Breeding Of High-yield Rapamycin-producing Strains And Metabolic Profiling-based Medium Optimization

Rapamycin, an important natural macrolide antibiotic which is produced by Streptomyces hygroscopicus, possesses various biological and pharmacological activities. Especially, as a potent immunosuppressive agent, rapamycin has been approved for the clinical treatment of organ transplant rejection and autoimmune disease. The breeding of high-yeilding strain and fermentation optimization is necessary to improve the production of rapamycin, which would have great significance for the industrialization of rapamycin, as well as the reduction of production costs.Ultraviolet mutagenesis is one of the most commn and effective mutagenesis methods in the mutation breeding of wild microorganism. In this study, the wild strain S. hygroscopicus ATCC29253was used as the starding strain and UV mutagenesis was performed to screen high-yielding rapamycin strain. A strain named S. hygroscopicus U1-6E7was isolated which presented rapamycin production of159mg/L, displaying a109.2%increment compared with the wild-type strain. Moreover, after fermentation optimization, the production of rapamycin by strain U1-6E7was improved to196mg/L. Additionally, taking consideration of the limted time during master’s study, we performed the investigation of comparative metabolic profiling-based improvement of rapamycin and screening of lysine resistance strains, simultaneously, using strain U1-6E7as the starting strain.To rationally guide the improvement of rapamycin production, comparative metabolic profiling analysis was performed in this work to investigate the intracellular metabolic changes in S. hygroscopicus U1-6E7fermentation in medium M1and derived medium M2. A correlation between the metabolic profiles and rapamycin accumulation was revealed by partial least-squares to latent structures analysis, and16key metabolites that most contributed to the metabolism differences and rapamycin production were identified. Most of these metabolites were involved in tricarboxylic acid cycle, fatty acids, shikimic acid and amino acids metabolism. Based on the analysis of key metabolites changes in the above pathways, corresponding exogenous addition strategies were proposed as:1.0g/L methyl oleate was added at0h;0.5g/L lysine was added at12h;0.5g/L shikimic acid was added at24h;0.5g/L sodium succinate,0.1g/L phenylalanine,0.1g/L tryptophan and0.1g/L tyrosine were added at36h, successively, and a redesigned fermentation medium (M3) was obtained finally on the basis of M2. The production of rapamycin in M3was increased by56.6%compared with it in M2, reaching307mg/L at the end of fermentation (120h). These results demonstrated that metabolic profiling analysis was a successful method applied in the rational guidance of the production improvement of rapamycin, as well as other industrially or clinically important compounds, which provides a new direction for fermentation medium optimization.Lysine exhibited a striking promotion on rapamycin biosynthesis. However, strain U1-6E7was very sensitive to lysine. Here, using U1-6E7as straing strain, the lysine enduring strain was isolated through lysine ending mode combained with UV mutagenesis. A strain named U2-3D9was obtained which showed14-fold increament of lysine tolerance compared with strain U1-6E7, and rapamycin production by strain U2-3D9was enhanced to324mg/L. After optimization of lysine addition, when5.0g/L lysine was added at12h, rapamycin production peaked at417mg/L at the end of fermentation (120h), achieving a112.7%improvement compared with rapamycin production of196mg/L by strain U1-6E7. In the near future, using strain U2-3D9as the starting strain, comparative metabolic profiling-based medium optimization would be performed in our laboratory to further improve rapamycin production.