Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors

Abstract

Recent advances in synthetic biology and protein engineering have increased the number of allosteric transcription factors used to regulate independent promoters. These developments represent an important increase in our biological computing capacity, which will enable us to construct more sophisticated genetic programs for a broad range of biological technologies. However, the majority of these transcription factors are represented by the repressor phenotype (BUFFER), and require layered inversion to confer the antithetical logical function (NOT), requiring additional biological resources. Moreover, these engineered transcription factors typically utilize native ligand binding functions paired with alternate DNA binding functions. In this study, we have advanced the state-of-the-art by engineering and redesigning the PurR topology (a native antirepressor) to be responsive to caffeine, while mitigating responsiveness to the native ligand hypoxanthine—i.e., a deamination product of the input molecule adenine. Importantly, the resulting caffeine responsive transcription factors are not antagonized by the native ligand hypoxanthine. In addition, we conferred alternate DNA binding to the caffeine antirepressors, and to the PurR scaffold, creating 38 new transcription factors that are congruent with our current transcriptional programming structure. Finally, we leveraged this system of transcription factors to create integrated NOR logic and related feedback operations. This study represents the first example of a system of transcription factors (antirepressors) in which both the ligand binding site and the DNA binding functions were successfully engineered in tandem.