The aim of the Seedfund project XyloSens was to support the HyImPAct project with FRET-based biosensors for continuous non-invasive measurement of C-source concentration (D-glucose, D-xylose) in design and process development of new microbial strains.
The starting point of the studies was a previously published glucose biosensor consisting of a glucose-binding protein flanked by two fluorescent proteins forming a FRET pair that responds to conformational changes of the binding protein upon glucose binding with an altered FRET efficiency. At the beginning of the project, the existing glucose sensor was further optimized. The project partner RWTH focused on its use in a microbioreactor to enable high-throughput characterization of production strains. First, optimal excitation and emission wavelengths were identified at which the strongest and most stable FRET signal can be recorded. Furthermore, the sensor formulation, stability and concentration were optimized. As a result, glucose consumption of an E. coli culture in a BioLector could be successfully and reproducibly tracked.
The analogous design of a xylose sensor appeared almost trivial due to the high similarity of the two sugar-binding proteins, according to the literature. However, despite intensive efforts, it was not yet possible to arrange the FRET partners in such a way that the binding of xylose is detectable based on a change in FRET intensity. Neither the insertion of linkers nor the stepwise shortening of the N- and C-termini of the xylose-binding protein have so far led to the desired effect. However, it is undisputed that our xylose sensor variants bind xylose. This was clearly demonstrated by project partner HHU using saturation transfer difference NMR (STD-NMR). Since STD-NMR also showed a weak affinity of the glucose sensor for xylose, directed mutagenesis was used to increase the xylose binding affinity, eventually leading to a very weak FRET effect towards xylose, while glucose was no longer recognized. NMR studies showed that the affinity for xylose and glucose could be successfully affected independently of the FRET event.
Prof. Martina Pohl
Forschungszentrum Jülich GmbH (FZJ)
phone: +49 2461 614388
Prof. Dr. Wolfgang Wichert & Prof. Dr. Martina Pohl, IBG-1, Forschungszentrum Jülich GmbH
Prof. Dr. Dieter Willbold & Dr. Bernd König, Biophysics of Protein Interactions, Heinrich Heine University Düsseldorf
Prof. Dr. Jochen Büchs, Biochemical Engineering, RWTH Aachen University
01.10.2018 - 31.03.2021
The total budget of XyloSenS is 339,542 €. XyloSenS is is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia.