*3.3. Bacterial Isolation*

Approximately 1 cm<sup>3</sup> (2 g) of the sea anemone *Gyractis sesere* (wet weight) were thawed and homogenized with a sterile mortar and pestle. Subsequently, the homogenized tissue was mixed with 9 mL of Ringer's buffer 1 4 strength [31] to produce a final solution of 1:10. This solution was incubated at 56 ◦C for 10 min with the aim of reducing the viability of non-actinobacterial microbes. After the incubation, 1 min of vortex was applied. The inoculation of the culture media was done by adding 50 μL of the dilution into 15 cm diameter Petri dishes containing the media. The inoculum was spread out on the plate with a triangular cell spreader made of glass. Finally, plates were incubated at 25 ◦C in darkness. Darkness was chosen as a filtering factor to eliminate potential microalgae contamination. Four different media were prepared for the isolation of Actinobacteria from the sea anemone *Gyractis sesere*. Medium SIMA1 (*Salinispora* isolation media A1) was selected from literature and slightly modified as follows: 2.5 g starch, 1 g yeas<sup>t</sup> extract, 0.5 g peptone, 1 L deionized water, and 25 g Tropic Marin ™ salt (Wartenberg, Germany), 15 g/L agar [32]. The other media (BCM, BTM, and BSEM) were generated for this study as follows: BCM, 3 g chitin, 0.5 g N-acetyl glucosamine, 0.2 g K2HPO4, 0.25 g KNO3, 0.25 g casein, 5 mL of mineral solution, 4 mL vitamin solution, 1 L deionized water, 15 g/L Tropic Marin ™ salt (Wartenberg, Germany), 12 g/L Gellan gum, pH = 7.35; BTM, 1 g trehalose, 0.25 g histidine, 0,25 g proline, 0.2 g MgCl2\*6H2O, 4 mL vitamin solution, 12 g/L Gellan gum, 1 L deionized water, 15 g Tropic Marin™ salt (Wartenberg, Germany), pH = 7.2; and BSEM, 0.1 g tyrosine, 0.1 g D-galactose, 4 mL vitamin solution, 5 mL mineral solution, 1 L Baltic Sea water, 16 g/L agar, pH = 7.4. Mineral salt solution contained 1 L distillated water, 50 mg FeSO4\*7H2O, 50 mg ZnCl2, and 50 mg CuSO4. Vitamin solution contained 1 L distillated water, 5 mg thiamine\*HCl, 5 mg riboflavin, 5 mg niacin, 5 mg pyrodoxine HCl, 5 mg inositol, 5 mg Ca-pantothenate, 5 mg p-amino benzoic acid, and 2.5 mg biotin.

The media were autoclaved for 35 min at 121 ◦C. Subsequently, the culture media were supplemented with 50 mg/L of nalidixic acid (Sigma-Aldrich, St. Louis, MO, USA) and 100 mg/L of cycloheximide (Carl Roth GmbH, Karlsruhe, Germany) [33], and poured into petri dishes. Once the sample was inoculated onto the petri dish, they were incubated for six weeks. When bacterial colonies were visually evident, we proceeded with the purification of the bacteria until obtaining an axenic culture. The isolated bacteria were conserved using Cryobank™ (Mast Diagnostica GmbH, Reinfeld, Germany) bacterial storage system.

### *3.4. Molecular Characterization and Phylogenetic Analysis*

DNA was extracted from bacterial cells by use of a DNA isolation kit, DNeasy™ (Qiagen, Hilden, Germany), following the manufacturer instructions. Subsequently, the 16S rRNA gene sequence was amplified with PCR and the use of general bacterial primers in a concentration of 10 pmol/μL, i.e., 27f and 1492r [34], 342f and 534r [35], 1387r [36] as well as 1525r [37]. PCR reagents were obtained from GE Healthcare Illustra™ PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare, Glattbrugg, Switzerland) containing DNA polymerase, MgCl2, and dNTPs. The PCR conditions were the same as reported by Staufenberger et al. [35]. Once the PCR amplification process was terminated, a quality check of the PCR products was performed by gel electrophoresis. The sequencing process was run at the Centre for Molecular Biology at Kiel University (IKMB). The 16S rRNA gene sequences were manually curated using Chromas pro software, version 1.7.6 (Technelysium Pty Ltd., Tewantin QLD, Australia), and saved in FASTA format. Sequences were aligned with nucleotide BLAST [38] and EZbiocloud [39]. Phylogenetic analysis involved the alignment of the sequences with related reference strains in the web platform SILVA-SINA [40]. MEGA was used to delete gap sites and to run bootstrapped phylogenetic trees using a neighbor-joining model [41].

### *3.5. Bacterial Growth for Secondary Metabolites Production*

For the evaluation of the secondary metabolites production, we grew the Easter Island isolated strain *Verrucosispora* sp. SN26\_14.1 in 10 × 2.5 L Thomson Ultra Yield® flasks (Thomson Instrument, Oceanside, CA, USA), which contained 1 L each of a modified starch-glucose-glycerol (SGG) liquid medium [31]. The composition of the production medium was: 5 g glucose, 5 g soluble starch, 5 g glycerol, 1.25 g cornsteep powder, 2.5 g peptone, 1 g yeas<sup>t</sup> extract, 1.5 CaCO3, and 1 L deionized water. The medium was also supplemented with 15 g/L Tropic Marin™ salt (Wartenberg, Germany). The pH was adjusted to 7.7 using 1 M HCl and NaOH. The culture was kept in orbital agitation at 240 RPM, 28 ◦C, for 14 days in darkness.

