Construction of a 5′-controllable stabilizing element (CoSE) for over-production of heterologous proteins at high levels in Bacillus subtilis

• Production of recombinant proteins intracellularly in Bacillus subtilis.
• Increasing the half-life of mRNAs to more than 60 min.
• DNA sequence acting both as a binding site for a repressor protein and as a 5′ stabilizing mRNA element

Different mRNA stabilizing elements including the 3′-stem-loop, the ribosome binding sites (RBS), the 5′-stem-loop and the spacer region between the RBS and the 5′-stem-loop were analysed in detail to increase mRNA stability resulting in enhanced expression of heterologous proteins. In addition, in combination with mRNA stabilizing elements, we propose a new class of 5′-mRNA controllable stabilizing element (CoSE) which is composed of a transcriptional operator such as lacO of the Escherichia coli lac operon and a suitable RBS followed by an optimal spacer length. Such a CoSE allowed Bacillus subtilis cells to synthesize extraordinary stable transcripts with a half-life of the model bgaB reporter transcript (codes for an β-galactosidase gene derived from Bacillus stearothermophilus) of more than 60 min. This CoSE will be an important tool to control mRNA stability in cells for both research and biotechnological applications. For example, this CoSE can be used in inducible expression vectors offering two major advantages: (i) controlling transcription of target genes by the inducer and (ii) enhancing the stability of the transcript allowing the production high levels of recombinant proteins.