Chinese Journal of Natural Medicines  2019, Vol. 17Issue (6): 475-480  

Cite this article as: 

HAN Hao, GUO Zhi-Kai, ZHANG Bo, ZHANG Mei, SHI Jing, LI Wei, JIAO Rui-Hua, TAN Ren-Xiang, GE Hui-Ming. Bioactive phenazines from an earwig-associated Streptomyces sp.[J]. Chinese Journal of Natural Medicines, 2019, 17(6): 475-480.

Research funding

This work was supported by Ministry of Science and Technology of China (No. 2018YFC1706205), the Natural Science Foundation of China (Nos. 81522042, 21572100, 81773591, 81530089, 21672101, 81673333, 21861142005, 21761142001, 21661140001, J1210026, and J1103512), Fundamental Research Funds for the Central Universities (Nos. 020814380092 and 020814380103), Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630052019011 and 19CXTD-32) and the Financial Fund of the Ministry of Agriculture and Rural Affairs, China (No. NFZX-2018)

Corresponding author

GE Hui-Ming, E-mail:

Article history

Received on: 27-Apr-2019
Available online: 20 June, 2019
Bioactive phenazines from an earwig-associated Streptomyces sp.
HAN Hao1 , GUO Zhi-Kai2 , ZHANG Bo1 , ZHANG Mei1 , SHI Jing1 , LI Wei1 , JIAO Rui-Hua1 , TAN Ren-Xiang1,3 , GE Hui-Ming1     
1 State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210023, China;
2 Hainan Provincial Key Laboratory for Functional Components Research and Utilization of Marine Bio-resources, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China;
3 State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
[Abstract]: Three new phenazine-type compounds, named phenazines SA-SC (1-3), together with four new natural products (4-7), were isolated from the fermentation broth of an earwig-associated Streptomyces sp. NA04227. The structures of these compounds were determined by extensive analyses of NMR, high resolution mass spectroscopic data, as well as single-crystal X-ray diffraction measurement. Sequencing and analysis of the genome data allowed us to identify the gene cluster (spz) and propose a biosynthetic pathway for these phenazine-type compounds. Additionally, compounds 1-5 exhibited moderate inhibitory activity against acetylcholinesterase (AChE), and compound 3 showed antimicrobial activities against Micrococcus luteus.
[Key words]: Earwig-associated actinomycete     Phenazine     Biosynthetic pathway     Antimicrobial activity     Acetylcholinesterase inhibitory activity    

Phenazines constitute a large family of nitrogen-containing natural products featuring a typical pyrazine ring (1, 4-diazabenzene) with two annulated benzenes and different types of side chains [1]. The first phenazine-type natural products, pyocyanin and chlororaphin, were isolated from severe purulent wounds of patients in the 19th century [2]. Subsequently, more than 150 phenazine-type natural products have been isolated from Gram-positive (e.g. Streptomyces) [3] and Gram-negative bacteria (e.g. Pseudomonas) [4], or from archaeal Methanosarcina species [5]. Phenazines display a broad range of biological functions including antibacterial [6], cytotoxic [7], and antimalarial activities [8]. In our continuing efforts to discover novel/bioactive natural products from microorganisms living in special niches [9-10], previously we investigated secondary metabolites from an earwig-associated actinomycete Streptomyces sp. NA04227 which led to the discovery of aurachin SS, a new aurachin-type antibiotic together with two known compounds [11]. Besides aurachin SS, HPLC analysis of its culture extract showed the presence of a series of unidentified metabolites with long UV wavelength absorption. After large-scale fermentation, extraction with ethyl acetate and repeated combinatorial chromatographic purification, seven phenazine-type compounds (1-7) were isolated, in which 1-3 were identified as new compounds on the basis of HRESIMS, NMR, as well as single-crystal X-ray diffraction analysis, while 4-7 were firstly isolated as natural products [12-13]. Herein, we report the isolation, structure elucidation and bioactivities of these phenazine compounds (Fig. 1) from the fermentation broth of Streptomyces sp. NA04227, as well as their plausible biosynthetic pathway based on the bioinformatic analysis.

