Developing Quorum Sensing‐Based Collaborative Dynamic Control System in <i>Halomonas</i> TD01

Dynamic control exhibits increasing significance in microbial cell factory engineering by precisely manipulating gene expression over time and levels. However, the practical uses of most dynamic control tools still remain challenging because of poor scale-up robustness, especially for non-model chassis. Herein, a quorum sensing (QS)-based collaborative dynamic control system is constructed in Halomonas TD by regrouping two orthogonal quorum-sensing modules into two cell types, namely cell-A harboring cinR-luxI and cell-B harboring luxR-cinI together with sfGFP driven by P_cin and P_lux promoters, respectively. Effective gene expression control with over 15-time dynamic foldchange is achieved by mixing cells A and B at different ratios and time points in a lab-scale fed-batch study. Besides, dynamic inhibitory and amplified control is further developed by cascading CRISPRi/dCas9 system and MmP1 RNA polymerase, respectively, yielding up to 80% repression efficiency and 30-time amplification foldchange under high cell density fermentation. Moreover, 500 mg L^-1 indigo and 4.7 g L^-1 superoxide dismutase (SOD) are obtained by engineered Halomonas using QS-based control tools in the fed-batch study, showing 1.5- and 1.0-fold higher, respectively, than the yields by recombinants induced by IPTG. This study exemplifies a standardized and streamlined inducer-free dynamic control pattern for metabolic engineering with promising robustness in scale-up fermentation contexts.