Mutagenesis of the Rapamycin Producer Streptomyces hygroscopicus FC904.

Abstract

Rapamycin (RPM)is produced by Streptomyces hygroscopicus FC904 isolated from soil in Fuzhou, China. It is a triene macrolide antibiotic with potential application as an immunosuppressant and drug for human gene therapy. In an attempt to improve rapamycin production, mutation and screening of the parent culture have been carried out. Thousands of survivors were obtained after mutagenesis by NTG (3mg/ml) and UV (30W, 15cm, 30 seconds) of spore suspensions. None showed improved production of RPM. Wedetermined the susceptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions of antibiotics in oatmeal agar plates. It was found that the strain was resistant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, but susceptible to mitomycin C (MIC, lO^g/ml) and aminoglycosides such as gentamicin (MIC, O.l^g/ml), kanamycin (MIC, 0.1^g/ml) and streptomycin (MIC, 0.3 /ig/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 RPMproducing mutants. Mutant C14 was selected by exposing the protoplasts of the parent strain FC904 to 1 /ig/ml ofgentamicin at 28°C for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, and its titer was 60% higher than that of the parent strain FC904 by HPLCanalysis. Another improved mutant (C14-2) was obtained from the spores of mutant C14 treated with 1 /ig/ml ofgentamicin plus 2 mg/ml ofNTG at 28°C for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possible mechanismfor the effect of gentamicin by using protoplasts or spore suspensions will be discussed, i.e. the possibility of gentamicin being a mutagen or a selective agent.

Enhancement of clavulanic acid production by Streptomyces sp MU-NRC77 via mutation and medium optimization

“…The antitumor glycopeptide antibiotic zorbamycin produced from Streptomyces flavoviridis ATCC 21892 was enhanced through strain improvement by using UV radiation [22]. In another study, mutation and screening of the parent culture to improve rapamycin production were carried out [23]. It was reported previously that the lethality rate should be extremely high in order to have powerful mutations and effective screening of mutants.…”

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

“…Therefore, further improvement of the rapamycin titer and generation of diverse derivatives of rapamycin in S. hygroscopicus has been imperative for its commercial development and clinical applications. 62 In an attempt to improve rapamycin productivity, fermentation medium optimization as described in the previous section, 16,18,53,54 in addition to classical strain improvement, 63 protoplasts-related techniques 64 and high-throughput screening method 65 have been developed. Demain's research group obtained a higher rapamycin-producing mutant from the protoplasts of S. hygroscopicus FC904 mutants treated with 1 mg ml À1 of gentamicin, widely used aminoglycosides, where an HPLC analysis showed that the rapamycin titer for this mutant (139 mg l À1 ) was 60% higher than that for the parent strain.…”

“…An enhanced productivity of rapamycin (194 mg l À1 ; 124% higher than that of starting strain) was also obtained when exposing the spores of the S. hygroscopicus FC904 strain to gentamicin and N-methyl-N¢-nitro-N-nitrosoguanidine. 63 Another attempt to generate high-yield rapamycin-producing strains was based on the protoplast-related techniques such as protoplasts mutation, intra-and interspecies fusion. When using mutation by UV, N-methyl-N¢-nitro-N-nitrosoguanidine, diethyl sulfates treatment or fusion of different parental protoplasts of S. hygroscopicus, progress was not achieved.…”

Multistage selection of soil actinomycete Streptomyces albus as a producer of antimicrobial substances

“…The most useful combination of affecting factors is the chemical and physical mutagens (Rasouli and Kulkarni, 1994;Matseliukh, 1998;Cheng et al, 2001;Liu et al, 2003). Usually, the effectiveness of applied scheme of microbial producer selection is defined as a value of culture productivity increasing that varied from 1.5 to several tens of times.…”