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Rapamycin (Rapamycin) and Mingxiluomo Division (Sirolimus), by Streptomyces (S. hygroscopicus) generates a new leukotriene nitrogenous macrolide antibiotics. Except as an immunosuppressant rapamycin, but is still widely used in organ transplantation, skin diseases, heart disease and other fields. In this paper, the laboratory saved rapamycin producing strain of low-yielding strain through traditional mutation breeding technology, combined with high-throughput screening methods, supplemented by medium optimization, substantially increased the rapamycin producing strain production capacity and conducted a pilot fermenter and amplification. Meanwhile, the use of protoplasm sports kind and genomic rearrangements and other new technologies on rapamycin producing bacteria were bred and initially explored rapamycin heterologous synthesis. First, a preliminary test rapamycin producing strains optimized medium and culture conditions to determine the optimum medium for fermentation: 4% glucose, 1% glycerol, 1% soybean meal, 0.15% L-Lys, 0.5% K2HPO4, 0.5% KH2PO4, 0.5% sodium chloride, 0.2% magnesium sulfate, 0.15% ferrous sulfate. The optimum fermentation conditions are: Shake bottle liquid volume 20ml/250ml, culture temperature is 280C, harvested 5 days after inoculation cultured cells. In the above optimal conditions, the raw fermentation broth yields up rapamycin 120mg / L. To improve yield rapamycin, rapamycin producing strain launched Mutation Breeding. First investigated UV, DES, NTG, such as different mutagen dose effects. Then through a single mutagen continuous mutation, alternating different mutagen mutagenesis, mutagenesis of different mutagens and other mutagenesis methods continuously screened to yield 279mg / L of the mutant strain D7-804, than the starting strain increased by 156%. Further development of the training based on the rapamycin 96-mutants of new technologies for high-throughput screening, about 100,000 by the rapid and efficient screening single colonies were screened to yield a mutant strain N5632, the highest yield 445mg / L in These high-yielding strain selection and optimization of fermentation on the basis of carried out in 120L fermenter rapamycin fermentation Amplification. The results showed that the initial stirring speed is set to 200rpm, after entering the product synthesis phase speed rose to 400rpm, dissolved oxygen control of the fermentation process is not less than 10%, the highest yield rapamycin reach 412mg / L, basically reached the shake bottle fermentation average. On this basis, carried out using a variety of ways to enhance the level of fermentation feeding studies. The results showed that, 40h feeding is the best time to make use of multi-component composition greatly improved feeding method can best level of fermentation, in which a combination of glycerol and phosphate feeding way, not only to maintain the pH of the fermentation broth is relatively stable, and Rapamycin can effectively improve the production level of fermentation and up to 800mg / L or more. Finally, this new batch fermentation process fermentor at 20 tons on the industrial scale, the level of fermentation rapamycin also reached 700mg / L, output was initially the subject at the beginning of fermentation level increased more than four times. In order to maximize the yield of rapamycin bacteria breeding efficiency, carried out genomic rearrangements of genetic transformation Streptomyces performance, increase the level of rapamycin avermectin fermentation. First identified rapamycin producing strain protoplast preparation, regeneration and fusion of optimum conditions; then Streptomyces protoplast mutagenesis, blending well with the erythromycin-producing bacteria species protoplast fusion technology, having Different genetic characteristics of high yield production of rapamycin streptozotocin mutants. Finally, seven rapamycin for a high-yielding mutant genomic rearrangements breeding results filter to a higher yield strain (GS-1437), rapamycin production reached 445mg / L, than the initial yield strain increased 59.5%, it shows that genomic rearrangements technology in the transformation of Streptomyces genetic characteristics enormous potential, compared with the traditional method of mutation breeding, with fast and efficient and other significant advantages. . Conducted rapamycin biosynthetic genes arrowheads in heterologous expression host bacteria and rapamycin biosynthetic studies. Has been constructed in three rapamycin heterologous synthesis system, Streptomyces albus S. albus 28-1-1 fermentation product samples showed similar rapamycin standard antibacterial biological activity. Through the initial identification and analysis of the structure of the fermentation product of the unknown sample is a biological active substances biological activity similar to rapamycin, but is quite different from the structure of the new compounds. Further from the transcriptional level heterologous host bacteria arrowhead rapamycin biosynthetic genes in each gene transcription analysis. 72h fermentation of biomass by extracting RNA, technical analysis by RT-PCR at the transcriptional level of each gene differences, S. albus 28-1-1 in the vast majority of rapamycin biosynthetic gene transcription can not be normal, only There downstream rapG, rapF and rapD reference system displays the same transcription signal. The findings in heterologous biosynthesis host bacteria and other novel antibiotic rapamycin has important reference significance.
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