The genetic manipulations required for constructing metabolic engineering strains often result in significant reduction in fitness, and poor production capabilities. Here, we present a strategy for improving growth-coupled bacterial strain designs through adaptive evolution, whole-genome resequencing, and characterization of individual adaptive mutations. We demonstrate the ability to identify genetic changes that lead to increased substrate uptake and/or production of desirable by-products through adaptation to specific environmental conditions. Constructing strains that carry specific combinations of adaptive mutations allows studying the physiological effects of mutations, and identification of epistatic effects. This information can be used to develop strategies for further iterative strain improvement through genetic manipulation. The specific mutant enzymes and regulators identified through this process will additionally form a valuable toolbox for future metabolic engineering applications.