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

Jae Sung Cho, Dongsoo Yang, Cindy Pricilia Surya Prabowo, Mohammad Rifqi Ghiffary, Taehee Han, Kyeong Rok Choi, Cheon Woo Moon, Hengrui Zhou, Jae Yong Ryu, Hyun Uk Kim and Sang Yup Lee ()
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Jae Sung Cho: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Dongsoo Yang: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Cindy Pricilia Surya Prabowo: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Mohammad Rifqi Ghiffary: Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
Taehee Han: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Kyeong Rok Choi: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Cheon Woo Moon: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Hengrui Zhou: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Jae Yong Ryu: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)
Hyun Uk Kim: Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST
Sang Yup Lee: Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST)

Nature Communications, 2023, vol. 14, issue 1, 1-13

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-38119-y

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