Gene Expression Knockdown by Modulating Synthetic Small RNA Expression in Escherichia coli

Escherichia coli gene expression knockdown using synthetic small RNA (sRNA) can be fine-tuned by altering sRNA sequences to modulate target mRNA-binding ability, but this requires thorough checking for off-target effects. Here, we present an sRNA gene expression knockdown system fine-tuned by using different promoters to modulate synthetic sRNA abundance. Our approach entails selecting knockdown target genes resulting from in silico flux response analysis and those related to product biosynthesis then screening strains transformed with a library of synthetic sRNA-promoter combinations for enhanced production. We engineered two E. coli strains, both utilizing fine-tuned repression of argF and glnA through our approach; one produced putrescine (42.3 +/- 1.0 g/L) and the other L-proline (33.8 +/- 1.6 g/L) by fed-batch culture. Fine-tuned gene knockdown by controlling sRNA abundance will be useful for rapid design of microbial strains through simultaneously optimizing expression of multiple genes at a systems level, as it overcomes the difficulties of constructing and testing many different sRNAs and checking their cross-reactivity.