To create bacterial transcription “circuits” for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coliAraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. For an RhaR-AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation.

Generation and optimization of AraC/RhaRchimeric transcription factors for biotechnology

ACS Spring 2022

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