An extension of directed evolution strategies to genome-wide variations increases the chance of obtaining metabolite-overproducing microbes. However, a general high-throughput screening platform for selecting improved strains remains out of reach. Here, 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. Using L-lysine-producing Escherichia coli as a model system, we demonstrated that this RNA device could enrich pathway-optimized strains to up to 75% of the total population after four rounds of enrichment cycles. Furthermore, the potential applicability of this device was examined by successfully extending its application to the case of L-tryptophan. When used in conjunction with combinatorial mutagenesis for metabolite overproduction, our synthetic RNA device should facilitate strain improvement.

Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase). Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux. Design of allosterically regulatable enzyme is essential to develop a highly efficient metabolite production. However, mutations on allosteric ligand binding sites often disrupt the catalytic activity of enzyme. To aid the design process of allosterically controllable enzymes, we develop an effective computational strategy to deregulate the allosteric inhibition of enzymes based on sequence evolution analysis of allosteric ligand-binding sites. We analyzed the molecular evolution and amino acid composition of catalytic and allosteric sites of enzymes, and discovered that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We then experimentally tested our strategy of enzyme allosteric regulation and found that the designed mutations effectively deregulated allosteric inhibition of FBPase. We believe that our method will aid the rational design of enzyme allosteric regulation and help to facilitate control of metabolic flux.