory Effects of Diketopiperazines from Marine-Derived Streptomyces puniceus on the Isocitrate Lyase of Candida albicans.

The glyoxylate cycle is a sequence of anaplerotic reactions catalyzed by the key enzymes isocitrate lyase (ICL) and malate synthase, and plays an important role in the pathogenesis of microorganisms during infection. An icl-deletion mutant of Candida albicans exhibited reduced virulence in mice compared with the wild type. Five diketopiperazines, which are small and stable cyclic peptides, isolated from the marine-derived Streptomyces puniceus Act1085, were evaluated for their inhibitory effects on C. albicans ICL. The structures of these compounds were elucidated based on spectroscopic data and comparisons with previously reported data. Cyclo(L-Phe-L-Val) was identified as a potent ICL inhibitor, with a half maximal inhibitory concentration of 27 µg/mL. Based on the growth phenotype of the icl-deletion mutants and icl expression analyses, we demonstrated that cyclo(L-Phe-L-Val) inhibits the gene transcription of ICL in C. albicans under C2-carbon-utilizing conditions.

Seco-Tetracenomycins from the Marine-Derived Actinomycete Saccharothrix sp. 10-10.

Six new tetracenomycin congeners, saccharothrixones E⁻I (1⁻5) and 13-de-O-methyltetracenomycin X (6), were isolated from the rare marine-derived actinomycete Saccharothrix sp. 10-10. Their structures were elucidated by spectroscopic analysis and time-dependent density functional theory (TDDFT)-electronic circular dichroism (ECD) calculations. Saccharothrixones G (3) and H (4) are the first examples of tetracenomycins featuring a novel ring-A-cleaved chromophore. Saccharothrixone I (5) was determined to be a seco-tetracenomycin derivative with ring-B cleavage. The new structural characteristics, highlighted by different oxidations at C-5 and cleavages in rings A and B, enrich the structural diversity of tetracenomycins and provide evidence for tetracenomycin biosynthesis. Analysis of the structure⁻activity relationship of these compounds confirmed the importance of the planarity of the naphthacenequinone chromophore and the methylation of the polar carboxy groups for tetracenomycin cytotoxicity.

Anthocidins A⁻D, New 5-Hydroxyanthranilic Acid Related Metabolites from the Sea Urchin-Associated Actinobacterium, Streptomyces sp. HDa1.

Four new 5-hydroxyanthranilic acid related compounds, named anthocidins A⁻D (1⁻4), two known analogues n-lauryl 5-hydroxyanthranilate (5) and isolauryl 5-hydroxyanthranilate (6), together with benzamide (7), 3-hydroxy-4-methoxycinnamamide (8), and (3S-cis)-hexahydro-3-[(3,4-dihydroxyphenyl)methyl]pyrrolo[1,2-a]pyrazine-1,4-dione (9), were isolated from the fermentation broth of the marine-derived actinomycete, Streptomyces sp. HDa1, which was isolated from the gut of a sea urchin, Anthocidaris crassispina, collected from Hainan Island, China. The structures of these secondary metabolites were elucidated on the basis of their 1D and 2D-NMR and mass spectroscopic data, and anthocidin A was confirmed by single-crystal X-ray diffraction with Cu Kα radiation. Anthocidins A⁻D (1⁻4) feature an acetyl group substitution at the amino group and varying alkyl side chains at the carboxyl group of 5-hydroxyanthranilic acid, and compound 5 was isolated as a natural product for the first time. The cytotoxic and antibacterial activity of compounds 1⁻9 were evaluated.

QM-HiFSA-Aided Structure Determination of Succinilenes A-D, New Triene Polyols from a Marine-Derived Streptomyces sp.

Based on profiles of secondary metabolites produced by marine bacteria obtained using LC/MS, succinilenes A-D (1-4), new triene polyols, were discovered from a culture of a Streptomyces strain SAK1, which was collected in the southern area of Jeju Island, Republic of Korea. The gross structures of 1-4 were primarily determined through analysis of NMR spectra. The double bond geometries of the succinilenes, which could not be established from conventional ¹H NMR spectra because of the highly overlapped olefinic signals, were successfully deciphered using the recently developed quantum-mechanics-driven ¹H iterative full spin analysis (QM-HiFSA). Succinilenes A-C (1-3) displayed inhibitory effects against lipopolysaccharide (LPS)-induced nitric oxide (NO) production, indicating their anti-inflammatory significance. These three compounds (1-3) commonly bear a succinic acid moiety, although succinilene D (4), which did not inhibit NO production, does not have this moiety in its structure. The absolute configurations of succinilenes A-D (1-4) were established through J-based configuration analysis, the modified Mosher's method following methanolysis, and CD spectral analysis.

