Discovery of Streptomyces marinisediminis sp. nov., a new thiolutin producing actinomycete isolated from Thai marine sediment

Polyphasic taxonomic characterization of strain JHD1^T

The marine environment undeniably acts as a prolific reservoir for the identification of novel bioactive secondary metabolite-producing actinomycetes. Marine ecosystems harbor a diverse array of microorganisms, including actinomycetes, which thrive under unique and often extreme conditions. These organisms are well-known for their capacity to synthesize a wide variety of bioactive secondary metabolites, many of which demonstrate promising antibacterial, antifungal, anticancer, and antiviral properties. The relatively underexplored nature of marine environments renders them an invaluable and largely untapped source of novel bioactive compounds with substantial therapeutic potential^8,9. Hence, actinomycetes residing in marine ecosystems are anticipated to exhibit distinct characteristics compared to those found in other environments. Strain JHD1^T was isolated from marine sediment collected in Chonburi Province, Thailand (12°52′54.3″N, 100°52′54.0″E). The marine sediment sample was initially air-dried at ambient temperature (30 °C) and subsequently subjected to heat treatment at 100 °C for 45 min. Following this, the sample was serially diluted to a 10⁻³ dilution (1,000-fold) using 0.01% sterile sodium dodecyl sulfate (SDS) in seawater. Aliquots of the diluted suspension were then spread onto modified starch-casein nitrate seawater agar supplemented with 50 mg/l nalidixic acid and 200 mg/l nystatin. The strain grew well on International Streptomyces Project number 2 (ISP 2), ISP 4, and ISP 6 media. Moderate growth was observed on ISP 5, ISP 7, and nutrient agar (NA), while poor growth occurred on Czapek’s sucrose and ISP 3. The substrate mycelium was light yellowish-brown on ISP 2, ISP 4, and ISP 6. Greyish white to yellowish white aerial mycelium was clearly visible on ISP 2, ISP 4, ISP 6, and NA after 14 days of incubation at 30 °C. Greenish yellow diffusible pigments were detected on ISP 2, ISP 4, ISP 5, ISP 7, and NA media (Table S1). Strain JHD1^T produced spiral chains of ovoid-shaped spores (0.4–0.5 × 0.8–1.0 μm) with smooth surfaces, borne directly on the aerial mycelium (Fig. 1). Whole-cell hydrolysates of strain JHD1^T contained LL-diaminopimelic acid, and ribose, galactose, and glucose were detected as cell wall sugars. This chemotaxonomic profile is commonly observed in Streptomyces species¹⁰. The polar lipid profile included diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylinositol mannoside (PIM), two unidentified phospholipids (PLs), and two unidentified lipids (Ls) (Fig. S1). The major menaquinones (MK) were MK-9(H₈) (44.4%), MK-9(H₆) (30.9%), and MK-9(H₄) (16.3%), while MK-9(H₂) (8.4%) was a minor component. Cellular fatty acid analysis revealed that the major fatty acids (> 10%) were anteiso-C₁₅:₀ (21.3%), iso-C₁₆:₀ (33.3%), and anteiso-C₁₇:₀ (14.9%). Minor components included iso-C₁₄:₀, iso-C₁₅:₀, C₁₆:₀, iso-C₁₇:₀, C₁₈:₁ ω9c, iso-C₁₈:₀, anteiso-C₁₉:₀, and anteiso-C₁₇:₁ ω9c (Table S2). The fatty acid profile was similar to those of closely related Streptomyces type strains, Streptomyces alkaliterrae OF1^T and Streptomyces chumphonensis KK1-2^T. The morphological and chemotaxonomic characteristics of strain JHD1^T are consistent with those of the genus Streptomyces¹⁰. The 16S rRNA gene sequence comparison is a foundational method for the identification and classification of novel actinomycete and bacterial species. A sequence similarity of ≤ 98.65% with any validly published species indicates the potential discovery of a previously unidentified taxon¹¹. The 16S rRNA gene sequence of strain JHD1^T (1,503 bp) showed that it belongs to the genus Streptomyces. It shared the highest sequence similarity with S. alkaliterrae OF1^T (98.48%), which is below the species delineation threshold of 98.65%. The next closest relatives were Streptomyces lycii TRM 66187^T (98.35%) and Streptomyces qinglanensis 172205^T (98.34%), further supporting the novelty of the strain. Phylogenetic analysis using neighbor-joining (NJ) and maximum-likelihood (ML) methods placed strain JHD1^T in a clade with Streptomyces gobiensis 1–25, although the bootstrap support was weak (< 50%) (Figs. S2 and S3). In contrast, the maximum-parsimony (MP) tree showed that JHD1^T formed a distinct clade with S. alkaliterrae OF1^T and S. chumphonensis KK1-2^T (Fig. S4), suggesting phylogenetic separation from these close relatives. While 16S rRNA gene analysis provides initial insights, genome-based metrics such as average nucleotide identity based on MuMmer ultra-rapid aligning tool (ANIm) and digital DNA–DNA hybridization (dDDH) offer greater resolution and are now considered gold standards for species delineation in actinomycetes and other bacteria. Thus, the genome of strain JHD1^T was analyzed in comparison with its closest Streptomyces type strains. The draft genome sequence of strain JHD1^T, deposited in GenBank under accession number JAPKFL000000000, comprises 5.6 Mbp across 141 contigs, with an N50 of 170.8 kbp and a G + C content of 74.7%. A total of 4,888 predicted genes were identified, including 4,689 protein-coding genes (Table S3). Phylogenomic analysis using Automated Multi-Locus Species Tree (autoMLST) and the Type (Strain) Genome Server (TYGS) revealed that strain JHD1^T clustered closely with S. chumphonensis KK1-2^T with strong bootstrap support (100%) (Fig. 2; Fig. S5), but was clearly separated from the clade containing S. gobiensis 1–25^T. According to current taxonomic standards, species-level boundaries are defined by ANIm values below 95%¹² and dDDH values below 70%¹³. Strain JHD1^T exhibited ANIm values of 85.7–86.2% and dDDH values of 23.4–25.0% compared to S. alkaliterrae OF1^T and S. chumphonensis KK1-2^T (Table 1), supporting its classification as a distinct species. In addition to genomic data, comparative phenotypic characterization further distinguished strain JHD1^T from its closest type strains. Notable differences included tolerance to a maximum growth temperature of 40 °C, nitrate reduction capability, oxidase production, and the utilization of dextran, D-lactose, and xylitol. Unlike S. alkaliterrae OF1^T, strain JHD1^T was unable to assimilate inulin, D-mannitol, D-melibiose, D-raffinose, L-rhamnose, or sucrose, and did not produce melanin. Compared to S. chumphonensis KK1-2^T, strain JHD1^T could hydrolyze urea and gelatin and utilize D-cellobiose, D-fructose, glycerol, 4-hydroxyproline, L-methionine, L-proline, L-serine, L-valine, DL-2-aminobutyric acid, L-arginine, and L-phenylalanine, whereas S. chumphonensis KK1-2^T could not (Table 2). Taken together, the genotypic and phenotypic data clearly support the conclusion that strain JHD1^T represents a novel species of the genus Streptomyces, for which the name Streptomyces marinisediminis sp. nov. is proposed. The type strain is JHD1^T (= TBRC 15653^T = NBRC 115639^T).

