A new macrolactin antibiotic from deep sea-derived bacteria Bacillus subtilis B5

Abstract A new macrolactin derivate, 7-O-2′E-butenoyl macrolactin A (1), together with three known macrolactin compounds, macrolactin A (2), 7-O-malonyl macrolactin A (3) and 7-O-succinyl macrolactin A (4), was isolated from the bacterial strain Bacillus subtilis B5, which was isolated from the 3000 m deep sea sediment of the Southwest Pacific Ocean. The structures of the new compounds were assigned by spectroscopic methods including 1-D/2-D NMR and MS analysis techniques. Compounds 1 and 2 demonstrated antifungal activities against tea pathogenic fungi Pestalotiopsis theae and Colletotrichum gloeosporioides.

bacterial genus was also reported to biosynthesis cellulose economically by using Saccharum spontaneum (Abdullah et al. 2015). Up till now, nearly all the macrolactins isolated from microorganism had shown antimicrobial activity. For example, 7-O-methyl-5-hydroxy-3heptenoate macrolactin showed broad spectrum activity against human opportunistic clinical pathogens (Chakraborty et al. 2014). Furthermore, macrolactin S exhibited antibacterial activity against E. coli (Lu et al. 2008) and macrolactin A showed antibacterial activity against Gram positive bacteria Staphylococcus aureus (elkahoui et al. 2013). The product of Bacillus chlorotetaine also had antimicrobial activity against six common fungal pathogens (Wang et al. 2015). ib recent years, some other activities had been found, such as antiangiogenic activity of macrolactin A (Kang et al. 2015) and inhibiting TNF-α activity of 7-O-succinyl macrolactin A (Park et al. 2014).
Marine microorganisms were demonstrated as a new source of novel natural products, due to their extreme living conditions and their special physiological metabolism manner (Zhuang et al. 2012). Bacillus subtilis B5, a bacterial strain derived from deep sea sediments, showed antifungal activities against tea tree leaf pathogens Pestalotiopsis theae and Colletotrichum gloeosporioides. Bioactivity guided isolation of antibiotic macrolactin lead to the isolation of a new macrolactin derivate, 7-O-2′E-butenoyl macrolactin A (1), together with three known macrolactin compounds, macrolactin A (2), 7-O-malonyl macrolactin A (3) and 7-O-succinyl macrolactin A (4) (Figure 1).

Results and discussion
Compound 1 (7-O-2′E-butenoyl macrolactin A) was isolated as a yellow amorphous powder. The molecular formula of C 28 H 38 O 6 , which gave 11 unsaturation degrees, was established by the HR-eSi-MS showing an ion peak at m/z 493.2559 [M + Na] + . The iR spectrum showed the presence of OH groups (3436 cm −1 ), olefinic protons (1450, 1104 cm −1 ) and carbonyls (1664 cm −1 ). The UV maximum absorption wave length at λ max (log ε): 229 (2.1) nm indicated the presence of conjugated carbonyls. The 1 H and 13 C NMR spectra, including DePT, clearly showed two carbonyl carbons and 14 olefinic methines belonging to seven double bonds, in the sp 2 low field region. The sp 3 high field region showed the existence of two methyls, four oxygenated methines and six methylenes.
Most 1-D NMR spectral data, except for the 1 H and 13 C at the end of the side chain, approach to those of macrolactin A (2), a typical macrolactin isolated from culture of Bacillus amyloliquefaciens (Lee et al. 2004), indicated the presence of a macrolactin nucleus. The 13 C NMR signal assigned to C-7 of macrolactin A had downfield shifted from δ 70.2 to δ 72.4 in 1, as well as C-6, 8 upfield shifted from δ 42.3, δ 136.5 to δ 38.8, δ 131.7, respectively, indicating that the two compounds were different in their side chains at C-7. The acylation shifting was also observed in the 1 H NMR spectra, in which the H-7 signal of macrolactin A was downfield shifted from δ 4.19 to δ 5.43 in compound 1. A set of carbon signals of 2-butenoyl moiety at δ 165.2 (C-1′), 122.6 (C-2′), 146.1 (C-3′) and 18.2 (C-4′) was found in the 13 C NMR spectrum of 1. The corresponding 1 H NMR data of 2-butenoyl moiety could also be found at δ 5.91 (1H, dd, J = 15.6, 1.7 Hz, H-2′), 6.92 (1H, dd, J = 15.4, 6.8 Hz, H-3′) and 1.85 (3H, br.d, J = 6.2 Hz, H-4′), which were confirmed by HSQC and 1 H-1 H COSY spectra. The linkage of 2-butenoyl to C-7 was further concluded by HMBC spectrum, in which the correlation between H-7 at δ 5.43 to the carbonyl carbon at δ 165.2 (C-1′) was observed. Uptil now, the structure of compound 1 was elucidated as 7-O-2′E-butenoyl macrolactin A (Figure 1).
The antifungal activity of compounds 1-4 was evaluated by the paper disc inhibition assay and the minimum inhibitory concentration (MiC) was determined by paper disc dilution method (Woo et al. 2002). Compounds 1 and 2 demonstrated activities against tea pathogenic fungi P. theae and C. gloeosporioides (Supplementary material). The MiCs of compounds 1 and 2 are 500 and 125 μg/disc, respectively, to both pathogenic fungi.

General experimental procedures
Positive HR-eSi-MS were measured on a Thermo Q exactive LC-MS/MS spectrometer. The infrared (iR) and ultraviolet (UV) spectra were measured using a Perkin-elmer 683 (Perkinelmer, Norwalk, CT, USA) infrared spectrometer in KBr pellets and Shimadzu UV-260 spectrometer (Shimadzu, Tokyo, Japan), respectively. 1 H, 13 C NMR and 2-D NMR spectra were recorded on a Bruker Avance 600 FT-NMR spectrometer (Bruker, Bremen, Germany) with TMS as internal standard. A Cosmosil C18 column (20 mm × 250 mm, Kyoto, Japan) was used for preparative HPLC with Acetonitrile-H 2 O as the solvent at a flow rate of 8 mL min −1 . Column chromatography was performed on silica gel (Yantai Chemical industry Research institute, Yantai, China).

Bacteria and fermentation
The bacterial strain B5 was isolated from deep-sea sediments which were collected at water depths of 3000 m Pacific Ocean by the Third institute of Oceanography of China, and was identified as B. subtilis B5 by complete 16S rRNA gene sequence. This bacterium was cultivated on 80 L scale twice by using 100 L fermentation tank. Shake flasks containing 250 mL of the seed medium (tryptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.4) were cultured at 37 °C for 18 h at 200 rpm. 1% of the seed culture was inoculated into 80 L fermentation medium consisting of soluble starch 0.5%, yeast extract 1%, K 2 HPO 4 1%, NaNO 3 0.5%, pH 6.8-7.0 for 36 h at the same condition mentioned above.

Antifungal activity assay and MIC ditermination
The antifungal assay of all the four compounds was performed against tea pathogenic fungi P. theae and C. gloeosporioides. The tests were carried out in PDA Petri plates in which a 0.6 cm diameter piece of tested fungal strains cylinder agar was placed on the centre and sterile blank paper discs of 0.65 cm diameter were placed at a distance of 2 cm away from the growing mycelial colony. Approximately 50 μg compound was added to each paper disc.
DMSO without compound was used as the blank control. The plates were incubated at 28 °C until mycelial growth enveloped discs containing the control disc.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The project was supported by the National Natural