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On-Chip Analysis, Indexing and Screening for Chemical Producing Bacteria in Microfluidic Static Droplet Array


Combinatorial engineering is known as one of the most powerful ways to create microbes for economic production of chemicals. The importance of efficient screening has been emphasized to investigate vast phenotypic diversity provided by advanced technologies. However, current methods place constraints on monitoring of intracellular concentration of diverse target products at the level of single cell. Here, we established a high-throughput screening platform for assessment of metabolites based on microfluidic static droplet array (SDA) and artificial riboswitch. In this system, a genetically encoded riboswitch visualizes intracellular concentration of a target molecule in a quantitative manner. Then, single cells from a library are individually entrapped in SDA to detect and collect highly productive variants. Using this approach, we analyzed intracellular L-tryptophan concentrations of single Escherichia coli cells. Fluorescence from an artificial L-tryptophan riboswitch enabled easy measurement of the metabolite at single cell resolution. Then, a mutant E. coli library was screened for improved L-tryptophan production and superior strains were obtained exhibiting up to 145% productivity in comparison with their parental strain. It is concluded that the combination of SDA and artificial riboswitch was successfully applied to high-throughput screening of microbial cell library for combinatorial metabolic engineering. This platform will be widely applied to a variety of screening projects by developing new artificial riboswitches for new target molecules.