A, Colonial appearance of Streptomyces sp. JHD1^T; B, Scanning electron micrograph of strain JHD1^T grown on ISP 2 seawater agar for 21 days at 30 °C. Bar, 2 μm.

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Phylogenomic analysis of Streptomyces sp. JHD1^T and type strains affiliated to the genus Streptomyces based on 100 bacterial conserved single copied gene sets of the members. The bootstrap values on the nodes are displayed by > 50.

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The prediction for secondary metabolite production in the genome of strain JHD1^T

The biosynthetic gene clusters (BGCs) in the genome of strain JHD1^T and its closely related type strains were identified using antiSMASH version 7.0. Comparative analysis with S. chumphonensis KK1-2^T and S. alkaliterrae OF1^T revealed that strain JHD1^T possesses a diverse array of BGCs, including those predicted to encode butyrolactone, cyclodipeptide synthase (CDPS), ectoine, type III polyketide synthase (type III PKS), hydrogen cyanide, lanthipeptide classes I–III, lassopeptide, lipolanthine, non-ribosomal peptide synthetase-independent siderophore (NI-siderophore), heterocyst glycolipid synthase-like ketide synthase (hgIE-KS), NRP-metallophore, phosphonate, and trans-acyltransferase polyketide synthase (trans-AT PKS) gene clusters. Notably, strain JHD1^T was particularly rich in clusters associated with type I polyketide synthase (type I PKS), non-ribosomal peptide synthetase (NRPS), and terpene biosynthesis. The hgIE-KS and phosphonate gene clusters were uniquely detected in the genome of strain JHD1^T and were absent in its closely related strains (Fig. 3). The genome of strain JHD1^T, unlike those of S. chumphonensis KK1-2^T and S. alkaliterrae OF1^T, contained BGCs with high similarity confidence associated with the synthesis of several notable bioactive compounds: 2-Methoxy-5-methyl-6-(13-methyltetradecyl)-1,4-benzoquinone, a derivative of 1,4-benzoquinone, is known for its antimicrobial activity¹⁴. SapB, a lantibiotic-like peptide, is important for aerial mycelium formation in the filamentous bacterium Streptomyces coelicolor. It also plays a critical role in sphingolipid metabolism by acting as an activator protein for specific lysosomal hydrolases involved in glycosphingolipid degradation¹⁵. Isorenieratene, a carotenoid pigment belonging to the aryl carotenoid class, is primarily found in green sulfur bacteria (Chlorobiaceae). This compound exhibits strong radical scavenging activity, often greater than that of β-carotene, and can neutralize reactive oxygen species (ROS), thereby reducing oxidative damage to DNA, proteins, and lipids¹⁶. Naringenin, a typical plant secondary metabolite, is produced by Streptomyces clavuligerus. This flavonoid, commonly found in citrus fruits such as grapefruits and oranges, is well known for its diverse bioactivities, including antioxidant, anticancer, antimicrobial, and neuroprotective effects¹⁷. Sibiromycin is an antitumor antibiotic belonging to the pyrrolobenzodiazepine (PBD) family. It is recognized for its ability to bind to DNA and inhibit essential cellular processes. This compound is naturally produced by Streptosporangium sibiricum and is structurally related to other DNA-alkylating agents with potent antitumor potential¹⁸. These findings underscore the biosynthetic potential of strain JHD1^T for producing medically relevant secondary metabolites (Table S4). Similarly, there are numerous documented examples where novel Streptomyces strains produce only known compounds despite having distinct BGCs. For example, S. coelicolor A3 possesses over 20 BGCs but produces only a few known compounds under normal cultivation¹⁹. Marine Streptomyces collections show that phylogenetically distinct strains with different BGC profiles often produce overlapping sets of known metabolites like actinomycin and streptomycin derivatives²⁰.

Biosynthetic gene clusters (BCGs) presented in Streptomyces sp. JHD1^T and the closest type strains. Cluster type designations provided by antiSMASH 7.1 analysis.

