Study On The High-yielding Strain Improvement And Culture Condition Optimization For Rapamycin

Rapamycin is an triene macrolide antibiotics produced by Streptomyces hygroscopicus which was isolated from a soil sample collected on Easter Island. It is considered as a potent immunosuppressant which also has antifungal and antitumor activities. Strain improvement of rapamycin producer and fermentation of rapamycin have been systematically studied in this paper. And finally several high-yielding mutants which increased rapamycin productivity obviously were isolated.The determination method of rapamycin by HPLC and bioassay activity were established. The relation between two methods was studied and it confirmed that they had linear correlation in a certain range. A novel high-throughput screening method was developed to rapidly screen large numbers of rapamycin-producing mutants based on the relationship.We studied the slant medium, seed culture medium and fermentation medium separately, observed the growth character of the strain and optimized all the mediums. After optimization, the productivity of rapamycin was 97% higher than origin.According to the study of biosynthesis pathway of rapamycin, we designed continuous mutations by mono-mutagen, alternat mutations by physical and chemical mutagens, continuous mutations by physical and chemical mutagens and so on. After many runs continuous mutations, one mutant produced 279mg rapamycin /l, which was 156% higher than the original strain used in the submerged fermentation process.The optimum conditions for preparing and regenerating rapamycin producer's protoplast were determined. The effects of heat inactive and ultraviolation inactive were studied. And both the single-parent inactive and double-parents fusion for rapamycin producer were also studied. According to the comparability of rapamycin and erythromycin in biosynthesis path, the protoplast fusion for rapamycin and erythromycin producers was studied and a high-producing strain was obtained, with which the rapamycin productivity was 345mg·l^-1.Through three runs of multi-parents protoplast fusion and regeneration based on the theory of whole genome shuffling, a high-yielding strain was obtained, with which rapamycin productivity reached 445mg·l^-1, which was 28.9% higher than the strain before genome shuffling.