Fig. 1 Structures of compounds 1-7
Results and Discussion

Compound 1 was isolated as a brown powder. The molecular formula was determined to be C19H20N2O2 on the basis of HR-ESI-MS data ([M + H]+, m/z 309.1604, Calcd. for [C19H20N2O2H]+, 309.1603), indicating eleven degrees of unsaturation. The 1H, 13C and HSQC NMR spectra of 1 (Table 1) revealed the presence of two methyls, two methoxyls, one methylene, and six olefinic/aromatic protons. The 1H-1H COSY correlations of H-2 (d, δH 7.14) and H-3 (d, δH 7.85), together with the HMBC correlations of H-2 with C-4 (δC 132.3) and C-12 (δC 136.9) and of H-3 with C-1 (δC 153.5) and C-5 (δC 141.3), indicated the presence of a 1, 2, 3, 4-tetrasubstituted benzene ring (ring A in 1). The 1H-1H COSY correlations of H-8 (d, δH 7.24, J = 8.2 Hz)/H-9 (t, δH 7.82, J = 8.2 Hz)/H-10 (d, δH 7.81, J = 8.2 Hz), and HMBC correlations of H-8 and H-10 with C-6 (δC 135.7), and of H-9 with C-7 (δC 155.7) and C-11 (δC 142.6) revealed a 1, 2, 3-trisubstituted benzene ring (ring C in 1). Furthermore, the 13C NMR spectrum also suggested that six quaternary carbons are not identified, along with the 1H-1H COSY and HMBC experiment of 1 displayed a typical pattern of phenazine skeleton. Two putative methoxyl groups resonance at δH 4.07/δC 56.4 and δH 4.13/δC 56.3 were connected to the phenazine skeleton based on HMBC correlations (Fig. 2), however, their relative position on the A and C rings cannot be established. The remaining 13C NMR signals at δC 28.7, 123.3, 132.1, 25.1 and 17.1 comprised a characteristic isopentenyl group, which located at the opposite position of CH3-19 (δH 4.13, δC 56.3), based on HMBC correlations of H-13 (δH 4.02) with C-3 (δC 128.1) and C-5 (Fig. 2). Since the relative location of two methoxyl groups was difficult to be fully assigned by NMR interpretation, we attempted to crystalize 1 for single-crystal X-ray diffraction analysis. Fortunately, a high quality single crystal of 1 was obtained from MeOH/H2O 3 : 1 (V/V) (Fig. 3), leading to the complete determination of structure for 1.

Table 1 1H and 13C NMR data for compounds 1-3
Fig. 2 The Key 2D NMR correlations of 1-3
Fig. 3 X-ray crystal structure of compound 1

Compound 2 was obtained as yellow powder and had a molecular formula of C18H18N2O2 ([M + H]+, m/z 295.1449, Calcd. for [C18H18N2O2H]+, 295.1447), according to their HR-ESI-MS data. The 1H and 13C NMR data of 2 resembled those of 1, indicating 2 possessed the same phenazine skeleton as 1. The major difference between 2 and 1 observed in the NMR spectra was the absence of one oxygenated methyl signal at C-7 (δC 151.5) in 2. In the phenazine skeleton, HMBC correlations of H-2 (δH 7.03) with C-12 (δC 137.9), of H-3 (δH 7.56) and H2-13 (δH 4.00) with C-5 (δC 140.5), of H-8 (δH 7.25) and H-10 (δH 7.93) with C-6 (δC 133.5), and of H-9 (δH 7.75) with C-11 (δC 142.1) allowed the assignment of the C-12, C-5, C-6 and C-11, respectively. Furtherly combined with detailed 2D NMR data analysis (Table 1, Fig. 2), the structure of compound 2 was elucidated as shown in Fig. 1.

