| Doramectin is a new generation of macrocyclic lactones antibiotics, belonging to third- generation derivatives of avermectin. It has a broad-spectum antiparasitic, a unique role and no cross-resistance with other antiparasitics, and it is a potential veterinary antiparasitic. This study improved the production of doramectin by genome shuffling, optimized the fermentation medium by Response Surface Methodology (RSM) and optimized the fermentation conditions by one-factor-at-a-time method, providing the reliable technological parameters for large scale production.Firstly, initial strain was treated with traditional ultraviolet radiation and nitrosoguanidine mutagenesis, the highest doramectin-producing strain in them was screened and purified as starting strains for genome shuffling. Then the conditions for protoplasts preparing and fusion were optimized and the result showed YEME medium with 0.5% glycine added was best mycelia cultivation medium, mycelia were harvested after 20 h and smashed by 250 rpm vibration in 250 ml flask with 35 glass beeds. Protoplasts were obtained from mycelia using 3mg/ml lysozyme at 30°C for 30 min and the frequency of protoplasts formation and regeneration reached 88.9% and 4.4% respectively. While the fusion rate reached 8.3‰with 40% PEG4000 trated for 3min. Equal number of protoplasts from starting strains was mixed and devided into two identical parts. One part was inactivated with UV for 200 s, and the other was heat treated at 60°C for 40 min. The first round of genome shuffling strains was screened by 5μg/ml streptomycin resistance, and the screened high yield strains were used as the second round of shuffling starting strains. Four rounds of genome shuffling were carried out and the streptomycin resistance were 5μg/ml, 10μg/ml, 15μg/ml, 20μg/ml respectively. A stable strain F4-120 with 20μg/ml streptomycin resistance was obtained after four rounds of genome shuffling and its production of doramectin reached 547 mg/l, which was increased by 372.11% and 531.08% in comparison with that of the highest parent strain UV-100(147 mg/l) and the original strain NEAU1069(103 mg/l), respectively. Time courses of doramectin fermentation by recombinant and initial strain were compared. Fermentation medium components of the recombinant strain F4-120 was optimized by response surface methodology. The results showed variables that had significant effect on doramectin production were corn starch, peptone and calcium carbonate, the optimized concentration were 118.9g/l,17.16 g/l and 10.18 g/l respectively. Doramectin production was increased by 23% and reached 672 mg/l. In addition, this dissretation determined the fermentation temperature, the initial pH, the container of shaking flask, inoculation time and the volum of vaccination. Sensitivity to sheer stress and precursor adding time and volumes were investigated, also effect of water-feeding in fermention flask was studied. The results showed doramectin-producing strain was sensitive to sheer stress, the best way of providing cyclohexane sodium salt was a final concentration of 0.1% and 0.06% at 1d and 6d respectively. Water-feeding at the fourth day with equal evaporation volum could improve total doramectin. |