Isolation, stereochemical study, and cytotoxic activity of isobenzofuran derivatives from a marine Streptomyces sp.

A new 1,3-dihydroisobenzofuran derivative (), together with its epimer (), was isolated from marine Streptomyces sp. W007. The structure of the two compounds was established by extensive spectroscopic analysis and comparison with reported data. The absolute configurations of and were determined by a combination of experimental and computational means, including J-coupling analysis and nuclear Overhauser effect spectroscopy (NOESY) spectra, nuclear magnetic resonance (NMR) calculations, electronic circular dichroism (ECD), and optical rotation (OR) calculations. Compound had no cytotoxicity against human lung adenocarcinoma cell line A549, while compound exhibited weak activity, suggesting that the biological activity depends on the configuration of a single chirality center.

Hormaomycins B and C: New Antibiotic Cyclic Depsipeptides from a Marine Mudflat-Derived Streptomyces sp.

Alterations in microbial culture conditions may trigger the production of diverse bioactive secondary metabolites. While applying various culture conditions and monitoring secondary metabolite profiles using LC/MS, hormaomycins B and C (1 and 2) were discovered from a marine mudflat-derived actinomycete, Streptomyces sp., collected in Mohang, Korea. The planar structures of the hormaomycins, which bear structurally-unique units, such as 4-(Z)-propenylproline, 3-(2-nitrocyclopropyl)alanine, 5-chloro-1-hydroxypyrrol-2-carboxylic acid and b-methylphenylalanine, were established as the first natural analogues belonging to the hormaomycin peptide class. The absolute configurations of 1 and 2 were deduced by comparing their CD spectra with that of hormaomycin. These hormaomycins exhibited significant inhibitory effects against various pathogenic Gram-positive and Gram-negative bacteria.

Bahamaolide A from the marine-derived Streptomyces sp. CNQ343 inhibits isocitrate lyase in Candida albicans.

Bahamaolide A, a new macrocyclic lactone isolated from the culture of marine actinomycete Streptomyces sp. CNQ343, was evaluated for its inhibitory activity toward isocitrate lyase (ICL) from Candida albicans. These studies led to the identification of bahamaolide A as a potent ICL inhibitor with IC50 value of 11.82 µM. The growth phenotype of ICL deletion mutants and quantitative RT-PCR analyses indicated that this compound inhibits the ICL mRNA expression in C. albicans under C2-carbon-utilizing conditions. The present data highlight the potential for bahamaolide A treatment of C. albicans infections via inhibition of ICL activity.

Antiproliferative metabolites against glioma cells from the marine-associated actinomycete Streptomyces sp. ZZ735.

Metabolites produced by the genus Streptomyces are the most important resource for discovering bioactive compounds. In this study, chemical investigation on the metabolites produced by the marine-derived Streptomyces sp. ZZ735 in rice solid medium led to the isolation of eighteen compounds (1-18). Chemical structures of the isolated compounds were determined based on their HRESIMS data and the extensive NMR spectral analyses. Streptonaphthothiazines A (1), B (2), 2-(2-hydroxy-2-methylpropanoylamino)-benzoic acid (7), and streptomycinoic acids A (17), B (18) are characterized as five previously undescribed compounds. The structural backbones of streptonaphthothiazines A (1), B (2) and streptomycinoic acids A (17), B (18) are found from a natural resource for the first time. It is also the first report of 2-(2-methylpropanoylamino)-benzoic acid (3), 2-(2-methylpropanoylamino)-benzamide (4), methyl 2-(3-hydroxypropanoylamino)-benzoate (5), 2-propionylaminobenzamide (6), and (2E)-3-(3-hydroxy-4,5-dimethoxyphenyl)-2-propenoic acid (15) as natural products. Streptonaphthothiazines A (1), B (2) and streptomycinoic acids A (17), B (18) have antiproliferative activity against human glioma U87MG or U251 cells with IC50 values ranging from 31.8 to 37.9 µM.

