Medium And Process Optimization For Production Enhancement Of Antimicrobial Macrotetrolides From Arctic Actinomycete R-527F

Streptomyces sp. R-527F, isolated from sediments of the north off 73°N of the Arctic ocean by Polar Research Institute of China, was identified as actinomycete. Its secondary metabolite Dinactin and its homologues, belonging to macrotetrolides antibiotic, showed significantly antibacterial, immunosuppressive, anti-tumor, insecticidal, and coccidiostatic activities. In this work, we developed a high-yield fermentation medium of R-527F for Dinactin. Then,5-L bioreactor fermentation and the metabolic regulation by precursor-feeding strategy was performed for further improving production of Dinactin.Firstly, a statistical design was conducted to optimize the fermentation medium. One-factor-at-a-time design selected initial carbon source, nitrogen source and artificial seawater to construct the primary components of the medium for Dinactin biosynthesis. Then, Plackett-Burman design determined four critical factors that significantly affected Dinactin production. After that, the central composite design confirmed the optimal concentration of the four critical factors by the fitted multiple regression equation of Dinactin production and critical factors. Ultimately, with the optimized medium 160.8 mg/L Dinactin was produced, 47-fold higher than the original one.Secondly, fermentation conditions were explored for Dinactin production of R-527F, such as the seed culture medium and its culture conditions, pH, shear force. Consequently, the LB medium was selected as the optimal seed medium with two colonies from solid plate for 48 h. For fermentation medium, the optimal initial pH was 5.0. Moreover, it was found in flasks that Streptomyces sp. R-527F was of shear-resistance, which provided guidance on agitation speed or shear control for bioreactor fermentation.Furthermore, metabolic regulation by precursor-feeding strategy for Dinactin production of R-527F was conducted. When adding exogenous short-chain fatty acid, such as acetate, succinate, malonate and citrate, Dinactin production was obviously promoted. By feeding 1.5 mmol/L acetate,24 mmol/L malonate,6 mmol/L succinate, and 6 mmol/L citrate, the Dinactin production reached 184.1 mg/L,215.0 mg/L,286.8 mg/L,209.1 mg/L, which was increased by 42.7%,66.7%,122.3%,62.1% respectively, as compared with 129.0 mg/L of the control. By precursor-feeding strategy, the utilization of glucose was promoted and the synthesis of Dinactin was stimulated.Finally, the precursor-feeding strategy was performed in 5-L bioreactor. The maximalDinactin production of 279.0 mg/L was achieved and a very low residual glucose (0.75 g/L) remained by six-pulse feeding of total 6 mmol/L succinate,124.1% higher than control. Repeated fed-batch fermentation suggested that the smooth modification of physiological environment might play a positive role on the biosynthesis of antibiotics. Too much precursor feeding might cause metabolism alienation.