Discovery of a Novel Chromone Enantiomer and the Precursors of Nonactic Acid from the Coral-Reef-Derived Streptomyces sp. SCSIO 66814

Three pairs of enantiomers (1–3)—the new 12R-aloesol (1a) and two new fatty acids (2 and 3)—and one new natural product (4) together three known compounds (5–7) were isolated from a coral-reef-derived Streptomyces sp. SCSIO 66814. Their structures were determined through extensive spectroscopic analysis, chiral analysis, and single-crystal X-ray diffraction data. Compounds 2 and 3 were presumed to be intermediates for further generating homononactic acid (5) and nonactic acid, and the latter two molecules were able to act as precursors to form macrotetrolides with remarkable biological activity. The isolation of related precursors, compounds 2–5, provided more evidence to support the proposal of a plausible biosynthetic pathway for nonactic acid and its homologs. Additionally, (+)-1 exhibited a weak activity against DPPH radicals.


Introduction
Nonactin and its homologs (Figure 1) are a class of ionophore antibiotics known as the macrotetrolides [1], which are composed of four monomers, the latter being either nonactic acid or homononactic acid.These antibiotics are mainly synthesized by Streptomyces strains, including Streptomyces griseus subsp.griseus ETH A7796.Nonactin exhibits significant antibacterial activity and has been shown to be effective in combating tumors and inhibiting the P-glycoprotein-mediated drug resistance in cancer cells [2].The biosynthetic pathway for nonactin was originally established by stable isotope feeding experiments with primary metabolites and more advanced precursors [3].However, some steps of the pathway remain unconfirmed, mainly due to a lack of direct isolation of intermediates in the pathway.The two important precursors, nonactic acid and homononactic acid, are assembled from acetate, propionate, and succinate through a pathway based on polyketide biosynthesis [4].Nevertheless, the precursors for nonactic acid and homononactic acid are not directly isolated from microorganisms to the best of our knowledge, instead relying mainly on chemical synthesis of substances such as (6R, 8R)-and (6S, 8S)-2-methyl-6, 8-dihydroxynon-2E-enoic acids [5] and 4, 6-diketoheptanoate derivatives [3].The discovery of intermediates plays an important role in establishing the complete biosynthetic process for nonactin and its analogs.As part of a continuous exploration in search of new metabolites of biological significance from marine microorganisms, we found a coral-reef-derived actinomycete Streptomyces sp.SCSIO66814.This strain possessed 57 biosynthetic gene clusters (BCGs), as determined using antiSMASH with a "loose" detection strictness setting, including a potential nonactin BGC (namely hmn) (Figure 2, Supplementary Materials: Tables S1 and  S2).Interestingly, the LC-MS/MS analysis, coupled with molecular networking (MN), revealed the potential production of nonactin, monactin, and trinatin by the strain (Supplementary Materials, Table S4, Figure S5), aligning with the hmn BGC.The result suggested that the MRA medium could activate the hmn BGC.Chemical investigation of the strain led to the isolation of three pairs of enantiomers (1-3)-(±)-aloesol (1) and two fatty acids (2 and 3)-and one new natural product (4), along with four known compounds (5-7) (Figure 3).Their structures were established by a combination of extensive spectroscopic analysis, chiral analysis, and single-crystal X-ray diffraction data.Herein, we report the isolation, structural elucidation, and biologic activity of these compounds in addition to the plausible biosynthetic pathway of compounds 2-5.As part of a continuous exploration in search of new metabolites of biological significance from marine microorganisms, we found a coral-reef-derived actinomycete Streptomyces sp.SCSIO66814.This strain possessed 57 biosynthetic gene clusters (BCGs), as determined using antiSMASH with a "loose" detection strictness setting, including a potential nonactin BGC (namely hmn) (Figure 2, Supplementary Materials: Tables S1 and S2).Interestingly, the LC-MS/MS analysis, coupled with molecular networking (MN), revealed the potential production of nonactin, monactin, and trinatin by the strain (Supplementary Materials, Table S4, Figure S5), aligning with the hmn BGC.The result suggested that the MRA medium could activate the hmn BGC.Chemical investigation of the strain led to the isolation of three pairs of enantiomers (1-3)-(±)-aloesol (1) and two fatty acids (2 and 3)-and one new natural product (4), along with four known compounds (5-7) (Figure 3).Their structures were established by a combination of extensive spectroscopic analysis, chiral analysis, and single-crystal X-ray diffraction data.Herein, we report the isolation, structural elucidation, and biologic activity of these compounds in addition to the plausible biosynthetic pathway of compounds 2-5.As part of a continuous exploration in search of new metabolites of biological significance from marine microorganisms, we found a coral-reef-derived actinomycete Streptomyces sp.SCSIO66814.This strain possessed 57 biosynthetic gene clusters (BCGs), as determined using antiSMASH with a "loose" detection strictness setting, including a potential nonactin BGC (namely hmn) (Figure 2, Supplementary Materials: Tables S1 and  S2).Interestingly, the LC-MS/MS analysis, coupled with molecular networking (MN), revealed the potential production of nonactin, monactin, and trinatin by the strain (Supplementary Materials, Table S4, Figure S5), aligning with the hmn BGC.The result suggested that the MRA medium could activate the hmn BGC.Chemical investigation of the strain led to the isolation of three pairs of enantiomers (1-3)-(±)-aloesol (1) and two fatty acids (2 and 3)-and one new natural product (4), along with four known compounds (5-7) (Figure 3).Their structures were established by a combination of extensive spectroscopic analysis, chiral analysis, and single-crystal X-ray diffraction data.Herein, we report the isolation, structural elucidation, and biologic activity of these compounds in addition to the plausible biosynthetic pathway of compounds 2-5.As part of a continuous exploration in search of new metabolites of biological significance from marine microorganisms, we found a coral-reef-derived actinomycete Streptomyces sp.SCSIO66814.This strain possessed 57 biosynthetic gene clusters (BCGs), as determined using antiSMASH with a "loose" detection strictness setting, including a potential nonactin BGC (namely hmn) (Figure 2, Supplementary Materials: Tables S1 and  S2).Interestingly, the LC-MS/MS analysis, coupled with molecular networking (MN), revealed the potential production of nonactin, monactin, and trinatin by the strain (Supplementary Materials, Table S4, Figure S5), aligning with the hmn BGC.The result suggested that the MRA medium could activate the hmn BGC.Chemical investigation of the strain led to the isolation of three pairs of enantiomers (1-3)-(±)-aloesol (1) and two fatty acids (2 and 3)-and one new natural product (4), along with four known compounds (5-7) (Figure 3).Their structures were established by a combination of extensive spectroscopic analysis, chiral analysis, and single-crystal X-ray diffraction data.Herein, we report the isolation, structural elucidation, and biologic activity of these compounds in addition to the plausible biosynthetic pathway of compounds 2-5.