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The high number of BGCs identified in the genome of strain JHD1^T suggests that it was a promising actinomycete capable of producing a wide variety of valuable secondary metabolites. Notably, some of these gene clusters exhibited low sequence similarity to those associated with known bioactive compounds, indicating the potential for novel metabolite discovery. For example, echoside A–E, a group of structurally diverse natural products produced by Streptomyces sp. LZ35, exhibit various biological activities, including antimicrobial, anticancer, and neuroprotective effects, as well as inhibitory activity against DNA topoisomerase I and IIα²¹. Phthoxazolin, an oxazole-containing polyketide produced by Streptomyces species such as Streptomyces avermitilis, displays broad-spectrum anti-oomycete and herbicidal activities. Its biosynthesis involves a trans-acyltransferase (trans-AT) type I polyketide synthase (PKS) system. Phthoxazolin A’s unique mode of action as a cellulose biosynthesis inhibitor makes it a promising candidate for developing new herbicides, particularly for combating herbicide-resistant weeds²². Lavendiol, a bisabolane-type sesquiterpenoid diol, has been isolated from both Lavandula species and S. lavendulae FRI-5. It demonstrates antimicrobial, anticancer, antioxidant, and anti-inflammatory properties²³. Terpenomycin, a rare polyketide-terpenoid hybrid antibiotic isolated from Streptomyces sp. DSM 4137 and Nocardia terpenica AUSMDU00012715, contains both terpenoid and polyketide moieties. It has attracted attention for its potent antimicrobial and antitumor activities²⁴. Lasalocid, a polyether ionophore antibiotic produced by several Streptomyces species including S. lasaliensis, is widely studied for its ion-transporting capabilities and broad biological activities, such as antibacterial, anticoccidial, antiviral, and anticancer effects²⁵. Pyrroloformamide A–D, members of the dithiolopyrrolone family of natural products, were isolated from Streptomyces sp. CB02980. These compounds are distinguished by a unique bicyclic core featuring a disulfide or thiosulfonate bridge, which is essential for their biological activity, including strong antibacterial and cytotoxic effects. Their biosynthesis involves the pyf gene cluster, including pyfE, a nonribosomal peptide synthetase that assembles the core Cys–Cys dipeptide precursor for the dithiolopyrrolone scaffold, as well as pyfC and pyfN, which are standalone type II thioesterases involved in pathway optimization. Notably, overexpression of pyfN has been shown to increase production of pyrroloformamides A and B by more than twofold²⁶. Overall, the genomic data suggest that strain JHD1^T harbored significant biosynthetic potential for the production of diverse and novel secondary metabolites, especially those originating from PKS and NRPS pathways, which are major sources of bioactive compounds (Table S5).

The analysis of the Streptomyces sp. JHD1^T genome sequence revealed a biosynthetic gene cluster responsible for producing thiolutin, a member of the dithiolopyrrolone family that has promising clinical applications as an antitumor agent²⁷. At the heart of this molecular assembly line lies an architecturally NRPS containing the cyclization (Cy), adenylation (A), and thiolation (T) domain triad (Table S6). We identified this specialized Cy-A-T arrangement within the homE gene, which produces a remarkably large protein (1,077 amino acids, HomE) sharing significant family resemblance (69.15% sequence identity) with its relative EntF from Streptomyces sp. CAI 127 (Table 3). The cluster encompasses nine functionally distinct genes (Table 3) that orchestrate the assembly of the characteristic pyrrolinonodithiole scaffold of thiolutin. As illustrated in Table 3, this gene cluster encodes a sophisticated enzymatic consortium: HomA (acylation), HomD (redox chemistry), HomF (decarboxylation), and HomC (chain release), collectively orchestrating the precise molecular assembly of thiolutin²⁸. The Cy-A-T domain arrangement plays a pivotal role in catalyzing heterocyclization reactions that generate distinctive ring structures in the peptide backbone, a structural feature responsible for the broad-spectrum antimicrobial and potential anticancer properties that characterize dithiolopyrrolones^28,29. The notable sequence conservation observed across diverse Streptomyces species (with identity values ranging from 69.15 to 100%, Table 3) underscores the evolutionary significance of this biosynthetic pathway, aligning with previously characterized dithiolopyrrolone clusters in other organisms, such as S. clavuligerus and various marine bacteria^30,31. Our chemical analysis provided definitive confirmation that Streptomyces sp. JHD1^T actively synthesizes thiolutin and its derivative (2,2-dioxidothiolutin), establishing a clear functional connection between the identified genetic elements and the resultant bioactive metabolite.

Pairwise amino acid sequence alignment revealed 68.46% sequence identity and 78.87% similarity between the two proteins (Fig. 4), demonstrating significant conservation of this essential biosynthetic enzyme across different Streptomyces species. The alignment showed particularly high conservation throughout the protein sequence, with extensive stretches of identical and similar residues spanning the entire length of the 1,077 amino acid protein. This level of sequence conservation supports the functional assignment of HomE in strain JHD1^T and confirms the close evolutionary relationship between thiolutin and holomycin biosynthetic pathways. The high similarity is consistent with their shared dithiolopyrrolone scaffold, where both compounds contain the characteristic pyrrolinonodithiole core structure. The observed conservation suggests that the NRPS machinery for dithiolopyrrolone biosynthesis has been well-preserved across marine and terrestrial Streptomyces species, reflecting the fundamental importance of this enzymatic system in secondary metabolite production. Based on the identified gene cluster and existing literature on dithiolopyrrolone biosynthesis²⁸we propose the following thiolutin biosynthetic pathway for strain JHD1^T: The pathway begins with L-cysteine activation and dimerization catalyzed by HomE (NRPS), which utilizes its Cy-A-T domain architecture to form a Cys–Cys dipeptide. Core scaffold formation proceeds through oxidative cyclization mediated by HomD (oxidoreductase) and HomB (acyl-CoA dehydrogenase), generating the characteristic pyrrolinonodithiole structure, followed by decarboxylation by HomF to remove carboxyl groups. N-acetylation is then performed by HomA (N-acyltransferase), using acetyl-CoA as a cofactor, to produce the acetylated intermediate. The final disulfide bond formation is catalyzed by HomI (thioredoxin reductase), yielding mature thiolutin. Product release and transport involve HomC (thioesterase) for enzyme complex release and HomH (efflux protein) for cellular export, with HomG (globin) potentially providing oxygen for oxidative steps. This proposed pathway (L-cysteine → Cys–Cys dipeptide → holothin core → N-acetyl-holothin → thiolutin) is consistent with established dithiolopyrrolone biosynthetic mechanisms and is supported by the functional annotation of all identified genes within the cluster (Fig. 5). These findings provide valuable insights into the evolutionary relationship of strain JHD1^T’s thiolutin pathway with those of other characterized dithiolopyrrolone producers, supporting both the taxonomic and biosynthetic characterization of this novel marine-derived Streptomyces species.

Pairwise sequence alignment of HomE proteins from Streptomyces sp. JHD1^T and S. clavuligerus ATCC 27064. High conservation is observed in the central catalytic domains essential for dithiolopyrrolone biosynthesis.