### *3.6. Chemical Extraction, Purification and Structure Elucidation*

After the growth period, 20 g/L amberlite XAD-16 (Sigma-Aldrich, St. Louis, MO, USA) was added to each culture medium flask and mixed for one hour using orbital agitation with 120 rpm. Subsequently, the resin was separated through cheesecloth filtration [42], and the liquid was discarded. Afterwards, amberlite plus cheesecloth was mounted on a glass funnel, washed with 3 L of deionized water, and eluted with 1 L of acetone [42]. Acetone was then concentrated under reduced pressure until an aqueous residue was obtained. One liter of deionized water was added to the acetone residue, and it was brought to a separation funnel. The organic molecules were extracted using 3 × 1 L of ethyl acetate. The organic phase was concentrated under reduced pressure until dryness.

For the evaluation of the produced metabolites, we used HPLC-DAD (Merck Hitachi LaChrom Elite, Darmstadt, Germany) and a 30 min gradient of H2O-acetonitrile supplemented with 0.1% of formic acid. The gradient was developed as following: 0 min: 90% water, 10% acetonitrile, 25 min: 0% water, 100% acetonitrile, 28 min: 0% water, 100% acetonitrile, 30 min: 90% water, 10% acetonitrile. The gravity SB™ C-18 column was obtained from Macherey-Nagel (Düren, Germany).

The purification of chemicals involved three different steps: 1) Flash chromatography using standard silica gel 60, pore size ~ 60 Å (Macherey-Nagel, Düren, Germany) as a stationary phase, mounted in a glass Buchner funnel (D = 70 mm, H = 180 mm). The mobile phase solvents were iso-octane and ethyl acetate. The chromatographic process was developed in a stepwise increase of polarity (10% each), starting with 100% iso-octane, and 0% of ethyl acetate, and ending in 0% iso-octane and 100% ethyl acetate, resulting in 10 different fractions. 2) The fraction that contained compound (**1**) and (**2**) was selected and worked in HPLC (Merck Hitachi LaChrom Elite, Darmstadt, Germany) using a normal phase NUCLEODUR® 100-5 column (4.6 × 250 mm) from Macherey-Nagel (Düren, Germany). The method used for the purification was a combination of isocratic and gradient solvent mix, with a flow rate of 1 mL/min, where A: iso-octane, B: ethyl acetate, and C: dichloromethane/methanol (50:50). The method was developed as following: 0 min: 100% A and 0% B, 3 min: 100% A and 0% B, 5 min: 95% A and 5% B, 9 min: 95% A and 5% B, 11 min: 0% A and 100% B, 13 min: 0% A and 100% B, 14 min: 10% A, 50% B, and 40% C, 16 min: 10% A, 50% B, and 40% C, 18 min: 50% A and 50% B, 19 min: 100% A and 0% B, 21 min: 100% A and 0% B. 3) The semi-purified compounds were purified through HPLC (Merck Hitachi LaChrom Elite, Darmstadt, Germany using a reverse phase C-18 column, 10 × 250 mm (YMC, Kyoto, Japan). The method used for the purification was a combination of isocratic and gradient solvent mix, with a flow rate of 2.5 mL/min. The method was developed as following: 0 min: 90% A and 10% B, 5 min: 20% A and 80% B, 9 min: 20% A and 80% B, 13 min: 0% A and 100% B, 19 min: 0% A and 100% B, 23 min: 90% A and 10% B, 25 min: 90% A, 10% B (A. water, B: acetonitrile).

After these purification steps, Lupinacidin A (**1**) and Galvaquinone B (**2**) were obtained with high purity to perform structural elucidation experiments. HRLCMS was performed with a Thermo Scientific™ Q Exactive™ Hybrid-Quadrupol-Orbitrap (Thermo Scientific, Bremen, Germany), positive mode, and a 30 min gradient of H2O and acetonitrile supplemented with 0.1% of formic acid. The gradient was developed as follows: 0 min: 90% water, 10% acetonitrile, 25 min: 0% water, 100% acetonitrile, 28 min: 0% water, 100% acetonitrile, 30 min: 90% water, 10% acetonitrile. Mass spectroscopic data was evaluated with Xcalibur® (Thermo Fisher Scientific, San Jose, CA, USA), and the compared with online databases (MarinLit, and Scifinder), and literature.

Additionally, 1H and 13C NMR and two-dimensional NMR experiments (HMBC, HSQC, COSY) were acquired to characterize the main components of crude extract, and their chemical functionality. For this, compound (**1**) and (**2**) were redissolved in CDCl3 (Eurisotop™, Saint-Aubin, France), and transferred to NMR tubes (178 × 5.0 mm). Experiments were acquired on a Bruker (Rheinstetten, Germany) Avance spectrometer operating at 600 MHz proton frequency equipped with a cryogenically cooled triple resonance z-gradient probe head using stand pulse sequences from the Bruker experiment library. Spectra were referenced against tetramethylsilane (Sigma-Aldrich, St. Louis, MO, USA) as internal standard.
