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Exploring and Engineering Novel Strong Promoters for High-Level Protein Expression in Bacillus subtilis DB104 through
Ji-Su Jun1, Hyang-Eun Jeong1, Kwang-Won Hong1
1Department of Food Science and Biotechnology, College of Life Science and Biotechnology, Dongguk University, Goyang-si 10326, Republic of Korea.
Microorganisms
|December 23, 2023
Summary
This study engineered a potent Bacillus subtilis expression system for rapid, inducer-free recombinant protein production. Optimized promoters like Psdp-4 achieve high yields of bioactive human epidermal growth factor (hEGF) within 24 hours.
Area of Science:
- Microbial biotechnology and synthetic biology.
- Recombinant protein expression and optimization.
Background:
- Bacillus subtilis is a key host for recombinant protein production.
- DB104, a protease-deficient derivative, offers advantages for extracellular protein expression.
- Transcriptome data provides insights into highly expressed genes for promoter discovery.
Purpose of the Study:
- To identify and characterize strong promoters from Bacillus subtilis DB104 transcriptome data.
- To engineer enhanced promoters for improved recombinant protein expression.
- To develop a highly efficient, inducer-free expression system for industrial applications.
Main Methods:
- Time-course transcriptome analysis of Bacillus subtilis DB104.
- Identification of highly expressed genes and assessment of their promoter strengths using eGFP reporter.
- Promoter engineering through optimization of regulatory elements and sequence elements.
- Construction and evaluation of an expression cassette for human epidermal growth factor (hEGF).
Main Results:
- Psdp and PskfA promoters showed significantly higher strength compared to the control P43 promoter.
- Engineered Psdp-4 promoter demonstrated a 3.84-fold increase in strength over the original Psdp.
- The Psdp-4 cassette achieved a high yield of 103.9 μg/mL for His-tagged hEGF within 24 hours.
- Expressed hEGF protein was purified and confirmed to be bioactive via cell proliferation assay.
Conclusions:
- A highly efficient, transcriptome-driven expression system for Bacillus subtilis DB104 was successfully constructed.
- The engineered Psdp-4 promoter enables rapid, inducer-free protein expression.
- This system serves as a valuable tool for industrial-scale recombinant protein production.
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