Metabolic analysis of Streptomyces heterologous hosts

For analysis of macrolactam production in the heterologous hosts carrying the integrative and inducible expression vector pAsa. This vector is equipped with two Streptomyces-specific, inducible promoters flanking the inserted BGC. Potr⁴⁷, upstream of the cluster, can be activated by oxytetracycline dihydrate (OXT) and PnitA^{48, 49}, downstream of the cluster, can be activated by ε -caprolactam (ε-CL). To test whether macrolactam production can be induced using one or both promotors, Streptomyces were tested in four different conditions: ISP2 + no inducer (basal expression), ISP2 + OXT (final concentration 2.5 µM), ISP2 + ε-CL (final concentration 0.1%) or ISP2 + both inducers simultaneously. First 20 ml pre-cultures of S albus::pAsa, S. coelicolor::pAsa, and S. lividans::pAsa in ISP2 (+ 50 µg/mL Apr, 25 µg/mL NA) were grown at 30 °C and 150 rpm for 7 days. Afterwards, 100 µL of the preculture were inoculated on ISP2 agar (plates 90 × 15 mm) with different inducer combinations and incubated at 30 °C for another 7 days in the dark. One plate of each Streptomyces strain and each treatment was cut into small cubes (1 × 1 cm) and extracted with 30 mL EtOAc overnight. The solvents were evaporated under reduced pressure and the dried samples were dissolved in MeOH to a concentration of 50 µg/mL and analyzed by HR-UPLC-MS/MS. To compare macrolactam production with producer strains MS/MS data was also used for GNPS analysis.