Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs

Abstract

Synthetic sRNAs allow knockdown of target genes at translational level, but have been restricted to a limited number of bacteria. Here, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. BHR-sRNA is tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria, with >50% of target gene knockdown achieved in 12 bacterial species. For medical applications, virulence factors in Staphylococcus epidermidis and Klebsiella pneumoniae are knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, high performance Corynebacterium glutamicum strains capable of producing valerolactam (bulk chemical) and methyl anthranilate (fine chemical) are developed by combinatorial knockdown of target genes. A genome-scale sRNA library covering 2959 C. glutamicum genes is constructed for high-throughput colorimetric screening of indigoidine (natural colorant) overproducers. The BHR-sRNA platform will expedite engineering of diverse bacteria of both industrial and medical interest.

Process Systems Engineering Approach for the Sustainable Design of Cyanobacteria-Based Integrated Biorefineries and Their Heat Exchanger Network

“…Also, it is important to note that the in silico strains S2 and S3 of Synechocystis are favored due to their superior biomass and ethanol productivities, emphasizing the significance of strain engineering for optimal performance in biorefineries. In the context of current research, gene editing and precise post-transcriptional control over gene expression tools, such as CRISPR-Cas9 − and small regulatory RNAs (sRNAs), − respectively, have emerged as revolutionary techniques for cyanobacteria strain design. This combined approach offers researchers unprecedented capabilities to engineer cyanobacteria with enhanced metabolic pathways, increased productivity, and specialized adaptability to varying environmental conditions.…”

Development of synthetic small regulatory RNA for Rhodococcus erythropolis

“…In this study, a novel synthetic sRNA based on the RhlS scaffold from P. low at approximately 30%. [26] In contrast, the RhlS gfp only resulted in 55.9% repression of sfgfp in R. erythropolis XP. Although the repression efficiency of RhlS gfp in R. erythropolis XP (55.9%) was lower than that of MicC-based sRNAs in E. coli, it was higher than the repression efficiency observed with synthetic sRNAs in most other non-E.…”

“…[24] In Corynebacterium glutamicum, the RoxS-BsHfq system showed the repression efficiency of 64.6% while the RoxS only resulted in 58.0% repression of gfp. [26] The differential effect of Hfq introduction could be attributed to the distinct mechanisms of action of synthetic sRNAs in different host species. [22,35] In Gram-negative bacteria, sRNAs regulate gene expression at the post-transcription level with the assistance of the RNA chaperone Hfq, which blocks ribosome binding and affects RNA stability.…”

“…[22] Additionally, E. coli SgrS scaffold, Pseudomonas aeruginosa PrrF1-2 scaffold, and Bacillus subtilis RoxS scaffold have also been employed as part of synthetic sRNAs. [24][25][26] Among these synthetic sRNAs, only the sRNA platform based on the RoxS scaffold and the Hfq chaperone from B. subtilis (RoxS-BsHfq system) has been tested for its usability in R. opacus PD630 DSM 43205. However, there is still a need to improve the efficiency of gene repression for practical applications.…”

Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology

“…For sRNA knock-down tools in diverse bacteria, Cho et al designed broad-host-range sRNA system (BHR-sRNA system) base-on sRNA scaffold of RoxS from B. subtilis [ 120 ]. Translationally, BHR-sRNA system achieved knockdown of reporter genes in 12 strains out of 16 strains, with slight modification in each strain.…”