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Facilitating Protein Expression with Portable 5'-UTR Secondary Structures in Bacillus licheniformis.
Jun Xiao1, Bing Peng2, Zhaowei Su1
1State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, Hubei University, Wuhan 430062, PR China.
ACS Synthetic Biology
|April 18, 2020
Summary
Researchers engineered a novel 5' untranslated region (UTR) for Bacillus species, enhancing protein production by up to 50-fold. This sequence improves translation initiation and mRNA stability, offering a versatile tool for microbial cell factories.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Microbial Biotechnology
Background:
- The 5'-untranslated region (5'-UTR) is crucial for post-transcriptional regulation in prokaryotes.
- Bacillus species are industrial microbial cell factories for producing valuable chemicals and proteins.
- Optimizing gene expression in Bacillus is key for industrial applications.
Purpose of the Study:
- To develop a portable 5'-UTR sequence for significantly enhanced protein output in Bacillus licheniformis.
- To investigate the mechanism of enhanced translation initiation and mRNA stability conferred by the designed 5'-UTR.
- To demonstrate the adaptability of the 5'-UTR for diverse target proteins.
Main Methods:
- Design and synthesis of a short (∼30 nt) 5'-UTR sequence featuring a hairpin structure.
- Optimization of the Shine-Dalgarno (SD) sequence within the hairpin loop for ribosome binding.
- Thermodynamic folding energy optimization of the 5'-UTR.
- Expression analysis of enhanced green fluorescent protein (eGFP) and other proteins (RFP, nattokinase, keratinase) using the designed 5'-UTR.
Main Results:
- The engineered 5'-UTR enhanced eGFP expression by approximately 50-fold.
- The 5'-UTR demonstrated broad applicability, successfully boosting the expression of RFP, nattokinase, and keratinase.
- The hairpin structure improved accessibility of the SD sequence and start codon, enhancing translation initiation efficiency.
- The 5'-UTR conferred increased mRNA stability by protecting against 5'-exonucleases.
Conclusions:
- A novel, portable 5'-UTR sequence was successfully developed for significantly boosting protein production in Bacillus licheniformis.
- The designed 5'-UTR functions by enhancing translation initiation and mRNA stability, overcoming previous concepts about RBS structure limitations.
- This rationally designed 5'-UTR offers a versatile and tunable tool for optimizing protein expression in various Bacillus species for industrial biotechnology.
