*3.1. Bacterium Culture Condition, Isolation, and Identification of Bioactive Compounds*

*Streptomyces* sp. SV 21 was isolated from the sea cucumber *Stichopus vastus* in Lampung, Indonesia. Sequences from the 16S rRNA of the bacterium has been submitted to the NCBI database under the accession number MK696479. It showed 100% similarity sequence to *Streptomyces cavourensis* with the type strain accession number NR\_043851.1. Further information on bacterial isolation can be found in [5]. After successful isolation, the bacterium was grown from the glycerol stock on Marine Agar that was made from Marine Broth (MB, Carl Roth, Karlsruhe, Germany) according to manufacturer's instruction, with addition of 9 g/L agar (Agar-agar Bacteriological, Carl Roth, Karlsruhe, Germany). A single colony of the bacterium was transferred into 10 mL of MB and incubated at room temperature (~22 ◦C) for three days and used as seeding broth. For that, 1 mL of the seed broth was used to inoculate 800 mL of fresh MB media stored in a sterile 2 L Erlenmeyer flask. The culture was grown for 14 days at room temperature (~22 ◦C) under constant shaking (100 rpm) to keep the media aerated.

A total of 2.4 L of broth culture were filtered through a 65 g/m2 paper filter grade 3 hw (Sartorius, Goettingen, Germany) to separate the cell mass from the remaining MB media. Subsequently, the resulting cellular material on the paper filter was extracted exhaustively with methanol (MeOH; HPLC grade VWR International GmbH, Darmstadt, Germany). The remaining broth media was extracted with ethyl acetate (EtOAc; HPLC grade VWR International GmbH, Darmstadt, Germany) in a 1 to 1 ratio by using an Ultra-Turrax (T25, IKA, Staufen, Germany) set at 12,000 rpm and applied for approximately 30 s. Both organic extracts were finally mixed and dried using a rotary evaporator (Rotavapor R II, Büchi, Flawil, Switzerland).

The bacterial crude extract was fractionated into seven fractions (Fr.) using a C18-based solid phase extraction (SPE) cartridge (10 g) with a column capacity of 75 mL (HyperSep C18, Fischer Scientific, Leicestershire, UK) with a mixture of DI water (Arium 611, Sartorius, Goettingen, Germany) and MeOH in different ratios starting with 70% and ending with 100% MeOH. However, only Fr. 3, 4 and 5 (85, 90, and 95% MeOH) showed high antiinfective activities. The target compounds were further purified by high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity, Agilent, Santa Clara, SA, USA), using a C18 (Pursuit XRs 5 μm C18 250 × 10.0 mm, Agilent, Santa Clara, SA, USA) semipreparative column. A linear gradient (sequence: 10 min from 70:30 MeCN:H2O to 100:0

MeCN:H2O, 30 min 100:0 MeCN:H2O, then 20 min 70:30 MeCN:H2O) was applied. Formic acid (FA, 98%, Carl Roth, Karlsruhe, Germany) was added at a concentration of 0.1% to both the solvent MeCN and H2O. The flow rate was set at 1.5 mL min−1, the column temperature was set at 40 ◦C, and the detection wavelength of the Diode Array Detector (DAD; Agilent 1260 Infinity Diode Array Detector (G4212-60008), Agilent, Santa Clara, SA, USA) was set to 210 nm. With each semi-preparative run, 100 μL of a 10 mg mL−<sup>1</sup> solution was injected. The target compounds eluted between 28 and 36 min. Each individual compound was collected: compound **1** (0.6 mg, RT 29.1 min), compound **2** (1.3 mg, RT 30.4 min), compound **3** (23.2 mg, 32.5 min), and compound **4** (1.8 mg, 34.6 min).