Anti-inflammatory Oleanane-Type Triterpenoids Produced by Nonomuraea sp. MYH522 through Microbial Transformation.

Eleven oleanane-type triterpenoids named soyasapogenols B1-B11 have been obtained unexpectedly from a marine actinomycete Nonomuraea sp. MYH522. Their structures have been determined by extensive analysis of spectroscopic experiments and X-ray crystallographic data. Soyasapogenols B1-B11 exhibit subtle differences in the positions and degrees of oxidation on an oleanane skeleton. The feeding experiment suggested that soyasapogenols might be derived from soyasaponin Bb through microbial-mediated conversion. The biotransformation pathways from soyasaponin Bb to five oleanane-type triterpenoids and six A-ring cleaved analogues were proposed. The assumed biotransformation involves an array of reactions including regio- and stereo-selective oxidation. These compounds alleviated the 5,6-dimethylxanthenone-4-acetic acid-induced inflammation in Raw264.7 cells via the stimulator of interferon genes/TBK1/NF-κB signaling pathway. The present work provided an efficient approach for rapid diversification of soyasaponins and for developing food supplements with potent anti-inflammatory effects.

Draft genome sequence data of Gordonia hongkongensis strain EUFUS-Z928 isolated from the octocoral Eunicea fusca.

Octocorals are among the most prolific sources of biologically active compounds. A significant part of their specialized metabolites richness is linked to the abundance of their associated microbiota. Consequently, research on the bioprospecting potential of microorganisms associated with these marine invertebrates has gained much interest. Here, we describe the draft genome of Gordonia hongkongensis strain EUFUS-Z928 isolated from the octocoral Eunicea fusca. The genome was assembled de novo from short-read whole-genome sequencing data. Additionally, functional annotation of predicted genes was performed using the RAST tool kit, including genome mining for specialized metabolite biosynthetic gene clusters using the antiSMASH v6.0 tool. The genome sequence data of G. hongkongensis EUFUS-Z928 can provide information for further analysis of the potential biotechnological use of this microorganism and guide the characterization of other related actinobacterial isolates. Likewise, this information increases the analytical capacity for studying the genus Gordonia.

Octyl substituted butenolides from marine-derived Streptomyces koyangensis.

A new butenolide derivative (1) featuring octyl substitution at γ-position, together with four known analogues (2-5) were isolated from marine-derived Streptomyces koyangensis SCSIO 5802. The structure of 1 was elucidated by HR-MS and NMR spectroscopic data analyses. The absolute configuration of the stereo centre in lactone ring of 1 was determined by comparison of CD spectrum with those of known compounds. Compound 1 exhibited mild antiviral activity against herpes simplex virus with EC50 value of 25.4 µM.

Biochemical, thermodynamic and structural characteristics of a biotechnologically compatible alkaline protease from a haloalkaliphilic, Nocardiopsis dassonvillei OK-18.

This report describes purification strategies, biochemical properties and thermodynamic analysis of an alkaline serine protease from a marine actinomycete, Nocardiopsis dassonvillei strain OK-18. The solvent tolerance, broad thermal-pH stability, favourable kinetics and thermodynamics suggest stability of the enzymatic reaction. The enzyme was active in the range of pH 7-12 and 37-90 °C, optimally at pH 9 and 70 °C. The deactivation rate constant (Kd), half-life (t½), enthalpy (ΔH*), entropy (ΔS*), activation energy (E) and change in free energy (ΔG*) suggested stability and spontaneity of the reaction. ß-Sheets as revealed by the Circular dichroism (CD) spectroscopy, were the major elements in the secondary structure of the enzyme, while Fourier-transform infrared spectroscopy (FTIR) indicated the presence of amide I and amide II. Based on the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis, the amino acid sequence had only 38% similarity with other proteases of Nocardiopsis strains, suggesting its novelty. The Ramachandran Plot revealed the location of the amino acid residues in the most favored region. The blood de-staining, gelatin hydrolysis, silver recovery and deproteinization of crab shells established the biotechnological potential of the enzyme.