Compound 3 was isolated as red powder with molecular ion peaks at m/z 371.1733 [M + Na]+ and Calcd. for [C22H24N2O2Na]+, 371.1735. The additional proton chemical shifts at δH 2.10 (H-16), δH 2.08 (H-17), δH 5.09 (H-18), δH 1.67 (H-20), δH 1.60 (H-21) and the 1H-1H COSY correlations between H-16/H-17/H-18 indicated that the isopentenyl group in 5 is replaced by a geranyl group. In the HMBC spectrum, characteristic J3 HMBC correlations of H-2 (δH 7.25) with C-12 (δC 134.5), of H-3 (δH 7.09) with C-5 (δC 132.0), of H-7 (δH 7.94) and H-9 (δH 7.73) with C-11 (δC 137.9), and of H-8 (δH 7.24) and H-10 (δH 7.73) with C-6 (δC 137.8) were observed. The downfield chemical shifts for C-4 (δc 154.4) and C-13 (δc 66.8) also suggested that the phenazine skeleton and geranyl group could be connected through an oxygen bridge, which was also confirmed by the HMBC correlation of H2-13 (δH 4.94) with C-4 and the NOESY correlation of H2-13 with H-3. Thus, the structure of 3 was established as shown.

As the whole genome of Streptomyces sp. NA04227 was fully sequenced and analyzed by antiSMASH previously [11]. One of the gene clusters was annotated as putative phenazine biosynthetic gene cluster (accession No. MH990330) as it contained the conserved proteins for phenazine biosynthesis. This gene cluster showed identical genetic organization, with 60%-90% sequence identity, to that phenazine gene cluster in Streptomyces sp. SpC080624SC-11 (Fig. 4) [13].

Fig. 4 Biosynthesis of the phenazine compounds in S. sp. NA04227. (A) The biosynthetic gene cluster of the phenazine compounds; (B) Plausible biosynthetic pathway of the phenazine compounds

This cluster spanned a 17.9 kb contiguous DNA region consisting of 16 open reading frames. Six genes, from spzB to spzG, exhibited high sequence identity to phzBCDEFG for phenazine biosynthesis in Pseudomonas fluorescens [14], while the homologous gene of phzA was missing in the spz gene cluster. An additional set of genes located at downstream of the spz cluster, spzH to spzM, which were tentatively annotated as enzymes for supplying dimethylallyl pyrophosphate and isopentenyl pyrophosphate in mevalonate pathway [15]. Besides the phenazine and mevalonate synthesis gene, additional four genes, spzO1, spzO2, spzS and spzP, were annotated as tailoring enzymes. SpzS was annotated as a flavin dependent hydroxylase and showed 53% protein sequence identity to PhzS [16], which can convert PCA to 1-hydroxyl phenazine (1-HP) by hydrolyzation and decarboxylation. SpzO1 was a monooxygenase and showed 58% protein sequence identity to LaPhzNO1, which can catalyze phenazine N-oxidation in Lysobacter antibioticus [17]. Similarly, SpzP exhibited 82% protein sequence identity to Mpz10 and was responsible for prenylations of 1, 6-dihydroxyphenazine (DHP) [13]. The remaining SpzO2 only showed 39% identity to ElmH, which was a monooxygenase catalyzing naphthacenone into naphthacenequinone in elloramycin biosynthesis [18].

