A new sRNA‐mediated posttranscriptional regulation system for Bacillus subtilis

Abstract

Many genetic tools for gene regulation have been developed during the past decades. Some of them edit genomic DNA, such as nucleotides deletions and insertions, while the others interfere with the gene transcriptions or messenger RNA translation. Here, we report a posttranscriptional regulation tool which is termed "Modulation via the small RNA (sRNA)-dependent operation system: MS-DOS" by engineering the type I toxin-antitoxin system in Bacillus subtilis. MS-DOS depends simply on insertion of an operation region (OPR; partial toxin-encoding region) downstream of a genomic open reading frame of interest and overexpression of the coupling antitoxin sRNA from a plasmid. Pairing between the OPR and the sRNA will trigger the RNAse degradation of the transcripts of selected genes. MS-DOS allows for the quantitative, specific, and reversible knockdown of single or multiple genomic genes in B. subtilis. We also showed that the truncated antitoxin SR4 with 53 nt length is sufficient to repress gene expression. Superior to other existing RNA based interfering systems, MS-DOS allows simultaneous knockdown of multiple genes with effortless expression of a single antitoxin RNA. This sRNA-guided repression system will further enrich the gene regulation tools and expand the gene regulation flexibility.

Genetic strategies for improving hyaluronic acid production in recombinant bacterial culture

“…The downregulation of pfkA through a posttranscriptional regulation approach also improved the HA titre (1.6‐fold) in another B . subtilis HA‐producing strain (Yang et al, 2018), confirming that the attenuation of glycolysis is a valuable strategy to improve HA production.…”

“…Redirecting the carbon flux to the biosynthesis of HA via downregulation of glycolysis generated the strain E168T/pP43-DU-PBMS with a slight increase in HA titre (3.16 g L −1 ) and similar MW. The downregulation of pfkA through a posttranscriptional regulation approach also improved the HA titre (1.6-fold) in another B. subtilis HA-producing strain (Yang et al, 2018),…”

Type I Toxin-Antitoxin Systems in Clostridia

“…For example, type II RelBE TA was used to develop the toxin counter-selectable cassette regulated using an antitoxin switch for a wide variety of genetic modifications in Gram-positive bacteria including large-scale deletions and insertions, and gene knockdown and replacement, as well as point mutations [100]. In addition, a well-characterized B. subtilis type I TA module bsrG -SR4 has been recently used to design a new antitoxin RNA-guided gene repression system in this model bacterium [101].…”

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

“…Naturally, ncRNAs are widely involved in sensing intracellular and extracellular environmental changes and rapidly responding via altering the expression of other RNA or protein species [ [1] , [2] , [3] ]. Because of their versatile functions in a variety of cell processes, ncRNAs have attracted intense studies on their working mechanisms [ 4 ] and the standardization as artificial synbio parts [ [5] , [6] , [7] , [8] ]. Generally, such RNA modules contain antisense RNAs [ 9 ], small regulatory RNAs [ 10 ], and a variety of 3′ or 5′ untranslated regions [ 11 , 12 ] for controlling mRNA stability, transcription, or translation [ 4 ].…”