Tunicamycin

Antimicrobial tunicamycin derivatives from the deep sea-derived Streptomyces xinghaiensis SCSIO S15077

Shanwen Zhang, Chun Gui, Mingwei Shao, Pachaiyappan Saravana Kumar, Hongbo Huang & Jianhua Ju

To cite this article: Shanwen Zhang, Chun Gui, Mingwei Shao, Pachaiyappan Saravana Kumar, Hongbo Huang & Jianhua Ju (2018): Antimicrobial tunicamycin derivatives from the deep sea-derived Streptomyces xinghaiensis SCSIO S15077, Natural Product Research, DOI: 10.1080/14786419.2018.1493736 To link to this article: https://doi.org/10.1080/14786419.2018.1493736

KEYWORDS : Tunicamycin; nucleotide antibiotics; Streptomyces xinghaiensis; antimicrobial activity

1. Introduction

Tunicamycins are natural nucleoside antibiotics that can block both bacterial cell wall biosynthesis and eukaryotic protein N-acetylglucosamine-1-phosphate transfer by inhibiting the phospho-N-acetylmuramyl-pentapeptide translocase (Heifetz et al. 1979; Tsvetanova and Price 2001; Funabashi et al. 2013). Tunicamycins can be used as biological experimental tools, such as exploring the pathway of the lipid-linked oligo- saccharides formation and inducing unfolded protein response (Hemming 1977; Mahoney and Duksin 1979; Chan and Egan 2005). Structurally, tunicamycin com- pounds consist of an uracil unit, an N-acetylglucosamine (GlcNAc) unit and a fatty acid chain linked via an amide bond to the amino group of the central C11 aminosugar (tunicamine) moiety (Ito et al. 1980; Tsvetanova and Price 2001; Price and Tsvetanova 2007). Tunicamycins were originally isolated from Streptomyces lysosuperficus nov. sp., in 1971 (Takatsuki et al. 1971). To date, more than 43 tunicamycins have been isolated from Clavibacter species, Streptomyces species and Corynebacterium rathayi. The natural tunicamycins contain tunicamycins (Mizuno et al. 1971; Mahoney and Duksin 1979), streptovirudins (Eckardt et al. 1975), mycospocidins (Thrum et al. 1975), MM 19290 (Kenig and Reading 1979) and corynetoxins (Vogel et al. 1981; Edgar et al. 1982). Investigations revealed that tunicamycin is derived from 6- and 5-carbohydrate precur- sors by 13C isotope labelling experiments (Tsvetanova et al. 2002). In 2010, the biosyn- thetic gene cluster responsible for tunicamycin production was identified (Chen et al. 2010). More recently, the total synthesis of tunicamycin V has been described by Yamamoto et al. (2018).

As part of our on-going efforts to search structurally unique and biologically active molecules from the deep sea derived microorganisms (Song et al. 2014; Gui et al. 2015; Luo et al. 2016; Sun et al. 2018), we report herein the isolation, structural elucidation and activities of the antibiotic components 1–7 produced by Streptomyces xinghaiensis SCSIO S15077. Tunicamycin E (1) was elucidated as a new member of the tunicamycin antibiotics, together with six known tunicamycin analogues (2–7) (Figure 1).

Figure 1. Chemical structures of the compounds 1–7.

2. Results and discussion

Strain SCSIO S15077 was isolated from a sediment sample collected in the northern South China Sea at a depth of —3536 m. The isolate was identified on the basis of 16S rRNA sequence and blast analysis. It was confirmed as Streptomyces species and also had 99% sequence similarity with Streptomyces xinghaiensis (Zhao et al. 2009). Further, phylogenetic relatedness also revealed that the isolate was clustered within the lineages of the Streptomycetaceae with 99% sequence similarity to Streptomyces xinghaiensis S187 (CP023202.1) (Zhao et al. 2009).

The strain SCSIO S15077 was cultivated on a 15 L scale, and then the fermentation extract was purified by a series of purification procedures including silica gel chroma- tography, Sephadex LH-20 chromatography, reverse-phase medium-pressure chroma- tography, semi-preparative and preparative HPLC which rendered one new compound tunicamycin E (1, 10 mg) and six known compounds, tunicamycin B (2, 4 mg) (Takatsuki et al. 1977), tunicamycin X (3, 5 mg) (Edgar et al. 1982), tunicamycin A (4, 2 mg) (Takatsuki et al. 1977), streptovirudin D2 (5, 10 mg) (Eckardt et al. 1981), tunicamy- cin C (6, 3 mg) (Takatsuki et al. 1977), and tunicamycin C3 (7, 5 mg) (Quan et al. 2011). The structures of 2–7 were elucidated by comparisons of spectroscopic data with those previously reported in literatures.

