Mutation Breeding And Optimization Of Fermentation Process Optimization On Producing Demethylchlortetracycline

This paper reported the mutagenesis by UV and LiCl/EMS on Streptomyces aureofaciens. Then the mutants were selected by demethylchlortetracycline,2-DG and malonic acid. The potency of the mutant1-E1-12was5102u/ml which was20.3%higher than the original strain.The fermentation medium was optimized by Plackett-Burman experiment, steepest ascent search and response surface optimization. First we found three important factors corn starch, soybean meal and CaCCO3which influenced the potency obviously by Plackett-Burman experiment. The first optimization was achieved by response optimization which was a part of Plackett-Burman experiment. Then the steepest ascent search based on the Plackett-Burman experiment was used to find the center concentration of three factors. Finally the optimal concentration was determined by the response surface optimization and the result was corn starch110g/L, soybean meal43g/L and CaCO37g/L.The average chemical potency was5435u/ml which was25-31%higher than the initial fermentation medium.A preliminary study was carried out to find the reason which resulted in the increase of the potency. The relevant experiments were designed on the basis of the component and C/N before and after optimization. The result indicated that the increase of the potency was possibly caused by the interaction of amino acid, C/N and other factors.Analyzed three important organic acids in and out of the cells, reducing power and CoA carboxyl transferase in the cells. The result indicated that more extracellular oxaloacetic acid and malonic acid were accumulated in optimized medium in the middle of fermentation process. Both of them entered into the cells at the end of fermentation process and the demethylchlortetracycline was synthetized greatly. The reducing power was higher in the optimized medium throughout the fermentation process, so the sufficient reducing power was supplied in the optimized fermentation medium. CoA carboxyl transferase intuitively reflected the capability of synthetizing demethylchlortetracycline in the Streptomyces aureofaciens and the result indicated that the trend of enzyme activity was anastomotic with it of the potency.On the basis of metabolic pathway in the Streptomyces aureofaciens, sodium citrate, BT and NAA,the inhibitors of glycolytic pathway, were added to the fermentation medium. The results indicated that the potency increased about8%by adding0.02%sodium citrate at48-72h, about10%by adding50-100ug/mlNAA and13.3%by adding0.26ug/mlBT at the beginning.In addition, the potency increased13.4%by adding0.01%malonic acid at48h.