Facilitating Protein Expression with Portable 5′-UTR Secondary Structures in Bacillus licheniformis

The 5′-untranslated region (5′-UTR) of prokaryotic mRNAs plays an essential role in post-transcriptional regulation. Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, have gained considerable attention as microbial cell factories for the production of various valuable chemicals and industrial proteins. In this work, we developed a portable 5′-UTR sequence for enhanced protein output in the industrial strain B. licheniformis DW2. This sequence contains only ∼30 nt and forms a hairpin structure located right before the open reading frame. The optimized Shine–Dalgarno (SD) sequence was presented as a single strand on the loop of the hairpin for better ribosome recognition and recruitment. By optimizing the free energy of folding, this 5′-element could effectively enhance the expression of eGFP by ∼50-fold and showed good adaptability for other target proteins, including RFP, nattokinase, and keratinase. This 5′-UTR could promote the accessibility of both the SD sequence and start codon, leading to improved efficiency of translation initiation. Furthermore, the hairpin structure protected mRNA against 5′-exonucleases, resulting in enhanced mRNA stability. It is well-known that the stable structure at a ribosome binding site (RBS) impedes initiation in Escherichia coli. In this study, we presented a unique structure at a RBS that can effectively enhance protein production, which is an exception of this prevailing concept. By adjusting a single thermodynamic parameter and holding the other factors affecting protein output constant, a series of 5′-UTR elements with different expression strengths could be rationally designed for wide use in Bacillus sp.