Fine-tuning knockdown system using synthetic regulatory small RNA and its application in metabolic engineering

Abstract

Fine-tuning of gene expression is essential for optimization of metabolic and genetic networks, but conventional methods are time-consuming and laborious. Here, we report the development of a fine-tunable knockdown system by modulating synthetic small RNA (sRNA) expression levels in Escherichia coli. A library of 75 synthetic sRNAs-promoter combinations was constructed for the enhanced production of putrescine, an engineering plastic monomer. Optimal repression of argF and glnA using sRNAs led to rapid development of an engineered E. coli strain capable of producing 43.0 g/l of putrescine by fed-batch cultivation. Similarly, through fine-tuned repression of argF and glnA by applying 25 sRNAs-promoter combinations, an engineered E. coli strain capable of producing 32.7 g/l of L-proline by fed-batch culture could be developed. The fine-tuning system of modulating synthetic sRNA expression levels reported here will be useful for the optimal and rapid design of microbial strains through simultaneous optimization of multiple gene expression levels at the systems level. This strategy will serve as a good complementing alternative to individually re-designing sRNAs in an sRNA-based gene expression knockdown system. The dissection of small regulatory RNA scaffold structure was also performed to enhance the knockdown activity of sRNA and to develop a tuned knockdown tool in the absence of series of synthetic promoters. The mutational study was carried out based on SgrS, one of well-studied sRNAs, and the importance of stem length and sequence of a loop of Hfq, an RNA chaperone binding site, was found. Based on these observations, the knockdown activity of sRNA is able to be strengthened and weakened by inserting mutations in the scaffold.