Analysis of Molecular Networking
The MN displayed a large cluster presumably associated with nonactin and its homologs, comprising 33 nodes (Figure 4).Within this cluster, six nodes corresponding to parent ions at m/z 785.3, 768.5, 432.4,418.3 (and 400.8), and 404.8 were annotated by GNPS as compound NP-002862, monactin, compound NP-002857, bonactin, and compound NP-003108, respectively.These compounds are nonactin derivatives, corresponding to the hmn BGC.Furthermore, three key ions were detected in our total ion chromatogram (TIC) at 37.8, 39.4, and 42.2 min, respectively (Supplementary Materials, Figure S5).Detailed MS/MS analysis showed that the parent ion at m/z 754.4755 was nonactin, based on its four key fragments at m/z 553.3369, 369.2274, 185.1177, and 167.1058 (Supplementary Materials, Figure S6).Similarly, the parent ions at m/z 768.4909

Analysis of Molecular Networkin
The MN displayed a large cluster presumably associated with nonactin and its homologs, comprising 33 nodes (Figure 4).Within this cluster, six nodes corresponding to parent ions at m/z 785.3, 768.5, 432.4,418.3 (and 400.8), and 404.8 were annotated by GNPS as compound NP-002862, monactin, compound NP-002857, bonactin, and compound NP-003108, respectively.These compounds are nonactin derivatives, corresponding to the hmn BGC.Furthermore, three key ions were detected in our total ion chromatogram (TIC) at 37.8, 39.4, and 42.2 min, respectively (Supplementary Materials, Figure S5).Detailed MS/MS analysis showed that the parent ion at m/z 754.4755 was nonactin, based on its four key fragments at m/z 553.3369, 369.2274, 185.1177, and 167.1058 (Supplementary Materials, Figure S6).Similarly, the parent ions at m/z 768.4909 S5).

