| Spinosad is a kind of macrocyclic lactone insecticidal substance produced by aerobic fermentation of Saccharopolyspora spinosa, it possesses a unique mechanism of action, and is characterized by high insecticidal activity, high selectivity, safety on non targeted animal, it demonstrates the safety of biological pesticide and the rapidity of chemical synthesized pesticide. The research on spinosad in order to increase fermentation yield and realize the industrialization scale, can produce significant economic and social benefits. In this article, the fermentation yield of spinosad got increased more than 6 times through mutagenesis breeding technology combined with fermentation control technology, and the spinosad fermentation of 1000 L pilot scale was investigated, the strain S. spinosa s4 maintained the high performance of spinosad yield in this pilot-scale fermentation.Microbial strain is the source of all kinds of fermentation technology, and the level of fermentation is one of the key factors to determine whether the product is competitively in the market. The competition of strains is also the competition of breeding technology, high-throughput screening technology can greatly improve the screening efficiency of strains, and it is a hot topic of research in recent years. Based on the preliminary screening technology developed in our group and the correlations of mosquito larvae fatality and spinosad titer, the high-throughput screening model of high yield strain of spinosad was established. The mutagenesis methods such as the commonly used ultraviolet (UV), nitrosoguanidine (NTG),60Co ray, and the recent popular Atmospheric and Room Temperature Plasma (ARTP), space flight were preliminarily evaluated, and the mutagenesis breeding efficiency of several mutagenesis methods of S. spinosa were compared. By using the method of space flight mutation, nearly 15000 mutated strains of S. spinosa were screened through the combination of mosquito survival screening technology and shake flask re-screening verification, one high yield strain was obtained, whose fermentation yield in shake flask culturing increased more than 2 times, the spinosad fermentation yield of shake flask reached 573.8 μg/ml.The data of spinosad high yield strains, obtained from space flight mutation was studied, the correl ation between spinosad yield and biomass of S. spinosa fermentation in shaking flask was then analyzed. The shake flask fermentation biomass of strain s4 increased by 25% through the methods of optimizing the medium proportion with central combination test and increasing vegetable oil components; Substance A which could induce the synthesis of spinosad was obtained by screening, and the concentration of the added substances A was preliminarily studied; the effects of ventilation efficiency on the yield and biomass of spinosad were investigated; the effects of fifteen defoaming agents on the yield of spinosad fermentation were compared and screened; The suitable and proper amount of defoaming agent for tank fermentation production of spinosad was determined, which laid the foundation for the regulation and the improvement of the yield of spinosad in tank in later stage.The obtained high yield strains were systematically studied in the aspects of metabolic characteristics, changes of mycelial morphology, analysis of metabolite and analysis of metabolic flux, respectively, the intracellular and extracellular changes of three key organic acids were explored during the processes of fermentation of high yield strain and low yield strain on the basis of 30 L and 50 L fermentor, the key point of fermentation control in tank was verified, and provided the theory and basis for multi-scale regulation of spinosad. The curve variations of oxygen consumption rate (OUR), carbon dioxide releasing rate (CER) and respiratory quotient (RQ) were analyzed in-depth, and the intuitive parameter node which regulated the fermentation process of spinosad was determined. The effects of pH control strategy and glucose fed batch strategy on the yield of spinosad fermentation were studied, the pH value was controlled between the range of 6.5-7.0, the rate of glucose fed batch was 2 g/h, which was benefit to the accumulation of spinosad. The regulation strategy of dissolved oxygen (DO) in the process of the fermentation of S. spinosa in tank was studied, the critical value of oxygen was determined between 25-30% in the process of fermentation, all these works provided theoretical and practical basis for pilot-scale fermentation of S. spinosa.The culture medium components for spinosad fermentation were replaced with the raw materials of agricultural and sideline products, the variations of metabolic curves for the tank before and after substitution of the raw materials were analyzed in-depth, the control strategy was adjusted according to the variations trend of OUR and CER, the yield of spinosad in small-scale of 50 L fermentor reached to 1878.8 μg/mL. The pilot-scale test of spinosad fermentation in 1000L scale of fermentor was processed based on small-scale fermentation, the yield of spinosad reached to 1426 μg/mL. According to the separation and extraction technologies of spinosad developed by the research group, the filter aid and decolorizing resin with excellent performance were screened for the issues of fermentation broth pre-treatment and spinosad refining, the extraction rate and product purity of spinosad were improved, the extraction rate of spinosad in pilot-scale was more than 60%, and the product purity reached to 93.5%. |