Thus, taking all genetic information together, a biosynthetic pathway was proposed for biosynthesis of phenazine SA-SE in NA04227 (Fig. 4). Chorismic acid was used as the starting unit, and converted to HHPDC (Hexahydro-PDC) by SpzD, SpzE, SpzF and SpzB, which was identical to the phenazine pathway described before [14]. Subsequently, HHPDC could be directly catalyzed by SpzG to generate PDC (phenazine-1, 6-dicarboxylic acid) and further hydrolyzed and decarboxylated by SpzS to generate DHP (1, 6-dihydroxyl phenazine). Finally, SpzP catalyzed the addition of DMAPP at C-4 to produce 4, followed by one unidentified methyltransferase, which might locate outside of this cluster, transferring two methyl group step by step to afford 2 and 1. Alternatively, HHPDC could also go through spontaneous decarboxylation to THPCA (tetrahydrophenazine-1, 6-carboxylic acid) and further be oxidized by SpzG and SpzS to generate 1-HP (1-hydroxyl phenazine). Similar to 1, 2, 4, 1-HP could firstly and directly go through a prenylation to form 5. Secondly, 1-HP could also be hydroxylated by SpzO2 to afford 1, 4-DHP (1, 4-dihydroxyl phenazine) and SpzP might also accept GPP instead of DMAPP as substrate to produce 3, due to the substrate flexibility.

The isolated compounds 1-5 were evaluated for their biological activities. As shown in Table 2 and Table 3, only compound 3 showed moderate bioactivity against Micrococcus luteus with a MIC value of 4.0 μmol·L-1, indicating the importance of geranyl group for antibacterial activity. Furthermore, all of five compounds showed moderate inhibitory activity against acetylcholinesterase with IC50 values ranging from 2.32-3.62 μmol·L-1.

Table 2 The antimicrobial activity of compounds 1-5 (MIC, μmol·L-1)
Table 3 In vitro AChE inhibitory activity of compounds 1-5

In conclusion, three new phenazine-type compounds (1-3) along with four new natural products (4-7) were isolated and characterized from an earwig-associated Streptomyces sp. NA04227. A unified biosynthetic pathway was proposed based on the deduced function of the corresponding gene cluster. Additionally, compounds 1-5 exhibited moderate AchE inhibitory activities, and 3 showed antimicrobial activities against M. luteus.

Experimental General experimental procedures

Silica gel (200-300 mesh) for CC (column chromatography) and GF254 (10-20 μmol·L-1) for TLC (thin layer chromatography) were purchased from Qingdao Marine Chemical Company, China. Sephadex LH-20 was purchased from GE Biotech, USA. Semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) was accomplished on an Eclipse XDB-C18 column (5 μm, 250 mm × 9.4 mm) from Agilent Technologies Inc. USA (Santa Clara, CA, USA). Mass spectra were acquired on an Agilent 6250 TOF LC-MS instrument equipped with electrospray ionization (ESI) probe operating in positive-ion mode with direct infusion. NMR spectra were acquired on Bruker Avance Ⅲ 600 MHz or 400 MHz spectrometer with TMS or solvent signals adopted as internal standards: acetone-d6H 2.05; δC 29.82), and CDCl3H 7.26; δC 77.06).

Culture media

ISP4 (Soluble starch 10 g·L-1, MgSO4·7H2O 2 g·L-1, (NH4)2·SO4 2 g·L-1, NaCl 1 g·L-1, KH2PO4 1.36 g·L-1, CaCO3 2 g·L-1, trace elements 1 mL·L-1, Agar powder 15 g·L-1) agar plates were used for strain NA04227 growth and sporulation. The Tryptic Soy Broth (TSB) medium was used to incubate seed cultures, and the fermentation medium was YEME medium (yeast extract 4 g·L-1, glucose 4 g·L-1, malt extract 10 g·L-1).

Cultivation and isolation

The purified strain was cultured in ISP4 agar plates at 28 ℃ for 6 d. The seed cultures were incubated in TSB medium at 28 ℃ with 200 r·min-1 for 2 d (50 mL media in 250 mL erlenmeyer flask). The seed cultures were used to inoculate in the 300 mL fermentation broth at 28 ℃ with 150 r·min-1 for 8 days [19]. After 8 d cultivation, the filtrate (30 L) were adsorbed by XAD16N resin, washed by water and eluted with methanol. The methanol extract was concentrated in vacuum to obtain 4.6 g of crude paste, which was fractionated by column chromatography (CC) over silica gel using a gradient elution of petroleum ether/EtOAc (V/V, 100 : 0, 50 : 1, 20 : 1, 10 : 1, 5 : 1, 2 : 1, 1 : 1) to give seven fractions (Frs.1-7). These fractions were then separated by Sephadex LH-20 CC using MeOH as eluents and further purified by semi-preparative HPLC (Agilent Technologies 1260 Infinity Ⅱ) with CH3CN (67%). Finally, new compounds 1 (22 mg), 2 (6 mg) and 3 (11 mg) were obtained.

