Real-time monitoring of subcellular states with genetically encoded redox biosensor system (RBS) in yeast cell factories

During industrial fermentation, microbial cell factories are usually confronted with environmental or metabolic stresses, leading to the imbalance of intracellular redox and the reduction of cell metabolic capacity. Here, we constructed the genetically encoded redox biosensor system (RBS) based on redox-sensitive fluorescent proteins to detect redox metabolites, including reactive oxygen species (ROS), oxidized glutathione, NADH, and NADPH in Saccharomyces cerevisiae. The functional biosensors were quantitatively characterized and the orthogonal redox biosensor system (oRBS) was designed for detecting multiple redox metabolites. Furthermore, the compartment targeted redox biosensor system (ctRBS) was constructed to detect ROS and NADPH, revealing the distribution and spatiotemporal dynamics of ROS in yeast under various stress conditions. As a proof-of-concept, RBS was applied to evaluate the redox states of engineered yeast with stress resistance and heterogenous tri-terpene synthesis in vivo, elucidating the redox balance significantly affecting the growth and production phe-notypes. The RBS in this study allowed the exploration of the diversity of compartmental redox state and real-time monitoring of the production process of yeast, providing a reliable and effective approach for accurate and in-depth profiling of bottlenecks of yeast cell factories.