Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 program), Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
Nat Biotechnol. 2013 Feb;31(2):170-4. doi: 10.1038/nbt.2461. Epub 2013 Jan 20.
Small regulatory RNAs (sRNAs) regulate gene expression in bacteria. We designed synthetic sRNAs to identify and modulate the expression of target genes for metabolic engineering in Escherichia coli. Using synthetic sRNAs for the combinatorial knockdown of four candidate genes in 14 different strains, we isolated an engineered E. coli strain (tyrR- and csrA-repressed S17-1) capable of producing 2 g per liter of tyrosine. Using a library of 130 synthetic sRNAs, we also identified chromosomal gene targets that enabled substantial increases in cadaverine production. Repression of murE led to a 55% increase in cadaverine production compared to the reported engineered strain (XQ56 harboring the plasmid p15CadA). The design principles and the engineering strategy using synthetic sRNAs reported here are generalizable to other bacteria and applicable in developing superior producer strains. The ability to fine-tune target genes with designed sRNAs provides substantial advantages over gene-knockout strategies and other large-scale target identification strategies owing to its easy implementation, ability to modulate chromosomal gene expression without modifying those genes and because it does not require construction of strain libraries.
小分子 RNA（sRNA）调控细菌中的基因表达。我们设计了合成 sRNA 来鉴定和调节大肠杆菌代谢工程的靶基因的表达。使用合成 sRNA 对 14 种不同菌株中的四个候选基因进行组合敲低，我们分离出了一种能够生产 2 克/升酪氨酸的工程大肠杆菌菌株（tyrR 和 csrA 抑制的 S17-1）。使用 130 个合成 sRNA 文库，我们还鉴定了染色体基因靶点，使 cadaverine 的产量大幅增加。与报道的工程菌株（携带质粒 p15CadA 的 XQ56）相比，murE 的抑制使 cadaverine 的产量增加了 55%。本文报道的使用合成 sRNA 的设计原则和工程策略可推广应用于其他细菌，并适用于开发更优的生产菌株。与基因敲除策略和其他大规模靶标鉴定策略相比，使用设计的 sRNA 微调靶基因具有显著优势，因为它易于实施，能够在不修饰这些基因的情况下调节染色体基因表达，而且不需要构建菌株文库。
