A part toolbox to tune genetic expression inBacillus subtilis

Abstract

Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis. We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis.

Pathway Engineering of Bacillus subtilis for Enhanced N‐Acetylneuraminic Acid Production via Whole‐Cell Biocatalysis

“…AGE and NanA are the two key enzymes that catalyzed the conversion of GlcNAc to ManNAc and ManNAc to NeuAc, respectively (Figure A). To strengthen the expression of AGE and NanA for efficient NeuAc production, seven strong constitutive promoters, including P lytR , P veg , P serA , P 43 , P sigX , P hbs , and P trnQ , which have been characterized with strong strength for controlling gene expression without the need of addition of chemical inducers, were selected for controlling the expressions of AGE and NanA . To facilitate NeuAc production and prevent substrate GlcNAc degradation, B. subtilis B6CG, which was previously constructed by blocking entire GlcNAc catabolic pathways, was selected as the expression host .…”

“…To strengthen the expression of AGE and NanA for efficient NeuAc production, seven strong constitutive promoters, including P lytR , P veg , P serA , P 43 , P sigX , P hbs , and P trnQ, which have been characterized with strong strength for controlling gene expression without the need of addition of chemical inducers, were selected for controlling the expressions of AGE and NanA. [31,32] To facilitate NeuAc production and prevent substrate GlcNAc degradation, B. subtilis B6CG, which was previously constructed by blocking entire GlcNAc catabolic pathways, was selected as the expression host. [24] The plasmid harboring the genes encoding AGE and NanA was transformed into B6CG to generate the recombinant strain B6CG/p43AN as a whole-cell biocatalyst.…”

A new sRNA‐mediated posttranscriptional regulation system for Bacillus subtilis

“…n the constructed MS‐DOS, OPR that base‐pairs with SR4 were inserted immediately behind stop codon of the target gene (Figure ) but not in the open reading frame. As a consequence, the properties of the target proteins are not affected which is quite superior to the SsrA degradation system (Griffith & Grossman, ; Guiziou et al, ). Moreover, MS‐DOS is also more controllable and feasible comparing with the CRISPR‐dCas9 system in B. subtilis (Qi et al, ).…”

“…By modifying the SsrA degradation tag (GKTNSFNQNVALAA) and controlling the expression of the adaptor protein SsrB, an inducible protein degradation system has been constructed in B. subtilis (Griffith & Grossman, ). With the help of the adaptor protein SsrB, enzymes fusing with different SsrA tags are degraded by the ClpXP protease at various rates (Guiziou et al, ). However, introducing a 14 amino acid peptide to a protein might alter its properties or even lead to inactivation.…”

“…of the adaptor protein SsrB, enzymes fusing with different SsrA tags are degraded by the ClpXP protease at various rates (Guiziou et al, 2016). However, introducing a 14 amino acid peptide to a protein might alter its properties or even lead to inactivation.…”