Study On The Breeding Of Spinosad Producing Strain And Optimization Of Fermentation Conditions

Spinosad, a novel kind of biopesticide, has following excellent characteristics: effective on a wide variety of crops pest, unique mode of action, active to economically important pests, short half-life, low residue, non-drug resistant, large margins of safety to mammals, mankind and environmental profile. Spinosad will be widely used in agriculture and stockbreeding, and its exploitation has good economic and societal benefit. Spinosyn A and spinosyn D are the most active compositions of spinosad, which are producted by the aerobic fermentation of Saccharopolyspora spinosa. Nowadays, breeding high-producing strains and optimizing culture conditions are the research focus in the field of increasing the yield of spinosad. In this paper, the productivity of spinosad was improved by 438.5% through breeding high-producing strains and optimizing culture conditions.A rational selection procedure was developed based on the biosynthetic pathway and metabolic regulation of spinosad. The wild type strain of Saccharopolyspora spinosa was treated with UV irradiation. A new mutant U-4213 was obtained using 2mg/L rhamnose and 1.5% sodium propionate as screening agents by resistant selection strategies in series. The results of experiment on tolerance to propyl alcohol showed that, in the mutant strain, the tolerance to precursor was improved and the level of producing spinosad was increased, the productivity of spinosad reached 120 mg/L, which was improved by 269.2% compared with that in the original strain (32.5 mg/L).The optimization of the fermentation conditions for U-4213 was investigated by series single factor experiments, Plackeet-Burman selective experiment and central composite design experiment. The final obtained optimal condition was: glucose 68.5 g/L, maltose 10 g/L, cottonseed protein 26.0 g/L, corn steep liquor 17.8 g/L, beef extract 2.0 g/L, zinc sulfate 0.2 g/L, ammonium sulfate 1.5 g/L, calcium carbonate 5 g/L, the liquid volume 30 mL in 250 mL shaking flask, the inoculum ratio 10%, the initial pH 7.0,shaker speed 220 rpm, cultivate for 7 days. The productivity of spinosad under this optimal fermentation condition by shaking-flask batch fermentation reached 175 mg/L, which was increased by 45.8% compared with that under the initial fermentation condition (120 mg/L). The kinetic models for biomass, substrate consumption and spinosad production were studied for the mutant strain U-4213 and the wild strain by conducting batch fermentation. The models performed well in simulating the cell, spinosad and carbohydrate concentrations of both wild strain and mutant strain with high accuracy (the correlative coefficient R2 were 0.9961, 0.9955, 0.9944 and 0.9984, 0.9934, 0.9938 respectively for wild strain and mutant strain). The result suggests that the kinetic models can describe well the process of spinosad fermentation, and will be a basic for the research of scaling up.