Real-time simultaneous monitoring of multiple analytes in bacterial cultures

ABSTRACT Bacterial metabolites are essential for biological processes, influencing human health, ecosystems, and various industrial applications. Simultaneous real-time monitoring of these metabolites is critical in understanding microbial dynamics, particularly in bioreactors and food or drug manufacturing. Current approaches often rely on offline methods, which are labor-intensive and susceptible to contamination, or genetic engineering techniques limited to single-analyte monitoring. Here, we present a novel method utilizing engineered periplasmic binding proteins (PBPs) conjugated with fluorophores to track multiple metabolites simultaneously in bacterial cultures, including Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , Enterococcus faecalis , and Clostridium striatum . This system continuously monitors the levels of multiple analytes, such as glucose, arabinose, ribose, glutamate, and arginine, providing high temporal resolution while maintaining sensor stability over a period of 24 h. Our findings confirm hierarchical substrate utilization in E. coli and other bacterial species and demonstrate the versatility of PBP-based multi-sensor arrays. This approach offers a non-invasive, modular, and scalable tool for bacterial metabolite analysis, paving the way for advances in both fundamental discoveries and practical applications in microbiology. IMPORTANCE Real-time monitoring of metabolites in bacterial cultures is crucial for advancing our understanding of microbial physiology, metabolic fluxes, and dynamic responses to environmental changes. This capability enables researchers to capture transient metabolic states that are often missed in endpoint measurements. The use of engineered periplasmic binding proteins as biosensors for this real-time metabolite monitoring represents a groundbreaking approach. By leveraging the natural specificity and high affinity of PBPs for small molecules, these biosensors can be engineered to detect a wide range of metabolites with exceptional sensitivity and temporal resolution. The integration of PBP-based biosensors into microbial research not only enhances our ability to study real-time metabolism but also provides a versatile tool for optimizing industrial bioprocesses and exploring bacterial infections and complex microbial ecosystems