伽马射线辐射结合亚硝基胍诱变选育他克莫司高产菌株  

Breeding of tacrolimus high-producing strains by gamma-ray radiation and nitrosoguanidine mutagent

作　　者：严凌斌[1] 张文州 张祝兰[1] 张引[1] 邱观荣[1] 王德森[1] 连云阳[1] YAN Lingbin;ZHANGWenzhou;ZHANG Zhulan;ZHANG Yin;QIU Guanrong;WANG Desen;LIAN Yunyang(Fujian Provincial Key Laboratory of Screening for Novel Microbial Products, Fujian Institute of Microbiology, Fuzhou 350007, China;Quanzhou Medical College, Quanzhou 362100, China)

机构地区：[1]福建省微生物研究所福建省新药(微生物)筛选重点实验室,福州350007 [2]泉州医学高等专科学校,泉州362100

出　　处：《辐射研究与辐射工艺学报》2019年第5期49-56,共8页Journal of Radiation Research and Radiation Processing

基　　金：福建省属公益类研究所基本科研专项(2019R1005-8、2017R1009-1);福州市科技计划项目(2018-G-48);福清市科技计划项目[福清市2018(6)]资助~~

摘　　要：为了选育稳定高产的他克莫司菌株,提高发酵水平,对他克莫司出发菌株采用450 Gy剂量的^60Co γ辐射诱变及3 mg/mL亚硝基胍(NTG)处理30 min诱变,并分别结合链霉素和庆大霉素进行抗性筛选.经选育获得一株高产、遗传稳定的他克莫司突变株FIM-17-17,该菌株的摇瓶发酵水平较出发菌株提高了65%.考察该突变株的稳定性,及树脂AB-8、D101、HT60、XAD16、HP20和XDA-8对发酵水平的影响,并在容量1 t的发酵罐进行中试发酵验证.结果表明,突变株FIM-17-17遗传稳定,且在发酵培养基中添加2%的D101树脂后,发酵水平又提高了29.8%,发酵罐中试发酵水平平均达1 319 μg/mL.结果提示,利用^60Co γ射线辐射和NTG复合诱变能有效获得他克莫司高产菌株.To enhance the tacrolimus fermentation titer of the production strain, original strains were treated using ^60Co γ-ray radiation at a dose of 450 Gy and nitrosoguanidine mutagen (NTG) at a concentration of 3 mg/mL for 30 min when combined with a streptomycin and gentamycin resistance screening technique. A mutant strain named FIM-17-17 showed a 65% higher-yield tacrolimus than that of the original strain in a flask culture. The stability of the high-yield mutants and the effects of different resins (AB-8, D101, HT60, XAD16, HP20, and XDA- 8) on the tacrolimus fermentation titer were investigated, and the optimized conditions of the mutant strain were tested in a 1-ton fermentation tank. The mutant strain FIM-17-17 showed high genetic stability. When 2% D101 resin was added to the culture medium, the tacrolimus titer increased by 29.8%, and the production of tacrolimus reached 1 319 μg/mL in the 1-ton fermentor. These results indicate that a tacrolimus high-producing strain can be obtained efficiently by compounding mutagenesis of ^60Co γ-ray radiation and NTG.

关 键 词：链霉菌 他克莫司 发酵 辐射诱变 亚硝基胍 

分 类 号：TL99[核科学技术—核技术及应用] TQ465.92[化学工程—制药化工]