Facilitating Protein Expression with Portable 5′-UTR Secondary Structures in <i>Bacillus licheniformis</i>

Abstract

The 5'-untranslated region (5'-UTR) of prokaryotic mRNAs plays an essential role in post-transcriptional regulation. <i>Bacillus</i> species, such as <i>Bacillus subtilis</i> and <i>Bacillus licheniformis</i>, 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 <i>B. licheniformis</i> 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 <i>Escherichia coli</i>. 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 <i>Bacillus</i> sp.