Opportunities for bioprocess monitoring using FRET biosensors

Abstract

Bioprocess monitoring is used to track the progress of a cell culture and ensure that the product quality is maintained. Current schemes for monitoring metabolism rely on offline measurements of samples of the extracellular medium. However, in the era of synthetic biology, it is now possible to design and implement biosensors that consist of biological macromolecules and are able to report on the intracellular environment of cells. The use of fluorescent reporter signals allows non-invasive, non-destructive and online monitoring of the culture, which reduces the delay between measurement and any necessary intervention. The present mini-review focuses on protein-based biosensors that utilize FRET as the signal transduction mechanism. The mechanism of FRET, which utilizes the ratio of emission intensity at two wavelengths, has an inherent advantage of being ratiometric, meaning that small differences in the experimental set-up or biosensor expression level can be normalized away. This allows for more reliable quantitative estimation of the concentration of the target molecule. Existing FRET biosensors that are of potential interest to bioprocess monitoring include those developed for primary metabolites, redox potential, pH and product formation. For target molecules where a biosensor has not yet been developed, some candidate binding domains can be identified from the existing biological databases. However, the remaining challenge is to make the process of developing a FRET biosensor faster and more efficient.

Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

“…It is also routinely used to monitor and control the trajectory of cultures during production to prolong the life of cells and maximize the yield of the product (Constantinou and Polizzi, 2013; Wuest et al, 2012). One particular metabolite of interest is lactic acid (lactate), a metabolic waste product produced as a byproduct of glycolysis.…”

Applications and advances of metabolite biosensors for metabolic engineering

“…5b). Although the ratiometric FRET signals provide higher accuracy than the use of a single fluorophore, both signal strength and ratios are still very sensitive to environmental changes (Constantinou and Polizzi, 2013). Great care must be taken to ensure changes in factors such as pH, salt, and temperature are not misinterpreted as changes in the metabolite of interest (Moussa et al, 2014).…”

Synthetic protein switches: design principles and applications

“…Based on these initial blueprints, more than 100 intramolecular FRET sensors have been developed to monitor a variety of molecular queues, ranging from protein-peptide, antibody-epitope, and protein-small molecule interactions to the detection of proteolysis, post-translational modifications, and mechanical properties, such as tension at cell-cell junctions [17][18][19]. Biophysical studies demonstrated that intramolecular FRET sensors with the largest signal change exploit the natural propensity of FPs to dimerize, which enables them to toggle between two defined molecular states in the presence and absence of a molecular queue.…”