OSMAC-Based Discovery and Biosynthetic Gene Clusters Analysis of Secondary Metabolites from Marine-Derived Streptomyces globisporus SCSIO LCY30

The one strain many compounds (OSMAC) strategy is an effective method for activating silent gene clusters by cultivating microorganisms under various conditions. The whole genome sequence of the marine-derived strain Streptomyces globisporus SCSIO LCY30 revealed that it contains 30 biosynthetic gene clusters (BGCs). By using the OSMAC strategy, three types of secondary metabolites were activated and identified, including three angucyclines, mayamycin A (1), mayamycin B (2), and rabolemycin (3); two streptophenazines (streptophenazin O (4) and M (5)); and a macrolide dimeric dinactin (6), respectively. The biosynthetic pathways of the secondary metabolites in these three families were proposed based on the gene function prediction and structural information. The bioactivity assays showed that angucycline compounds 1–3 exhibited potent antitumor activities against 11 human cancer cell lines and antibacterial activities against a series of Gram-positive bacteria. Mayamycin (1) selectively exhibited potent cytotoxicity activity against triple-negative breast cancer (TNBC) cell lines such as MDA-MB-231, MDA-MB-468, and Bt-549, with IC50 values of 0.60–2.22 μM.


Introduction
Marine-derived Streptomyces are an important source of secondary metabolites with biological activities and diverse chemical structures due to their unique habitats and metabolic pathways [1][2][3].Although genome sequencing and bioinformatic analyses revealed that there are about 20-40 biosynthetic gene clusters (BGCs) encoding the secondary metabolism in each Streptomyces genome, most of the gene clusters are "silent" or have low expression under the traditional culture conditions [4].The OSMAC strategy has proven to be an effective method for discovering new cryptic natural products in microorganisms through modification of the growth conditions without genetic manipulation of the producing strains.The OSMAC strategy was first developed by Zeeck's group in 2002 [5] and included methods such as changing nutritional conditions, trace elements, and physical parameters (i.e., pH and temperature), adding chemical composition (i.e., inhibitors and communication molecules), and co-culture [5][6][7].By using the OSMAC strategy, Zeeck's group discovered more than 100 secondary metabolites representing 25 structural types parameters (i.e., pH and temperature), adding chemical composition (i.e., inhibitors and communication molecules), and co-culture [5][6][7].By using the OSMAC strategy, Zeeck's group discovered more than 100 secondary metabolites representing 25 structural types from 6 microorganisms [5].In recent years, the OSMAC strategy has a racted large number of scholars, who apply it to the study of microbial secondary metabolites discovery because it has the advantages of easy operation, short cycle, low cost, and remarkable results.For instance, three rare anti-infective 22-membered macrolactone polyketides were isolated from Streptomyces sp.strain C34 using a range of cultivation media [8].Twelve new cytochalasins were discovered from the fungus Phomopsis sp.QYM-13 by changing the media nutrients [9]; a coculture of Streptomyces sp. with Pandoraea sp.produced antitumor 6-deoxy-α-L-talopyranose-bearing aromatic metabolites [10]; and so on.
While searching for new secondary metabolites of anti-infective and anti-tumor bioactivities from marine actinomycetes, we encountered a marine-derived actinomycete Streptomyces globisporus SCSIO LCY30.By using the OSMAC strategy, three angucyclines mayamycin A-B (1-2) and rabelomycin (3), two streptophenazines (4)(5), and a macrolide dimeric dinactin (6) were produced by Streptomyces globisporus SCSIO LCY30.Compounds 1-3 exhibited potent cytotoxicity against several human cancer cell lines and antibacterial activity against a series of Gram-positive bacteria.Herein, the whole genome sequence of Streptomyces globisporus SCSIO LCY30, the isolation, structure elucidation, and bioactivity of compounds 1-6, and the biosynthetic pathway analysis of these secondary metabolites were reported.

