The engineering of microbial strains for the production of small molecules of biotechnological interest is a time-consuming, laborious and expensive process. In this context, transcription factor-based biosensors represent powerful tools for the rapid screening of large and genetically diverse strain libraries at the single cell level by translating the intracellular concentration into a fluorescence signal.
In this study, such a biosensor for the detection of intracellular chorismate concentrations in Corynebacterium glutamicum was constructed on the basis of the cell-own LysR-type regulator QsuR and its cognate target promoter. Subsequently, the biosensor was characterized, and combined with fluorescence-activated cell-sorting (FACS) with the aim to improve the carbon flux into the shikimate pathway.
The dynamic and operational range of the chorismate biosensor was determined by supplementation experiments using the chorismate precursor quinate, since C. glutamicum cannot take up chorismate. An increased fluorescence signal was detected for quinate concentrations ranging from 250 µM to 64 mM, showing a 150-fold increase in specific fluorescence at a quinate concentration of 64 mM. The ligand spectrum was determined through the addition of molecules with a structural similarity to chorismate as well as other, chemically more different intermediates of the shikimate pathway. A fluorescence signal was only detectable in response to the supplementation of quinate, which is intracellularly converted to chorismate, indicating a high specificity of the QsuR-based sensor.
For the isolation of C. glutamicum strains with increased carbon flux into the shikimate pathway, the biosensor was used to screen genetically diverse strain variants generated through random chemical mutagenesis of the whole genome or PCR-based targeted mutagenesis of specific genes. Here, FACS screening enabled the isolation of strain variants with increased chorismate accumulation based on their specific fluorescence signal.