Design and Application of Transcription Factor Based Biosensors

Transcription-factor-based biosensors are an emerging technology to accelerate cell factory production and optimization. We describe several successful applications of those biosensors, e.g. for the optimization of an NADPH-dependent enzyme (Siedler et al. 2014a) and detection of flavonoid production in Escherichia coli (Siedler et al. 2014b). Furthermore, a lysine biosensor was combined with the recombineering technology in Corynebacterium glutamicum for the efficient identification of beneficial mutations at the genome level by detection of the lysine concentration in vivo (Binder et al. 2013). Direct transplantation of heterologous transcriptional regulators into new hosts for the generation of functional biosensors is often challenging and expression of the regulator needs to be fine-tuned. We analyzed the effect of different translational strengths, introduced by variation of the RBS, to the dynamic range of a biosensor. The best variant showed an up to 40-fold increased fluorescent signal upon induction. Another challenge of biosensor application is that intracellular concentration of small molecules is not always correlating with the extracellular concentration. We describe a novel screening method for identification of a producer strain in co-cultivation experiments of a producer strain and a biosensor containing strain in microfluidic droplets.

References

 

Binder, S; Siedler, S; Marienhagen, J; Bott, M; Eggeling, L (2013) Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. In: Nucleic Acids Res.41(12): 6360–6369.

Siedler, S; Schendzielorz, G; Binder, S; Eggeling, L; Bringer, S; Bott, M (2014a) SoxR as a Single-Cell Biosensor for NADPH-Consuming Enzymes in Escherichia coli. In: ACS Synth Biol (3), S. 41–47.

Siedler, S; Stahlhut, S. G.; Malla, S; Maury, J; Neves, A. R. (2014b) Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli. In: Metab. Eng. 21, S. 2–8.