Journal
ACS SYNTHETIC BIOLOGY
Volume 11, Issue 12, Pages 4156-4170Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.2c00464
Keywords
Bacillus subtilis; poly-gamma-glutamic; acid; PhrQ-RapQ-DegU; dynamic regulation
Categories
Funding
- National Nature Science Foundation of China
- Science and Technology Project of Wuhu
- National Undergraduate Innovation and Entrepreneurship Program
- Anhui Provincial Undergraduate Innovation and Entrepreneurship Program
- [31871781]
- [31772081]
- [2020yf62]
- [201910363042]
- [S202010363255]
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As a natural biological macromolecule, gamma-polyglutamic acid (gamma-PGA) plays a significant role in medicine, food, and cosmetic industries due to its unique properties. Current methods for increasing gamma-PGA yield are not effective due to the viscosity issue. This study successfully improved the yield of gamma-PGA by using a dynamic regulation strategy based on quorum sensing.
As a natural biological macromolecule, gamma-polyglutamic acid (gamma-PGA) plays a significant role in medicine, food, and cosmetic industries owing to its unique properties of biocompatibility, biodegradability, water solubility, and viscosity. Although many strategies have been adopted to increase the yield of gamma-PGA in Bacillus subtilis, the effectiveness of these common approaches is not high because the strong viscosity affects cell growth. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. A modular PhrQ-RapQ-DegU QS system is developed based on promoter P-D4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system in B. subtilis 168. We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU QS system to dynamically control the synthesis of gamma-PGA in BS168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of gamma-PGA to 6.53-fold of that by static regulation in a 3 L bioreactor, which verified the effectiveness of this strategy. In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which can be stretched to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.
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