Effect Of Different Feeding Strategies On Metabolic Regulation Of Streptomyces Cyaneogriseus Ssp.Noncyanogenus And Nemadectin Biosynthesis

Nemadectin,produced by Streptomyces cyaneogriseus ssp.noncyanogenus,is commonly used as a starting material for the synthesis of commercial anthelmintic moxidectin.Unfortunately,yet little is known about its metabolic regulation and control in the process of nemadectin fermentation,which lead to significant difficulty of yield improvement.To address these drawbacks and have further research on metabolic regulation and control,this research developed a novel chemical defined medium for nemadectin fermentation by high-throughput screening method and then had a preliminary study on the effect of different feeding processes on the metabolic regulation of Streptomyces cyaneogriseus ssp.noncyanogenus and nemadectin biosynthesisFirstly,a high-throughput culture system and a high-throughput detection system were established in this paper.The novel chemical defined medium was optimized by single factor,Plackett-Burman design,and response surface optimization methods.The yield of nemadectin can reach 150.33 mg/L in the optimized chemical defined medium.And then,the experiments in shake flasks with glass bead and different working volume were used to study the demand for shear and dissolved oxygen in the nemadectin fermentation process.Finally,in the 5 L bioreactor,high dissolved oxygen and low shear were achieved by changing the stirring speed and impeller combination,which made the final yield of nemadectin reach 191.59 mg/L.By optimizing the feeding strategies,the biosynthesis of nemadectin was further increased.Finally,it was found that when the residual glucose level was controlled at 2 g/L by adding glucose solution after 96 h,the maximum yield of nemadectin reached 268.9 mg/L.When the ammonium ion in the fermentation broth were controlled at 10 mg/L after the mycelial differentiation by feeding ammonium sulfate solution,the maximum yield of nemadectin was 378.2 mg/L.In the end,based on the method of metabolomics,the measure and analysis of the concentration of intracellular metabolites of Streptomyces cyaneogriseus ssp.noncyanogenus in the biosynthesis phase of nemadectin were conducted.Finally,it was found that adding ammonium sulfate after mycelial differentiation facilitated the synthesis of alanine,valine,leucine and isoleucine,which can transform to acyl-CoAs,the precursors of nemadectin biosynthesis,by catabolism.Addition of ammonium sulfate before mycelial differentiation enhanced the primary metabolism of the cell and thus increased the biomass of this bacteria.