Analysis of the SPE fractions was performed on an ultra-high-performance liquid chromatography–high-resolution mass spectrometer (UPLC-HRMS; Waters Synapt G2-Si, Milford, MA, USA). Chromatographic separation was achieved on a BEH C18 column (Waters ACQUITY, Milford, MA, USA; 1.7 μm, 2.1 × 50 mm) with the column temperature set to 40 ◦C. The mobile phase was a linear gradient (sequence: 30 min from 5:95 MeCN:H2O to 95:5 MeCN:H2O, 10 min 95:5 MeCN:H2O, then 10 min 5:95 MeCN:H2O). The flow rate was set at 0.3 mL min<sup>−</sup>1. Formic acid (FA, 98%, Carl Roth, Karlsruhe, Germany) was added at a concentration of 0.1% to both the solvent MeCN and H2O. Analytes were detected by ESI-MS in the positive ionization mode (POS) by monitoring the mass range of *m/z* 50 to 2000 Dalton (Da). The target compounds showed clear peaks in the total ion chromatogram (TIC), with *m/z* of 1100.633, 1114.650, 1128.673, and 1142.685 Da.

For structure verification purposes, we analyzed the MS<sup>2</sup> data by using MASST GNPS to identify the likely fragments of the target compounds or its analogues with the set parameters described in [5]. In addition, we also measured the NMR spectra on a Bruker 700 MHz cryo NMR spectrometer (Avance III HD) with Topspin 3.6.2 for analysis. For compounds **1** to **3**, the structures were verified with the following NMR experiments: 1D proton, 1H,13C-HSQC (pulse program: hsqcedetgpsisp2.3) and 1H,13C-HMBC (pulse program: hmbcgplpndqf). For structure elucidation of compound **4**, the following additionally experiments were conducted: 1D carbon, 1H,1H-COSY (pulse program: cosygpppqf) and 1H,1H-TOCSY (pulse program: mlevphpp). The following parameters were used for compound **4**: COSY (data acquisition: 2K/512 points, relaxation delay D1 1.5 s, acquisition time AQ 133 ms, number of scans NS 32), TOCSY (data acquisition: 2K/512 points, relaxation delay D1 2 s, acquisition time AQ 133 ms, number of scans NS 32), HSQC (data acquisition: 2K/256 points, relaxation delay D1 2 s, acquisition time AQ 146 ms, number of scans NS 16, delay <sup>1</sup>*J*CH 145 Hz) and HMBC (data acquisition: 2K/256 points, relaxation delay D1 1.5 s, acquisition time AQ 146 ms, number of scans NS 40, delay *nJ*CH 8 Hz). All samples were dissolved in 0.6 mL CDCl3. Optical rotation was measured on a polarimeter (Anton Paar, Graz, Austria) with a 100 mm path length and sodium D line at 589 nm.

Streptodepsipeptide P11B (**1**): white amorphous powder; molecular formula C52H86N6O18; UV (MeOH, photodiode array), *λ*max 220 nm; [*α*] 20 *<sup>D</sup>* +23.3◦ (*c* 0.03, CHCl3) (lit. +26.7◦, [6]); HR-ESI-MS *m/z* 1100.6332 [M + NH4] <sup>+</sup> (calcd. for C52H90N7O18+, 1100.6337).

Streptodepsipeptide P11A (**2**): white amorphous powder; molecular formula C53H88N6O18; UV (MeOH, photodiode array), *λ*max 220 nm; [*α*] 20 *<sup>D</sup>* +34.0◦ (*c* 0.05, CHCl3) (lit. +21.6◦, [6]); HR-ESI-MS *m/z* 1114.6486 [M + NH4] <sup>+</sup> (calcd. for C53H92N7O18+, 1114.6493).

Valinomycin (**3**): white amorphous powder; molecular formula C54H90N6O18; UV (MeOH, photodiode array), *λ*max 210 nm; [*α*] 20 *<sup>D</sup>* +29.7◦ (*c* 0.60, CHCl3) (lit. +18.6◦, [6]); HR-ESI-MS *m/z* 1128.6659 [M + NH4] <sup>+</sup> (calcd. for C54H94N7O18+, 1128.6650).

Streptodepsipeptide SV21 (**4**): white amorphous powder; molecular formula C55H92N6O18; UV (MeOH, photodiode array), *λ*max 210 nm; [*α*] 20 *<sup>D</sup>* +15.0◦ (*c* 0.10, CHCl3); HR-ESI-MS *m/z* 1142.6804 [M + NH4] <sup>+</sup> (calcd. for C55H96N7O18+, 1142.6806); for the 13C and 1H NMR data, see Table 2.
