食品与发酵工业2023，Vol.49Issue(23)：25-32,8.DOI:10.13995/j.cnki.11-1802/ts.035178
谷氨酸棒杆菌人工核糖体结合位点(RBS)文库的建立与应用
Construction and application of an artificial ribosomal binding site(RBS)library in Corynebacterium glutamicum
摘要
Abstract
Ribosome binding site(RBS)is an important biological control element for fine-regulation of genes expression,which has been widely applied in microbial metabolic engineering and synthetic biology.Corynebacterium glutamicum is an important industrial mi-croorganism for industrial fermentation of several amino acids and organic acids.However,there were few researches on the RBS element for C.glutamicum.In this study,a random RBS library was designed and constructed based on the common RBS characteristics in C.glutamicum.Then,a high-throughput screening technology was developed by coupling flow cytometry and 96-well plate screening technol-ogy for screening of the artificial RBS library.After two-step screening,an artificial RBS element library with wide range of regulation and uniform coverage was constructed,and the regulation strength range of the library was 29.04-fold.Next,the artificial RBS library was fur-ther characterized by α-amylase expression system,and the results showed that the RBS library had high stability and versatility.Subse-quently,the artificial library was applied for the regulation and optimization of the L-homoserine biosynthetic pathway.The expressions of LysCr and Homr,the key enzymes in L-homoserine synthesis pathway,were fine-regulated using RBS elements with different intensities.Finally,the best combination of LysCr and Homr for L-homoserine synthesis was obtained,and the resulted strain accumulated 12.7 g/L of L-homoserine,which was 3.6 times higher than that of the wild-type strain.Therefore,an artificial RBS library was constructed and ex-hibited wide regulation range and high stability in this study,which provided the effective control elements for metabolic engineering and gene expression regulation in C.glutamicum.关键词
谷氨酸棒杆菌/人工核糖体结合位点文库/高通量筛选/L-高丝氨酸Key words
Corynebacterium glutamicum/artificial RBS library/high-throughput screening technology/L-homoserine引用本文复制引用
张悦,张继伟,吴硕,徐宁,刘君,魏亮..谷氨酸棒杆菌人工核糖体结合位点(RBS)文库的建立与应用[J].食品与发酵工业,2023,49(23):25-32,8.基金项目
国家重点研发计划项目(2021YFC2100700) （2021YFC2100700）
国家自然科学基金项目(32001671,31972061) （32001671,31972061）
