Fabrication, Evolution, and Mutual Conversion of d-Fucose-Activatable and -Repressible Acetyltransferase upon...

The fusion of different proteins can result in the linkage-dependent emergence of molecular switches. We inserted chloramphenicol acetyltransferase (CAT) from Escherichia coli into a loop of a d-fucose-responsive mutant of transcription factor AraC, using linker libraries with various lengths. We found that on-switches tend to emerge when two proteins are fused with linkers long enough to fill the gap of the distance of residues to be connected, while fusing with short or zero linkers results in the frequent emergence of off-switches. Both types of switches rapidly evolved their switching efficiency upon mutations, establishing the d-fucose-on and -off regulations of CAT activity without disrupting the d-fucose-inducible logic of AraC function. To our surprise, both one-input/two-output split gates thus obtained could be easily interconverted upon mutations. Through mutations, fusion proteins rapidly establish and evolve mutual regulatory relationships with unrelated partner proteins, enabling diverse functional outcomes. Furthermore, random mutagenesis can alter the behavior of these emergent regulatory relationships, such as interconverting the activation or deactivation of the partner protein upon ligand binding, sometimes at a surprisingly high frequency.