基因组重排选育胸苷磷酸化酶高产菌株

Screening and breeding of high thymidine phosphorylase-producing strains by genome shuffling

以短乳杆菌为研究对象,通过基因组重排技术选育胸苷磷酸化酶高产菌株。首先采用紫外复合诱变筛选出EA_(42)、EB_(27)作为基因组重排育种的亲本并制备成原生质体,分别采用紫外照射50 min和60℃水浴加热60 min双亲灭活原生质体,然后用质量分数40%PEG 6000,30℃恒温诱导融合10 min进行基因组重排。经过3轮基因组重排育种,成功选育出3株胸苷磷酸化酶高产菌株,其中菌株F3-36在菌体发酵量提高的前提下,进行5次传代测试其胸苷磷酸化酶活均在2.500U/mg湿茵体,比原始茵株酶活提高了260%。

Genome shuffling was utilized to screen out the thymidine phosphrylase high-product strains. Firstly, Lactoba- ciUus brevis mutants, EA42 and EB27 strains, obtained by UV-irradiation, were served as the parents of genome shuffling breeding. The protoplasts of mutants mentioned above were irradiated by UV-irradiation for 50 rain, or heated at 60 ℃ for 60 min. Then, the inactivated parental protoplasts were fused with 40% PEG 6000 at 30 ℃ for 10 min. The fusants with high thymidine phosphorylase activity were as the parents for the next round of protoplast fusion. After three rounds of re- cursion fusion, three mutants with high thymidine phosphorylase activity and genetic stability were successfully screened. And the highest thymidine phosphorylase activity of the fusant F3-36 could reach 2. 500 U/mg wet cells, which was in- creased by 260% than that of the original strain.