Riboselector: riboswitch-based synthetic selection device to expedite evolution of metabolite-producing microorganisms.

Many successful metabolic engineering projects have utilized evolutionary approaches, which consist of generating phenotypic diversity and screening for desired phenotype. Since conventional screening methods suffer from low throughput and limited target metabolites, a universal high-throughput screening platform for selection of improved strains should be developed to facilitate evolution of metabolite high producer. Recently, riboswitches have received attention as attractive sensor-actuator hybrids that can control gene expression in response to intracellular metabolite concentration. Our group developed a riboswitch-based selection device called "Riboselector" which can give a growth advantage to metabolite-overproducing strains by modulating expression of a selectable marker gene. We applied the device to expedite evolution of lysine producing Escherichia coli, and the selected strain showed a dramatic improvement of lysine production compared to its parental strain. Moreover, a tryptophan-responsive Riboselector was also developed using synthetic tryptophan aptamer. In this chapter, we provide a step-by-step overview of developing synthetic RNA devices comprising a riboswitch and a selection module that specifically sense inconspicuous metabolites and enrich high producer strains out of library.