Genetically engineered rpsL merodiploidy impacts secondary metabolism and antibiotic resistance in Streptomyces

Properties of Multidrug-Resistant Mutants Derived from Heterologous Expression Chassis Strain Streptomyces albidoflavus J1074

“…E. coli DH5α was used for routine cloning purposes. E. coli ET12567 (pUZ8002) was used to transfer cosmid pOJara-harboring aranciamycin BGC [ 10 ] and other plasmids according to the mating protocol described in [ 13 ]. Solid media SG2, SFM, R5S (see the recipe below), TSA, MM, and GYM [ 14 ] were used in the agar plug assays of antibiotic activity of S. albidoflavus strains.…”

“…Organic phase was evaporated in vacuo, and dry residue from a 5 mL fraction was dissolved in 100 µL of methanol. A volume of 12 µL of the methanol solution was used in disk assay of antibiotic activity, and 1 µL was subjected to LC-MS analysis, as described in [ 10 ]. MS analysis was carried out both in negative and positive ionization modes.…”

“…MS analysis was carried out both in negative and positive ionization modes. Spectrophotometric quantification of aranciamycin production by recombinant S. albidoflavus strains followed the protocol described in [ 10 ].…”

“… The pedigree of Streptomyces albidoflavus J1074 strains described in this work (adapted from [ 12 ]). SAM2 is a derivative of J1074, carrying the deletion of the pseudo attB φC31 site [ 10 ]. R94G is an initial pure rpsL mutant that served to generate multiple resistant spontaneous mutants.…”

“…of different types [ 5 , 6 ]; genome minimization [ 7 ]; and the introduction of beneficial genes and mutations [ 8 ]. We have previously described a set of genetically engineered J1074 mutants carrying point mutations within the rpsL gene encoding the ribosomal protein S12 [ 9 , 10 ]. Such mutations are known to enhance the production of SMs by streptomycetes [ 11 ].…”