Identification and Genome Analysis of the Strain SCSIO LCY30
SCSIO LCY30 is a marine-derived actinomycete from Weizhou Island, Guangxi Beibu Gulf, China.Analysis of the 16S rDNA and morphological characteristics suggested that SCSIO LCY30 was likely a Streptomyces sp.The 16S rRNA gene sequence was deposited in GenBank under accession number OR342326.The whole genome sequencing of SCSIO LCY30 was carried out by PacBio RS and Illumina sequencing technologies.The genome sequence results showed that SCSIO LCY30 has a linear chromosome with the length of 8,077,634 bp and that the average G+C content is 71.56%.The genome was predicted to contain 6990 protein-coding genes, 18 rRNA genes, and 66 tRNA genes (Figure 1).By using autoMLST to construct a phylogenomic tree of SCSIO LCY30 in the "denovo mode" with the default settings [11], the results showed that the sequence similarities be-tween SCSIO LCY30 and Streptomyces globisporus C-1027 (accession number: GCF 000261345, ANI: 97.5%, AAI: 96.77%) were the highest (Figure S1).Based on phylogenomic analysis of the whole genome sequences, the strain SCSIO LCY30 was identified as Streptomyces globisporus.To identify the secondary metabolite BGCs of SCSIO LCY30, the whole genome sequence was analyzed by antiSMASH version 7.1.0online tools [12].The analysis results revealed that there are at least 30 putative BGCs, including 3 nonribosomal peptide clusters (NRPS), 2 polyketide clusters (Type I or Type III PKS), 5 hybrid PKS-NRPS clusters, 4 terpene clusters, and several other gene clusters, such as butyrolactone, lassopeptide, ribosomal peptide (RiPP), and so on (Table S1).

Production of Angucyclines and Streptophenazines via OSMAC Strategies
In order to explore the secondary metabolites produced by SCSIO LCY30, we used conventional actinomycetes media, such as P2 [13], HMT [14], etc., to ferment the SCSIO LCY30 strain; at first, very few secondary metabolites were detected (Figure 2).However, the genome analysis of the SCSIO LCY30 strain showed that it contained 30 BGCs, which indicated that the strain had the potential to be further explored.The OSMAC strategy has proven to be an effective method for discovering new cryptic natural products, and the change in nutrient regimes is regarded as one of the effective methods in the OSMAC strategy [6].Five other different culture media (N4 [15], Am2ab [16], Am3 [16], Am6-1 [17], and SCAS [18]) were used to ferment the SCSIO LCY30 strain, and the fermentation extracts were analyzed by HPLC.Fortunately, the UV absorption peaks of two groups, like angucyclines and streptophenazines, were mainly observed in three culture media (N4, Am3, and Am6-1) (Figure 2).The fermentations carried out in Am6-1 media by adding 2% XAD16N resins presented optimum yields.Among the 30 putative BGCs of SCSIO LCY30, cluster 20 is predicted to be a T1PKS, T2PKS, and phenazines hybrid cluster.Based on the above results and the analysis by antiSMASH, we speculated that cluster 20 might be activated due to the co-expression of adjacent gene clusters in bacteria [19,20].
By using autoMLST to construct a phylogenomic tree of SCSIO LCY30 in the "denovo mode" with the default se ings [11], the results showed that the sequence similarities between SCSIO LCY30 and Streptomyces globisporus C-1027 (accession number: GCF 000261345, ANI: 97.5%, AAI: 96.77%) were the highest (Figure S1).Based on phylogenomic analysis of the whole genome sequences, the strain SCSIO LCY30 was identified as Streptomyces globisporus.To identify the secondary metabolite BGCs of SCSIO LCY30, the whole genome sequence was analyzed by antiSMASH version 7.1.0online tools [12].The analysis results revealed that there are at least 30 putative BGCs, including 3 nonribosomal peptide clusters (NRPS), 2 polyketide clusters (Type I or Type III PKS), 5 hybrid PKS-NRPS clusters, 4 terpene clusters, and several other gene clusters, such as butyrolactone, lassopeptide, ribosomal peptide (RiPP), and so on (Table S1).

Production of Angucyclines and Streptophenazines via OSMAC Strategies
In order to explore the secondary metabolites produced by SCSIO LCY30, we used conventional actinomycetes media, such as P2 [13], HMT [14], etc., to ferment the SCSIO LCY30 strain; at first, very few secondary metabolites were detected (Figure 2).However, the genome analysis of the SCSIO LCY30 strain showed that it contained 30 BGCs, which indicated that the strain had the potential to be further explored.The OSMAC strategy has proven to be an effective method for discovering new cryptic natural products, and the change in nutrient regimes is regarded as one of the effective methods in the OSMAC strategy [6].Five other different culture media (N4 [15], Am2ab [16], Am3 [16], Am6-1 [17], and SCAS [18]) were used to ferment the SCSIO LCY30 strain, and the fermentation extracts were analyzed by HPLC.Fortunately, the UV absorption peaks of two groups, like angucyclines and streptophenazines, were mainly observed in three culture media (N4, Am3, and Am6-1) (Figure 2).The fermentations carried out in Am6-1 media by adding 2% XAD16N resins presented optimum yields.Among the 30 putative BGCs of SCSIO LCY30, cluster 20 is predicted to be a T1PKS, T2PKS, and phenazines hybrid cluster.Based on the above results and the analysis by antiSMASH, we speculated that cluster 20 might be activated due to the co-expression of adjacent gene clusters in bacteria [19,20].

