Genome Mining of Marine-Derived Streptomyces sp. SCSIO 40010 Leads to Cytotoxic New Polycyclic Tetramate Macrolactams.

Polycyclic tetramate macrolactams (PTMs) biosynthetic gene cluster are widely distributed in different bacterial types, especially in Streptomyces species. The mining of the genomic data of marine-derived Streptomyces sp. SCSIO 40010 reveals the presence of a putative PTM-encoding biosynthetic gene cluster (ptm' BGC) that features a genetic organization for potentially producing 5/5/6 type of carbocyclic ring-containing PTMs. A fermentation of Streptomyces sp. SCSIO 40010 led to the isolation and characterization of six new PTMs 1-6. Comprehensive spectroscopic analysis assigned their planar structures and relative configurations, and their absolute configurations were deduced by comparing the experimental electronic circular dichroism (ECD) spectra with the reported spectra of the known PTMs. Intriguingly, compounds 1-6 were determined to have a trans-orientation of H-10/H-11 at the first 5-membered ring, being distinct from the cis-orientation in their known PTM congeners. PTMs 1-5 displayed cytotoxicity against several cancer cell lines, with IC50 values that ranged from 2.47 to 17.68 µM.

1. Introduction

Polycyclic tetramate macrolactams (PTMs) are a unique class of natural products that consist of a tetramate-embedding macrocyclic lactam core and a varying carbocycle with 5/6, 5/5, 5/6/5, or 5/5/6 ring system [1]. PTMs display a wide range of antifungal, antibiotic, antiprotozoal, and antitumor properties [2,3,4,5], and they have significant potential for applications in agricultures and medicines [1,6]. HSAF (also known as dihydromaltophilin) [7], a typical representative of 5/5/6 type of PTMs, exhibits a broad spectrum of antifungal activities and it has been used as an antifungal agent to control plant diseases [8]. The anticancer agent ikarugamycin [9], a typical 5/6/5 type of PTMs, shows activity as an inhibitor of clathrin-mediated endocytosis [10]. Therefore, PTMs draw the attention of synthetic chemists; however, multiple chiral centers in PTMs greatly enhance the structure diversity and increase the difficulty for the total synthesis [11,12,13,14]. In a sharp contrast, in nature, a conserved and compact biosynthetic pathway has been evolved to simply assemble such kinds of complex structures [1]. Recent studies reveal that PTMs are derived from a conserved hybrid polyketide synthase (PKS)/non-ribosomal peptide synthethase (NRPS) pathway [1,15]. The PKS portion of the hybrid PKS/NRPS enzyme is iteratively used to produce two separate polyketide chains, which are respectively condensed with the α- and δ-amino groups of an l-orinithine that is tethered in the NRPS portion to generate a common polyene tetramate precursor [7,15,16,17,18,19,20,21]. Afterwards, a set of oxidoreductases catalyzes divergent cyclization reactions to control the formation of diverse carbocyclic ring systems in PTMs [18,20,22,23,24]. Particularly, the iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase has been demonstrated in vitro in the biosynthesis of HSAF [16], and the biocatalytic total synthesis of ikarugamycin has been recently achieved [22].

Genome Mining has been successfully utilized to discover new PTMs [9,15,17,20,25,26]. For example, we have reported the activation of a silent PTM biosynthetic gene cluster (BGC) by promoter engineering in marine-derived Streptomyces pactum SCSIO 02999 to produce a series of new PTMs pactamides with a 5/5/6 ring system [20]. In addition, we have characterized three new PTMs containing a 5/6/5 ring system from a South China Sea-derived Streptomyces sp. SCSIO 40060 while using a genomics-guided approach [26]. We identified a mangrove-derived Streptomyces sp. SCSIO 40010 harboring a putative PTM BGC during our continuous search for PTM-producing strains. Herein, we reported the isolation, structural elucidation and biological evaluation of six new PTMs 1–6 (Figure 1).

Figure 1

Chemical structures of polycyclic tetramate macrolactams (PTMs). Compounds 1–6 were isolated from Streptomyces sp. SCSIO 40010. The known compounds 7–12 with the same planar structures as those of 1–6, respectively, are shown here for comparison.

2. Results and Discussion

2.1. Genome Mining of a PTM Biosynthetic Gene Cluster

The strain SCSIO 40010 was isolated from the mangrove sediment in Penang, Malaysia, and it was identified to be a Streptomyces species on the basis of its 16S rDNA sequence (GenBank accession number MN224032). The mining of the sequenced genome of Streptomyces sp. SCSIO 40010 reveals the presence of a putative PTM BGC (ptm′, GenBank accession number MN234160) that displays high similarity to the ptm BGC in S. pactum SCSIO 02999 (Figure 2a) [20]. This ptm′ BGC encodes six conserved enzymes, including the hybrid PKS/NRPS PtmA′, the FAD-dependent oxidoreductase PtmB1′, PtmB2′, the alcohol dehydrogenase PtmC′, the hydroxylase PtmD′, and the P450 enzyme PtmE′. In addition to the scaffold constructing enzymes PtmA′, PtmB1′, PtmB2′, and PtmC′, two modifying enzymes PtmD′ (resembling the C-25 hydroxylase FtdA [15], 63% identity) and PtmE′ (resembling the P450 enzyme FtdF [15], 59% identity) were also found in the ptm′ BGC in Streptomyces sp. SCSIO 40010.

Figure 2

(a) Bioinformatics analysis of 5/5/6 type of PTM biosynthetic gene clusters (BGCs). (b) The proposed biosynthetic pathway for six new 5/5/6 type of PTMs.

Our preliminary genome mining of Streptomyces sp. SCSIO 40010 indicates that it should be a potential producer of PTMs with a 5/5/6 carbocyclic ring system [1,15]. Thus, we mined the available genome sequences for PTM BGCs and made a bioinformatics analysis of the PTM BGCs, typically for 5/5/6 type of PTMs [1,15]. Our analysis shows that the BGCs for 5/5/6 type of PTMs should fall into two categories (Figure 2a), depending on the number of oxidoreductases that are involved in the construction of the 5/5 ring system (two for Group I and three for Group II).

The PTM BGCs of Group I are mainly distributed in Streptomyces species (Figure 2a). Some of these Streptomyces strains have been demonstrated to produce 5/5/6 and/or 5/5 type of PTMs, such as pactamides in S. pactum SCSIO 02999 [20], compounds a–d in S. griseus NBRC 13350 [17], alteramides in S. albus J1074 [25], and frontalamides in Streptomyces sp. SPB78 [15]. In contrast, no PTMs have been reported from S. flavogriseus ATCC 33331 and S. roseosporus ATCC 11379 that contain Group I of PTM BGCs (Figure 2a) [15]. In addition to Streptomyces species, Actinoalloteichus sp. ADI127-7 and AHMU CJ201 also contain Group I of PTM BGCs (Figure 2a), while no PTMs have been reported from them. Actinoalloteichus cyanogriseus WH1-2216-6 was reported to produce HSAF and its analogues [27]; however, its genome sequence is not yet publicly available.

Biosynthetically, it has been experimentally demonstrated in S. pactum SCSIO 02999 that two oxidoreductases PtmB1 and PtmB2 are responsible for the sequential formation of the 5/5 ring system in pactamide A, with the formation of the first 5-membered ring by PtmB2 and the second 5-membered ring by PtmB1 [20]. Subsequently, formation of the inner 6-membered is catalyzed by the alcohol dehydrogenase PtmC [20].

The PTM BGCs of Group II are mainly found in bacterial strains of Lysobacter (Figure 2a). The strains Lysobacter enzymogenes C3 and strain YC36 are validated to produce HSAF and related PTMs [7,16,19,23]. The strains Lysobacter gummosus strain 3.2.11 and Lysobacter capsici strain 55 are indicated as HSAF producers [28]. Unlike Group I PTM BGCs, three oxidoreductases (such as OX1, OX2, and OX3 in Lysobacter enzymogenes) from the PTM BGCs of Group II are involved in the formation of the 5/5 ring system [23]. It has been shown that OX3 is responsible for the first 5-membered ring formation in lysobacterene A, while OX1 and OX2 catalyze the formation of the second 5-membered ring, but with different stereo selectivity [23]. In a similar fashion to PtmC, OX4 catalyzes the formation of the inner 6-membered ring in HSAF (7, Figure 1) [23,24].

The metabolite profiles of Streptomyces sp. SCSIO 40010 were investigated by cultivation in four different media, including modifed-A1BFe+C [29], AM6, AM6-4, and modifed-ISP3 [26]. HPLC analyses showed that compounds exhibiting UV-visible absorption spectra that were similar to PTMs were better produced in the modifed-A1BFe+C medium (Supplementary Figure S1). Subsequently, a 20-L fermentation of Streptomyces sp. SCSIO 40010 was performed in the modifed-A1BFe+C. Butanone extracts of the 20-L fermentation cultures were subjected to multiple chromatographic methods to provide six new PTMs 1–6.

Compound 1 was isolated as a white powder. The molecular formula of 1 was determined as C29H40N2O6 by HRESIMS ([M + H]+, m/z 513.2960, calcd for 513.2965, Supplementary Figure S2). The planar structure of 1 was determined to be the same as that of HSAF (7, Figure 1) [27,30], by comparing NMR spectroscopic data of 1 (Table 1 and Table 2; Supplementary Figures S3–S9) and HSAF (7) [27,30]. The geometries of double bonds in 1 were determined to be trans (E) or cis (Z) on the basis of their coupling constants (Z∆2,3 J2,3 11.5 Hz; E∆17,18 J17,18 15.5 Hz; Table 1). The relative configurations of 1 were assigned by NOESY correlations and then compared with pactamide A [20] and HSAF (7) [30] (Figure 3a). It should be noted that a trans-orientation of H-10 and H-11 was assigned for 1, because of the obvious NOESY correlations of H-8/H-10, H10/H-12, and H-11/H-29b (Figure 3). Previously, a trans-orientation of H-10 and H-11 was reported for pactamide A [20], aburatubolactam A (X-ray crystallography structure available [31]), combamide D [32], deOH alteramides, and lysobacterene B [33]. In the recently reported 5/5 type of PTMs umezawamides, the relative orientation of H-10 and H-11 was not determined [34]. However, a cis-orientation of H-10 and H-11 was determined in 7 [30]. As described by Cao et al. [9] and Hoshino et al. [34], the small vicinal coupling constant between H-23 and H-25 strongly indicated the relative configuration between H-23 and H-25 to be (23S*, 25S*) in 1 (3JH-23/H-25 1.2 Hz, Table 1). The configuration of C-23 was deduced to be 23S upon the proposed biogenesis from an l-orinithine [7,15,16,17,18,19,20,21]. Recently, a crystallographic study unequivocally determined the absolute configuration as 23S, 25S in hydroxylikarugamycin A [26]. Thus, when considering the biosynthetic similarity between 1 and hydroxyikarugamycin A, 1 was deduced to also have the stereochemistry of 23S, 25S. Given that 1 and 7 (5R, 6S, 8S, 10R, 11R, 12R, 13S, 14R, 16R, 23S, 25S [30]) displayed an almost identical electronic circular dichroism (ECD) spectra (Supplementary Figure S10), the absolute configuration of 1 was deduced as 5R, 6S, 8S, 10S, 11R, 12R, 13S, 14R, 16R, 23S, 25S, which was only different from 7 by adopting an opposite configuration at C-10 (Figure 1). Thus, compound 1 was designated 10-epi-HSAF.

Table 1

1H NMR (700 MHz) Data for PTMs 1–6 in dimethyl sulfoxide (DMSO)-d6 (δH, mult, J in Hz).

No.	1	2	3	4	5	6
2	5.70, dd, (2.3, 11.5)	5.73, dd, (1.9, 11.4)	5.72, dd, (2.2, 11.3)	5.75, dd, (2.0, 11.5)	5.75, dd, (2.1, 11.3)	5.77, dd, (2.0, 11.5)
3	5.90, td, (1.8, 11.2)	5.9, td, (2.2, 11.1)	5.92, td, (2.1, 11.0)	5.92, td, (2.1, 11.1)	5.96, td, (2.1, 11.1)	5.96, td, (2.2, 11.1)
4a	1.89, m	1.92, m	2.00, m	2.04, m	2.03, m	2.05, m
4b	3.52, m	3.49, m	3.62, m	3.62, m	3.65, m	3.64, m
5	1.28, m	1.27, m	1.90, m	1.90, m	1.87, m	1.88, m
6	1.64, m	1.27, m	2.09, m	2.10, m	2.56, m	2.57, m
7a	0.86, m	0.87, m	1.05, m	0.85, m	0.98, m	1.01, m
7b	1.96, m	1.97, m	2.05, m	2.06, m	2.11, m	2.14, m
8	2.35, m	2.35, m	2.35, m	2.35, m	2.03, m	2.03, m
9a	0.80, m	0.79, m	0.84, m	1.06, m	0.68, m	0.69, m
9b	2.01, m	2.01, m	2.05, m	2.06, m	2.02, m	2.03, m
10	1.33, m	1.32, m	1.37, m	1.37, m	1.53, m	1.53, m
11	1.27, m	1.64, m	1.09, m	1.09, m	1.21, m	1.22, m
12	1.75, m	1.77, m	2.21, m	2.21, m
13	1.10, m	1.09, m	2.37, m	2.37, m	2.32, m	2.32, m
14	3.25, m	3.25, m
15a	1.24, m	1.24, m	2.11, m	2.11, m	2.12, m	2.13, m
15b	1.74, m	1.75, m	2.59, m	2.59, m	2.59, m	2.60, m
16	2.06, m	2.07, m	2.38, m	2.40, m	2.44, m	2.47, m
17	6.57, dd, (10.5, 15.5)	6.55, dd, (10.5, 15.5)	6.63, dd, (10.3, 15.6)	6.61, t, (15.5)	6.60, dd, (10.3, 15.5)	6.59, dd, (11.5, 15.5)
18	6.86, d, (15.5)	6.95, d, (15.5)	6.87, d, (15.6)	6.96, d, (15.5)	6.88, d, (15.5)	6.96, d, (15.5)
22	NH, 8.68, s	NH, 8.68, s	NH, 8.95, s	NH, 8.73, brs	NH, 8.96, s	NH, 8.75, s
23	3.86, d, (1.2)	3.81, d, (5.7)	3.87, d, (1.4)	3.83, s	3.87, d, (1.1)	3.84, d, (6.1)
25a	3.81, dt, (1.5, 6.1)	1.74, m	3.81, dt, (1.4,6.2)	1.74, m	3.82, dt, (2.0, 6.4)	1.74, m
25b		1.84, m		1.86, m		1.86, m
26a	1.18, m	1.15, m	1.18, m	1.17, m	1.20, m	1.15, m
26b	1.38, m	1.32, m	1.39, m	1.35, m	1.39, m	1.34, m
27a	2.57, m	2.39, m	2.59, m	3.23, m	2.58 m	2.40, m
27b	3.25, m	3.22, m	3.26, m	2.39, m	3.24, m	3.23, m
28	NH, 7.96, t, (5.7)	NH, 7.82, t, (5.3)	NH, 7.98, t, (5.6)	NH, 7.86, s	NH, 8.00, t, (5.6)	NH, 7.88, t, (5.6)
29a	1.04, m	1.04, m	1.03, m	1.03, m	0.99, m	1.02, m
29b	1.55, m	1.55, m	1.53, m	1.54, m	1.51, m	1.52, m
30	0.85, t (7.4)	0.85, t (7.4)	0.84, t (7.4)	0.84, t (7.4)	0.84, t (7.4)	0.84, t (7.4)
31	1.06, d (6.4)	1.06, d (6.4)	0.96, d (6.5)	0.96, d (6.5)	0.94, d (6.7)	0.94, d (6.8)

Table 2

13C NMR (176 MHz) Data for PTMs 1–6 in DMSO-d6, (δC, type).

