Implementation of Synthetic Small Regulatory RNAs in Metabolic Engineering

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Tong Un Chae, Dokyun Na, Seung Min Yoo, Hannah Chung, Hyegwon Park, Jin Hwan Park and Sang Yup Lee

Korea Advanced Institute of Science and Technology, South Korea

Regulatory noncoding RNAs, such as small regulatory RNAs (sRNAs), are fast-acting and less-energy-consuming regulator widely used in many organisms to precisely control gene expression together with transcriptional control, and therefore they could be a vulnerable regulator for gene expression control. Here we present an approach for constructing synthetic small regulatory RNAs for controlling gene expression. We developed synthetic small regulatory RNAs repressing the translation of DsRed2 mRNA at various levels and also constructed three different sRNAs for the mRNAs of LuxR, AraC, and KanR without cross-reactivity. The results suggest that gene expression can be fine-tuned by designed artificial small RNAs. Synthetic sRNAs can be implemented for metabolic engineering. 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. 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). [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012-C1AAA001-2012M1A2A2026556); the Intelligent Synthetic Biology Center through the Global Frontier Project (2011-0031963) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea; and the World Class University program (R32-2008-000-10142-0) of MEST.]