Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector^® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

In most microbial cultivations d-glucose is the main carbon and energy source. However, quantification of d-glucose especially in small scale is still challenging. Therefore, we developed a FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwiched between two fluorescent proteins, constituting a FRET pair. Upon d-glucose binding the sensor undergoes a conformational change which is translated into a FRET-ratio change. The selected sensor shows an apparent Kd below 1.5 mM d-glucose and a very high sensitivity of up to 70% FRET-ratio change between the unbound and the glucose-saturated state. The soluble sensor was successfully applied online to monitor the glucose concentration in an Escherichia coli culture. Additionally, this sensor was utilized in an at-line process for a Corynebacterium glutamicum culture as an example for a process with cell-specific background (e.g. autofluorescence) and medium-induced quenching. Immobilization of the sensor via HaloTag® enabled purification and covalent immobilization in one step and increased the stability during application, significantly. A FRET-based glucose sensor was used to quantify d-glucose consumption in microtiter plate based cultivations. To the best of our knowledge, this is the first method reported for online quantification of d-glucose in microtiter plate based cultivations. In comparison to d-glucose analysis via an enzymatic assay and HPLC, the sensor performed equally well, but enabled much faster measurements, which allowed to speed up microbial strain development significantly.

Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein.