Luna 5 μm c18 2

For structure elucidation, each mutant strain was cultivated in 4 × 1 L LB medium (with the exception of bgc33, which was cultivated on 16 L scale) containing 100 μg/mL of the corresponding antibiotic and incubated at 30 °C (25 °C for bgc33) with shaking at 225 rpm. After 48 hr (28 hr for bgc33), the culture supernatant was extracted 2x with an equal volume of EA, and the solvent was removed from the combined extracts by rotary evaporation. The dried material was dissolved in 80% MeOH/DMSO and separated by reverse-phase HPLC (Agilent 1200 series) for small molecule purification. NMR spectra were collected on either a Bruker Avance DRX500 or a Bruker AvanceIII 600-I spectrometer. Purification of EA fraction was carried on by gradient HPLC on a C18 reverse phase column (Phenomenex Luna 5 μm C18 (2), 250 × 10 mm) with a flow rate of 5.0 ml/min. The gradient system was MeCN (solvent B) and H₂O (solvent A).

The activity assay samples were analyzed using an Agilent 1200 series HPLC with a Phenomenex Luna 5 μm C18(2), 100 Å, 150 × 4.6 mm column. The following method was used: 20 min at 52% solvent B, gradient of 52% to 95% solvent B over 1 min, 95% solvent B for 3 min, gradient of 95% solvent B to 52% solvent B over 1 min, and 2 min at 52% solvent B. Solvent A is HPLC grade water + 0.1% trifluoroacetic acid (TFA), and solvent B is HPLC grade acetonitrile + 0.1% TFA. Absorbance was monitored at 220 nm, and the tribromopyrrole product peak was integrated and normalized to the amount produced by WT Bmp8.

A. wentii strains were cultured in 40 mm × 150 mm diameter Petri dishes with a total volume of 2 L of PDA medium at 30 °C for 5 days. The agar was chopped into pieces and extracted with methanol and dichloromethane:methanol (1:1) followed by 1 h of sonication as described above. The residue was extracted three times with EtOAc, and the combined EtOAc layers were evaporated in vacuo to yield a crude extract. Silica gel column chromatography was performed using dichloromethane and methanol as eluent (starting with 100% dichloromethane). Each fraction was further separated through preparative HPLC [Phenomenex Luna 5 μm C₁₈ (2), 250 × 21.2; flow rate of 5.0 mL min⁻¹; UV detector at 280 nm]. Nuclear magnetic resonance (NMR) spectral analysis was performed using a Varian Mercury Plus 400 spectrometer. High-resolution electrospray ionization mass spectra (HRESIMS) were obtained on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer at a flow rate of 5 μL min⁻¹. MS conditions included a spray voltage of 5 kV, sheath gas flow rate at 15 au, auxiliary gas flow rate at 5 au, capillary temperature at 320 °C, s-lens RF level 60, scan range of 100.0–500.0 m/z, resolution of 140,000, AGC target of 5 × 10⁵, and maximum injection time of 50 ms.