Broad-Spectrum Gene Repression Using Scaffold Engineering of Synthetic sRNAs

Abstract

Gene expression regulation in broad-spectrum range is critical for constructing cell factories and genetic circuits to balance and control system-wide fluxes. Synthetic small regulatory RNAs (sRNAs) effectively regulate gene expression at the translational level by modulating an mRNA-binding chance and sRNA abundance; however, it can control target gene expression only within the limit of the intrinsic repression ability of sRNAs. Here, we systematically mutated a SgrS scaffold as a model sRNA by dividing the Hfq-binding module of the sRNA into the three regions: the A/U-rich sequence, the stem, and the hairpin loop, and examined how efficiently the mutants suppressed DsRed2 expression. By doing this, we found that a scaffold with an altered A/U-rich sequence (CUUU) and stem length and that with altered A/U-rich sequence (GCAC) showed a 3-fold stronger and a 3-fold weaker repression than the original scaffold, respectively. For practical application of altered scaffolds, proof-of-concept experiments were performed by constructing a library of 67 synthetic sRNAs with the strongest scaffold, each one targeting a different rationally selected gene, and using this library to enhance cadaverine production in Escherichia coli, yielding in 27% increase (1.67 g/L in flask cultivation, 13.7 g/L in fed-batch cultivation). Synthetic sRNAs with engineered sRNA scaffolds could be useful in modulating gene expression for strain improvement.

Keywords: Hfq-binding region; gene repression; knockdown; synthetic sRNA.