An N-N linked dimeric indole alkaloid from the marine sponge-associated rare actinomycetes Kocuria sp. S42

Abstract Marine derived rare actinomycetes is emerging as one of the new sources for various natural products for further drug discovery. Dimeric indole alkaloids represent a group of structurally diverse natural products and N-N linkage is a special dimerization mode. Here, we report the isolation of 1,1'-([1,1'-biindole]-3,3'-diyl) bis (ethane-1,2-diol), a new tryptophan-derived indole alkaloid from the marine sponge-derived Kocuria sp. S42. The structure was established based on extensive spectroscopic analyses, including nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass (HR-ESI-MS) spectrometry. The new dimeric indole alkaloid via N-N linkage exhibits moderate antimicrobial activity. Graphical Abstract


Introduction
Actinomycetes are the most important group of microorganisms with a great ability to produce diverse bioactive secondary metabolites (Wang et al. 2020).The marine ecosystem covers nearly 71% of the earth's surface, harboring tremendous underexplored microbial communities.Due to the extreme environmental challenges, such as limited light, nutrients, pressure and predation, a large number of microorganisms evolve complex symbiosis to accommodate to these variations (Roue et al. 2012).These marine-derived microorganisms including actinomycetes are found to be associated with marine lives like sponges, corals, and ascidians (Blunt et al. 2012).The marine-derived actinomycetes feature special biochemical characteristics and unique metabolic pathways, leading to production of diverse secondary metabolites to assure survival in marine habitats (Montaser and Luesch 2011).Hence, actinomycetes from marine ecosystems also constitute the novel sources of diverse and potentially bioactive natural products, aside from the terrestrial actinomycetes (Subramani and Sipkema 2019;Newman and Cragg 2020).
Rare actinomycetes are specifically defined as actinomycete strains less frequently reported than that of Streptomyces species (Subramani and Aalbersberg 2013).Over the past three decades, a substantial number of rare actinomycetes have been isolated from the marine ecosystem and are believed to be potentially new resources of structurally diverse natural products (Subramani and Sipkema 2019).For example, from the rare actinomycete Kocuria palustris, a new compound named PM18110448 (Kocurin) was discovered and demonstrated to have a broad spectrum of antibacterial activity (Martin et al. 2013).
Indole heterocycle alkaloids present a vital group in natural product chemistry and indole alkaloids those made up of two or more indole subunits are an important subset (Netz and Opatz 2015).Most of these alkaloids express the C-C or C-N linkage (Newhouse et al. 2010;Adak et al. 2022) while a rarity among such structures involves linkages between nitrogen atoms of the indole (Figure 1).Dixiamycin B, the pentacyclic indolesesquiterpene possesses an N-N dimeric linkage, which exhibits more potent than the monomeric xiamycin (Jin et al. 2021).In our work, we selected Kocuria sp.S42, a rare marine-derived actinomyces as the target strain for new natural products discovery.An N-N linked dimeric indole alkaloid has been isolated and structurally elucidated as 1,1 0 -([1,1 0 -biindole]-3,3 0 diyl) bis (ethane-1,2-diol) by spectroscopic analysis.The evaluation of bioactivities of this compound (JIA-X21) exhibited moderate antibacterial activity.

Cultural condition of strain S42
DNA sequencing of the housekeeping 16S rRNA gene showed significantly homologous (99.30%) to those of the Kocuria sp.strain SS263-23 (Accession Number: JX007975) in the GenBank database of NCBI.Phylogenetic analysis based on the 16S rRNA genes assigned species of S42 as Kocuria sp.(Accession Number: OM436921) (Figure S1).To explore the cultural conditions of Kocuria sp.S42, we carried out fermentation using selected five fermentation media according to the literature (Li and Zhang 2020), with metabolites varying in different fermentation media (Table S1 and Figure S2).The growth in M1 media is extremely well and gives the most prolific HPLC profile.We therefore chose M1 media for large-scale fermentation.2.2.Isolation and structure elucidation of a new product from kocuria sp.S42 A special metabolite (peak ᭜) in the M1 medium encouraged a large-scale fermentation of Kocuria sp.S42 to isolate the corresponding compound (Figure S2).Extraction of 12.0 L culture with organic solvent extraction and subsequent column chromatography resulted in the isolation of the compound JIA-X21.
A substituted indole substructure was constructed by analysis of the COSY correlation between H-5 and H-6; H-6 and H-7; H-7 and H-8, and combined with the HMBC correlation from H-5 to C-3 (d C 109.0) and C-9 (d C 135.6); H-6 to C-4 (d C 127.5); H-7 to C-9 (d C 135.6); H-8 to C-4 (d C 127.5); H-2 to C-4 (d C 127.5) and C-9 (d C 135.6).Another substructure was constructed by the COSY correlation between H-10 and H-11.Finally, the HMBC correlation of H-11 to C-3 connects the two substructures together to give the partial structure 1a (Figure S10) (Narumiya et al. 1979).

