一种谷氨酸棒杆菌4-异丙基苯甲酸诱导型启动子的设计与应用  

Design and Application of a Cumate-inducible Promoter for Corynebacterium glutamicum

作　　者：毋舒宁 苏永平 李冬雪 柏映国 刘波[2] 张志伟[1] WU Shu-ning;SU Yong-ping;LI Dong-xue;BAI Ying-guo;LIU Bo;ZHANG Zhi-wei(College of Forestry,Shanxi Agricultural University,Taigu 030801;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193)

机构地区：[1]山西农业大学林学院,太谷030801 [2]中国农业科学院生物技术研究所,北京100081 [3]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《生物技术通报》2024年第7期108-116,共9页Biotechnology Bulletin

基　　金：国家重点研发计划(2022YFD1300703);国家自然科学基金项目(32171806)。

摘　　要：【目的】谷氨酸棒杆菌是一种重要的工业微生物,挖掘可调控性元件可有效拓展谷氨酸棒杆菌研究和应用的广度和深度。【方法】在谷氨酸棒杆菌组成型强启动子P_(H10)上嵌入阻遏蛋白CymR结合的操纵序列CuO,构建了4-异丙基苯甲酸(Cumate)为诱导剂的启动子P_(H10-CuO)。【结果】绿色荧光蛋白作为报告基因的实验结果显示,无诱导剂时,谷氨酸棒杆菌工程菌相对荧光强度较低;以25μg/mL 4-异丙基苯甲酸诱导12 h时,谷氨酸棒杆菌工程菌荧光强度达62000,证明P_(H10-CuO)具有很好的严谨性和诱导表达强度。构建了以启动子P_(H10-CuO)调控recET和Cas12a表达的谷氨酸棒杆菌基因编辑质粒,实现谷氨酸棒杆菌染色体上靶标基因的精准编辑和外源基因插入。【结论】诱导型启动子P_(H10-CuO)具有表达强度高且渗漏表达低的特点,可用于谷氨酸棒杆菌中被其调控基因的时序性表达。【Objective】Corynebacterium glutamicum is an important industrial microorganism,and its metabolic engineering modification by gene editing may effectively broaden the diversity of its fermentation products.The lack of high-intensity,low-leakage,and low-cost inducible promoters limits the metabolic engineering modification,thereby the design and application of novel inducible promoters are necessary.【Method】An cumate-induced promoter P_(H10-CuO )was constructed by embedding the operator sequence CuO on the constitutive promoter P_(H10).【Result】Using green fluorescent protein as a quantitative reporter,the relative fluorescence intensity was low due to the basal leakage of P_(H10-CuO)when without the inducer 4-isopropylbenzoic acid;and gfp expression significantly increased when the fluorescence intensity was up to 62000 at presence of 25μg/mL 4-isopropylbenzoic acid for 12 h.This proved that P_(H10-CuO) has very good rigor and induced expression intensity.Meanwhile,in Constructing the gene editing plasmid for C.glutamicum with P_(H10-CuO) regulating the expression of recET and cas12a,The accurate editing of target gene and insertion of foreign genes in C.glutamicum chromosome.【Conclusion】The inducible promoter P_(H10-CuO) presented the advantages with high intensity and low leakage,which enables it as an useful element to regulate temporal expression of specific genes in C.glutamicum.

关 键 词：谷氨酸棒杆菌 诱导型启动子 阻遏蛋白CymR 4-异丙基苯甲酸 

分 类 号：Q78[生物学—分子生物学] Q93