Broad-Spectrum Gene Repression Using Scaffold Engineering of Synthetic sRNAs

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.