A dual-fluorescent biosensor for in vivo analysis of protein secretion in Escherichia coli and Corynebacterium glutamicum

A flavin-binding fluorescent protein (FbFP)-based biosensor was developed to enable real-time monitoring of protein expression and secretion in Gram-negative and Gram-positive bacteria. The applicability of this biosensor was first demonstrated in Escherichia coli and Corynebacterium glutamicum as a proof of concept. Both species are frequently used in industrial biotechnology, with C. glutamicum valued for its ability to secrete proteins directly into the culture supernatant, simplifying downstream processing.

This biosensor enables non-invasive, continuous tracking of both intracellular and extracellular protein levels during bacterial cell growth, providing detailed insights into secretion dynamics. When FbFPs remain in the cytoplasm after expression, they incorporate the endogenous chromophore FMN (flavin mononucleotide), resulting in a specific cyan-green fluorescence signal. In contrast, secretion of the biosensor protein into the periplasm or supernatant via the Sec secretion pathway requires an externally supplied FMN derivative. This addition results in a red-shifted fluorescence signal, enabling fluorescence-based differentiation between cytoplasmic and secreted proteins and providing real-time insights into protein localization and secretion pathways.

After demonstrating its functionality, the biosensor was used to comparatively analyze the expression and secretion of recombinant proteins consisting (i) different signal peptides from Bacillus subtilis, (ii) the cutinase from Fusarium solani pisi, and (iii) the FbFP domain. For this purpose, corresponding E. coli and C. glutamicum expression strains were cultivated, and green and red-shifted fluorescence signals as well as cutinase activities were subsequently measured. The results demonstrated that a direct correlation between fluorescence signals and cutinase activity could be achieved, validating the biosensor's ability to reliably report on protein localization and secretion.

Overall, this biosensor system expands the applicability of FbFPs by enabling in vivo investigation of Sec-dependent secretion processes, thereby providing valuable information for the optimization of biotechnological production processes in bacteria.