*2.2. Biological Activity*

These compounds were evaluated for their antimicrobial activities against *C. albicans* ATCC 10231, *S. aureus* ATCC 25923, *Mycobacterium bovis* (BCG Pasteur 1173P2), *M. tuberculosis* H37Rv (ATCC27294), and *E. coli* ATCC 25923. Compound **1** exhibited antibacterial activities against BCG, *M. tuberculosis*, and *S. aureus* with MIC values of 10, 10, and 100 μg/mL, respectively. Thiolutin (**3**) showed antibacterial activities against *E. coli*, BCG, *M. tuberculosis*, and *S. aureus* with MIC values of 6.25, 0.3125, 0.625, and 3.125 μg/mL, respectively (Table 3).

**Table 3.** Antibacterial activities of compounds **1**–**3** (MIC, μg/mL).


a Rapamycin, b vancomycin, c isoniazid, d ciprofloxacin.

#### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

NMR spectra were obtained on a Bruker Avance 500 spectrometer with residual solvent peaks as references (DMSO-*d*6: *δ*H 2.50, *δ*C 39.52). High-resolution ESIMS measurements were obtained on an Accurate-Mass-Q-TOF LC/MS 6520 instrument (Santa Clara, CA, USA) in the positive ion mode. HPLC was performed using an Agilent 1200 Series separation module equipped with an Agilent 1200 Series diode array, Agilent 1200 Series fraction collector, and Agilent ZORBAX SB-C18 column (250 × 9.4 mm, 5 μm).

#### *3.2. Microbial Material, Fermentation, Extraction, and Purification*

Strain *Streptomyces sp.* BTBU20218885 was isolated from a mud sample collected from the intertidal zone, Xiamen, China, and grown on an ISP2 (yeast extract 0.4%, malt extract 1%, dextrose 0.4%, agar 2%; pH 7.2) agar plate at 28 ◦C. Colony characteristics of BTBU20218885 are shown in Figure S17. The genomic DNA of BTBU20218885 was

extracted using a TINAamp Bacteria DNA Kit. PCR amplification of 16S rDNA was carried out by using universal primers (27f:5--GAGAGTTTGATCCTGGCTCAG-3-; 1492r: 5--CTACGGCTACCTTGTTACGA-3-). PCR amplification of the 16S rDNA was performed on TaKaRa PCR Thermal Cycler with the initial denaturation at 94 ◦C for 5 min, 30 cycles of denaturation (94 ◦C, 1 min), annealing (55 ◦C, 1 min), and elongation (72 ◦C, 1 min 15 s), and a final elongation at 72 ◦C for 10 min, in a 25 μL system (0.4 μL 20 μM of each primer, 2.5 μL 10× buffer, 2.5 μL 2.5 nM dNTP, 2 U rTap polymerase, and 1 μL DNA template). BTBU20218885 was identified as *Streptomyces* sp. by comparing the 16S rDNA sequence with the GenBank database using the BLAST program. A neighbor-joining (NJ) tree (Figure S18) was constructed using the software package Mega version 5 [26]. The strain was assigned the accession number BTBU20218885 in the culture collection at Beijing Technology and Business University, Beijing. The strain BTBU20218885 was inoculated on an ISP2 agar plate and cultured for 7 days. A 250 mL Erlenmeyer flask containing 40 mL of ISP2 medium was inoculated with BTBU20218885 and incubated at 28 ◦C (160 rpm) for 36 h. Aliquots (9 mL) of the seed cultures were aseptically transferred to 20 × 1 L Erlenmeyer flasks, each containing 300 mL of MPG media (glucose 1.0%, millet meal 2.0%, cotton seed gluten meal 2.0%, and MOPS 2.0%; pH 7.0), and the flasks were incubated at 28 ◦C, 160 rpm for 7 days. The culture broths were combined and centrifuged to yield a supernatant and a mycelial cake. The supernatant was extracted by equal volume of ethyl acetate (EtOAc, ×3 times), and the combined EtOAc extracts were evaporated to dryness in vacuo to give a dark residue. The residue was sequentially triturated with hexane, CH2Cl2, and MeOH to afford, after concentration in vacuo, hexane, CH2Cl2, and MeOH soluble fractions and precipitate. The precipitate was further purified by HPLC (Agilent ZORBAX SB-C18, 250 × 9.4 mm, 5 μm column, 3.0 mL/min, elution with 30% to 100% acetonitrile/H2O (0–20 min) to yield **1** (3.5 mg), **3** (2.6 mg), and **2** (13.2 mg).

Thiolopyrrolone A (**1**): Light-yellow amorphous powder; 1H and 13C NMR data, Table 1; HRESIMS *m*/*z* 621.0712 [M + H]+ (calcd for C24 H25 N6O6S4, 621.0713).

2,2-Dioxidothiolutin (**2**): Yellow amorphous powder; 1H and 13C NMR data, Table 1; HRESIMS *m*/*z* 260.9996 [M + H]+ (calcd for C8H9N2O4S2, 260.9998).
