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

摘要

img src="http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/asbcd6/2017/asbcd6.2017.6.issue-12/acssynbio.7b00247/20171215/images/medium/sb-2017-00247n_0006.gif">We have developed a novel selection circuit based on carbon source utilization that establishes and sustains growth鈥損roduction 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鈥損henotype associations at the single-gene and whole-genome levels.