Cui Shixiu, Lv Xueqin, Wu Yaokang, Li Jianghua, Du Guocheng, Ledesma-Amaro Rodrigo, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China.
Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University , Wuxi 214122 , China.
ACS Synth Biol. 2019 Aug 16;8(8):1826-1837. doi: 10.1021/acssynbio.9b00140. Epub 2019 Jul 1.
Quorum sensing (QS)-based dynamic regulation has been widely used as basic tool for fine-tuning gene expression in response to cell density changes without adding expensive inducers. However, most reported QS systems primarily relied on down-regulation rather than up-regulation of gene expression, significantly limiting its potential as a molecular switch to control metabolic flux. To solve this challenge, we developed a bifunctional and modular Phr60-Rap60-Spo0A QS system, based on two native promoters, (down-regulation by Spo0A-P) and (up-regulation by Spo0A-P). We constructed a library of promoters with different capacities to implement down-regulation and up-regulation by changing the location, number, and sequences of the binding sites for Spo0A-P. The QS system can dynamically balance the relationship between efficient synthesis of the target product and cell growth. Finally, we validated the usefulness of this strategy by dynamic control of menaquinone-7 (MK-7) synthesis in 168, a model Gram-positive bacterium, with the bifunctional Phr60-Rap60-Spo0A quorum sensing system. Our dynamic pathway regulation led to a 40-fold improvement of MK-7 production from 9 to 360 mg/L in shake flasks and 200 mg/L in 15-L bioreactor. Taken together, our bilayer QS system has been successfully integrated with biocatalytic functions to achieve dynamic pathway regulation in 168, which may be extended for use in other microbes to fine-tune gene expression and improve metabolites production.
基于群体感应（QS）的动态调控已被广泛用作一种基本工具，用于在不添加昂贵诱导剂的情况下，根据细胞密度变化对基因表达进行微调。然而，大多数已报道的QS系统主要依赖于基因表达的下调而非上调，这显著限制了其作为控制代谢通量的分子开关的潜力。为了解决这一挑战，我们基于两个天然启动子（Spo0A-P介导下调和Spo0A-P介导上调）开发了一种双功能模块化的Phr60-Rap60-Spo0A QS系统。我们通过改变Spo0A-P结合位点的位置、数量和序列，构建了具有不同下调和上调能力的启动子文库。该QS系统能够动态平衡目标产物高效合成与细胞生长之间的关系。最后，我们通过使用双功能Phr60-Rap60-Spo0A群体感应系统动态控制模式革兰氏阳性菌枯草芽孢杆菌168中甲基萘醌-7（MK-7）的合成，验证了该策略的有效性。我们的动态途径调控使摇瓶中MK-7的产量从9 mg/L提高到360 mg/L，提高了40倍，在15-L生物反应器中达到200 mg/L。综上所述，我们的双层QS系统已成功与生物催化功能整合，以实现枯草芽孢杆菌168中的动态途径调控，这可能会扩展到其他微生物中，用于微调基因表达和提高代谢产物产量。
