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

Area of Science:

• Synthetic biology
• Bacterial genetics
• Gene regulation

Background:

• Synthetic small RNAs (sRNAs) enable gene knockdown at the translational level but are limited to specific bacterial species.
• Developing versatile tools for bacterial gene manipulation is crucial for both medical and industrial advancements.

Purpose of the Study:

• To develop a broad-host-range synthetic sRNA (BHR-sRNA) platform.
• To demonstrate the efficacy of BHR-sRNA in diverse bacterial species for various applications.

Main Methods:

• Utilized the RoxS scaffold and Bacillus subtilis Hfq chaperone to create the BHR-sRNA platform.
• Tested BHR-sRNA in 16 bacterial species, including commensal, probiotic, pathogenic, and industrial strains.
• Applied BHR-sRNA for virulence factor knockdown in Staphylococcus epidermidis and Klebsiella pneumoniae, and for metabolic engineering in Corynebacterium glutamicum.

Main Results:

• Achieved >50% target gene knockdown in 12 out of 16 tested bacterial species.
• Successfully mitigated virulence phenotypes in pathogenic bacteria.
• Engineered high-performance Corynebacterium glutamicum strains for chemical production and developed a genome-scale sRNA library for high-throughput screening.

Conclusions:

• The BHR-sRNA platform offers a versatile tool for engineering a wide range of bacteria.
• This technology has significant potential to accelerate advancements in both industrial biotechnology and medical applications.
• BHR-sRNA facilitates precise gene knockdown, enabling the development of novel bacterial strains for diverse purposes.