Abstract
BackgroundIn 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.ResultsThe 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.ConclusionA 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.
Highlights
In most microbial cultivations d-glucose is the main carbon and energy source
Even though enzymatic assays are routinely used to quantify d-glucose in samples from microbial cultivations [2, 3], application in small scale is still limited to at-line processes
Protein design The biosensor without HaloTag® (Glu[−]) used in this study is based on sensor no. 2 in a recent publication [20] with a modification of the hexahistidine tag (His-tag)
Summary
In most microbial cultivations d-glucose is the main carbon and energy source. quantification of d-glucose especially in small scale is still challenging. A broad variety of chemical compounds is already produced via microbial cultivation, the development of new processes and strains for the production of, e.g., non-natural high value products using synthetic biology approaches and microbial communities is gathering momentum. In this respect, bioprocess development at small scales is becoming ever more important. Even though enzymatic assays are routinely used to quantify d-glucose in samples from microbial cultivations [2, 3], application in small scale is still limited to at-line processes. In general FRET-based biosensors consist of two fluorescent probes (donor and acceptor) fused to a central metabolite binding protein (BP). Extracellular applications of such sensors in the fermentation broths of producer cells enables quantitative detection of the target metabolite due to an easier calibration of such systems, as we have recently demonstrated for l-lysine [9]
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