Natural products biosynthesized by type II polyketide synthases (PKSs) comprise many important clinical drugs or drug candidates [1]. The angucycline-type polycyclic compounds, representing one of the largest family of type II polyketides, features a characteristic tetracyclic benz[a]anthracene scaffold, which are derived via successive decarboxylative Claisen condensations of an acetyl-CoA starter unit and nine methylmalonyl-CoA extender units [2,3,4]. The glycosylation using a combination of diverse sugar units at different angucyline aglycone position creates a great structural diversity for angucycline-type compounds [2, 5, 6]. These include landomycins, urdamycins, and saquayamycin, which are well known for their unusual structural features and potent antibacterial or antitumor bioactivities [7,8,9].

During our effort to discover new/bioactive natural products from microbes, we found that S. sp. 120454 strain mainly produce two major peaks based on chemical profiling when using B medium (dextrin 40 g, tomato paste 7.5 g, NZ Amine 2.5 g, primary yeast 5 g in 1 L distilled water) as fermentation medium. The large-scale fermentation was carried out at 30 °C for 7 days. The broth was harvested, and extracted by ethyl acetate, yielding 3.62 g brown crude extract, which was then fractionated and purified to afford compounds 1 and 2 (Fig. 1).

Fig. 1
figure 1

Structures of compounds 1 and 2

Compound 1 was isolated as a brown amorphous solid with the molecular formula of C25H23NO7, as determined by high-resolution ESIMS ([M+H]+ m/z = 450.1497), indicating 15° of unsaturation. Initial interpretation of its MS, 1H, 13C NMR spectra (Table 1) indicated the structure of 1 was highly similar to mayamycin (2), an aguacycline-type compound firstly isolated from a marine Streptomyces strain [10], with the exception of lacking an N-methyl group on its aminosugar moiety. Further elucidation of the 1D and 2D NMR spectra confirmed the presence of an identical anguacycline aglycone to that in mayamycin (2). An aminosugar moiety was evident by 1H-1H COSY correlations of H-1′/H-2′/H-3′/H-4′/H-5′/H-6′ from its 1H-1H COSY spectrum, and HMBC correlation of H-5′ (δH 3.56) with C-1′ (δC 72.5); as well as the NMR data comparison with those in 2. The linkage of this aminosugar moiety to angucycline was through a rare C–C bond according to the HMBC correlations of H-1′ with C-4a and C-6.

Table 1 1H (600 MHz) and 13C (125 MHz) NMR data of 1 and 2 [10] in methanol-d4

The stereochemistry of the sugar moiety was determined by interpretation of its NOESY spectrum and J-coupling constant. A large coupling constant (10.2 Hz) between H-1′ and H-2′a indicated an axial configuration of H-1′. The strong NOE correlations of H-1′ with H-3′ and H-5′ revealed that all of these three protons are in axial positions. Furthermore, H-4′ showed NOE correlations to H-2′a suggested that H-4′ and H-2′a are also in axial position. Thus, the relative configuration of the deduced aminosugar is 1′R*, 3′R*, 4′S, and 5′R. The result indicates that compound 1 is an N-desmethyl derivative of mayamycin (2), accordingly, compound 1 is designated as mayamycin B (Fig. 2).

Fig. 2
figure 2

Key 2D NMR correlations of 1

Compound 2 was also isolated as a brown amorphous solid, its structure is identified as mayamycin based on the comparison of its NMR and HRESIMS data with the published data [10, 11].

Mayamycin has been reported to show antibacterial activities against a panel of pathogenic bacteria [10, 11]. Therefore, the antimicrobial activities of compounds 1 and 2 were tested against six pathogenic bacteria as shown in Table 2. The result showed mayamycin B (1) showed potent bioactivity against Micrococcus luteus with MIC value of 2.0 μM, whereas, its congener mayamycin A (2) only has MIC value of 8.0 μM, suggesting the N-methyl group is important to its activity.

Table 2 Antibacterial activities of 1–2 (MIC, μM)

We next aimed to identify mayamycin biosynthetic gene cluster. To facilitate the process, the strain of Streptomyces sp. 120454 was subjected to whole-genome sequencing by Pacbio, yielding a 7.8 Mb linear chromosome with 71.5% GC content. AntiSMASH analysis indicated at least thirty one distinct secondary metabolite gene clusters were encoded in its genome with only one type II polyketide synthetic gene cluster, whose gene products show high similarity to proteins involved in other angucycline biosynthesis [2]. The putative may gene cluster spans a 23.2 kb contiguous DNA region consisting of 20 genes responsible for biosynthesis, regulation, and transporter. The nucleotide sequence have been deposited in the Genbank under accession number MG601230.

Functional assignments for the gene products within may gene cluster were made by sequence analysis through BLAST comparison. Biosynthesis of angucycline aglycone was initiated by the minimal type II PKS cassette, May16 (KSα), May15 (KSβ), and May14 (ACP) which utilize one acetyl CoA and nine malonyl-CoA to form a linear polyketide backbone. May12, May13, and May17 are homologues to bifunctional cyclase/dehydrase JadD, ketoreductase JadE, and polyketide cyclase JadI, respectively, in jadomycin biosynthesis. It is thus anticipated that the products of may12, may13, and may17 are sufficient for the formation of tetracyclic ring [12,13,14,15]. The subsequent two steps of dehydration followed by oxidation led to the formation of desired aglycone (Fig. 3).

Fig. 3
figure 3

Putative biosynthetic gene cluster for mayamycins

Six genes including may5, may6, may7, may9, may10, and may22 encode proteins similar to Med-ORF16, Med-ORF15, Med-ORF20, Med-ORF17, Med-ORF18, Med-ORF14, which are essential for the biosynthesis of aminosugar moiety of medemycin (Table 3) [16], suggesting these proteins were responsible for synthesizing aminosugar moiety in compounds 1 and 2. A C-glycosyltransferase, May21, catalyzes the final C-glycosylation step using either 3 or 4 as aminosugar donors to finally form 1 and 2 [16,17,18,19].

Table 3 Biosynthetic gene clusters of aminosugar moiety in mayamycins

In summary, a new type II polyketide, mayamycin B (1), together with its known congener mayamycin (2) were isolated and characterized from Streptomyces sp. 120454. Compound 1 showed potent antibacterial bioactivity against Micrococcus luteus. Sequencing and bioinformatics analysis of S. sp. 120454 allow us to propose the biosynthetic pathway of mayamycin for the first time.

Mayamycin B (1): dark brown amorphous solid; UV (MeOH) λmax (lgε) 440 (2.83), 328 (3.02), 298 (2.98), 236 (3.26); IR (KBr) νmax cm−1: 3250, 2922, 2857, 2360, 1608, 1420; 1H (600 MHz) and 13C (150 MHz) NMR data see Table 1; HRESIMS m/z: 450.1497 [M+H]+ (calcd. for C25H24NO7, 450.1508).