Isolation and Characterization of Compounds 1-6
A 20-liter fermentation of SCSIO LCY30 was performed in AM6-1 media adding XAD16N resins, which resulted in the isolation and characterization of compounds 1-6.HRESIMS analysis showed that the molecular formula of compounds 1 and 2 was found to be C 26 H 26 NO 7 and C 25 H 23 NO 7 , respectively.Combined with 1D ( 1 H, 13 C) NMR, 2D (HMBC, HSQC, NOESY, and COSY) NMR spectroscopic data, and HRESIMS data, the structures of compounds 1-2 were identified as the angucycline compounds mayamycin (1) and mayamycin B (2), according to the literature, respectively [21,22].The molecular formula of compound 3 was found to be C 19 H 14 O 6 on the basis of the HRESIMS peak at m/z 339.0858 [M+H] + and 361.0690 [M+Na] + .Compared with the reported literature, the structure of compound 3 was identified as the angucycline compound rabelomycin (3) based on its HRESIMS data and 1D ( 1 H, 13 C) NMR spectroscopic data [23].The 1 H and 13 C NMR spectra of compound 4 showed two groups of signals with an integration ratio of 1:4; their structures were identified as streptophenazine O (4) and dimeric dynactin (6) based on the comparisons with the previous literature [24,25].Like compound 4, the 1 H and 13 C NMR spectra of compound 5 also showed two groups of signals with an integration ratio of 1:5; their structures were identified as streptophenazine M (5) and dimeric dynactin (6) [24,25].The structures of these compounds are shown in Figure 3.

Isolation and Characterization of Compounds 1-6
A 20-liter fermentation of SCSIO LCY30 was performed in AM6-1 media adding XAD16N resins, which resulted in the isolation and characterization of compounds 1-6.HRESIMS analysis showed that the molecular formula of compounds 1 and 2 was found to be C26H26NO7 and C25H23NO7, respectively.Combined with 1D ( 1 H, 13 C) NMR, 2D (HMBC, HSQC, NOESY, and COSY) NMR spectroscopic data, and HRESIMS data, the structures of compounds 1-2 were identified as the angucycline compounds mayamycin (1) and mayamycin B (2), according to the literature, respectively [21,22].The molecular formula of compound 3 was found to be C19H14O6 on the basis of the HRESIMS peak at m/z 339.0858 [M+H] + and 361.0690 [M+Na] + .Compared with the reported literature, the structure of compound 3 was identified as the angucycline compound rabelomycin (3) based on its HRESIMS data and 1D ( 1 H, 13 C) NMR spectroscopic data [23].The 1 H and 13 C NMR spectra of compound 4 showed two groups of signals with an integration ratio of 1:4; their structures were identified as streptophenazine O (4) and dimeric dynactin (6) based on the comparisons with the previous literature [24,25].Like compound 4, the 1 H and 13 C NMR spectra of compound 5 also showed two groups of signals with an integration ratio of 1:5; their structures were identified as streptophenazine M (5) and dimeric dynactin (6) [24,25].The structures of these compounds are shown in Figure 3.

Proposed Biosynthetic Pathway of Angucycline Compounds 1-3
Type II polyketide synthases are responsible for the biosynthesis of the angucycline natural products skeleton.The biosynthesis of angucycline compounds 1-3 also follows this paradigm.In the genome of Streptomyces SCSIO LCY30, only one type II polyketides gene cluster was found, which showed high similarity with the reported mayamycin BGC of Streptomyces sp.120454 [22].The putative mry gene cluster (accession number: OR345931) covers a 23.2 kb contiguous DNA region, including six type II PKS genes, six sugar biosynthetic genes, five modified genes, a glycotransferase gene, one transport gene, one regulatory gene, and two other genes (Figure 4a, Table S2).

