Aurachins comprise a small family of natural products featuring a characteristic quinoline chromophore substituted in position 3 or 4 by a farnesyl chain.1, 2, 3 Owing to their structural similarity to analogs of the physiological ubiquinol and vitamin K, aurachins act as potent inhibitors of electron transport in the respiratory chain, more specifically as inhibitors of various cytochrome complexes.4, 5 With these structural properties, aurachin-type compounds are strong antimicrobial, antifungal and antiplasmodial agents. Aurachins were first isolated in 1987 from myxobacterium Stigmatella aurantiaca Sg a15.6 Since then, additional members of aurachin-type compounds were isolated from the same strain and also from an actinobacterial strain, Rhodococcus sp.7 During our continuous search for bioactive secondary metabolites from microorganisms living in special niche,8, 9, 10, 11 aurachin SS, a new aurachin-type compound together with two known compounds were isolated from Streptomyces sp. NA04227 (Figure 1).

Figure 1
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Structures of 1–3. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Strain NA04227 was isolated from an earwig (Forficula auricularia) collected in Qixia Mountain, Nanjing, China. This strain grows and sporulates well on CPA agar medium, and was identified as a Streptomyces sp. on the basis of its 16 s ribosomal RNA gene sequence (accession number: KY465886). The preliminary antibacterial assay of the crude extract from NA04227 in YEME medium showed strong antibacterial activity. To isolate and identify the bioactive components, we did a large-scale fermentation, and subjected the extract to a combination of chromatographic methods including normal phase, C-18 silica gel and Sephadex LH-20, leading to isolation and characterizations of a new compound, named as aurachin SS (1), together with two known compounds, aurachin C (2)12 and aurachin D (3)12 (Figure 1). Herein, we report the isolation, structure elucidation, biological activity and biosynthesis of these aurachins.

Compound 1 was isolated as a brown oil, and the molecular formula was determined as C21H27NO2 on the basis of HRESIMS analysis, indicating 9° of unsaturation. The 1H, 13C and HSQC NMR spectra indicated five methyl (with one of them oxygenated), three methylene, six methine groups and seven sp2 hybridized quaternary carbons (Table 1). The 1H-1H COSY correlations of δ 8.67 (d, H-5)/7.77 (t, H-6)/7.70 (t, H-7)/8.12 (d, H-8) suggested the presence of an ortho-substituted aromatic ring (Figure 2). A methyl substituted quinoline moiety was disclosed by the HMBC correlations of H-5/C-4 and C-8a, H-8/C-4a, H-7/C-8a, and H-9/C-2 and C-3 (Figure 2). The 1H-1H COSY spectrum also revealed two other spin systems of H-1′/H-2′ and H-4′/H-5′/H-6′. The HMBC correlations of H-6′/C-8′ and C-10′; H-5′/C-7′ and C-3′, H-9′/C-2′ and C-4′, and H-1′/C-3′ suggested the presence of a geranyl group, which was anchored at C-3 according to the HMBC correlations of H-1′/C-2 and C-4, and H-2′/C-3. The methoxyl group was assigned to C-4 on the basis of HMBC correlation of H-10/C-4. Finally, the unusual downfield chemical shifts for H-8 (δH 8.12) and H-9 (δH 2.62) indicated that the presence of N-oxide group to consume one remaining oxygen atom.12 Thus, the structure of 1 was established, and we named it as aurachin SS.

Table 1 1H (600 MHz) and 13C (150 MHz) NMR data of 1 in acetone-d6
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Figure 2
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Key 2D correlations of compound 1. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Inspired by the reported aua (in S. aurantiaca Sg a15) and rau (in Rhodococcus erythropolis JCM 6824) gene clusters, encoding the biosynthesis of aurachins1 and aurachin RE,7 respectively, we next aimed to identify Streptomyces aurachin biosynthetic genes (sau genes). The whole genome of Streptomyces sp. NA04227 was sequenced and then analyzed by antiSMASH online software,13 revealing a mixed biosynthetic gene cluster that showed identical genetic organization, with 30–40% sequence identity, to the aua gene cluster1 (Figure 3a; Table 2). Two genes auaEII and auaF, encoding an anthranilate-CoA ligase for activation of anthranilate14 and a Rieske [2Fe–2 S] oxygenase for N-hydroxylation,15 respectively, were missing from the sau cluster. The sau cluster has an additional set of genes, sauL, -M and -N, which were annotated as trans-isoprenyl diphosphate synthase, DXP synthase and HMBDP synthase, respectively, for supplying isopentenyl pyrophosphate and dimethylallyl pyrophosphate in MEP pathway.16 At both end of sau gene cluster, there are two P450 genes, sauPI and sauPII, showing 45 and 38% protein sequence identity to rauA (Figure 3a).7 Moreover, a thioesterase gene, sauK, was found in the middle of sau gene cluster, which is unique and missing in aua and rau gene clusters, and predicted to help the chain release from type II PKS and cyclization.

Figure 3
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Putative biosynthetic gene cluster for aurachin production in S. sp. NA04227. (a) Genetic organization and comparison of aurachin gene cluster in Stigmatella aurantiaca Sg a15 (aua cluster) and Rhodococcus erythropolis JCM 6824 (rau cluster). (b) Proposed biosynthetic pathway for aurachins in S. sp. NA04227. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Table 2 Deduced functions of ORFs in the aurachin biosynthetic gene cluster
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On the basis of the genetic organization and sequence identities, a biosynthetic pathway was proposed for biosynthesis of aurachins in NA04227 (Figure 3b). Anthranilic acid is activated by SauE,14 followed by extension of 5 with two malonyl-CoA units catalyzed by minimal PKS to form 6.1 Prenylation with farnesyl diphosphate catalyzed by SauA would yield aurachin D (3),17 and then hydroxylation presumably by SauPI or SauPII led to afford aurachin C (2). However, the substrate flexibility of SauA might also accept geranyl pyrophosphate instead of farnesyl pyrophosphate as substrate to produce 7. Finally, one unidentified methyltransferase will recognize the geranyl pyrophosphate attached product (8) as substrate to generate auracin SS (1).

The isolated compounds 1 –3 were evaluated for their antimicrobial activities. As shown in Table 3, compounds 2–3 showed potent bioactivities against Staphylococcus aureus, S treptococcus pyogenes, B acillus subtilis and M icrococcus luteus with MICs ranging from 4.0 to 16.0 μm. However, aurachin SS (1), which has a short side chain, only showed moderate activities against them with MICs from 32.0 to 64.0 μm.

Table 3 Antibacterial activities of 1–3 (MIC, μm)
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In summary, one new (1) and two known (2 and 3) aurachins were isolated and identified from an earwig-associated Streptomyces sp. NA04227. The new compound, aurachin SS (1), features a unique geranyl side chain, differing from typical aurachin-type compounds discovered so far. A unified biosynthetic pathway was proposed based on the identified gene cluster. Antibacterial assay showed compounds 1–3 exhibited moderate to potent activity against four tested bacteria.