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Proposed thiolutin biosynthetic pathway in Streptomyces sp. JHD1^T. The pathway proceeds through sequential enzymatic steps starting from L-cysteine substrate and culminating in thiolutin production. Gene names indicate the predicted enzymes catalyzing each biosynthetic step based on functional annotation and homology to characterized dithiolopyrrolone pathways.

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Structure Elucidation of secondary metabolites isolated from the marine-derived Streptomyces sp. JHD1^T

The detection of numerous biosynthetic gene clusters within the genome of strain JHD1^T highlights its considerable potential for the production of novel secondary metabolites. This genetic richness suggests that strain JHD1^T may serve as a valuable resource in the ongoing search for promising biologically active compounds. Accordingly, the crude extract obtained from the culture broth of strain JHD1^T grown in ISP 2 seawater medium was initially evaluated for antimicrobial activity and demonstrated strong inhibitory effects against Bacillus cereus ATCC 11778 and Staphylococcus aureus ATCC 29213. This prompted further fractionation and purification of the extract using various chromatographic methods. Subsequently, the isolated compounds were structurally characterized through spectroscopic analyses.

Six compounds (1–6) were isolated from the marine-derived Streptomyces sp. JHD1^T. Chemical structures (Fig. 6) were determined by using NMR spectroscopy and identified as thiolutin (1), 2,2-dioxidothiolutin (2), 8-amino-2 H-benz[e][1,3]oxazine-2,4(3 H)-dione (3), benadrostin (4), 2,3-dihydroxybenzamide (5), and 2-hydroxy-3-methoxybenzamide (6). The¹H and¹³C NMR spectral data of compounds 1, 2, 4, and 5 were identical to those reported in the literature^32,33,34 (Tables S7 and S8, Figs. S6, S7, S10, S11, S21, S22, S25, and S26 ). Compound 2 was structurally confirmed for the first time by X-ray crystallographic analysis (Fig. 7). Compounds 3 and 6 were naturally isolated for the first time from Streptomyces sp. and up to date, their¹H and¹³C NMR spectral data have not yet been documented. Herein¹, H and¹³C NMR data together with their physico-chemical data are recorded and the chemical structures were confirmed by HRESIMS data (Figs. S6-S35).

Chemical structures of compounds 1–6 isolated from the marine Streptomyces sp. JHD1^T.

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ORTEP Drawing of compound 2 (2,2-dioxidothiolutin) with atomic numbering and 50% displacement ellipsoids.

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Compound 3 was obtained as a colorless solid and in HRESIMS data showed a pseudo-molecular ion peak at m/z 177.0303 [M−H]⁻. The¹H and¹³C NMR spectra were comparable to those of benadrostin (4)³⁵ but the mass spectrum showed even number, suggesting the presence of two nitrogen atoms in the molecule. The evidence pointed out that a hydroxyl group in compound 4 was replaced with an amine group. Moreover, the HMBC correlations from H-5 (δ_H 7.28) to C-4 (δ_C 166.3), C-7 (δ_C 119.6) and C-8a (δ_C 142.6) indicated an amine group at C-8. Thus, compound 3 is 8-amino-2 H-benz[e][1,3]oxazine-2,4(3 H)dione (Fig. 6).

Likewise¹, H and¹³C NMR spectra of compound 6 were closely related to those of compound 5. HRESIMS spectrum showed 14 mass unit higher than that of compound 5, suggesting the extra methyl group in the molecule. Also, the¹H NMR spectrum of compound 6 displayed an additional methoxy group in the molecule. According to the evidence from HMBC correlations, the hydroxyl group at C-3 in compound 5 was replaced with the methoxy group in compound 6. Thus, compound 6 is 2-hydroxy-3-methoxybenzamide (Fig. 6).

Compounds 3 and 6 are initially reported from the marine-derived actinomycetes, Streptomyces sp. JHD1^T, while compounds 1, 2, and 4 were previously isolated from Streptomyces spp. ^33,35.

Biological activity of the isolated compounds

The isolated compounds were evaluated for a range of biological activities, including antiplasmodial activity against multidrug-resistant Plasmodium falciparum K-1, antitubercular efficacy against Mycobacterium tuberculosis H37Ra, antibacterial effects against Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 29213, and Acinetobacter baumannii, antifungal effect against Candida albicans ATCC 90028, as well as cytotoxicity toward both cancerous cell lines (MCF-7 and NCI–H187) and a non-cancerous cell line (Vero). Thiolutin (1) and 2,3-dihydroxybenzamide (5) showed antimalarial activity with IC₅₀ value of 5.92 and 40.1 µM, respectively. Compound 1 exhibited strong cytotoxicity against both malignant (MCF-7 and NCI-H187) and non-malignant (Vero) cell lines, with IC₅₀ values ranging from 0.35 to 5.61 µM, whereas the other compounds showed only weak cytotoxic effects (Table 4). In addition, compound 1 displayed antibacterial activity against Staphylococcus aureus ATCC 29213 and Bacillus cereus ATCC 11778, with minimum inhibitory concentration (MIC) values of 3.13 and 1.56 µg/ml, respectively. In addition, all tested compounds were inactive at the maximum tested concentration (50 µg/ml) in antibacterial and antifungal assays against the Gram-negative bacterium Acinetobacter baumannii and the fungus Candida albicans ATCC 90028. Thiolutin, originally isolated from the culture broth of Streptomyces species, has demonstrated broad-spectrum antibacterial activity against both Gram-positive (S. aureus, MIC 3.125 µg/ml and Mycobacterium bovis, MIC 0.313 µg/ml) and Gram-negative bacteria (Escherichia coli, MIC 6.25 µg/ml), while 2,2-dioxidothiolutin exhibited weak anti-bacterial activity against S. aureus (MIC 50 µg/ml)³⁴. Benadrostin (4) inhibited poly(ADP-ribose)synthetase with IC₅₀ 35 mM³² and 2,3-dihydroxybenzamide (5) had antioxidant property³⁴. Numerous Streptomyces strains, including the marine-derived Streptomyces sp. BTBU20218885, S. albus, and S. luteosporeus, have been identified as prolific producers of thiolutin^29,33,36,37. Moreover, thiolutin production has also been reported in Saccharothrix algeriensis³⁸. In our study, thiolutin (1) and its oxidized derivative, 2,2-dioxidothiolutin (2), were successfully isolated from the marine-derived Streptomyces sp. JHD1^T, consistent with previous reported by Song et al.³³. Streptomyces species isolated from marine environments are increasingly recognized as valuable microbial sources for the discovery of novel secondary metabolites, and their importance in natural product research continues to grow. Numerous compounds derived from these marine strains have demonstrated potent antimicrobial activity against a broad range of pathogenic microorganisms. For instance, marinomycins A–D, a unique class of polyene macrolide antibiotics, were originally isolated from marine-derived Streptomyces species. These compounds are notable for their unusual dimeric macrodiolide structures and potent antimicrobial and anticancer activities³⁹. In 2011, Wei et al. reported lobophorins C and D, complex spirotetronate antibiotics, produced by a marine sponge-associated actinomycete strain AZS17. These compounds exhibited a wide range of biological activities, including antibacterial, antifungal, anti-inflammatory, and cytotoxic effects⁴⁰. Cyclomarins A–C, a distinctive group of cyclic heptapeptides, were isolated from the culture broth of marine-derived Streptomyces sp. CNB-982 and showed anti-inflammatory, antibacterial, and antimalarial activities⁴¹. Notably, cyclomarins are biosynthesized by NRPS, which are frequently enriched in marine actinomycete genomes. Surugamides A–E, cyclic octapeptides containing four D-amino acid residues, were isolated from the broth of marine-derived Streptomyces sp. Surugamide A exhibited antibacterial and cytotoxic properties and inhibited autophagy, a promising therapeutic strategy in cancer treatment^42,43. Collectively, these findings highlight marine-derived Streptomyces species, including Streptomyces sp. JHD1^T, as promising microbial reservoirs for the discovery of novel bioactive compounds with significant potential for biotechnological and pharmaceutical applications.

