Poly-γ-glutamic acid production by engineering a DegU quorum-sensing circuit in Bacillus subtilis
Poly-γ-glutamic acid production by engineering a DegU quorum-sensing circuit in Bacillus subtilis
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通过在枯草芽孢杆菌中设计 DegU 群体感应电路来生产聚γ-谷氨酸
DOI：
10.1021/acssynbio.2c00464
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通讯作者：
Liu Yan
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作者：
Hu Liuxiu;Zhao Ming;Hu Wensong;Zhou Mengjie;Huang Junbao;Huang Xiling;Gao Xuli;Luo Yani;Li Chuang;Liu Kun;Xue Zhenglian;Liu Yan
γ-聚谷氨酸（γ-PGA）作为一种天然生物大分子，具有生物相容性、生物降解性、水溶性和粘性等独特的性质，在医药、食品和化妆品等领域发挥着重要作用。虽然人们采用了许多方法来提高γ-PGA在枯草芽孢杆菌中的产量，但由于其较强的粘度影响了菌体的生长，这些方法的效果并不高。然而，基于群体感应（quorum sensing，QS）的动态调节已被广泛应用为在不添加昂贵的诱导剂的情况下针对细胞密度的变化微调基因表达的基本工具。基于启动子PD 4开发了模块化PhrQ-RapQ-DegU QS系统，其被磷酸化DegU（DegU-P）上调。在本研究中，我们首先分析了B.枯草杆菌168.我们构建了不同能力的启动子文库，从文库中筛选合适的启动子，并对所选启动子和degU区域进行突变筛选。在此基础上，我们构建了一个PhrQ-RapQ-DegU QS系统，对γ-PGA的合成进行了动态调控。细胞生长和目标产物的有效合成可以通过QS系统动态平衡。在3L生物反应器中，动态调控使γ-PGA产量提高到静态调控的6.53倍，验证了该调控策略的有效性。综上所述，PhrQ-RapQ-DegU QS系统已经成功地与生物催化功能整合，实现了在BS 168中的动态代谢途径控制，可以延伸到大量微生物中，微调基因表达，增强代谢产物的产生。
As a natural biological macromolecule, γ-polyglutamic acid (γ-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 γ-PGA inBacillus 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 PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system inB. subtilis168. 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 γ-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 γ-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.