常压室温等离子体(ARTP)诱变及高通量筛选维生素B_(12)高产菌株  

Mutation by Using Atmospheric Pressure and Room Temperature Plasmas and High-throughput Screening Method for Improving Vitamin B_(12) Production

作　　者：蔡莹瀛 夏苗苗[2] 董会娜[2] 张同存[1] 张大伟[2] CAI Yingying;XIA Miaomiao;DONG Huina;ZHANG Tongcun;ZHANG Dawei(Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308

出　　处：《天津科技大学学报》2018年第2期20-26,共7页Journal of Tianjin University of Science & Technology

基　　金：天津市自然科学基金资助项目(16JCYBJC23500);天津市杰出青年科学基金资助项目(17JCJQJC45300)

摘　　要：采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变脱氮假单胞菌(Pseudomonas denitrificans)并从中筛选高产维生素B12突变株,确定等离子诱变最优处理时间75,s、输出功率100,W.通过流式细胞仪并结合核糖开关(riboswitch)感应元件检测其荧光值以初筛诱变后高产菌株,并利用48孔板高通量培养发酵,酶标仪快速检测维生素B_(12)产量,建立完整的诱变后高通量筛选体系.通过4轮ARTP诱变,筛选得到的突变株PA320-M4-1B1在250,mL摇瓶发酵6,d的条件下,维生素B_(12)产量达到(103.2±2.1)mg/L,较初始菌株PA320的(71.9±1.8)mg/L提高了43.8%,且遗传性状稳定.Atmospheric pressure and room temperature plasma(ARTP)mutagenesis system was used to induce Pseudomonas denitrificans.In order to obtain mutant strains with enhanced ability to produce VB12,the optimum conditions for mutation were determined,which were 75,s as the mutagenesis time and 100,W as the output power.A method was constructed with flow cytometry and riboswitch to efficiently identify positive mutant strains which were cultivated with 48-well microplates and detected by microplate reader as high-yielding mutants.Thus,a screening system for vitamin B12 high throughput was built.Through four rounds of ARTP mutation and high-throughput screening,PA320-M4-1B1 mutants(103.2±2.1)mg/L were obtained,which increased the contents of VB12 by 43.8%,compared with the control strain PA320(71.9±1.8)mg/L,when the strains were cultivated on a rotary shaker at 30℃for 6,d.

关 键 词：常压室温等离子体(ARTP) 维生素B12 核糖开关 高通量筛选 

分 类 号：Q563.4[生物学—生物化学]