An important goal of synthetic biology is to create novel proteins that provide life-sustaining functions in living organisms. Recent attempts to produce novel proteins have focused largely on rational design involving significant computational efforts. In contrast, nature does not design sequences a priori. Instead, nature relies on Darwinian evolution to select biologically functional sequences from nondesigned sequence space. To mimic natural selection in the laboratory, we combed through libraries of novel sequences and selected proteins that rescue E. coli cells deleted for conditionally essential genes. One such gene, gltA, encodes citrate synthase, the enzyme responsible for metabolic entry into the citric acid cycle. The de novo protein SynGltA was isolated as a rescuer of ΔgltA. However, SynGltA is not an enzyme. Instead, SynGltA allows cells to recover from a defect in central carbon and energy metabolism by altering the regulation of an alternative metabolic pathway. Specifically, SynGltA dramatically enhances the expression of prpC, a gene encoding methylcitrate synthase in the propionate degradation pathway. This endogenous protein has promiscuous catalytic activity, which when overexpressed, compensates for the deletion of citrate synthase. While the molecular details responsible for this overexpression have not been elucidated, the results clearly demonstrate that non-natural proteins-unrelated to sequences in nature-can provide life-sustaining functions by altering gene regulation in natural organisms.