No.	1	2	3	4	5	6
1	165.5, C	165.5, C	165.5, C	165.6, C	165.5, C	165.6, C
2	124.1, CH	124.2, CH	124.4, CH	124.5, CH	124.5, CH	124.7, CH
3	139.1, CH	138.9, CH	138.4, CH	138.3, CH	138.2, CH	138.1, CH
4	28.0, CH2	28.1, CH2	27.7, CH2	27.7, CH2	27.4, CH2	27.4, CH2
5	43.5, CH	43.5, CH	43.2, CH	43.2, CH	43.0, CH	43.0, CH
6	47.4, CH	46.4, CH	51.1, CH	51.2, CH	47.9, CH	48.0, CH
7	37.2, CH2	37.3, CH2	38.4, CH2	39.7, CH2	36.5, CH2	36.5, CH2
8	41.5, CH	41.4, CH	40.4, CH	40.4, CH	51.3, CH	51.3, CH
9	40.3, CH2	40.3, CH2	39.6, CH2	38.5, CH2	38.1, CH2	38.1, CH2
10	53.5, CH	53.5, CH	53,2, CH	53,2, CH	50.4, CH	50.4, CH
11	46.5, CH	47.6, CH	46.7, CH	46.7, CH	49.3, CH	49.3, CH
12	58.1, CH	58.1, CH	50.4, CH	50.4, CH	89.7, CH	89.7, CH
13	59.1, CH	59.1, CH	63.0, CH	63.0, CH	64.2, CH	64.2, CH
14	72.7, CH	72.7, CH	207.4, C	207.4, C	210.1 C	210.2, C
15	41.9, CH2	41.9, CH2	45.6, CH2	45.6, CH	46.0, CH2	46.0, CH2
16	45.7, CH	45.6, CH	47.7, CH	47.8, CH	47.1, CH	47.2, CH
17	150.1, CH	149.6, CH	147.8, CH	147.4, CH	147.5, CH	147.2, CH
18	121.3, CH	121.5, CH	122.0, CH	122.1, CH	122.1, CH	122.2, CH
19	172.2, C	171.8, C	171.9, C	175.1, C	171.8, C	171.2, C
20	100.4, C	100.7, C	100.7, C	101.1, C	100.7, C	101.0, C
21	175.7, C	175.3, C	175.6, C	171.4, C	175.6, C	175.1, C
23	68.6, CH	61.0, CH	68.5, CH	61.1, CH	68.6, CH	61.1, CH
24	193.0, C	195.8, C	193.0, C	195.8, C	193.0, C	195.9, C
25	70.1, CH	26.2, CH2	70.1, CH	26.2, CH2	70.1, CH	26.1, CH2
26	31.1, CH2	20.4, CH2	31.0, CH2	20.4, CH2	31.0, CH2	31.1, CH2
27	36.4, CH2	38.0, CH2	36.4, CH2	38.0, CH2	36.4, CH2	36.4, CH2
29	25.8, CH2	25.8, CH2	25.5, CH2	25.5, CH2	25.3, CH2	25.3, CH2
30	12.6, CH3	12.6, CH3	12.4, CH3	12.4, CH3	12.0, CH3	12.0, CH3
31	18.4, CH3	18.4, CH3	17.6, CH3	17.6, CH3	11.5, CH3	11.5, CH3

Figure 3

(a) Key COSY, HMBC correlations for 1, and selected NOE correlations for 1 and 3. (b) Key NOE correlations to support a trans-orientation of H-10/H-11 in 1 and 3.

Compound 2 was obtained as a white powder and it was assigned the molecular formula of C29H40N2O5 on the basis of HRESIMS ([M + H]+, m/z 497.3010, calcd for 497.3015, Supplementary Figure S11). A detailed comparison of one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic data of 2 (Table 1 and Table 2, Supplementary Figures S12–S18) and deOH-HSAF (8, Figure 1) revealed the same planar structure for 2 and 8 [27,30]. The relative configuration of 2 was deduced by proton coupling constants (Z∆2,3 J2,3 11.5 Hz; E∆17,18 J17,18 15.5 Hz; Table 1) and careful analysis of NOESY correlations (Figure 4, Supplementary Figures S17 and S18). Similar to 1, a trans-orientation of H-10/H-11 was determined in 2 (Figure 1), by deducing from NOESY correlations of H-8/H-10, H10/H-12, H12/Me-31, and H-11/H-29b. This was different from the cis-orientation of H-10/H-11 in 8 (Figure 1) [30]. 2 was deduced to have the absolute configuration of 5R, 6S, 8S, 10S, 11R, 12R, 14R, 13S, 16R, and 23S because of the almost identical ECD spectra of 1 and 8 (Supplementary Figure S10), and thus compound 2 was designated 10-epi-deOH-HSAF.