Compound 1 was isolated as a white solid and the molecular formula was deter- mined as C37H60N4O16 on the basis of HR-ESI-MS (m/z 839.3895 [M + Na]+). Analysis of the 1D NMR spectroscopic data (Table S1) showed three methyls (dC 22.8, C-800; dC 22.4, C-11000; dC 13.9, C-14000), ten methylenes (dC 35.3, C-60; dC 60.5, C-600; dC 31.2, C-4000; dC 27.8, C-5000; dC 29.0-28.6, C-6000, C-7000, C-8000; dC 38.4, C-9000; dC 22.0, C-12000; dC 31.2, C- 13000), sixteen methines, four olefin carbons (dC 102.1, C-5; dC 140.3, C-6; dC 124.7, C-2000;dC 142.4, C-3000), and four carbonyls (dC 150.8, C-2; dC 163.0, C-4; dC 169.2, C-700; dC 166.1, C-1000). By comparing the 1H and 13C NMR spectroscopic data for 1 with those for the known compound 6, 1 was found to possess the identical uracil and GlcNAc moieties to that of 6, except that the signals of the anteisobutyl group in the terminal methyl branch was obviously different. The 1H NMR spectrum showed two methyl sig- nals at dH 0.79 (3H, 6.6, Me-11000) and dH 0.81 (3H, 6.9, Me-14000) in the fatty chain in 1 instead of the one doublet in 6, inferring the different methyl position in 1. The HMBC correlations from H-11000 to C-9000, C-10000 and C-12000, from H-14000 to C-12000 and C-13000, together with the 1H-1H COSY correlations of H-9000 /H-10000 /H-11000 and H-10000/H-12000/ H-13000/H-14000 finally located the methyl group (CH3-11000) at C-10 position (Figure S6–S8). Thus, compound 1 was identified as a new member of tunicamycins, desig- nated as tunicamycin E. The unusual fatty acid chain was proposed to be generated from the starter unit propionyl-CoA, and then followed by one methylmalonyl-CoA and four malonyl-CoA extender units (Yuzawa et al. 2017). The similar C-10000 substitu- tion methyl (Me-11000) was also observed in natural products, for example, the sulfated hybrid polyketide-nucleoside sphaerimicin A (Funabashi et al. 2013; Genilloud 2017) and odoamide (Kaneda et al. 2016; Sueyoshi et al. 2016; Blunt et al. 2018).

Tunicamycin E (1): white solid; [a]25D + 43.3 (c 0.07, MeOH); UV (MeOH) kmax (log e) 206 (3.88), 259 (3.98) nm; IR (ATR) vmax 3308, 2924, 2853, 1665, 1551, 1466, 1377, 1260, 1092, 1023 cm—1. 1H and 13C NMR spectroscopic data see Table S1; (+) HR-ESI-MS m/z839.3895 [M + Na]+ (calcd for C37H60N4O16Na, 839.3897, error 0.5 ppm).

All the isolates (1–7) were evaluated for their antimicrobial activities against six Gram-positive bacteria: Bacillus thuringiensis BT01, Bacillus thuringiensis W102, Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus shhs-A1 (clinical isolate), Enterococcus faecalis ATCC 29212 and Micrococcus luteus (Song et al. 2015); five Gram-negative bacteria: Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa (Song et al. 2015), Escherichia coli ATCC 25922, and drug resistant Escherichia coli E11 (aclinical isolate), as well as two fungi Candida albicans ATCC 96901 and Candida albicans CMCC (F) 98001. The previ- ous reports showed that tunicamycin homologues were highly active against B. subtilis (Eckardt et al. 1975; Thrum et al. 1975; Kamogashira et al. 1988). In this work, all compounds showed significant growth-inhibiting activities against B. thuringiensis with MIC values ranging from 0.008 to 2 lg/mL, which were superior to those of positive controls such as vancomycin, kanamycin and ampicillin. As indicated by MIC values, the longer fatty acid chain obviously increases the antibacterial activity against B. thur- ingiensis. Moreover, all compounds showed moderate growth inhibiting activities against fungi C. albicans CMCC (F) 98001 and a fluconazole resistant strain C. albicans ATCC 96901 with MIC values between 2 and 32 lg/mL (Table 1). Moreover, tunicamy- cins were previously reported to show antifungal activity against C. albicans KYU (Kamogashira et al. 1988). In present era, with the emergence of antimicrobial resist- ance microbes, tunicamycin analogues have potential applications in the field of bio- medical as antimicrobial agents to treat various infections caused by pathogenic microorganisms.

3. Conclusions

In summary, chemical investigation of the marine Streptomyces xinghaiensis SCSIO S15077 led to the discovery of new tunicamycin derivative, tunicamycin E (1), as well as six known tunicamycin antibiotics. All compounds showed significant growth-inhibiting activities against B. thuringiensis (MIC values, 0.008–2 lg/mL) and moderate anticandidal activity against C. albicans CMCC (F) 98001 and C. albicans ATCC 96901 (MIC values, 2–32 lg/mL).

Acknowledgements

This study was supported in part by the National Natural Science Foundation of China (81425022, U1501223 and U1706206), and Natural Science Foundation of Guangdong Province (2016A030312014). We thank Dr. Xiao, Ms. Sun, Ms. Zhang, Ms. Ma, and Mr. Li in the analytical facility at SCSIO for recording spectroscopic data.

Disclosure statement

No potential conflict of interest was reported by the authors.

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