多杀菌素生产菌原生质体的制备条件及其再生菌株生理特性

Conditions for protoplast preparation of spinosyn-producing strain and the physiological properties of protoplast-regenerated strains

摘要:

为了改良多杀菌素生产菌种, 提高多杀菌素产量, 研究了甘氨酸添加浓度、溶菌酶作用时间、温度和浓度对多杀菌素生产菌刺糖多胞菌Saccharopolyspora spinosa SP06081菌株原生质体制备和再生的影响, 并考察了不同再生培养基和渗透压稳定剂对其再生的影响, 确定了该菌株原生质体制备和再生的最佳条件。同时, 对原生质体再生菌株的形态与多杀菌素产量变化进行了比较研究。结果表明: 菌体在添加0.2%的甘氨酸的TSB培养基中培养48 h收集, 0.1 mg/mL 溶菌酶, 28oC作用20 min制备原生质体, 将原生质体涂布于以蔗糖为渗透压稳定剂的R2YE培养基中, 原生质体再生数目最多, 达108个/mL以上; 原生质体再生菌株在形态和抗生素产量上产生分化, 29.3%的再生菌株形态上保持与亲本菌株一致, 具有菌丝松散, 断裂分枝多的特点, 其中53.2%的再生菌株多杀菌素产量变异向正方向移动, 最高产量达到582.0 mg/L, 比亲本菌株提高85.6%。原生质体再生菌株的形态分化与多杀菌素产量具有重要相关性。

Abstract:

To improve spinosyn-producing strain and enhance spinosyns yield, we studied the effects of glycin concentration and the operational time, temperature and lysozyme concentration on protoplast preparation of Saccharopolyspora spinosa SP06081. We also studied different regeneration media and osmotic stabilizing agents. In addition, we compared the change of morphology and spinosyns yield of the regenerated strains. The results showed that the Saccharopolyspora spinosa SP06081 protoplast yield was the highest under these conditions: the collected mycelium from SP06081 grown in Tryptic Soy Broth (TSB) medium with 0.2% glycin for 48 h was treated by 0.1 mg/mL lysozyme at 28oC for 20 min, then plated on the R2YE medium with sucrose as osmotic stabilizer, the number of regeneration protoplast was up to 108/mL. The protoplast-regenerated strains exhibited changes in morphology and antibiotic production, 29.3% protoplast-regenerated strains was characterized by loose mycelium and abundant broken branches as did their parent. Among them, 58.2% strains presented the trend to positive variation in spinosad yield, with the highest spinosad yield of up to 582.0 mg/L, 85.6% higher than that of their parent. There is significant correlation between the morphological differentiation and antibiotic yield of the protoplast-regenerated strains from spinosyn-producing strain.