Figure 4

Key COSY, HMBC, and selected NOE correlations for 2–6.

Compound 3 was isolated as a reddish and amorphous powder. The molecular formula of 3 was determined as C29H38N2O6 by HRESIMS ([M−H]−, m/z 509.2642, calcd for 509.2657, Supplementary Figure S19). Careful analysis of the 1D and 2D NMR data of 3 (Table 1 and Table 2, Supplementary Figures S20–S26) revealed that 3 was an isomer of maltophilin (9, Figure 1) [27]. The trans-orientation of H-10/H-11 in 3, which differed from the cis-orientation of H-10/H-11 in 9, was supported by NOESY correlations of H-8/H-10, H-10/H-12, and H-12/Me-31 (Figure 3, Supplementary Figures S25 and S26). When considering the similar ECD spectra of 3 and 1 (Supplementary Figure S10), the absolute configuration of 3 was deduced as 5R, 6S, 8S, 10S, 11R, 12R, 13S, 16R, 23S, and 25S, and thus 3 was designated 10-epi-maltophilin.

Compound 4 was obtained as a white powder and it was assigned the molecular formula as C29H38N2O5 by HRESIMS ([M + H]+, m/z 495.2846, calcd for 495.2859, Supplementary Figure S27). Detailed comparison of NMR spectroscopic data of 4 (Table 1 and Table 2, Supplementary Figures S28–S34) and xanthobaccin C (10, Figure 1) uncovered that 4 was an isomer of 10 [27]. The key NOESY correlations of H-8/H-10, H-10/H-12, and H-12/Me-31 in 4 (Figure 4, Supplementary Figure S34) supported a trans-orientation of H-10/H-11 in 4. The absolute configuration of 4 was deduced as 5R, 6S, 8S, 10S, 11R, 12R, 13S, 16R, and 23S by comparing the ECD spectra of 4 and 2 (Supplementary Figure S10). Therefore, 4 was designated 10-epi-xanthobaccin C.

Compound 5 was obtained as a reddish powder. The molecular formula of 5 was assigned as C29H38N2O7 by HRESIMS ([M + H]+, m/z 527.2757, calcd for 527.2757, Supplementary Figure S35). A detailed comparison of NMR spectroscopic data of 5 and hydroxymaltophilin (11, Figure 1) suggested that both compounds should have the same planar structure (Table 1 and Table 2, Supplementary Figures S36–S42) [27]. However, distinct from the cis-orientation of H-10/H-11 in 11 [27], a trans-orientation of H-10/H-11 was indicated in 5 by key NOE correlations of H-8/H-10, H10-/H-12, and H-11/H-29b (Figure 4, Supplementary Figures S41 and S42). Based on the similar ECD spectra of 5 and 11 (Supplementary Figure S10), 5 was suggested to have the configuration of 5R, 6S, 8S, 10S, 11R, 12R, 13S, 16R, 23S, and 25S, and it was thus designated 10-epi-hydroxymaltophilin.

Compound 6 was isolated as a yellowish solid. The molecular formula of 6 was determined to be C29H38N2O6 by HRESIMS ([M + H]+, m/z 511.2800, calcd for 511.2808, Supplementary Figure S43). An analysis of 1D, COSY, and HMBC correlations (Supplementary Figures S44–S48) showed that the planar structure of 6 was the same as that of FI-2 (12, Figure 1), an intermediate in frontalamide biosynthesis [15,27]. A trans-orientation of H-10/H-11 was indicated in 6 by key NOE correlations of H-8/H-10, H-10/H-12, and H-11/H-29b (Figure 4, Supplementary Figures S49 and S50), different from the cis-orientation of H-10/H-11 in 12 [27]. The high similarity in the ECD spectra of 6 and 12 indicated that 6 should be a 10-epimer of 12, designated 10-epi-FI-2.

2.2. Biological Activities

The in vitro cytotoxicities of compounds 1–5 (compound 6 was not tested due to limited amount) were evaluated against four human cancer cell lines, including SF-268, MCF-7, A549, and HepG2, by the SRB method since most reported PTMs exhibits cytotoxic activities [1] (Table 3). Compounds 1–5 showed moderate activities against these four cancer cell lines with half inhibitory concentration (IC50) values of 2.47–17.68 μM, which were comparable to those of the positive control cisplatin (Table 3). It should be noted that pactamide A, differing from 2 only by lacking C-14 OH, displayed much better cytotoxicities (IC50 values ranging from 0.2–0.5 μM against these four cancer cell lines) than 2 [20].

Table 3

Cytotoxicities of PTMs 1–5.

	IC50 (μM)
	SF-268	MCF-7	A549	HepG2
1	3.83 ± 0.13	2.47 ± 0.05	5.99 ± 0.15	3.48 ± 0.17
2	10.62 ± 0.45	3.84 ± 0.07	11.01 ± 1.09	10.34 ± 0.88
3	4.57 ± 0.18	3.18 ± 0.13	3.75 ± 0.62	6.30 ± 0.34
4	7.53 ± 0.27	3.54 ± 0.24	10.45 ± 0.46	17.86 ± 0.62
5	3.21 ± 0.18	6.83 ± 0.36	3.28 ± 0.04	3.12 ± 0.11
a CP	3.26 ± 0.29	3.19 ± 0.12	1.56 ± 0.08	2.42 ± 0.14

Cisplatin, positive control.

