Deng Chen, Xin Ruijie, Li Xingjian, Zhang Jie, Fan Liqiang, Qiu Yongjun, Zhao Liming
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCICBT), East China University of Science and Technology, Shanghai 200237, China.
Nucleic Acids Res. 2024 Dec 11;52(22):14260-14276. doi: 10.1093/nar/gkae1149.
Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.
谷氨酸棒杆菌是生产氨基酸和高价值化学品的关键工业主力菌株。平衡细胞生长与产物合成之间的代谢流对于提高生产效率至关重要。为谷氨酸棒杆菌开发动态、广泛适用且毒性最小的基因调控工具仍然具有挑战性，因为尚未开发出适用于动态调控策略的光遗传学工具。本研究引入了一种先进的光控基因表达系统，该系统使用光控RNA结合蛋白（RBP），这在谷氨酸棒杆菌中尚属首次。我们建立了一个基因表达调控系统“LightOnC.glu”，利用光控RBP在谷氨酸棒杆菌中构建光控转录因子。同时，我们使用CRISPR/Cpf1工具开发了一种高性能的光控基因干扰系统。设计合成网络中的代谢流，首次在谷氨酸棒杆菌中实现了几丁寡糖（CHOSs）和硫酸软骨素寡糖A（CSA）的生产。此外，构建了一个光控生物反应器，实现了6.2 g/L的CHOSs生产浓度，这是迄今为止CHOSs生物合成记录的最高滴度。在此，我们在谷氨酸棒杆菌中建立了一个可编程的光响应遗传电路，推进了基于光信号的动态调控理论。这一突破在优化其他底盘细胞中的代谢模块和合成其他化合物方面具有潜在应用。
