Naringenin-Responsive Riboswitch-Based Fluorescent Biosensor Module for Escherichia coli Co-Cultures

Authors: 
Xiu, Y., Rensselaer Polytechnic Institute
Jang, S., POSTECH
Jones, J. A., Rensselaer Polytechnic Institute
Linhardt, R. J., Rensselaer Polytechnic Institute
Yuan, Q., Beijing University of Chemical Technology
Koffas, M. A. G., Rensselaer Polytechnic Institute
Jung, G. Y., POSTECH
Zill, N. A., Rensselaer Polytechnic Institute
The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of the synthetic biology and metabolic engineering fields. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate its ability to screen for high-titer naringenin-producing strains in an Escherichia coli co-culture system. Our data helped identify a number of important parameters that affect the biosensor performance of the co-culture, including the type of the promoter used at the 5’-untranslated region of the sensor-actuator domain and the linker sequence within the sensor-actuator domain. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain containing the riboswitch-actuating device and naringenin titer produced by the second member of the synthetic co-culture system, while only a small proportion (4% v/v) of the biosensor strain was required at inoculation. The designed co-culture system can be utilized in high-throughput, through the presented FACS-based screening technique, for in vivo naringenin detection and regulation of flavonoid biosynthesis. Application of the developed biosensor in a synthetic microbial consortium reduces the need for re-optimization of the producer module when the biosensor is subsequently removed. This is due to the compartmentalization of the two genetic constructs in separate cells and communication between the strains being controlled through the efficiently transported target analyte, naringenin. This technique represents a novel application for synthetic co-culture-based systems and can be widely applied to a variety of target metabolites using the SELEX approach for aptamer design.