Maltose Utilization as a Novel Selection Strategy for Continuous Evolution of Microbes with Enhanced Metabolite Production

We have developed a novel selection circuit based on carbon source utilization that establishes and sustains growth production coupling over several generations in a medium with maltose as the sole carbon source. In contrast to traditional antibiotic resistance-based circuits, we first proved that coupling of cell fitness to metabolite production by our circuit was more robust with a much lower escape risk even after many rounds of selection. We then applied the selection circuit to the optimization of L-tryptophan (L-Trp) production. We demonstrated that it enriched for specific mutants with increased L-Trp productivity regardless of whether it was applied to a small and defined mutational library or a relatively large and undefined one. From the latter, we identified four novel mutations with enhanced L-Trp output. Finally, we used it to select for several high L-Trp producers with randomly generated genome-wide mutations and obtained strains with up to 65% increased L-Trp production. This selection circuit provides new perspectives for the optimization of microbial cell factories for diverse metabolite production and the discovery of novel genotype phenotype associations at the single-gene and whole-genome levels.