*2.5. Albucidin Isolation and 1H-NMR Spectroscopy*

*Streptomyces albus* 1K1 was grown in 10 L of SG medium, and albucidin was extracted with butanol. The dry extract was dissolved in 50 mL of water containing 5% acetonitrile and 0.1% formic acid. The extract was loaded onto 3 C18 SPE columns (Discovery DSC-18 SPE 52607-U) equilibrated with 5% acetonitrile in water with 0.1% formic acid. The flowthrough was collected. The columns were washed twice with 12 mL of 5% acetonitrile containing 0.1% formic acid. The flowthrough and the wash fractions were combined and evaporated in a rotary evaporator. The presence of albucidin was detected by HPLC-MS.

The dry material after the SPE purification step was dissolved in methanol and used for size-exclusion chromatography. Separation was performed on a glass column (30 mm × 1000 mm) packed with Sephadex LH-20 and methanol as the mobile phase. Fractions containing albucidin were identified by HPLC-MS. Albucidin-containing fractions were combined and evaporated. The dry extract was dissolved in 5 mL of 5% methanol in water containing 10 mM potassium phosphate buffer pH 6.4.

HPLC separation was performed on a preparative HPLC Thermo Dionex Ultimate 3000 equipped with a Macherey Nagel Nucleodur HTec C18 column (5 μm, 21 mm × 150 mm). A 10 mM potassium phosphate buffer (pH 6.4) was used as solvent A, and 50% methanol in 10 mM phosphate buffer (pH 6,4) was used as solvent B. The following gradient at a flowrate of 15 mL/min was used for separation: 0 min–13% B, 20 min—25% B, 21 min–25% B, 24 min–100% B, 25 min–100% B, 28 min–13% B, 29 min–13% B. Albucidin eluted at 18 min. The albucidin-containing fractions were pooled and evaporated.

For the final purification step, the dry material after HPLC purification was dissolved in 5 mL of water and loaded onto a Sephadex LH-20 column (30 mm × 450 mm) previously equilibrated with water. Water was used as the mobile phase. The fractions containing albucidin were identified by HPLC-MS, pooled and evaporated.

The 1H-NMR spectra were recorded on a Bruker Avance 500 spectrometer (Bruker, BioSpin GmbH, Rheinstetten, Germany) at 300 K equipped with a 5 mm BBO probe using deuterated trifluoroacetic acid (Deutero, Kastellaun, Germany) as the solvent containing tetramethylsilane (TMS) as a reference. Albucidin was measured in deuterated water (Deutero, Kastellaun, Germany). The chemical shifts are reported in parts per million (ppm) relative to TMS. All spectra were recorded with the standard 1H pulse program using 128 scans. The structure of albucidin was confirmed by comparison of the recorded 1H NMR data (Figure S1) with published data [10].

#### *2.6. Construction of the 1K1 BAC Derivatives*

The derivatives of 1K1 BAC with gene deletions were constructed using the RedET approach. For this, the antibiotic resistance marker was amplified by PCR with primers harbouring overhang regions complementary to the boundaries of the DNA to be deleted. The amplified fragment was used for recombineering of the BAC. The recombinant BACs were analysed by restriction mapping and sequencing. The primers used for recombineering purposes are listed in Table S3.

For the construction of BAC 1K1\_LS, the ampicillin marker from pUC19 was amplified with the primers LS-F/LS-R. For the construction of the BAC 1K1\_RS, the hygromycin marker from pACS-hyg [16] was amplified with the primers RS-F/RS-R. For the construction of the BACs 1K1\_KO14, 1K1\_KO15 and 1K1\_KO16, the ampicillin cassette was amplified with the pairs of primers KO14-F/KO14-R, KO15-F/KO15-R and KO16-F/KO16-R, respectively. For the construction of the BACs 1K1\_KO7, 1K1\_KO8, 1K1\_KO9, 1K1\_KO10, 1K1\_KO11, 1K1\_KO12 and 1K1\_KO13, the ampicillin cassette was amplified with the pairs of primers KO7-F/KO7-R, KO8-F/KO8-R, KO9-F/KO9-R, KO10-F/KO10-R, KO11-F/KO11-R, KO12-F/KO12-R and KO13-F/KO13-R, respectively.

BAC 1K1\_alb\_act was constructed in two steps. First, 1K1\_RS2 BAC was constructed from 1K1 using an ampicillin marker amplified with primers RS2-F/RS2-R. Then BAC 1K1\_alb\_act was constructed by recombineering the BAC 1K1\_RS2 using a hygromycin marker from pACS-hyg amplified with the primers ACT-F/ACT-R.
