Science Center for Future Foods, Jiangnan University, Wuxi 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Science Center for Future Foods, Jiangnan University, Wuxi 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Enzyme Microb Technol. 2021 Jun;147:109782. doi: 10.1016/j.enzmictec.2021.109782. Epub 2021 Mar 18.
Menaquinone-4 (MK-4) plays a significant role in bone health and cardiovascular therapy. Although many strategies have been adopted to increase the yield of MK-4 in Bacillus subtilis 168, the effectiveness of MK-4 is still low due to the inherent limitations of metabolic pathways. 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. Nevertheless, in most reports, QS systems depend on down-regulated expression rather than up-regulated expression, which greatly limit their potential as molecular switches to control metabolic flux. To address this challenge, a modular PhrQ-RapQ-ComA QS system is developed based on promoter P, which is up-regulated by phosphorylated ComA (ComA-P). In this paper, firstly we analyzed the ComA-based gene expression regulation system in Bacillus subtilis 168. We constructed a promoter library of diff ;erent abilities, selected best promoters from a library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrQ-RapQ-ComA QS system to dynamically control the synthesis of MK-4 in B. subtilis 168. 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 MK-4 in shake flask from 120.1 ± 0.6 to 178.9 ± 2.8 mg/L, and reached 217 ± 4.1 mg/L in a 3-L bioreactor, which verified the effectiveness of this strategy. In summary, PhrQ-RapQ-ComA QS system can realize dynamic pathway regulation in B. subtilis 168, which can be stretched to a great deal of microorganisms to fine-tune gene expression and enhance the production of metabolites.
甲萘醌-4（MK-4）在骨骼健康和心血管治疗中发挥着重要作用。尽管已经采用了许多策略来提高枯草芽孢杆菌 168 中甲萘醌-4 的产量，但由于代谢途径的固有局限性，MK-4 的有效性仍然很低。然而，基于群体感应（QS）的动态调控已被广泛应用作为精细调节基因表达的基本工具，以响应细胞密度的变化，而无需添加昂贵的诱导剂。然而，在大多数报告中，QS 系统依赖于下调表达而不是上调表达，这极大地限制了它们作为分子开关控制代谢通量的潜力。为了解决这一挑战，我们基于受磷酸化 ComA（ComA-P）上调的启动子 P 开发了模块化的 PhrQ-RapQ-ComA QS 系统。在本文中，我们首先分析了枯草芽孢杆菌 168 中基于 ComA 的基因表达调控系统。我们构建了不同能力的启动子文库，从文库中选择最佳启动子，并对选定的启动子进行突变筛选。此外，我们构建了 PhrQ-RapQ-ComA QS 系统来动态控制枯草芽孢杆菌 168 中 MK-4 的合成。通过 QS 系统可以动态平衡细胞生长和目标产物的有效合成。我们的动态调整方法使摇瓶中 MK-4 的产量从 120.1±0.6 增加到 178.9±2.8 mg/L，在 3-L 生物反应器中达到 217±4.1 mg/L，验证了该策略的有效性。总之，PhrQ-RapQ-ComA QS 系统可以实现枯草芽孢杆菌 168 中的动态途径调控，可以扩展到大量微生物中，以精细调节基因表达并增强代谢产物的生产。