Structural Elucidation
Compound 1 was isolated as a colorless crystal (MeOH-H 2 O).It was assigned the molecular formula C 13 H 14 O 4 based on its HRESIMS (m/z 235.0968 [M + H] + , calcd for C 13 H 15 O 4 235.0965),suggesting seven degrees of unsaturation.The 1 H NMR spectrum (Table 1) showed three olefinic proton signals at δ H 6.65 (H-8, d, 2.4), δ H 6.63 (H-6, d, 2.4), and δ H 6.06 (H-3, s), one oxygenated methine proton signal at δ H 4.18 (H-12, dqd, 7.9, 6.2, 4.9), and two methyl proton signals at δ H 2.72 (H-14, s) and δ H 1.27 (H-13, d, 6.2).The 13 C and DEPT spectra displayed thirteen carbon signals, comprising two methyl carbons, one methylene carbon, four methine carbons, and six quaternary carbons.The NMR data were similar with those of aloesol [2-(2 ′ -hydroxypropyl)-5-methyl-7-hydroxychromone] [6], and the same planar structure was confirmed by using 2D NMR spectra (Figure 5).The opposite sign for the specific rotation of [α] 25 D − 11.4 (c 0.1, MeOH), in comparison to that of [α] 21 D + 38.4 (c 0.89, MeOH) reported in the literature, suggested that compound 1 may be the enantiomer of aloesol (12S), with the configuration of C-12 to be R. Interestingly, the space group of crystals of compound 1 was determined to be triclinic/P1, and one unit cell displayed four molecules containing three 12R-aloesol and one 12S-aloesol, indicating that compound 1 was a mixture (Figure 6).The chiral HPLC analysis (Supplementary Materials, Figure S1) revealed the ratio of 12R-aloesol and 12S-aloesol to be 3:1, in accordance with the results of single-crystal X-ray diffraction.The pair of enantiomers was further separated by a chiral column, and the novel 12R-aloesol (1a) exhibited specific rotation of [α] 25 D − 15.5 (c 0.06, MeOH).  ) reported in the literature, suggested that compound 1 may be the enantiomer of aloesol (12S), with the configuration of C-12 to be R. Interestingly, the space group of crystals of compound 1 was determined to be triclinic/P1, and one unit cell displayed four molecules containing three 12R-aloesol and one 12S-aloesol, indicating that compound 1 was a mixture (Figure 6).The chiral HPLC analysis (Supplementary Materials, Figure S1) revealed the ratio of 12R-aloesol and 12S-aloesol to be 3:1, in accordance with the results of single-crystal X-ray diffraction.The pair of enantiomers was further separated by a chiral column, and the novel 12R-aloesol (1a) exhibited specific rotation of [α] 25 D − 15.5 (c 0.06, MeOH).    ) reported in the literature, suggested that compound 1 may be the enantiomer of aloesol (12S), with the configuration of C-12 to be R. Interestingly, the space group of crystals of compound 1 was determined to be triclinic/P1, and one unit cell displayed four molecules containing three 12R-aloesol and one 12S-aloesol, indicating that compound 1 was a mixture (Figure 6).The chiral HPLC analysis (Supplementary Materials, Figure S1) revealed the ratio of 12R-aloesol and 12S-aloesol to be 3:1, in accordance with the results of single-crystal X-ray diffraction.The pair of enantiomers was further separated by a chiral column, and the novel 12R-aloesol (1a) exhibited specific rotation of [α] 25 D − 15.5 (c 0.06, MeOH).     1) displayed one olefinic proton signal at δ H 6.75 (H-3, tq, 7.6, 1.6), one oxygenated methine proton signal at δ H 4.04 (H-6, tt, 8.4, 4.4), and two methyl proton signals at δ H 1.82 (H-11, br s) and δ H 1.01 (H-10, t, 7.3).The 13 C and DEPT spectra exhibited eleven carbon signals, including two methyl carbons, four methylene carbon, two methine carbons, and three quaternary carbons.The two structural fragments of C3-C4-C5-C6-C7 and C9-C10 were uncovered by 1 H-1 H COSY correlations (Figure 5).The planar structure of 2 was constructed by the key HMBC correlations from H-11 to C-1 (δ C 172.4), C-2 (δ C 130.0), and C-3 (δ C 142.5), and from H-6, H-7, and H-10 to C-8 (δ C 212.9) (Figure 5).The E-configuration of the double bond between C-2 and C-3 was determined by the key NOE correlation of H-4 with H-11 (Figure 5).The structure of 2 was similar to (5R)-(2E)-5-hydroxy-6-keto-2methyl-2-heptenoic acid [7], with the only difference being the presence of three additional methylenes in 2. The specific rotation for 2 was measured to be [α] 25 D + 0.4 (c 0.1, MeOH), suggesting that 2 was a racemate.The profiles of chiral HPLC analysis (Supplementary Materials, Figure S2) further supported the above result.The mixture was further separated by a chiral column, resulting in 6R-2 (2a) with a specific rotation of [α] 2) displayed one olefinic proton signal at δ H 6.72 (H-3, tq, 7.5, 1.5), one oxygenated methine proton signal at δ H 4.04 (H-6, m), and two methyl proton signals at δ H 2.17 (H-9, s) and δ H 1.82 (H-11, br s).The 13 C and DEPT spectra exhibited ten carbon signals, including two methyl carbons, three methylene carbon, two methine carbons, and three quaternary carbons.The detailed NMR analysis revealed that the NMR data of 3 were similar to those of 2, with the only difference being the absence of one methylene in 3 compared to 2. The result was further supported by 2D NMR spectra (Figure 5).The key NOE correlation of H-4 with H-11 revealed the configuration of the double bond between C-2 and C-3 to be E. Similar to compound 2, the specific rotation of [α] 25 D + 3.7 (c 0.1, MeOH) in combination with the result for the chiral HPLC profile (Supplementary Materials, Figure S3) showed that compound 3 was also a racemate.Additionally, compound 3 was identified as a new natural product.Small quantities of it had been previously detected through the conversion of (6R, 8R)and (6S, 8S)-2-methyl-6, 8-dihydroxynon-2E-enoic acids in feeding experiments with shake cultures of S. griseus ETHA7796 [5].However, the original paper only provided the planar structure without detailed spectroscopic data.Furthermore, the mixture was separated by a chiral column, resulting in 6R-3 (3a) with a specific rotation of [α] 25 D − 7.2 (c 0.04, MeOH), and 6S-3 (3b) with a specific rotation of [α] 25 D + 9.5 (c 0.06, MeOH).2.17 (s) 30.7, q 10 1.82 (br s) 12.7, q a 1 H and 13 C NMR data were recorded at 700 MHz and 176 MHz, respectively; b 1 H and 13 C NMR data were recorded at 500 MHz and 126 MHz, respectively.

