盐霉素产生菌原生质体的制备与再生

Protoplasts prepartion and regeneration of Streptomyces albus

为获得盐霉素产生菌的原生质体制备与再生的最佳条件,本文考察了影响盐霉素产生菌原生质体制备与再生的各种因素。结果表明,盐霉素产生菌的原生质体制备与再生的最佳条件为:菌丝体培养基选择为R2YE,一级菌丝体最佳培养时间为22h,二级菌丝体最佳培养时间为18h,最佳蔗糖的添加量为100g/L,最佳甘氨酸质量浓度为5g/L,菌丝体在酶解前要经玻璃珠打碎,最佳溶菌酶质量浓度为4g/L,最佳酶解温度为33℃,最佳酶解时间为60min。原生质体制备与再生条件经优化后,盐霉素产生菌的原生质体释放率可达95%,原生质体再生率可达17%。较高的原生质本制备率与再生率使得利用原生质体技术进行微生物菌种选育成为可能,同时,这种成熟的原生质体制备与再生工艺,确保了经原生质体诱变育种后发生基因突变的遗传个体绝大部分都是纯合体,从而避免了高产性状经传代后发生性状回复的现象,为盐霉素工业生产菌种的选育工作奠定了基础。

Protoplast preparation and regeneration were prerequisites of genetic breeding by protoplast mutation. To obtain the optimal conditions of protoplasts formation and the ability of regeneration of Streptomyces albus, a salinomycin-producing strain. Effects of various factors on the protoplasts fromation and regeneration of Streptomyces albus were investigated by the method of losozyme hydrolyzation cell wall. Results showed that the optimal conditions for the protoplast formation and regeneration were as follows： primary mycelia were cultivated for 22h in R2 YE liquid medium, and then transferred to the same medium with 100g/L sucrose and 5g/L glycine for the second mycelia cultivation. After 18h cultivation, the mycelia were collected, washed, destroyed by glass beads and lysed by lysozyme at the optimal concentration of 4g/L for 60min at 33℃. Then spread the prepared protoplast on RM solid medium for regeneration. Finally, the protoplast formation rate and regeneration rate could respectively approach 95% and 17% under the optimal conditions. Higher protoplast formation rate and regeneration rate make it possibe to genetically improve microbal products by protoplast mutation. Meanwhile, it ensures that the genetic breeding mutations are mostly individuals homozygous and the flat response of the high yielding character after subculture is avoided on the basis of the mature protoplast preparation and regeneration process and lays a good foundation for the genetic improvement of industrial salinomycin-producing strain.