Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904

Rapamycin (RPM) is produced by Streptomyces hygroscopicus FC904 isolated fr om soil in Fuzhou, China. It is a triene macrolide antibiotic with potentia l application as an immunosuppressant and drug for human gene therapy. In a n attempt to improve rapamycin production, mutation and screening of the pa rent culture have been carried out. Thousands of survivors were obtained af ter mutagenesis by NTG (3 mg/ml) and UV (30 W, 15 cm, 30 seconds) of spore suspensions. None showed improved production of RPM. We determined the susc eptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions o f antibiotics in oatmeal agar plates. It was found that the strain was resi stant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, b ut susceptible to mitomycin C (MIC, 10 mug/ml) and aminoglycosides such as gentamicin (MIC, 0.1 mug/ml), kanamycin (MIC, 0.1 mug/ml) and streptomycin (MIC, 0.3 mug/ml). Protoplasts of strain FC904 were prepared after finding the best conditions for their formation. They were treated with gentamicin, erythromycin, mitomycin C and NTG. Surprisingly, gentamicin was especially effective for obtaining higher RPM-producing mutants. Mutant C14 was selec ted by exposing the protoplasts of the parent strain FC904 to 1 mug/ml of g entamicin at 28 degreesC for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, a nd its titer was 60% higher than that of the parent strain FC904 by HPLC an alysis, Another improved mutant (C14-2) was obtained from the spores of mut ant C14 treated with 1 mug/ml of gentamicin plus 2 mg/ml of NTG at 28 degre esC for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possi ble mechanism for the effect of gentamicin by using protoplasts or spore su spensions will be discussed, i.e. the possibility of gentamicin being a mut agen or a selective agent.