Description of Streptomyces marinisediminis sp. nov.

Streptomyces marinisediminis (ma.ri.ni.se.di’mi.nis. L. masc. adj. marinus of or belonging to the sea, marine; L. neut. n. sedimen, -imins sediment; N.L. gen. n. marinisediminis of marine sediment).

Cells are Gram-stain-positive and aerobic. It grows well on ISP 2, ISP 4, and ISP 6. Grows moderately on ISP 5, ISP 7, and nutrient agar. The poorly growth is observed on ISP 3, ISP 6, and Czapek’s sucrose. Light yellowish brown substrate mycelium is observed on ISP 2, ISP 4, and ISP 6 agar. Aerial spore masses is produced on ISP 2, ISP 4, ISP 5, ISP 6, ISP 7, and nutrient agar for 14 days of cultivation. The spiral chain of ovoid-shaped spores with smooth surfaces are detected. Greenish yellow diffusible pigments are found on ISP 2, ISP 4, ISP 5, ISP 6, ISP 7, and nutrient agar. The liquefaction of gelatin, hydrolysis of starch, the production of urease, catalase, oxidase, and milk peptonization are positive. Negative results are observed for the reduction of nitrate, and melanin production. Decomposes hypoxanthine, but not adenine, cellulose, L-tyrosine and xanthine. Utilizes D-cellobiose, dextran, D-fructose, D-glucose, glycerol, D-lactose, D-mannose, D-ribose, salicin, and xylitol; but does not utilize L-arabinose, D-galactose, myo-inositol, inulin, D-mannitol, D-melibiose, D–melezitose, D-raffinose, L-rhamnose, sucrose, D-trehalose, and D-xylose as sole carbon sources. Utilizes L-asparagine, 4-hydroxyproline, L-methionine, L-proline, L-serine, and L-valine; weally utilizes L-arginine, L-histidine, L-threonine, but does not utilize DL-2-aminobutyric acid, L-phenylalanine, and L-cysteine as sole nitrogen sources. The growth temperature is between 20 and 37 °C. Maximum NaCl for growth is 10% (w/v). The pH range for growth is 6–8. Cell wall peptidoglycan contains LL-diaminopimelic acid. The major menaquinones are MK-9(H₈), MK-9(H₆), and MK-9(H₄), while MK-9(H₂) is minor component. Glucose, galactose, and ribose, are detected as whole-cell sugars. The phospholipid profile contains diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, two unidentified phospholipids. The major fatty acids (> 10%) are anteiso-C_15:0, iso-C_16:0, and anteiso-C_17:0. The DNA G + C content of the type strain is 74.7%. The type strain, JHD1^T (= TBRC 15653^T = NBRC 115639^T), is an actinomycete isolated from marine sediment collected from Chonburi province, Thailand. The GenBank accession number for the 16S rRNA gene sequence of strain JHD1^T is OM755759. The whole-genome shotgun project has been deposited at GenBank under the accession JAPKFL000000000.

This study reports the identification of a novel species within the genus Streptomyces and its associated bioactive secondary metabolites. Streptomyces sp. JHD1^T was taxonomically characterized using a polyphasic approach, including genome-based analyses, leading to its designation as a new species for which the name Streptomyces marinisediminis sp. nov. is proposed.

This strain, JHD1^T, was found to encode a rich biosynthetic repertoire for synthesizing diverse secondary metabolites, mainly through PKS and NRPS pathways, well-known engines for generating bioactive natural products. The crude extract obtained from the culture broth of S. marinisediminis sp. nov. exhibited notable biological activity, enabling the isolation of several secondary metabolites: thiolutin (1), 2,2-dioxidothiolutin (2), 8-amino-2 H-benz[e][1,3]oxazine-2,4(3 H)-dione (3), benadrostin (4), 2,3-dihydroxybenzamide (5), and 2-hydroxy-3-methoxybenzamide (6). X-ray crystallography was employed in this study to confirm, for the first time, the structure of 2,2-dioxidothiolutin (2). Among these, thiolutin, benadrostin, and 2,3-dihydroxybenzamide exhibited a broad spectrum of biological activities, including antimalarial effects, antimicrobial activity against Gram-positive pathogens, and cytotoxicity against human cancer cell lines. These findings highlight Streptomyces marinisediminis sp. nov. as a promising microbial resource, and its metabolites as candidates for further development in biotechnological and pharmaceutical applications.