Proposed Biosynthetic Pathway of Streptophenazines O and M (4-5)
Streptophenazines are 1,6-disubstituted phenazines with a long alkyl chain at the C-6 position.The biosynthetic gene cluster of streptophenazines was identified in Streptomyces sp.CNB-091 [28].In the BGCs of Streptomyces globisporus SCSIO LCY30, cluster 20 is a T1PKS, T2PKS, and phenazines hybrid gene cluster.In the downstream of cluster 20 in Streptomyces sp.LCY30, a series of genes showed high similarity to those of the streptophenazine BGCs in Streptomyces sp.CNB-091 (Figure 5a and Table S3); we named it as an spn gene cluster (accession number: OR345932).The biosynthesis of the streptophenazines was divided into two parts: the phenazine skeleton structure synthesized by a set of contiguous phenazine core genes (spn16-21) and the alkyl chain at the C-6 position of streptophenazines synthesized by type I PKS genes (spn4-11).We proposed the biosynthetic pathway of streptophenazines O and M (4-5) produced in Streptomyces globisporus SCSIO LCY30 as follows (Figure 5b): phenazine-1,6-dicarboxylic acid (PDC) displays the ability to form a series of high-activity phenazine derivatives, which is also a key start unit for the biosynthesis of streptophenazines.Chorismic acid derived from the shikimate pathway in bacteria serves as a common branching point of PDC.The formation of 2-amino-4-deoxychorismic acid (ADIC) from chorismic acid is catalyzed by Spn18 (PhzE), a homolog of anthranilate synthase.Spn19 (PhzE) is predicted to be the homolog of isochorismatase, which could cleave the ADIC to trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) [29].Then, under the catalyzation of Spn19 (PhzE), the homolog of 2,3-Dihydro-3-hydroxylanthranilate isomerase, 6-amino-5-oxocyclohex-2-ene-1-carboxylic acid (AO-CHC), is formed [30].Spz21 (phenazine biosynthesis protein, PhzA/B) could catalyze the formation of hexahydrophenazine-1,6-dicarboxylic acid (HHPDC).The homolog of pyridoxamine 5′-phosphate oxidase, Spz16 (PhzG), catalyzes HHPDC to undergo a two-step oxidation reaction to generate PDC [29].One of the most interesting aspects in the biosynthesis of streptophenazines is the PKS extension at the C-6 position.It was deduced that PDC served as the starter unit and that intermediate 17 derived from the fatty acid biosynthetic pathway acted as the extension unit for the PKS extension.The carbonyl group was reduced to the hydroxyl group at C-1 ′ by Spn7 (ketoreductase) in intermediate 19; then, under the catalyzation of Spn8 (thioesterase), the polyketone chain was released to produce intermediate 20 [28].Finally, streptophenazines O and M (4-5) were formed under the catalyzation of methyltransferase Spn25 or Spn27 [28].However, the catalytic order of those two methyltransferases to the carboxyl group on the C-1 and C2' position is unclear, and more in vivo evidence is required to identify the BGC of streptophenazines.

Proposed Biosynthetic Pathway of Macrolide Dimeric Dinactin (6)
Considering the structural similarity between dinactin and nonactin, we speculated the biosynthetic pathway of dimeric dinactin (6) based on the biosynthetic characteristics of nonactin.(Figure 6) The structure of dimeric dynactin is composed of nonactic acid (23) and homononactic acid (24).The biosynthetic gene cluster of nonactin (non) in Streptomyces griseus DSM40695 revealed a novel type of PKS for polyketide biosynthesis that consists of five type II ketoacyl synthases (KSs) and four ketoreductases (KRs) but lacks acyl carrier protein (ACP) [31].Using local Blast, a gene cluster similar to a non-gene cluster was found in the SCSIO LCY30 genome; we named it as a din gene cluster (accession number: OR351017), which covers a 12.0 kb contiguous DNA region, including ten open reading frames (ORFs) (Table S4).Acetyl CoA or Malonyl CoA acts as a start unit for the biosynthesis of the polyketide chain 21 and 22 under the catalysis of KS and KR [32].Then, it is proposed that the furan cyclic formation is accomplished by the NonS (enoyl-CoA hydratase) homolog Dyn4.Dyn5 (homolog of nonactic acid: CoASH ligase, NonL) is responsible for the transformation of 23/(24) to CoA esters 25/(26) [33,34].Finally, the condensation between the -OH group of the distal nonactyl unit of 27 and the acyl-S-KS carbonyl group of 28 occurred, and the -OH group of the distal nonactyl unit of 28 and the acyl-S-KS carbonyl group of 27 also occurred to form dimeric dinactin (6) [34].Interestingly, this C-O bond condensation dimeric dinactin (6) might be catalyzed by the ketoacyl synthases Dyn10 (NonK homolog) and Dyn9 (NonJ homolog) in nonactin biosynthesis [34].However, the specific biosynthetic pathway of dimeric dynactin (6) and the catalytic mechanism of the enzyme need to be confirmed by further experimental data.One of the most interesting aspects in the biosynthesis of streptophenazines is the PKS extension at the C-6 position.It was deduced that PDC served as the starter unit and that intermediate 17 derived from the fa y acid biosynthetic pathway acted as the extension unit for the PKS extension.The carbonyl group was reduced to the hydroxyl group at C-1′ by Spn7 (ketoreductase) in intermediate 19; then, under the catalyzation of Spn8 (thioesterase), the polyketone chain was released to produce intermediate 20 [28].Finally, streptophenazines O and M (4-5) were formed under the catalyzation of methyltransferase Spn25 or Spn27 [28].However, the catalytic order of those two methyltransferases to the carboxyl group on the C-1 and C2' position is unclear, and more in vivo evidence is required to identify the BGC of streptophenazines.