2.3. Biosynthetic Implications

Based on bioinformatics analysis, the ptm′ BGC in Streptomyces sp. SCSIO 40010 was highly similar to that of frontalamides (ftd) in Streptomyces sp. SPB78 and it should be classified into the Group I of 5/5/6 type of PTM BGCs (Figure 2a). Subsequently, six new PTM analogues with moderate antitumor activities were isolated from Streptomyces sp. SCSIO 40010 and the absolute configuration at C-10 in these PTMs was identified as being 10S, opposite to their known PTM congeners. These observations further highlight the importance of Streptomyces species as prolific sources for bioactive compounds and they indicate the worth of genome mining in marine-derived Streptomycetes [35]. Similar to the well-established biosynthetic pathway for 5/5/6 type of PTMs [20,23,33], PtmA′ catalyzes the formation of a common polyene tetramate precursor, which is sequentially cyclized by PtmB2′/PtmB1′ into an intermediate with the 5/5 carbocyclic ring system (Figure 2b). It has been hypothesized that OX3, which is a PtmB2′ homologous enzyme, is involved in controlling the formation of products with both cis- and trans-orientated H-10/H-11 in HSAF (7) biosynthesis [23]. However, it appears that PtmB2′ only generates products with trans-orientated H-10/H-11. Additionally, it has been proposed that C-14 oxidation occurs during the OX2 (PtmB1′ analogue)-catalyzed formation of the second five-membered ring [23], and a recent in vivo combinatorial study has confirmed that the second ring formation is coupled with the C-14 hydroxylation in the biosynthesis of HSAF and analogues [33]. However, the detailed biochemistry and enzymology responsible for such transformations have not been elucidated. Next, PtmC’ generates the inner six-membered ring in 2 (Figure 2b). Finally, different oxidations of 2 by PtmD’ (a C-25 hydroxlase, analogous to FtdA for frontalamides [15], SD for HSAF [36]) and PtmE’ (a putative C-12 hydroxylase and C-14 dehydrogenase, analogous to FtdF for frontalamides [15]) lead to the formation of products 1 and 3–6 due to the substrate promiscuity of PtmD’ and PtmE’ (Figure 2b).

2.4. Conclusion

Conclusively, on the basis of a genome mining approach, we isolated six new PTMs 1–6 from the marine-derived Streptomyces sp. SCSIO 40010. The 10S absolute configuration is the unique feature of these new PTM analogues, which is distinct from the 10R configuration in their known congeners. PTMs 1–5 display moderate cytotoxic activities toward four human cancer cell lines. Although a biosynthetic pathway for PTMs 1–6 is proposed, the precise biochemistry and enzymology involved in the polycyclic ring formation and the stereochemistry selectivity remains elusive and awaits further investigations.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were measured using a 341 Polarimeter (Perkin-kinelmer, Inc., Norwalk, CT, USA). The CD spectra were measured on a Chirascan circular dichroism spectrometer (Applied Photophysics, Ltd., Surrey, UK). UV spectra were measured on a U-2900 spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were recorded on an Affinity-1 FT-IR spectrometer (Shimadzu, Tokyo, Japan). The 1D and 2D NMR spectra were recorded on a Bruker AV-700 MHz NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) with tetramethylsilane (TMS) as the internal standard. Mass spectrometric data were obtained on a quadrupole-time-of-flight mass spectrometry (Bruker Maxis 4G) for HRESIMS. Column chromatography was performed while using silica gel (100–200 mesh, 300–400 mesh; Jiangyou Silica gel development, Inc., Yantai, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). HPLC was carried out while using a reversed-phase column (Phenomenex Gemini C18, 250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) with UV detection at 270 nm and 320 nm. Semi-preparative HPLC was performed on a Hitachi HPLC station (Hitachi-L2130) with a Diode Array Detector (Hitachi L-2455) using a Phenomenex ODS column (250 mm × 10.0 mm, 5 mm; Phenomenex, Torrance, CA, USA) with UV detection at 320 nm.

