Improvement Of Spinosyn-producing Strain By Protoplast Technology

Spinosyns and its analogs, produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. Its many excellent features, containing favorable to mammalian and environmental, insect selectivity, unique mechanism of action and outstanding efficacy, lead to high benefit of economy and society. In this paper, we have tested the optimal conditions for the formation, regeneration and preservation of protoplasts, selected high spinosad-producing strain using mutation and interfamial/intraspecific protoplast fusion. The main results were as follows:1. All factors were tested on protoplast formation and regeneration including the preparation medium, regeneration medium, spawn age, glycine concentration and the treatment conditions of lysozyme. The maximum rate of protoplast formation and regeneration were under these conditions: the collected mycelia grown in EHC medium with 0.3% glycine for 72 h were treated by 2mg/mL lysozyme at 32℃for 40min, then plated on the R6 medium. The rate of protoplasts formation was more than 99% and the number of regeneration protoplast was up to 107cfu/mL. Method of short-term storage for protoplasts was 4℃for 72h, long-term storage needed -80℃.2. With UV mutation, one high spinosad-producing strain was achieved, which was 40% higher than the initial strain.3. Screen the recombinants by inactivatting parental strain protoplasts. S.spinosa NU-92 was inactivated by UV for 120s. S.spinosa C3-10-4 was heat-inactivated for 50min at 60℃. S.avermitilis 76-02-e was heat-inactivated for 30min at 60℃.4. The best conditions of pre-alignment electric field and pulse electric field in the electrofusion were tested between S.spinosa and S.avermitilis.The maximum rate of protoplast fusion were under these conditions: pre-alignment voltage 7V, time 50s, pulse voltage 200V, pulse time 40μs, pulse number 2. But the high spinosad-producing strain was non-achieved. Although the fusion rate of intraspecific was much higher, the spinosad-producing wasn't improved.5. The maximum rate of protoplast chemical fusion between interfamial/intraspecific protoplasts were achieved by using 50% PEG1000 and the fusion time was 2-5min. The rate of negative mutation was 94.7% in the protoplast fusion between S.spinosa and S.avermitilis.The positive mutation was 75% in the intraspecific protoplasts fusion. One high spinosad-producing strain was achieved, which was near to 200% higher than the initial strain.