Gene Expression Knockdown by Modulating Synthetic Small RNA Expression in Escherichia coli

被引:92
作者
Minho Noh [1 ,4 ]
Yoo, Seung Min [1 ,2 ,3 ,4 ]
Kim, Won Jun [1 ,4 ]
Lee, Sang Yup [1 ,2 ,3 ,4 ]
机构
[1] Korea Adv Inst Sci & Technol, Dept Chem & Biomol Engn, Metab & Biomol Engn Natl Res Lab, Plus Program BK21, Daejeon 34141, South Korea
[2] Korea Adv Inst Sci & Technol, BioProc Engn Res Ctr, Daejeon 34141, South Korea
[3] Korea Adv Inst Sci & Technol, Bioinformat Res Ctr, Daejeon 34141, South Korea
[4] Korea Adv Inst Sci & Technol, Inst BioCentury, Daejeon 34141, South Korea
基金
新加坡国家研究基金会;
关键词
SMALL REGULATORY RNAS; MEVALONATE PATHWAY; BIOGENIC-AMINES; MULTIPLE GENES; HFQ-BINDING; BIOSYNTHESIS; DERIVATIZATION; BACTERIA; SEQUENCE; DESIGN;
D O I
10.1016/j.cels.2017.08.016
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Escherichia coli gene expression knockdown using synthetic small RNA (sRNA) can be fine-tuned by altering sRNA sequences to modulate target mRNA-binding ability, but this requires thorough checking for off-target effects. Here, we present an sRNA gene expression knockdown system fine-tuned by using different promoters to modulate synthetic sRNA abundance. Our approach entails selecting knockdown target genes resulting from in silico flux response analysis and those related to product biosynthesis then screening strains transformed with a library of synthetic sRNA-promoter combinations for enhanced production. We engineered two E. coli strains, both utilizing fine-tuned repression of argF and glnA through our approach; one produced putrescine (42.3 +/- 1.0 g/L) and the other L-proline (33.8 +/- 1.6 g/L) by fed-batch culture. Fine-tuned gene knockdown by controlling sRNA abundance will be useful for rapid design of microbial strains through simultaneously optimizing expression of multiple genes at a systems level, as it overcomes the difficulties of constructing and testing many different sRNAs and checking their cross-reactivity.
引用
收藏
页码:418 / +
页数:13
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