Proposed Biosynthetic Pathway of Macrolide Dimeric Dinactin (6)
Considering the structural similarity between dinactin and nonactin, we speculated the biosynthetic pathway of dimeric dinactin (6) based on the biosynthetic characteristics of nonactin.(Figure 6) The structure of dimeric dynactin is composed of nonactic acid (23) and homononactic acid (24).The biosynthetic gene cluster of nonactin (non) in Streptomyces griseus DSM40695 revealed a novel type of PKS for polyketide biosynthesis that consists of five type II ketoacyl synthases (KSs) and four ketoreductases (KRs) but lacks acyl carrier protein (ACP) [31].Using local Blast, a gene cluster similar to a non-gene cluster was found in the SCSIO LCY30 genome; we named it as a din gene cluster (accession number: OR351017), which covers a 12.0 kb contiguous DNA region, including ten open reading frames (ORFs) (Table S4).Acetyl CoA or Malonyl CoA acts as a start unit for the biosynthesis of the polyketide chain 21 and 22 under the catalysis of KS and KR [32].Then, it is proposed that the furan cyclic formation is accomplished by the NonS (enoyl-CoA hydratase) homolog Dyn4.Dyn5 (homolog of nonactic acid: CoASH ligase, NonL) is responsible for the transformation of 23/(24) to CoA esters 25/(26) [33,34].Finally, the condensation between the -OH group of the distal nonactyl unit of 27 and the acyl-S-KS carbonyl group of 28 occurred, and the -OH group of the distal nonactyl unit of 28 and the acyl-S-KS carbonyl group of 27 also occurred to form dimeric dinactin (6) [34].Interestingly, this C-O bond condensation dimeric dinactin (6) might be catalyzed by the ketoacyl synthases Dyn10 (NonK homolog) and Dyn9 (NonJ homolog) in nonactin biosynthesis [34].However, the specific biosynthetic pathway of dimeric dynactin (6) and the catalytic mechanism of the enzyme need to be confirmed by further experimental data.

GNPS Molecular Network Analysis of Streptophenazines
Due to the instability of streptophenazines when mixed with compound 6 (UV absorption below 210 nm), we had only previously identified two streptophenazine structures (4-5) from SCSIO LCY30.To explore the possible streptophenazine analogues that might be neglected in the initial visual analysis, the crude extract of Streptomyces globisporus SCSIO LCY30 was fractionated by positive ion mode LC-MS/MS; then, a GNPS molecular network was generated using the tandem mass spectra of the two isolated streptophenazines (4-5) as an anchor.The network identified 17 nodes corresponding to streptophenazine-type compounds (Figure 7 and Table S6).Compared with the structures of the streptophenazine compounds we reviewed in recent years (Figure S3) [24,28,[35][36][37][38][39], the possible structures of the compounds corresponding to these nodes were speculated based on molecular weight and mass spectrum cleavage (Table S5).In the molecular network of streptophenazines, two nodes were identified as streptophenazin M (5) (m/z = 439.226)and O (4) (m/z = 425.21) in this study; the molecular weights of these 10 nodes highlighted in green were consistent with those reported for the streptophenazine compounds.In addition, four nodes marked in blue might be new compounds due to there being no matching streptophenazines compounds found for these molecular weights, and we proposed the possible structure according to the structure feature of streptophenazines (Table S6).