Antibacterial activity
The isolated JIA-X21 was tested for antimicrobial activity in vitro against E. coli, B. subtilis, P. aeruginosa, and S. aureus.The compound JIA-X21 inhibits E. coli, B. subtilis, P. aeruginosa at minimum inhibitory concentration (MIC) values of 31 mg/mL, 31 to 63 mg/ mL, and 63 to 125 mg/mL, respectively.It didn't show any antibacterial activity against S. aureus.
The 16S rRNA gene sequence of strain S42 was proofread using Chromas (version 2.6.5), and compared with the sequences available in NCBI (http://www.ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST).Multiple sequence alignment was performed using CLUSTALX.Phylogenetic tree was constructed using MEGA 11.

Bacterial strain, culture conditions and fermentation
Kocuria sp.S42 was isolated from a marine sponge Agelas cathodes sample, Yongxing Island, South China Sea (May 2011).Five fermentation media were tested for Kocuria sp.S42 (Table S1).All media were supplemented with sea salt (38.0 g L À1 ) to simulate the conductivity and salinity conditions of the sea water.Kocuria sp.S42 was grown on solid ISP2 (glucose 4.0 g L À1 , yeast extract 4.0 g L À1 , malt extract 10.0 g L À1 , sea salt 38.0 g L À1 , adjusted to pH 7.2 before autoclave) agar plate for 3 days at 30 C. Kocuria sp.S42 mycelium (about 1 cm 2 ) was inoculated into 250 mL Erlenmeyer flask containing 50 mL of ISP2 as seed medium and incubated at 30 C and 220 rpm for 2 days.500 mL M1 medium (starch 30.0 g L À1 , soybean powder 10.0 g L À1 , yeast extract 2.5 g L À1 , CaCO 3 3 g L À1 , sea salt 38.0 g L À1 , pH 7.0) in 2000 mL Erlenmeyer flasks was inoculated with 10% (v/v) seed culture and incubated at 30 C, 220 rpm for 5 days.

Extraction and isolation
After 5-day fermentation, 12.0 L of Kocuria sp.S42 fermentation broth were harvested by centrifugation (6500 rpm, 20 min).The separated supernatant was extracted three times with the same volume of ethyl acetate.The ethyl acetate extracts were combined and removed under the reduced pressure, and the crude extract (5.42 g) was subjected to Sephadex LH-20 column and eluted with methanol as the mobile phase to yield eight fractions (Frs.A-Frs.H).Fr.A was further chromatographed on semi-preparative reversed HPLC (XBridge C18,10 Â 250 mm, 5 lm, Fisher Wharton) using a gradient elution system of 55 to 60% acetonitrile in H 2 O containing 0.1% formic acid (t R ¼ 25.0 min).Each sub-fraction was tracked by HPLC analysis to afford a pure compound JIA-X21 (3.0 mg).

Antimicrobial assay
Antimicrobial assays were carried out using E. coli, P. aeruginosa, S. aureus, and B. cereus as the indicators.Stock solutions of the compound were prepared in methanol, and the antimicrobial assays were carried out in 96-well plates against the microbial strains (5 Â 10 5 CFU/mL) using a modification of the published method (Yang et al. 2006;Malapaka et al. 2007).After incubation for 14-16 h at 37 C, the absorbance at 600 nm was measured using a microplate reader (BioTek, Winooski, VT, USA).Kanamycin was used as the reference compound.

Conclusions
In summary, we isolated and identified a new N-N linked dimeric indole alkaloid from Kocuria sp.S42, a marine sponge-associate rare actinomycetes from the South China Sea.Until now, very few compounds have been reported from Kocuria sp. and our work provides an opportunity to investigate the metabolic potential of Kocuria sp.Besides, this is the first report of a dimeric indole alkaloid via N-N linkage from rare actinomycetes.The discovery of JIA-X21 from the strain would expand the family of dimeric indole alkaloids and further explore the N-N dimerization mode from genomic landscape.

Figure 1 .
Figure 1.Representative dimeric indole alkaloids from marine natural products.