3.2. Strain, Screening and Culture Methods

Streptomyces sp. SCSIO 40010 was isolated from the Mangrove sediment obtained from Penang, Malaysia, and it was identified by 16S rDNA sequence analysis. The strain SCSIO 40010 was maintained in 40% glycerol aqueous solution at −80 °C in Research Center for Marine Microbiology Culture Collection Center of South China Sea Institute of Oceanology, Chinese Academy of Sciences. It was found that the strain SCSIO 40010 was best maintained on 38#-Agar medium containing 3% sea salt for optimal growth and sporulation. A single colony was inoculated into 50 mL of four different media, including modifed-A1BFe+C (soluble starch 1.0%, yeast extract 0.4%, tryptone 0.2%, CaCO3 0.2%, sea salts 3%, pH 7.2–7.4) [29], AM6 (soluble starch 2.0%, glucose 1.0%, tryptone 0.5%, yeast extract 0.5%, CaCO3 0.2%, sea salts 3%, pH 7.2–7.4) [37], AM6-4 (glycerol 0.1%, bacterial peptone 0.5%, glycine 0.01%, alanine 0.01%, CaCO3 0.5%, sea salts 3%, pH 7.2–7.4) [37], and modifed-ISP3 (oat meal 1.5%, FeSO4 0.0001%, MnCl2 0.0001%, ZnSO4 0.0001%, sea salts 3%, pH 7.2–7.4) [37], in 250 mL Erlenmeyer flasks, and then incubated on a rotary shaker (200 rpm) at 28 °C for seven days. The culture broths were extracted with an equal volume of n-butanol and the extracts were then monitored by HPLC-DAD. HPLC analyses were carried out under the following program: solvent system (solvent A, 10% acetonitrile in water supplemented with 0.08% formic acid; solvent B, 90% acetonitrile in water); 5% B to 100% B (linear gradient, 0–18 min.), 100% B (18–23 min.), 100% B to 5% B (23–27 min.), 5% B (27–32 min.); flow rate at 1 mL/min. A single colony was inoculated into 30 mL of modifed-A1BFe+C medium and incubated at 28 °C for 2–3 days. Then, a total of 20 L fermentation cultures were performed by inoculating 30 mL of the seed culture into a 1000 mL Erlenmeyer flask containing 200 mL of the modifed-A1BFe+C medium to cultivate on a rotary shaker (200 rpm) at 28 °C for 7 days.

3.3. Genome Mining and Bioinformatics Analysis

The strain SCSIO 40010 was inoculated into modifed-A1BFe+C medium and incubated at 28 °C for 48 h. Then the mycelia were collected by centrifugation. Genomic DNA was released from the mycelia by lysozyme and proteinase K digestion, which was extracted with Phenol-chloroform, followed by anhydrous ethanol precipitation. The draft genome of Streptomyces sp. 40010 was sequenced by using Illumina HiSeq 2500. The reads were de novo assembled by using SOAPdenovo ver 2.04 (http://soap.genomics.org.cn/soapdenovo.html). Gene sequences were predicted and annotated by the Rapid Annotations using Subsystems Technology (RAST) server [38]. The putative PTM biosynthetic gene clusters in the genome were predicted with antiSMASH 4.0 [39]. The DNA sequences of the ptm′ gene cluster were deposited under GenBank accession number MN234160. The function of gene products was predicted with protein blast and/or blastx program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The PTM BGCs were obtained from GenBank database for bioinformatics analysis: Streptomyces sp. SCSIO 40010 (MN234160); Streptomyces pactum SCSIO 02999 (KU569222); Streptomyces griseus NBRC 13350 (AP009493); Streptomyces albus J1074 (ABYC01000481); Streptomyces flavogriseus ATCC 33331 (NZ_ACZH01000010); Streptomyces roseosporus ATCC 11379 (ABYX01000252); Streptomyces sp. SPB78 (NZ_ACEU01000453 and NZ_ACEU01000454); Actinoalloteichus sp. ADI127-7 (CP016076); Actinoalloteichus sp. AHMU CJ021 (CP025990.1); Lysobacter enzymogenes C3 (EF028635.2); Lysobacter enzymogenes strain YC36 (CP040656.1); Lysobacter gummosus strain 3.2.11 (CP011131.1); Lysobacter capsici strain 55 (CP011130.1).

3.4. Extraction, Isolation and Purification

The 20 L of culture broth of Streptomyces sp. SCSIO 40010 were pooled and centrifuged at 3900 rpm for 15 min. at 25 °C. The mycelia were extracted three times, each with 2 L acetone. The acetone extracts were concentrated under reduced pressure to afford an aqueous residue, which was extracted four times with equal volume of n-butanone. The supernatants were extracted four times with equal volume of n-butanone. The butanone extracts were combined and concentrated under reduced pressure to afford the crude extracts (11.5 g). The crude extracts were subjected to the column chromatography over silica gel eluting with a gradient of CHCl3/MeOH mixtures ranging from 100/0, 95/5, 90/10, 80/20,50/50 and 0/100 (v/v) yielded six fractions (Fr.1–Fr.6). Then Fr.2 (0.72 g) was further purified via MPLC (Medium Pressure Preparative Liquid Chromatography) with reverse phased C-18 column (14.5 × 2.5 cm i.d., 5 mm Agela Technologies) by eluting with a linear gradient of H2O/MeOH (0–100%, 15 mL/min, 300 min) give fractions Fr.2.1–Fr.2.18. Fractions Fr.2.14–15 (170 mg) were further purified by semi-preparative HPLC while using a mobile phase of MeCN-H2O (65:35, v/v) to give compounds 2 (3.4 mg), 3 (10.8 mg), and 4 (3.6 mg). The fraction Fr.3 (0.83 g) was purified by Sephadex LH-20 (120 × 3.5 cm i.d.), eluting with CHCl3/MeOH (5:5, v/v) to give fractions Fr.3.1–Fr.3.25. Fractions Fr.3.5–9 (300 mg) were further purified by semi-preparative HPLC while using a mobile phase of MeCN-H2O (45:55, v/v) to provide compounds 1 (4.1 mg), 5 (5.6 mg), and 6 (2.8 mg).