GNPS Molecular Network Analysis of Streptophenazines
Due to the instability of streptophenazines when mixed with compound 6 (UV absorption below 210 nm), we had only previously identified two streptophenazine structures (4-5) from SCSIO LCY30.To explore the possible streptophenazine analogues that might be neglected in the initial visual analysis, the crude extract of Streptomyces globisporus SCSIO LCY30 was fractionated by positive ion mode LC-MS/MS; then, a GNPS molecular network was generated using the tandem mass spectra of the two isolated streptophenazines (4-5) as an anchor.The network identified 17 nodes corresponding to streptophenazine-type compounds (Figure 7 and Table S6).Compared with the structures of the streptophenazine compounds we reviewed in recent years (Figure S3) [24,28,[35][36][37][38][39], the possible structures of the compounds corresponding to these nodes were speculated based on molecular weight and mass spectrum cleavage (Table S5).In the molecular network of streptophenazines, two nodes were identified as streptophenazin M (5) (m/z = 439.226)and O (4) (m/z = 425.21) in this study; the molecular weights of these 10 nodes highlighted in green were consistent with those reported for the streptophenazine compounds.In addition, four nodes marked in blue might be new compounds due to there being no matching streptophenazines compounds found for these molecular weights, and we proposed the possible structure according to the structure feature of streptophenazines (Table S6).S6).
The above data showed that the SCSIO LCY30 strain had great potential to produce streptophenazines.In recent years, based on the wide range of biological properties and medical applications of phenazines, the synthesis, structural modification, and structureactivity relationship of phenazines have been studied extensively [40][41][42], while the biological activity of streptophenazine compounds has rarely been reported to date.Among the streptophenazine compounds that have been reported, (-)-Streptophenazine B showed moderate activity against methicillin-resistant Staphylococcus aureus, with an MIC value of 4.2 µg/mL [24].Streptophenazines C and H showed moderate inhibitory activity against Bacillus subtilis (MIC value of 15.6 µg/mL), and streptophenazine C was also active against Staphylococcus lentus (MIC value of 46.9 µg/mL) [35].And other biological activities of streptophenazine-type compounds could be explored in the future.S6).
The above data showed that the SCSIO LCY30 strain had great potential to produce streptophenazines.In recent years, based on the wide range of biological properties and medical applications of phenazines, the synthesis, structural modification, and structureactivity relationship of phenazines have been studied extensively [40][41][42], while the biological activity of streptophenazine compounds has rarely been reported to date.Among the streptophenazine compounds that have been reported, (-)-Streptophenazine B showed moderate activity against methicillin-resistant Staphylococcus aureus, with an MIC value of 4.2 µg/mL [24].Streptophenazines C and H showed moderate inhibitory activity against Bacillus subtilis (MIC value of 15.6 µg/mL), and streptophenazine C was also active against Staphylococcus lentus (MIC value of 46.9 µg/mL) [35].And other biological activities of streptophenazine-type compounds could be explored in the future.

Antibacterial Activity of Compounds 1-3
As high amounts of compounds 1-3 were available, the antibacterial activities of angucycline compounds 1-3 against several Gram-positive bacteria were first tested.The minimum inhibitory concentration (MIC) values are shown in Table 1.The results showed that compounds 1-3 exhibited inhibitory activity against many Gram-positive bacteria, such as Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Enterococcus gallinarum, Bacillus subtilis, and methicillin-resistant Staphylococcus aureus (MRSA), as summarized in Table 1.It is worth noting that compounds 1-3 exhibited potent antibacterial activity against Micrococcus luteus ML01; the MIC values were 1 µg/mL, 1 µg/mL, and 2 µg/mL, respectively.Vancomycin and ampicillin were used as positive controls.

Tumor Cytotoxic Activity of Compounds 1-3
Given that angucycline compounds have potent antitumor activities, the half inhibitory concentration of angucycline compounds 1-3 against fourteen different human cell lines were then tested.The IC 50 values are shown in Table 2. Compounds 1-3 also showed potent in vitro cytotoxicity against fourteen human cell lines; the IC 50 values were in a range of 0.60~5.98µM, 2.05~7.16µM, and 1.57~16.13µM, respectively.Notably, the tumor cytotoxic activities against eight human cancer cell lines of mayamycin (1) was superior to that of mayamycin B (2), suggesting that the N-methyl group is important for its antitumor activity.In addition, the cytotoxicity of rabelomycin (3) against the colon cancer cell line SW480 (IC 50 = 1.57µM) was lower than that against the normal intestinal epithelial cell line NCM460 (IC 50 = 7.05 µM); the selective index (SI) was 4.49, which is expected to be developed as an anti-colorectal cancer drug lead.
Triple-negative breast cancer (TNBC), once known as "the most malignant breast cancer", is a great challenge for clinical management due to the lack of therapeutic targets and limited therapeutic drugs.The anti-TNBC activities of compounds 1-3 have not been reported.In this study, we also tested the inhibitory activity of compounds 1-3 against TNBC cell lines.It is worth noting that compounds 1-3 exhibited potent in vitro cytotoxicity against TNBC cell lines such as MDA-MB-231, MDA-MB-468, and Bt-549; the IC 50 values were in a range of 0.60~2.22µM, 3.01~6.08µM, and 2.18~8.67µM, respectively.Among compounds 1-3, mayamycin (1) showed the best cytotoxicity activity against the TNBC cell lines MDA-MB-231, MDA-MB-468, and Bt-549 (IC 50 = 0.60 µM, 2.22 µM, and 1.88 µM); the selective indexes were 9.97, 2.69, and 3.18, which indicated that the toxicity and side effects of mayamycin (1) on normal breast cells were relatively weak and that it has the potential to become a drug lead compound for the treatment of TNBC.

General Experiment Procedure
HRESIMS and HRESIMS/MS spectra were measured by a MaXis 4G UHR59 TOFMS spectrometer.All 1D and 2D NMR spectra were acquired by a Bruker Ascend 700 spectrometer (Bruker Company, Karlsruhe, Germany) with TMS as the internal standard (Sigma-Aldrich Inc., Vienna, Austria) Silica gel (100−200 mesh and 200−300 mesh; Yantai Jiangyou Silica Gel Development company, Yantai, China) was used for column chromatography.Analytic HPLC was performed by an Agilent 1260 HPLC system equipped with a G1311C isocratic pump and an Agilent G1315D diode array detector (DAD), using a reversed-phase column Basic C18 120A (Agilent company, 4.6 × 250 mm, 5 µm, Santa Clara, CA, USA).Semipreparative HPLC was performed by an Agilent 1260 HPLC system equipped with a 1110 isocratic pump and a 1430 DAD detector, using an ODS-A column (YMC company, 10 mm × 250 mm, 5 µm, Kyoto, Japan).

Producing Strain and Genome Scanning
The SCSIO LCY30 strain was isolated from Weizhou Island, Guangxi Beibu Gulf, China.The genome sequencing of SCSIO LCY30 was performed by 2nd Illumina sequencing technologies and 3rd PacBio RS platforms in Shanghai BIOZERON biotechnology CO., LTD (Shanghai, China).

Phylogenetic Construction and Bioinformatic Analysis
The phylogenomic tree of the SCSIO LCY30 whole genome sequences was completed by autoMLST in the "denovo mode" with default settings [11], and the phylogenomic tree was beautified by iTOL software (version 6.7.2) [43].The average amino acid identity of the different genomes was determined by ANI calculator [44].The secondary metabolite BGCs of SCSIO LCY30 were identified and analyzed using the online software antiSMASH version 7.1.0[12].The sequences alignment of the gene clusters was completed by clinker software (https://cagecat.bioinformatics.nl/tools/clinker,accessed on 9 May 2023).

Purification and Characterization of Compounds
About 20 L of fermentation culture was obtained, and the XAD16N resins were separated from the fermentation broth through a sieve.The fermentation broth was centrifuged at 3500 rpm for 15 min to separate the supernatant and the mycelia.The mycelia were extracted by acetone, and the XAD16N resins were extracted using EtOH by ultrasonication three times.The extract solution was combined, and 24 g crude material was obtained by vacuum concentration.The crude material was subjected to a silica gel CC using gradient elution with CHCl

Antibacterial Activity Assays
An antibacterial activity test of compounds 1-3 was conducted according to a standard protocol provided by the Clinical and Laboratory Standards Institute (CLSI) [45,46]  C for 16-18 h, the microdilution instrument was used to detect the minimum concentration of each tested compound that completely inhibited bacterial growth in the microdilution hole.All the assays were carried out in triplicate.