Putative Biosynthetic Pathway for Homononactic Acid and Nonactic Acid
The enantiomeric nonactic acid and its homologs have been confirmed to be of polyketide origin by isotope labeling experiments, and biochemical investigations have shown that NonS in a cell-free preparation can convert 2-methyl-6, 8-dihydroxynon-2E-enoic acid into nonactic acid [5,13].However, the lack of isolation of intermediates makes it difficult to establish the biosynthetic process for nonactic acid and its homologs.Based on the isolated intermediates (2-5) from Streptomyces sp.SCSIO 66814, we proposed a putative biosynthetic pathway for homononactic acid and nonactic acid (Figure 7).The three putative substrates, methylmalonyl-CoA, succinyl-CoA, and malonyl-CoA, undergoes successive condensation reactions to synthesize intermediate 9.The intermediate 9 sequentially undergoes decarboxylation, ketone reduction, and dehydration reactions to generate compound 4. Additionally, the formation of intermediate 10 occurs through a condensation reaction of 9 with either acetyl-CoA or propionyl-CoA.Similar to the formation of compound 4, compounds 2 and 3 are derived from intermediate 10 through ketone reduction and dehydration reactions.Subsequently, compounds 2 and 3 are subjected to a reductive reaction, converting the ketone at C-8 to a hydroxyl group, yielding intermediate 11.The formation of the furan ring in nonactic acid and its homolog are putatively catalyzed by HmnN, a NonS homolog enzyme (90% identity), wherein the substrate, a thioester analog of intermediate 11 instead of free acid, undergoes an intramolecular Michael addition reaction [14].Ultimately, the enzyme complex is hydrolyzed, releasing homononactic acid (5) and nonactic acid.
compounds 2 and 3 are subjected to a reductive reaction, converting the ketone at C-8 to a hydroxyl group, yielding intermediate 11.The formation of the furan ring in nonactic acid and its homolog are putatively catalyzed by HmnN, a NonS homolog enzyme (90% identity), wherein the substrate, a thioester analog of intermediate 11 instead of free acid, undergoes an intramolecular Michael addition reaction [14].Ultimately, the enzyme complex is hydrolyzed, releasing homononactic acid (5) and nonactic acid.