Phenazine SA (1)

Brown crystal; mp 107-108 ℃; UV (CH2Cl2) λmax (log ε) 272 (3.94), 364 (2.81) nm; IR (ZnSe) νmax 2955.2, 2922.4, 2850.6, 1712.5, 1463.2, 1377.4, 1270.6, 1153.1 cm-1; 1H and 13C NMR data, Table 1; HR-ESI-MS data [M + H]+, m/z 309.1604, Calcd. for [C19H20N2O2H]+, 309.1603.

Crystal data for 1

Molecular formula C19H20N2O2, Mγ =308.37, Monoclinic crystal, a = 15.996 (5) Å, b = 4.9479 (13) Å, c = 22.429 (6) Å, α = 90°, β = 100.32 (3)°, γ = 90°, Z = 4, μ = 0.394 mm-1, F (000) = 656.0 and T = 304 K; Crystal size: 0.15 × 0.1 × 0.05 mm3, Volume = 1746.5 (9) Å3, 8640 Reflections collected, 3219 independent reflections (Rint = 0.0735), the final R indices [I > 2sigma(I)] R1 = 0.0888, wR2 = 0.2583, R indices (all data) R1 = 0.1525, wR2 = 0.3342. The goodness-of-fit on F2 was 0.938. Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre (deposition no. CCDC 1864635). These data can be obtained free of charge via

Phenazine SB (2)

Yellow powder; mp 105-106 ℃; UV (CH2Cl2) λmax (log ε) 275 (4.06) nm; IR (ZnSe) νmax 2955.4, 2924.0, 2852.2, 1735.7, 1462.2, 1377.4, 1151.5 cm-1; 1H and 13C NMR data, Table 2; HR-ESI-MS data [M + H]+, m/z 295.1449, Calcd. for [C18H18N2O2H]+, 295.1447.

Phenazine SE (3)

Red powder; mp 113-114 ℃; UV (CH2Cl2) λmax (log ε) 273 (4.07) nm, 393 (2.87) nm; IR (ZnSe) νmax 2955.3, 2923.7, 2852.0, 1734.5, 1462.0, 1377.5, 851.6, 738.9 cm-1; 1H and 13C NMR data, Table 2; HR-ESI-MS data [M + Na]+, m/z 371.1723, Calcd. for [C22H24N2O2Na]+, 371.1735.

Antimicrobial assay

The MIC (minimum inhibition concentration) characters on the antifungal and antibacterial activities, which were determined in serial dilution assays against Bacillus subtilis CICC 10283, Staphylococcus aureus (MRSA) ATCC 43300, Streptococcus pyogenes ATCC 19615, Staphylococcus aureus CMCC (B) 26003 and Escherichia coli as described previously [20]. The sterilized YPM medium (49 μL) and different concentration of compounds (1 μL) were placed in 96-well microtiter plates and incubated at 37 ℃ for 24 h. The MIC values were determined as the lowest sample concentration, at which no bacterial growth could be discerned.

Acetylcholinesterase assay

An Ellman's method was applied to test the activity [21]. The phosphate buffer (130 μL, 0.1 mol·L-1, pH 8.0), AchE (20 μL, 0.035 U·mL-1), DTNB (20 μL, 0.333 mmol·L-1), and different concentration of compounds (20 μL) were placed in a 96-well microtiter plates and incubated at 37 ℃ for 15 min, and the inhibition rates were calculated by the absorbance in 412 nm. Finally, the IC50 values were determined by the standard curve line according to changes in concentration and inhibition rate.

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