Cytotoxic Activity Assays
The cell growth inhibitory activities of compounds 1-3 against fourteen different human cell lines (human liver cell line LX-2; hepatocellular cancer line HEPG2; human intestinal epithelial cell line NCM460; colorectal cancer cell lines HCT116 and SW480; normal human breast cell line MCF-10A; breast cancer cell line MCF7; triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-468, and Bt-549; human umbilical vein endothelial cell line; lung cancer cell line A549; cervical cancer cell line Hela; and cholangiocarcinoma cell line RBE) were tested according to the previously published methods [47].SPSS software version 22.0 was used to test the IC 50 values.Cisplatin and adriamycin were used as positive control agents, and all the experiments were carried out in triplicate.

Conclusions
In summary, the whole genome sequence of marine-derived Streptomyces globisporus SCSIO LCY30 was acquired, and the bioinformatic analysis showed at least 30 BGCs encoding various secondary metabolites, among which cluster 20 contains an angucycline biosynthesis gene cluster and a phenazine biosynthesis gene cluster.Three angucyclines, including mayamycin (1), mayamycin B (2), rabelomycin (3), two streptophenazines (streptophenazines O (4) and M (5)), and macrolide dimeric dinactin (6), were isolated from the marine-derived Streptomyces globisporus SCSIO LCY30 by using the OSMAC strategy under different culture conditions.The antibacterial bioassay results showed that compounds 1-3 showed potent bioactivity against Micrococcus luteus, with MIC values of 1.0 to 2.0 µg/mL.Furthermore, compounds 1-3 showed potent in vitro cytotoxicity against 11 human cancer cell lines.Mayamycin (1) selectively exhibited potent cytotoxicity activity against TNBC cell lines such as MDA-MB-231, MDA-MB-468, and Bt-549 (IC 50 = 0.60 µM, 2.22 µM, and 1.88 µM); the selective indexes were 9.97, 2.69, and 3.18, respectively, which indicated that mayamycin (1) is a promising drug lead compound for the treatment of TNBC.Rabelomycin (3) selectively exhibited potent cytotoxicity against colon cancer cell line SW480 (IC 50 = 1.57µM), with the selective index of 4.49, which is expected to be developed as an anti-colorectal cancer drug lead.In addition, the biosynthetic pathways of compounds 1-6 were determined by in vivo and in silico analysis, which provides ideas for the subsequent biosynthetic mechanism studies of these compounds.This study not only serves as a new strain resource for antitumor drug development but also lays a foundation for the mining of the rest of the other biosynthetic gene clusters that encode natural products.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/md22010021/s1, Figure S1 S1: The antiSMASH-predicted BGCs for Streptomyces globisporus SCSIO LCY30, Table S2: Deduced functions of orfs in the mry BGC, Table S3: Deduced functions of orfs in the spn BGC, Table S4: Deduced functions of orfs in the din BGC, Table S5: Summary of 1 H and 13 C NMR data for compounds 1-2 (δ in ppm), Table S6: High resolution ESI MS/MS (HRESIMS/MS), retention time, and predicted chemical formula for nodes in streptophenazine cluster from GNPS molecular network (related to Figures 3 and 5).

Figure 1 .
Figure 1.The complete genomic graph of Streptomyces globisporus SCSIO LCY30.These six circles (from outer to inner) represent the size of the genome, CDs on positive chains, CDs on negative chains (different colors indicate different COG functional classifications for CDS), rRNA and tRNA, GC content, and GC skew value.

Figure 1 .
Figure 1.The complete genomic graph of Streptomyces globisporus SCSIO LCY30.These six circles (from outer to inner) represent the size of the genome, CDs on positive chains, CDs on negative chains (different colors indicate different COG functional classifications for CDS), rRNA and tRNA, GC content, and GC skew value.

Figure 2 .
Figure 2. Metabolite analyses HPLC of Streptomyces globisporus SCSIO LCY30 fermented in seven different culture media using OSMAC strategy.(The product peaks of compounds 1-5 have been marked, streptophenazines product peaks have been marked with *).

Figure 2 .
Figure 2. Metabolite analyses HPLC of Streptomyces globisporus SCSIO LCY30 fermented in seven different culture media using OSMAC strategy.(The product peaks of compounds 1-5 have been marked, streptophenazines product peaks have been marked with *).

Figure 7 .
Figure 7. Streptophenazines cluster of nodes from the GNPS molecular network (related to Figures S2 and S3 and TableS6).

Figure 7 .
Figure 7. Streptophenazines cluster of nodes from the GNPS molecular network (related to Figures S2 and S3 and TableS6).