摘要
针对谷氨酸棒杆菌(Corynebacterium glutamicum)基因编辑效率较低和操作耗时长等问题,该研究采用诱导型启动子PrpR-PrpD2表达Cpf1,将重组和切割事件分离,建立了一种高效的诱导型CRISPR-Cpf1/dsDNA双质粒基因编辑系统,并通过比较组成型和诱导型启动子表达Cpf1蛋白的效果,统计该基因编辑系统的基因编辑效率。最后以不产L-丝氨酸的谷氨酸棒杆菌ATCC 13032为出发菌株,采用该系统构建高产L-丝氨酸重组菌株。结果表明,诱导型CRISPR-Cpf1/dsDNA双质粒基因编辑系统的基因敲除效率为(66.67±1.53)%,基因整合效率为(14.29±1.50)%,均显著高于组成型启动子组(P<0.05)。此外,利用诱导型CRISPR-Cpf1/dsDNA双质粒基因编辑系统成功构建了一株高产L-丝氨酸重组菌株SW-3,其L-丝氨酸产量为10.51 mg/L,说明诱导型CRISPR-Cpf1/dsDNA双质粒基因编辑系统在谷氨酸棒杆菌代谢工程改造中具有可行性,对加速谷氨酸棒杆菌代谢工程改造进程及推动其在氨基酸等高附加值产品研发上的应用将产生深远意义。
Aiming at the problems of low efficiency of gene editing and time-consuming operation in Corynebacterium glutamicum,the Cpf1 was expressed using the inducible promoter PrpR-PrpD2,the recombination and cleavage events were separated,and a high-efficiency inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system was established.The gene editing efficiency of the dual-plasmid gene editing system was statistically calculated by comparing the expression effects of Cpf1 protein with constitutive and inducible promoters.Finally,using C.glutamicum ATCC 13032 without L-serine production as starting strain,a high-producing L-serine recombinant strain was constructed using this system.The results showed that the gene deletion efficiency of the inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system was(66.67±1.53)%,and the gene integration efficiency was(14.29±1.50)%,both of which were significantly higher than those of the constitutive promoter group(P<0.05).In addition,an high-producing L-serine recombinant strain SW-3 was successfully constructed using the inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system with an L-serine yield of 10.51 mg/L,which indicated that the inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system was feasible in the metabolic engineering modification of C.glutamicum,and it would have far-reaching significance in accelerating the process of metabolic engineering modification of C.glutamicum and promoting its application in the research and development of amino acids and other high value-added products.
作者
王婷
石佳宝
高印
陈宁
WANG Ting;SHI Jiabao;GAO Yin;CHEN Ning(College of Agriculture,Henan University of Science and Technology,Luoyang 471000,China;College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China)
出处
《中国酿造》
北大核心
2025年第8期35-41,共7页
China Brewing
基金
河南省重点研发与推广专项(科技攻关)(252102110239)。
作者简介
王婷(1983-),女,讲师,博士,研究方向为工业微生物遗传育种;通讯作者:陈宁(1963-),男,教授,博士,研究方向为工业微生物组学与遗传育种。
