Daunorubicin And High-yielding Strain Selection And Its Biotransformation

Daunomycin is one of the antitumor antibiotics, which belongs to anthracyclines and is produced by Streptomyces peucetius or Streptomyces coeruleorubidus. Its bioactivities include antibacterial, antitumor and immunosuppression. Adriamycin (also named doxorubicin) is a daunomycin derivative obtained by hydroxylation at c-i 4 and is produced from daunomycin by semi-synthesis methods in industry. Adriamycin has wider antitumor spectrum and fewer side effects as compared with daunomycin. They are the first line antitumor antibiotics in the chemotherapy of various cancers at present. The main purpose of this study is to improve the productivity of daunomycin producer, Streptomyces coeruleorubidus SIPL 1482, as well as to develop a biological system so as to produce adriamycin by bio-conversion. In the study of strain breeding by a novel technology called ribosome engineering, a screening method based on the resistance of strain to streptomycin or kanamycin was applied to improve the productivity of daunomycin by? Streptomyces coeruleorubidus var. Zhengding SIPI 1482. Streptomycin-resistant (str~) colonies occurred on G1 medium containing 6p.g/ml streptomycin showed a high frequency of positive mutation (34%). The highest one named Str5 1 demonstrated about 1.63 folds increase in daunomycin productivity as compared with parent strain. Initiative time for daunomycin biosynthesis by mutant Str~5 1 is about 24 hours earlier than that of the parent strain. When kanamycin was used to screen resistant mutants, the mutation frequency is much less than that with streptomycin. A distinctive character between these mutants is that kanamyein resistant mutant secretes something to inactivate kanamycin into medium but not for streptomycin resistant mutants. A pair of PCR primers was used to clone ribosomal gene from mutant Str5 1, however none of the desired results was obtained. The relationship between the improvement of daunomycin productivityand the level of streptomycin resistance was also discussed in this section. A mutagenie method of ion-beam implantation was adopted in the breeding of SIPI 1482. The spores of parent strain were treated in the conditions as follows: vacuum degree (1O3Pa), the energy of ion (6Okev), dosage rate (1O拁 N~/s ) and total dosage (1 X lO N/cm2). A mutant named S1P11482M2 was selected and it was found that its productivity is 15-folds higher than that of parent strain based on flask fermentation and 1-LPLC analysis. TLC and HPLC analysis was used to identify whether or not mutant SIPI 1 482M 1 produces any adriamycin. The colony characters between parent and mutant were also compared and it was found that mutant SIPI 1 482M2 could secrete great deal of red anthracycline metabolites into medium. The total DNAs of both parent and mutant SIPI 1 482M2 were isolated and digested completely by BamI-ll, and the electrophoresis characteristic of the SJPI 1482 M2 is obviously different from that of parent. In order to increase the resistance of Streptomyces lividans TK24 to daunomyc in, two kinds of mutagen (UV and NTG) were used in the strain breeding. Positive mutants were selected by a method of critical drug lethal concentration on the G 1 medium. Finally a stable mutant which exhibits the level of resistance up to 20p.gIml daunomyciri was obtained through several generations of mutation and selection. Other conditions such as type of medium, cultural time, spore pre-germination and so on were thoroughly investigated. It was found that artificial mutagenesis...