Isolation, cultivation, and preservation of Streptomyces sp. JHD1^T

Streptomyces sp. JHD1^T was isolated from marine sediment collected from Chonburi province, Thailand (12°52’54.3”N100°52’54.0”E). The marine sediment sample was air-dried at 30 ˚C for 3 days and heated at 100 ˚C for 45 min. The sample was serial diluted (1000-fold) to 10^− 3 with 0.01% sterile SDS in seawater and spread onto modified starch-casein nitrate seawater agar containing 10 g soluble starch, 1 g sodium caseinate, 0.5 g KH₂PO₄, 0.5 g MgSO₄ and 18 g agar in 1 L seawater, pH 8.3, supplemented with antibiotics suggested by Thawai et al.⁴⁴. After 21 days of incubation at 30 ˚C, a yellowish-brown colony of Streptomyces sp. JHD1^T was picked and purified on yeast extract-malt extract seawater agar. The pure culture was maintained in glycerol solution (20%, v/v) at − 80 ˚C for short-term preservation or lyophilized for long-term preservation.

Polyphasic taxonomic characterizations

Morphological, cultural, physiological, and biochemical characteristics

To analyze the morphological characteristics, 21-day-old cultures of Streptomyces sp. JHD1^T were examined using scanning electron microscopy (JSM-6610 LV; JEOL). Cultural traits were assessed by cultivating the strain on International Streptomyces Project (ISP) media 2–7⁴⁷ (from Difco) for 14 days at 30 °C. The coloration of aerial and substrate mycelia, along with diffusible pigments, was recorded using the ISCC-NBS color charts⁴⁶. Growth range tests were conducted in ISP 2 broth over 14 days, evaluating temperature tolerance (10–50 °C), NaCl concentration tolerance (0–10% w/v), and pH range (4–12, in increments of 1 unit). Various phenotypic characteristics were examined following established procedures, including tests for the utilization of individual nitrogen sources, decomposition of insoluble compounds (adenine, hypoxanthine, xanthine, tyrosine, and cellulose), starch hydrolysis, nitrate reduction, milk peptonization, and gelatin liquefaction, based on methods by Arai⁴⁷Williams and Cross⁴⁸and Gordon et al.⁴⁹. Carbon source utilization (1% w/v) was determined using the protocol described by Supong et al.⁵⁰. For comparative purposes, reference strains S. alkaliterrae OF1^T and S. chumphonensis KK1-2^T were cultivated under identical conditions. All media used for testing phenotypic characteristics were prepared with seawater instead of distilled water.

Chemotaxonomic analyses

Chemotaxonomic characterization was performed using freeze-dried cells harvested from cultures grown in ISP 2 seawater broth on a rotary shaker at 200 r.p.m. and 30 °C for 7 days. The diaminopimelic acid isomer, presence of reducing sugars in cell hydrolysates, and the type of menaquinones were identified following established protocols by Hasegawa et al.⁵¹Komagata and Suzuki⁵²Minnikin et al.⁵³and Collins et al.⁵⁴respectively. To examine the polar lipid profile of Streptomyces sp. JHD1^T, lipids were extracted from dried cells and analyzed according to the method of Minnikin et al.⁵⁵ The cellular fatty acid composition was determined using gas chromatography (Agilent model 6890), following the guidelines provided by the Microbial Identification System (MIDI)^56,57.

Genome-based taxonomy and genomic analysis for secondary metabolite production

Genomic DNA for PCR amplification and whole-genome sequencing was extracted following the method of Tamaoka⁵⁸. Amplification of the 16S rRNA gene was performed using primers 9 F and 1541R⁵⁹ under conditions described by Kittiwongwattana et al.⁶⁰. Sequence similarity of the 16S rRNA gene from Streptomyces sp. JHD1^T was assessed using the EzBioCloud server⁶¹. For phylogenetic analysis, the 16S rRNA gene sequence of Streptomyces sp. JHD1^T and those of closely related Streptomyces type strains retrieved from the EzBioCloud database were used to construct phylogenetic trees in MEGA X⁶². Three tree-building methods, neighbor-joining⁶³maximum-parsimony⁶⁴and maximum-likelihood⁶⁵were employed. The confidence of tree topology was evaluated through bootstrap analysis with 1000 replicates⁶⁶. Whole-genome sequencing was conducted at Chulalongkorn University, Thailand, using the Illumina MiSeq platform (2 × 200 bp paired-end reads). Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Genomic similarity metrics, ANIm, AAI, and dDDH, were calculated using JSpecies⁶⁷the AAI calculator from the Kostas Lab⁶⁸and the Genome-to-Genome Distance Calculator (GGDC 2.1; BLAST + method)⁶⁹, respectively. A high-resolution species tree was generated using the autoMLST pipeline (https://automlst.ziemertlab.com)⁷⁰. The resulting tree was reconstructed using the maximum-likelihood algorithm in MEGA X, with Nocardia uniformis NBRC 13702^T (GCF_001613345) serving as an outgroup. To further confirm the taxonomic placement of Streptomyces sp. JHD1^T, phylogenomic analysis was performed using TYGS (https://tygs.dsmz.de)⁷¹. Secondary metabolite biosynthetic potential was explored using antiSMASH version 7.0⁷². Analyses included KnownClusterBlast, ActiveSiteFinder, ClusterBlast, ClusterPfam, and SubClusterBlast modules. Additionally, BGCs were manually examined in relation to the chemical structure of the compound isolated from Streptomyces sp. JHD1^T.

Gene prediction and in Silico NRPS domain analysis

The draft genome sequence of Streptomyces sp. JHD1^T was annotated using Bakta v1.10.4⁷³ with default parameters to identify protein-coding regions essential for downstream functional characterization. The protein sequences were analyzed for nonribosomal peptide synthetase (NRPS) domains with a focus on cyclization (Cy), adenylation (A), and thiolation (T) domain architectures using InterPro⁷⁴.

Comparative analysis of HomE protein sequences

HomE protein sequences from Streptomyces sp. JHD1^T and S. clavuligerus ATCC 27064 were aligned using Clustal omega⁷⁵ with default parameters, and the alignment was visualized using ESPript 3.0⁷⁶. For quantitative comparison, pairwise global alignment was performed using EMBOSS Needle⁷⁷ with default parameters to calculate sequence identity and similarity percentages. This analysis assessed the evolutionary relationship between the thiolutin biosynthetic machinery of our marine-derived strain and the characterized dithiolopyrrolone pathway from S. clavuligerus.

