Construction and application of theophylline-activated RNA switches in the regulation of expression of recombinant proteins in Bacillus subtilis

Abstract:

Bacillus subtilis can be widely used as an important microorganism for metabolic engineering and recombinant proteins expression in industrial biotechnology and synthetic biology. However, it is difficult to make accurate regulation of exogenous gene by biological tools in B. subtilis, which limits the application of B. subtilis in synthetic biology. The purpose of this study is to develop regulatory tools for precise control of gene expression by using non-coding RNAs, by which the activation of heterologous gene could be achieved without the auxiliary protein factors. We constructed the synthetic riboswitch E and aptazyme AZ using the theophylline aptamer. Six different native promoters from B. subtilis were functionally adapted with the E and AZ to fabricate an array of novel regulatory elements activated by theophylline. Then, we determined the performance of these elements using green fluorescence protein as reporter, and then further verified using red fluorescence protein and pullulanase as cargo proteins. Results showed that the same kind of RNA elements with different promoters showed different levels of efficiency. Promoter PsigW and E combination (sigWE) had the highest induction rate in B. subtilis. Compared with the control group, it can produce the induction rate of 16.8. Promoter PrpoB and AZ combination (rpoBAZ) showed the highest induction rate of 6.2. SigWE mediated mCherry induction rate was 9.2, and P43E mediated pullulanase induction rate was 32.8, in which enzyme activity reached 81 U/mL. This study confirmed that GFP, mCherry and pullulan can all be regulated by riboswitch and aptazyme, but there were differences between different combinations of promoters with RNA regulators.