Expedite evolution of metabolite-producing microbe equipped with synthetic RNA device

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Jina Yang, Sang Woo Seo, Gyoo Yeol Jung

Pohang University of Science and Technology, South Korea

An extension of directed evolution strategies to genome-wide variations increases the chance of obtaining metabolite-overproducing microbes. However, there is no general high-throughput screening platform to isolate improved strain from tremendous library. Among the various screening methods available currently, selection by the physiology associated with the growth is known to be the most efficient since high producer can be easily enriched simply by a serial culture. However, metabolite overproduction is usually not beneficial features for microbe and consequently, tedious screening process should be used for isolating improved strain from generated library. In this study, to expedite the evolution of metabolite-producing microbes, we utilize synthetic RNA devices comprising a riboswitch and a selection module that specifically sense inconspicuous metabolites. L-lysine-producing Escherichia coli, as a model system, we demonstrate that this RNA device could enrich pathway-optimized strains to up to 75% of the total population diversifying phosphoenolpyruvate carboxylase expression level using randomized promoter sequences after four rounds of enrichment cycles from library. When used in conjunction with combinatorial mutagenesis for metabolite overproduction, our synthetic RNA device strategy should facilitate strain improvement.