General chemical experimental procedures

FT-IR spectra were measured on a Bruker ALPHA spectrometer. UV spectra were performed in MeOH (HPLC-grade from Duksan) on a V-730 UV-Visible spectrophotometer, JASCO. Optical rotations were measured on a JASCO P-2000 digital polarimeter. NMR spectra were recorded either on a Bruker Avance III™ HD 500 MHz spectrometer or Bruker Avance III™ HD 400 MHz NMR spectrometer using a deuterated solvent as an internal standard, such as CD₃OD, CDCl₃, DMSO-d₆ (from Cambridge Isotope Laboratiories, Inc.). HRESIMS data were obtained from Bruker MicrOTOF spectrometer. Column chromatography was performed on Sephadex LH-20 (from GE Healthcare) column using 100% MeOH as an eluent. HPLC was performed on a Dionex-Ultimate 3000 series equipped with a binary pump, an autosampler, and diode array detector. Preparative HPLC was performed on either a Sunfire C18 OBD column from Waters (particle size 10 µm, diam. 19 mm × 250 mm) or a Sunfire C18 OBD column from Waters (particle size 5 µm, diam. 19 mm × 150 mm) at a flow rate of 10 mL/min and at the detection wavelength of 210 nm.

Large scale culture and isolation of secondary metabolites

Streptomyces sp. JHD1^T was grown on ISP 2 seawater agar at 30 °C for 4 days. The stock culture was inoculated into 250 ml Erlenmeyer flasks, containing 150 ml of seed medium (ISP 2 seawater broth, comprising (w/v) 0.4% dextrose, 1.0% malt extract, and 0.4% yeast extract in distilled water, pH 6.5). The seed culture of Streptomyces sp. JHD1^T was cultivated on a rotary shaker (200 rpm) at 30 °C for 4 days. Then, the seed culture (20 flasks) was transferred into 80 × 1 L Erlenmeyer flasks, which each contained 250 ml of ISP 2 seawater broth, pH 6.5. The production culture (20 L) was cultivated for 14 days at 30 °C on rotary shakers (160 rpm). Then the whole culture was extracted with an equal volume of EtOAc (from Duksan) three times and EtOAc was dried over anhydrous Na₂SO₄ (from Fisher Chemical). EtOAc was then evaporated to dryness to yield brown gum (2.3 g), which was triturated with MeOH (from Duksan) to give a solid (1.4 g) and filtrate (0.91 g). The filtrate was passed through a Sephadex LH20 column (4.5 cm × 40 cm), eluted with 100% MeOH, and 40–50 mL fractions were collected. Fractions were analyzed by HPLC and then combined based on HPLC analyses to give four main fractions (F1-F4). F1 and F4 were discarded due to no compound of interest. The second fraction (F2, 0.13 g) was purified by a preparative HPLC, using a SunFire C18 column (diam 19 mm × 150 mm), eluted with a linear gradient system of 5% − 30% CH₃CN in water over 20 min to yield compounds 5 (t_R 11.3 min, 18.1 mg), 4 (t_R 13.4 min, 93.7 mg), 6 (t_R 15.5 min, 99.2 mg), and 1 (t_R 16.8 min, 8.6 mg). The third fraction (F3, 0.46 g) was purified by a preparative HPLC, using a SunFire C18 column (diam 19 mm × 250 mm), eluted with the same linear gradient system as for F2 to afford compound 5 (t_R 21.8 min, 24.0 mg).

The solid (1.4 g) was purified by a preparative HPLC, using a SunFire C18 column (diam 19 mm × 250 mm), eluted with a step gradient system of 5% − 35% CH₃CN in water over 20 min, 35% CH₃CN in water 2 min, 55% − 100% CH₃CN in water over 3 min, and 100% CH₃CN for 10 min, to furnish 3 (t_R 14.7 min, 30.7 mg), 4 (t_R 18.4 min, 13.1 mg), 6 (t_R 20.6 min, 10.7 mg), 1 (t_R 21.8 min, 0.13 g), and 2 (t_R 24.9 min, 10.9 mg).

2,2-Dioxidothiolutin (2)

Orange prisms (from MeOH / H₂O); HRESIMS m/z 282.9813 [M + Na]⁺ (calcd for C₈H₈ N₂NaO₄S₂, 282.9818).

Single X-ray diffraction measurement was performed on a Bruker D8 venture equipped with Cu Ka as the radiation source at 273 K, mounted on Cu in Parabar 10,312. The data was reduced and refined by using SAINT (V8 40B). The structure was solved by using Olex2 program. All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were included in calculated positions with isotropic thermal parameters which were 1.2 and 1.5 times the equivalent isotropic thermal parameters of their parent atoms. All hydrogen atoms attached to N atom were located in the different fourier map.

X-ray data of 2 : C₈H₈O_3.454S₂⋅ C₈H₈O_3.748S₂, M_r = 507.77, Triclinic, dimensions: 0.05 × 0.05 × 0.05 mm³space group P1; a = 8.3690(5) Å; b = 10.8542(7) Å; c = 12.1457(7) Å; α = 82.214(2)°; β = 73.847(2)°; γ = 75.547(2)°; V = 1019.19(11) ų; Z = 2; T = 273 K; λ(Cu-Kα) = 1.54178 Å; µ(Cu-Kα) = 4.75 mm^− 1; ρ_calc = 1.655 g.cm^− 3; reflections collected/independent : 35,528/3883 (R_int = 0.033); final R indices [I > 2σ (I)]: R₁ = 0.0371, wR₂ = 0.1051; final R indices for all data : R₁ = 0.0379, wR₂ = 0.1056. Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under the reference number CCDC2296168. The data can be obtained free of charge at www.ccdc.cam.ac.ul/conts/retrieving.html [or from CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033; email: deposit@ccdc.cam.ac.uk]. The cif file is given as supplementary information.