3.5. Physical and Chemical Properties of New Compounds 1–6

10-epi-HSAF (1): White powder; [α + 50.7 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 322 (3.92) nm, 219 (4.18) nm; ECD (c 4.3 × 10−4 M, MeOH) λmax (Δε) 215 (+15.5), 241 (−18.1), 326 (+6.2) nm; IR νmax 3356, 2951, 2918, 2369, 2341, 1653, 1541, 1471, 1020, 679 cm−1; 1H NMR (700 MHz, DMSO-d6) and 13C NMR (176 MHz, DMSO-d6) data, see Table 1 and Table 2; (+)-HRESIMS m/z [M + H]+ 513.2960 (calcd for C29H41N2O6, 513.2965).

10-epi-deOH-HSAF (2): White powder; [α + 53.7 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 322 (4.03) nm, 212 (4.37) nm; ECD (c 2.2 × 10−4 M, MeOH) λmax (Δε) 214 (+7.8), 244 (−9.9), 326 (+4.4) nm; IR νmax 3356, 3334, 2953, 2868, 2358, 2341, 1647, 1541, 1506, 1203, 1024, 669 cm−1; 1H NMR (700 MHz, DMSO-d6) and 13C NMR (176 MHz, DMSO-d6) data, see Table 1 and Table 2; (+)-HRESIMS m/z [M + H]+ 497.3010 (calcd for C29H41N2O5, 497.3015).

10-epi-maltophilin (3): Reddish solid; [α + 42.4 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 322 (4.06) nm, 218 (4.31) nm; ECD (c 4.9 × 10−4 M, MeOH) λmax (Δε) 214 (+26.3), 238 (−23.1), 332 (+6.0) nm; IR νmax 3336, 2953, 2920, 2358, 2341, 1647, 1456, 1022, 679 cm−1; 1H NMR (700 MHz, DMSO-d6) and 13C NMR (176 MHz, DMSO-d6) data, see Table 1 and Table 2; (-)-HRESIMS m/z [M − H]− 509.2642 (calcd for C29H37N2O6, 509.2952).

10-epi-xanthobaccin C (4): White powder; [α + 8.31 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 322 (3.97) nm, 219 (4.27) nm; ECD (c 2.6 × 10−4 M, MeOH) λmax (Δε) 210 (+15.3), 247 (−16.9), 327 (+5.0) nm; IR νmax 3335, 2951, 2920, 2837, 2358, 2341, 1653, 1456, 1018, 758, 669 cm−1; 1H NMR (700 MHz, DMSO-d6) and 13C NMR (176 MHz, DMSO-d6) data, see Table 1 and Table 2; (+)-HRESIMS m/z [M + H]+ 495.2846 (calcd for C29H39N2O5, 495.2859).

10-epi-hydroxymaltophilin (5): Reddish powder; [α + 30.8 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 321 (4.02) nm, 216 (4.32) nm; ECD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 214 (+24.1), 238 (−18.4), 326 (+4.4) nm; IR νmax 3334, 3327, 2955, 2927, 2359, 2342, 1697, 1653, 1541, 1471, 1217, 1024, 754, 678 cm−1; 1H NMR (700 MHz, DMSO-d6) and 13C NMR (176 MHz, DMSO-d6) data, see Table 1 and Table 2; (+)-HRESIMS m/z [M + H]+ 527.2757 (calcd for C29H39N2O7, 527.2757).

10-epi-FI-2 (6): Yellowish solid; [α + 41.4 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 322 (3.96) nm, 226 (4.39) nm; ECD (c 2.9 × 10−4 M, MeOH) λmax (Δε) 209 (+12.0), 239 (−7.4), 332 (+2.7) nm; IR νmax 3321, 2957, 2926, 2359, 2342, 1684, 1647, 1541, 1456, 1238, 669 cm−1; 1H NMR (700 MHz, DMSO-d6) and 13C NMR (176 MHz, DMSO-d6) data, see Table 1 and Table 2; (+)-HRESIMS m/z [M + H]+ 511.2800 (calcd for C29H39N2O6, 511.2808).

3.6. Bioactivity Assays

The in vitro cytotoxic activities of PTMs 1–5 were evaluated against four tumor cell lines, SF-268 (human glioma cell line), HepG2 (human liver carcinoma cell line), and MCF-7 (human breast adenocarcinoma cell line), A549 (human lung adenocarcinoma cell) by the SRB method, according to a previously described protocol [40]. All of the cells were cultivated in RPMI 1640 medium [41]. Cells (180 μL) with a density of 3 × 104 cells/mL were seeded onto 96-well plates and incubated for 24 h at 37 °C, 5% CO2. Subsequently, 20 μL of different concentrations of PTM compounds, ranging from 0 to 100 µM in dimethyl sulfoxide (DMSO), were added to each plate well. Equal volume of DMSO was used as a negative control. After a further incubation for 72 h, the cell monolayers were fixed with 50% (wt/v) trichloroacetic acid (50 μL) and then stained for 30 min. with 0.4% (wt/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by repeatedly washing with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris-base solution (200 µL) for the determination of optical density (OD) at 570 nm while using a microplate reader. The cytotoxic compound cisplatin was used as a positive control. All of the data were obtained in triplicate and presented as means ± S.D. IC50 values were calculated with the SigmaPlot 14.0 software using the non-linear curve-fitting method.