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
毋舒宁 1苏永平 1李冬雪 2柏映国 2刘波 3张志伟4
作者信息
- 1. 山西农业大学林学院,太谷 030801;中国农业科学院生物技术研究所,北京 100081
- 2. 中国农业科学院北京畜牧兽医研究所,北京 100193
- 3. 中国农业科学院生物技术研究所,北京 100081
- 4. 山西农业大学林学院,太谷 030801
- 折叠
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PH10 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PH10-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 25 μg/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 62 000,证明PH10-CuO具有很好的严谨性和诱导表达强度.构建了以启动子PH10-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PH10-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.
Abstract
[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 PH10-CuO was constructed by embedding the operator sequence CuO on the constitutive promoter PH10.[Result]Using green fluorescent protein as a quantitative reporter,the relative fluorescence intensity was low due to the basal leakage of PH10-CuO when without the inducer 4-isopropylbenzoic acid;and gfp expression significantly increased when the fluorescence intensity was up to 62 000 at presence of 25 μg/mL 4-isopropylbenzoic acid for 12 h.This proved that PH 10-CuO has very good rigor and induced expression intensity.Meanwhile,in Constructing the gene editing plasmid for C.glutamicum with PH10-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 PH10-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Key words
Corynebacterium glutamicum/inducible promoter/repressor protein CymR/4-isopropylbenzoic acid引用本文复制引用
基金项目
国家重点研发计划(2022YFD1300703)
国家自然科学基金项目(32171806)
出版年
2024
国家科技期刊平台
NSTL
万方数据