8-Amino-2 H-benz[e][1,3]oxazine-2,4(3 H)dione (3)

Colorless solid; IR (ν_max, cm^− 1) 3430, 1705, 1644, 1631, 1587, 1544, 1489, 1417, 1391, 1357, 1301, 1194, 1018, and 749;¹H (400 MHz, DMSO-d₆) 7.13 (1 H, t, J = 7.7 Hz, 6-H), 7.15 (1 H, d, J = 7.9 Hz, 7-H), 7.28 (1 H, dd, J = 6.6, 2.7 Hz, 5-H), 8.06 (2 H, NH), 10.38 (1 H, NH);¹³C NMR (100 MHz, DMSO-d₆) 116.0 (5-C), 118.3 (4a-C), 119.6 (7-C), 124.5 (6-C), 142.6 (8a-C), 144.8 (8-C), 159.7 (2-C), 166.3 (4-C); HRESIMS m/z 177.0303 [M − H]⁻ (calcd. for C₈H₅N₂O₃, 177.0306).

2-Hydroxy-3-methoxybenzamide (6)

Colorless solid; IR (ν_max, cm^− 1) 3420, 3213, 1665, 1463, 1452, 1439, 1420, 1377, 1245, 1059, 746, and 715;¹H (400 MHz, CD₃OD) 3.87 (3 H, s, 3-OCH₃), 6.84 (1 H, t, J = 8.1 Hz, 5-H), 7.11 (1 H, dd, J = 8.0, 1.3 Hz, 4-H), 7.39 (1 H, dd, J = 8.2, 1.4 Hz, 6-H);¹³C NMR (100 MHz, CD₃OD) 56.7 (3-OCH₃), 116.0 (4-C), 116.6 (1-C), 119.6 (5-CH), 121.1 (6-C), 142.6 (6-CH), 149.9 (3-C), 151.5 (2-C), 173.5 (7-C); HRESIMS m/z 166.0508 [M − H]⁻ (calcd. for C₈H₈NO₃, 166.0510).

Biological activity tests

Antimalarial assay against Plasmodium falciparum (K-1, multi-drug resistant strain) was performed by using the microculture radioisotope technique⁷⁸. Dihydroartemisinin and mefloquine were used as positive controls. Antibacterial activity against Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii was determined by the optical density microplate assay^79,80. Vancomycin and rifampicin were used as positive controls for Gram-positive bacteria and rifampicin and erythronmycin were used as positive controls for Gram-negative bacteria. Cytotoxicity against Vero cells was done by using the green fluorescent protein microplate assay (GFPMA)⁸¹. Ellipticine was used as a positive control. Cytotoxicity against MCF-7 (human breast cancer, ATCC HTC-22) and NCI-H187 (human small-cell lung cancer, ATCC CRL-5804) cell lines and antifungal activity against C. albicans ATCC 90028 were evaluated by using the resazurin microplate assay (REMA)⁸². Amphotericin B was used as the positive control for anti-C. albicans. Tamoxifen and doxorubicin were used as the positive controls for anti-MCF-7 activity. Doxorubicin and ellipticine were used as positive controls for anti-NCI-H187 activity. IC₅₀ value was reported for the concentration that inhibits 50% of tested cells in accordance with dose-response curve, which is plotting between compound concentrations and % inhibition. Minimum inhibitory concentration (MIC) showed the concentration that could inhibit 90% growth of bacteria. Maximum concentration of all samples is done at 50 µg/ml for all tests, except for antimalarial activity (at 10 µg/ml). 0.5% DMSO was used as negative control.

Accession number of Streptomyces sp. JHD1^T

The GenBank accession number for the complete 16S rRNA gene sequence of Streptomyces sp. JHD1^T is OM755759. The Whole Genome Shotgun projects for Streptomyces sp. JHD1^T has been deposited at GenBank under the accession JAPKFL000000000. The strain is deposited in the Thailand Bioresource Research Center (TBRC) and NITE Biological Resource Center (NBRC) for code numbers TBRC 15653^T and NBRC 115639^T, respectively (Figs. S36, S37).

ANI:

Average nucleotide identity

AAI:

Average amino-acid identity

dDDH:

Digital DNA-DNA hybridization

DAP:

Diaminopimelic acid

DSM:

German Collection of Microorganisms and Cell Cultures GmnH

EtOAc:

Ethyl acetate

FT-IR:

Fourier transformation infrared spectroscopy

GC:

Gas chromatography

ISP:

International Streptomyces Project

GFPMA:

Green fluorescent protein microplate assay

MeOH:

Methanol

MS:

Mass spectrometry

REMA:

Resazurin-based microplate assay

NMR:

Nuclear magnetic resonance

NRPS:

Non-ribosomal peptide synthetase

BGCs:

Biosynthetic gene clusters

T1PKS:

Type I polyketide synthase

T2PKS:

Type II polyketide synthase

T3PKS:

Type III polyketide synthase

TBRC:

Thailand Bioresource Research Center

NBRC:

NITE Biological Resource Center

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Authors and Affiliations

1. Department of Biology, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand

   Rawirat Pansomsuay & Chitti Thawai

2. Actinobacterial Research Unit, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand

   Poowadon Fukasem & Chitti Thawai

3. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phaholyothin Road, Khlong Nueng, Khlong Luang, 12120, Pathum Thani, Thailand

   Pattama Pittayakhajonwut & Chakapong Intaraudom

4. Thailand Bioresource Research Center (TBRC), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phaholyothin Road, Khlong Nueng, Khlong Luang, 12120, Pathum Thani, Thailand

   Chanwit Suriyachadkun

5. Program on Environmental Toxicology, Chulabhorn Graduate Institute, Bangkok, Thailand

   Montri Yasawong

6. State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China

   Ya-Wen He

7. Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand

   Somboon Tanasupawat

8. Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, 150001, People’s Republic of China

   Yang Qian

Contributions

R. P., P. F., P. P., C. I., M. Y., C. S.: Data curation, Formal analysis, Methodology, Writing – original draft. Y-W. H., Y. Q., and S. T.: Writing – review & editing. C. T.: Conceptualization, Formal analysis, Methodology, Formal analysis, Funding acquisition, Supervision, Writing – review & editing.

Corresponding author

Correspondence to Chitti Thawai.

Competing interests

The authors declare no competing interests.

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