Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

Abstract

BackgroundSynthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.ResultsA novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43′-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43′-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.ConclusionThe constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

“…Enzymatic activity of GUS was determined with the chromogenic substrate p -nitrophenyl-glucuronide ( p NPG, Sigma-Aldrich/Merck, Darmstadt, Germany) as described by Cui et al [ 33 ]. For subsequent cell lysis, 30 µl of the GUS expression cultures were mixed with 85 µl PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 2H 2 O, 1.76 mM KH 2 PO 4 , pH 7.4) and 5 µl of a 1 mg/ml lysozyme solution in PBS and incubated at 37 °C for 30 min.…”

Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis

“…To verify the expression level of GusA, driven by P srfA and P BH4 , BSGus and BSBHGus were cultured in 250-mL conical flasks with a working volume of 50 mL of LB. The activity of GusA was measured with 4-nitrophenyl β- d -glucuronide (PNPG) as described previously [7]. Strains BSNK and BSBHNK were used to verify the expression level of NK, driven by P srfA and P BH4 and NK activity was determined by a modified fibrin degradation assay [65].…”

“…Recently, diverse biological parts, comprising synthetic promoters, Ribosome Binding Sites (RBS), protein degradation tags SsrA [31], and small RNA-based regulators and switches [7, 32, 33], were constructed and directedly engineered to precisely tune the gene expression in B. subtilis . Among these synthetic biological parts, the promoter is the primary genetic pivotal element for gene expression, since it controls gene expression at the most fundamental level and determines the spatiotemporal regulation.…”

Recent trends in biocatalysis

“…71,89 The dynamic range of the output signal can be expanded by TF, riboswitch, promoter, and RBS engineering. 94,[107][108][109][110][111][112][113][114] Curated databases for prokaryotic TFs, their associated regulatory elements and target genes may be helpful for initial designs but might not avoid the necessity of iterative rounds permutating different combinations of genetic parts. 71,99,115,116 All these strategies are often time-consuming and results nonintuitive.…”