In recent years, the transition from static design to dynamic control in genetic engineering has resulted in remarkable progress and is currently affecting a paradigm shift in microbial cell factory development using high-throughput genetic engineering (Jung et al., 2021, Yeom et al., 2023). The dynamic metabolic control strategy is employed to redistribute cellular resources and optimally regulate these pathways. Since living cell factories require a balance of growth and production, gene expression in metabolic flux should be optimized and finely controlled. For this reason, much effort has been put into the development of genetic engineering tools for fine-tuning gene expression and regulation at the genomic, mRNA, and protein levels (Jung et al., 2021, Yeom et al., 2023).

Among these genetic engineering tools, tailor-made synthetic sRNA systems rely on the capabilities of native sRNA as posttranscriptional regulators of gene expression by binding with complementary sequences on target mRNA (Na et al., 2013, Yoo et al., 2013). Like native sRNA, tailor-made synthetic sRNA engages an sRNA scaffold with a specific target mRNA-binding module and Hfq chaperon protein in ternary complex, preventing translation by mRNA masking, blocking access to the binding site on the ribosome, or degrading mRNA by RNase E. Due to its advantage of enabling easy and customizable regulation of target gene expression, many studies have been conducted on the regulation of gene expression by factors affecting the efficiency of sRNA; for example, mRNA-binding module in sRNA, Hfq-binding module in sRNA scaffold, and sRNA expression levels (Na et al., 2013, Noh et al., 2017, Noh et al., 2019, Yang et al., 2018a, Yang et al., 2018b). As changing the mRNA-binding sequence of the sRNA scaffold relies on calculating the binding energy between the target mRNA and the sRNA sequences (Yoo et al. 2013), changing mRNA binding module in the scaffold could modulate the level of target mRNAs repression (Na et al., 2013, Noh et al., 2017, Noh et al., 2019) in diverse microbes (Cho and Lee, 2017, Sun et al., 2018, Sun et al., 2019, Gao et al., 2019, Long et al., 2020, Cho et al., 2023) and using high throughput methods (Yang et al., 2018a, Yang et al., 2018b). Through the sRNA scaffold engineering, a novel scaffold with a specific gene repression ability have been created, enabling the modulation of target gene expression levels in a broad range beyond the intrinsic ability of sRNA (Noh et al. 2019). The sRNA abundance modulation could control the expression of desired target genes without disturbing sRNA scaffold, furthermore, it could save cellular resources by producing only the necessary amounts of sRNA, ensuring the maximum amount of resources available for chemical production (Noh et al. 2017). Despite of advancements in tailor-made synthetic sRNA system, they have mainly focused on the interaction between sRNAs and target mRNAs (Na et al., 2013, Yoo et al., 2013, Cho and Lee, 2017, Noh et al., 2017, Noh et al., 2019; Yang 2018; Yang 2019).

Hfq protein is another factor affecting the efficiency of tailor-made synthetic sRNA. The tailor-made synthetic sRNA-based system operates in a competitive mode of gene regulation, sharing intrinsic intracellular machinery, such as Hfq proteins, with intrinsic sRNA (Jung et al. 2021). The limited amount of intrinsic intracellular machinery can lower the tailor-made sRNA-repressing efficacy. Since in Hfq-dependent sRNAs, especially, Hfq affects the sRNAs’ repressive action against the target mRNA, regulating the expression level of the Hfq protein can be important for the efficient repression and modulation of target gene expression. The Hfq protein plays several roles in the mRNA-repression process: facilitating sRNA–mRNA binding in an ATP-independent manner, destabilizing mRNA by promoting polyadenylation, degrading mRNA by recruiting RNase E, and stabilizing sRNA against degradation (Hajnsdorf et al. 2000; Le Derout et al. 2003; Sledjeski et al. 2001; Møller et al. 2002; Massé et al. 2003; Zhang et al. 2002; Hämmerle et al. 2012; Morita et al. 2005). A recent study showed that the Hfq occupancy of the targets affects sRNA–target interaction frequency greatly, with definite effects on target mRNA levels (Faigenbaum-Romm et al. 2020). The tight autoregulation of hfq gene expression leads to limited amounts of cellular Hfq protein, causing competition among RNAs for Hfq to bind to (Morita et al. 2019), which can lead to a low repression efficiency against target genes in the sRNA system.

The modulation of Hfq amount to be produced can improve the tailor-made sRNA-repressing efficiency without any sRNA modification, which does not require additional consideration of potential cross-reactivity. This method can be also beneficial to improve the productivity of engineered chemical-producing strain as it was reported that the modulation of Hfq expression level can increase cell growth (Vo et al. 2021). However, the sRNA-based gene repression strategy by modulating Hfq expression level in engineered E. coli strain is not focused yet. In this regard, for a tunable knockdown using synthetic sRNAs, here, we report the effect of modulating the production of Hfq proteins on the efficiency of sRNAs and the application of this system in adjusting gene repression in Escherichia coli. To regulate the expression levels of hfq, we constructed an hfq gene expressed under the control of promoters with different strengths. The Hfq amount-mediated tunability of target gene expression levels was evaluated by observing the sRNA repression efficiencies against four fluorescent proteins as model proteins. Furthermore, the gene expression regulation system was employed to modulate the biosynthesis of two invaluable precursors of useful chemicals, 5-aminolevulinic acid (5-ALA) and L-tyrosine (L-Tyr), investigating its effect on the expression level of target proteins and chemical production. Our results reveal a tunable gene knockdown control, which opens the possibility of sophisticated gene expression control with tailor-made synthetic sRNA engineering-mediated strategies.