抗药标记酿酒酵母菌株间原生质体融合的研究

STUDIES ON INCREASING L-LYSINE PRODUCTION IN SACCHAROMYCES CEREVISIAE BY PROTOPLAST FUSlON

酵母属中的一些种是生产单细胞蛋白的主要菌株,通过遗传工程选育高赖氨酸酵母有重要的理论意义和价值,采用100mg/ml蜗牛酶制备亲株的原生质体,在35%聚乙二醇(MW6000)的作用下,成功地获得了酿酒酵母(Saccharomyces cerevisiae)的株间融合重组子,融合频率为10^(-8)-10^(-7),经过五代的移接验证,融合子稳定,融合子抗药性产生叠加效应,个体形态发生了变化,DNA含量加倍,赖氨酸含量有所提高.

LiCl-resistant and AEC-resistant Saccharomyces cerevisioe strains were obtained by treating the parent strain with LiCl and UV, and with NTG, UV and AEC, respectively. Protoplasts of both resistat strains were prepared through log-phase cells with snail enzyme. Protoplast formation achieved 90-95% efficiency in 45-60 minutes of treatment. Maximum regeneration of LiCl- resistant protoplast was 20-25% while that of AEC-resistant protoplast was 15-20%. Protoplast fusion was carried out in 35% PEG(MW6000) with 10mM CaCl2,and fusion products were grown in hypertonic CM medium containing LiCl and AEC. Protoplast fusion showed a frequency of 10-6-10-7. All fusion products showed hybrid properties. One hybrid strain was found to produce much higher level of L-lysine than its parents.