Biological Activities
All isolated compounds were tested for antibacterial activity against two Grampositive and two Gram-negative bacteria (Bacillus subtilis, Staphylococcus aureus, Vibrio alginolyticus, and Escherichia coli), and none of them presented any obvious antibacterial activity.Furthermore, the free radical scavenging activities of all compounds against DPPH radicals were also evaluated.The results (Table 3) revealed that only (+)-1 (1b) displayed a weak activity against DPPH radicals, with a scavenging rate of 34.42% at a concentration of 200 µg/mL.

Biological Activities
All isolated compounds were tested for antibacterial activity against two Grampositive and two Gram-negative bacteria (Bacillus subtilis, Staphylococcus aureus, Vibrio alginolyticus, and Escherichia coli), and none of them presented any obvious antibacterial activity.Furthermore, the free radical scavenging activities of all compounds against DPPH radicals were also evaluated.The results (Table 3) revealed that only (+)-1 (1b) displayed a weak activity against DPPH radicals, with a scavenging rate of 34.42% at a concentration of 200 µg/mL.a The final concentration of the tested compounds was 200 µg/mL; b the final concentration of ascorbic acid as the positive control was 10.6 µg/mL.

General Experimental Procedures
The same general procedures were used as those described previously [15].

Microorganism and Growth Conditions
SCSIO 66814 was obtained from stony coral collected from the South China Sea.The 16S rRNA sequence of SCSIO 66814 revealed that the strain belonged to Streptomyces sp. and exhibited 100% identity with Streptomyces cavourensis NBRC 13026(T) (GenBank accession number: AB184264.1).A 40 L scale fermentation was carried out in liquid medium (soluble starch 20 g/L, glucose 10 g/L, malt extract 10 g/L, maltose 10 g/L, corn steep liquor 5 g/L, CaCO 3 2 g/L, sea salt 30 g/L) using Erlenmeyer flasks at 28 • C for 7 days with a shaking rate of 180 rpm.

Bioinformatic Analysis
The complete genome of the strain SCSIO 66814 was sequenced using a combination of PacBio RS and Illumina sequencing platforms at Shanghai Biozeron Biotechnology Co., Ltd.(Shanghai, China).The hmn BCG predicted by the antiSMASH platform was deposited in GenBank under accession number PP098209.The detailed bioinformatic analysis of the hmn BCG was performed using the BLAST tool (Supplementary Materials, Table S2).

Extraction and Isolation
The fermentation broth (40 L) was subjected to three rounds of extraction with an equal volume of ethyl acetate (EtOAc) at room temperature.The resulting EtOAc layer was separated from the aqueous phase, and then evaporated in vacuo to obtain a dry EtOAc extract weighing 14.5 g.The EtOAc extract was initially separated on silica gel CC eluted with CH 2 Cl 2 /MeOH from 1:0 to 0:1 to afford seven subfractions, Fr.

Figure 1 .
Figure 1.Chemical structures of nonactin and its homologs.

Figure 1 .
Figure 1.Chemical structures of nonactin and its homologs.

Figure
Figure 2. Comparative analysis of hmn biosynthetic gene cluster (BGC) in Streptomyces sp.SCSIO 66814 with reported nonactin BGC from S. griseus subsp.griseus ETH A7796.Homologous genes are connected by black dashed lines.

Figure 4 .
Figure 4. Partially enlarged cluster-node graph of molecular networking.The numbers on nodes represent parent ions, and the nodes highlighted in yellow were annotated by GNPS.

Figure 4 .
Figure 4. Partially enlarged cluster-node graph of molecular networking.The numbers on nodes represent parent ions, and the nodes highlighted in yellow were annotated by GNPS.

2 .
Comparative analysis of hmn biosynthetic gene cluster (BGC) in Streptomyces sp.SCSIO 66814 with reported nonactin BGC from S. griseus subsp.griseus ETH A7796.Homologous genes are connected by black dashed lines.