An insoluble de novo protein enables survival of Escherichia coli by sequestering a gene repressor.

De novo proteins that share no ancestry with natural sequences can serve as additions to the evolved proteomes of living cells. Upon expression in cells, these novel proteins can provide biological functions that alter cell viability and growth. To isolate such proteins, we searched a combinatorial library of novel sequences by selecting for sequences that sustain the growth of Escherichia coli under conditions where the recipient cell would otherwise be inviable. This led to the identification of Resc4 (Rescuer 4), a de novo protein that sustains growth on minimal medium of an E. coli strain harboring a lethal deletion of metC, which encodes cystathionine [Formula: see text]-lyase, a conditionally essential enzyme in the biosynthesis of methionine. Surprisingly, despite its ability to rescue the deletion of a biosynthetic enzyme, Resc4 is insoluble. Nonetheless, Resc4 sustains the growth of [Formula: see text]metC cells by upregulating expression of metB, which encodes a different enzyme, cystathionine [Formula: see text]-synthase, which has a moonlighting activity that compensates for the deleted activity encoded by metC. Proteomic analysis revealed that Resc4 sequesters MetJ, the repressor of the methionine biosynthesis operon. Sequestration of MetJ leads to overproduction of cystathionine [Formula: see text]-synthase, thereby allowing it to rescue the deletion of metC. These results, taken together with previous findings on other de novo proteins, demonstrate that novel proteins added to a cell's proteome can perform life-sustaining functions, and may shed light on de novo gene birth-both in synthetic biology and in natural evolution.