Synchronized Regulation Strategy Of PH And Nutrition For Dinactin Production Enhancement By Arctic Actinomyces Streptomyces Sp.

Streptomyces sp. R-527F, which was isolated from the sediments of the Arctic Ocean, could produce a macrotetrolide antibiotic dinactin. Dinactin and its homologues exhibit a broad spectrum of biological activities, ranging from antibacterial, antifungal, antitumor, antiprotozoan, insecticidal activites to immunosuppressive activities. In this work, we aimed to enhance dinactin production for pharmaceutical research by the synchronized regulation of pH and nutrition.Firstly, balanced nutrition composition of the medium is the basis a high dinactin production. One-factor-at-a-time design was used to select the appropriate carbon source and nitrogen source based on the original medium. Then, orthogonal design and Plackett-Burman design were used to determine the optimal composition of the medium. Ultimately, dinactin production reached 252 mg/L under the optimized conditions, which increased by 6.6 times as compared to the original one.Secondly, the exogenous precursors are often directly involved in the synthesis of secondary metabolites and promote the yield of products. On the basis of the biosynthetic pathway, feedings of indirect precursors Met, Thr, Ile, Val, Glu were investigated, and feeding 0.50 mmol/L Thr,0.50 mmol/L Ile,0.25 mmol/L Glu improved the dinactin production by 25%,12%,13%, respectively, as compared to the control. Moreover, feeding the direct precursors sodium acetate, sodium malonate, sodium succinate, sodium methylmalonate, sodium propionate with different concentrations at different time points were studied. The results showed that sodium succinate (6.00 mmol/L) was divided into six identical aliquots and fed one aliquot every 12 h since 48 h, the dinactin production reached 274 mg/L,63% higher than the control.As we know, the variations of pH directly reflect the metabolic changes in the cells. By analyzing the variations of pH in 5 L bioreactor fermentation, it was found that the pH was closely related with the biosynthesis of dinactin. then utilizing NaOH to control the pH at 5.2-5.5 since pH 4.8, the dinactin production was increased by 103%. Then, the synchronized regulation of pH and precursors were proposed based on the above results, and the optimal strategy was determined. Six mmol/L sodium succinate was divided into six equal aliquots and fed since pH 4.8 and then one aliquot was added every 12 h. NaOH and HC1 were used to control pH at 5.2-5.5, the dinactin production reached 291 mg/L, increased by 108%. Considering the glucose depletion since the middle phase of the fermentation, the pH-precursor-glucose synchronized regulation strategy was proposed, the glucose (15 g/L) was fed in the broth once at 72 h, the optimal pH control and sodium succinate regulation were conducted synchronously, the resulted dinactin production reached 343 mg/L, which further increased by 18%.Furthermore, the product of nons gene could catalyze the succinyl-CoA to transform into tetrahydrofuran ring of dinactin and an exogenous supply of sodium succinate could increase the succinyl-CoA intracellular. Therefore, nons was overexpressed in the wild type strain and then a high yield recombinant strain Streptomyces sp. R-527F-N1 was constructed. The maximum dinactin production was increased by 59% in shake flasks compared to the wild type and it produced dinactin of 382 mg/L under the optimal conditions in 5 L bioreactor, which further increased by 11% as compared to the wild type.