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.

Biological signal processing filters via engineering allosteric transcription factors

“…To accomplish this, we leveraged the workflows that we developed in refs. 13 and 30 , which required an initial block in allosteric communication (i.e., via conferred superrepression S S YQR ) followed by one or more round(s) of error-prone PCR (EP-PCR). To identify putative superrepressor mutations, we performed a primary structure alignment between LacI and GalS ( SI Appendix , Fig.…”

“…Repressors are bound to DNA operators in the absence of the ligand, inhibiting transcription, and dissociate (are induced) when the ligand is present ( 21 , 23 ). The engineering of the transcriptional antirepressor (X A , where A indicates the antirepressor phenotype) has additionally resulted in increased computing capacity in synthetic biology ( 13 , 27 – 30 ). While repressors function as Boolean BUFFER gates ( 12 , 13 ), antirepressors function as Boolean NOT gates ( 13 , 25 , 30 ).…”

“…The engineering of the transcriptional antirepressor (X A , where A indicates the antirepressor phenotype) has additionally resulted in increased computing capacity in synthetic biology ( 13 , 27 – 30 ). While repressors function as Boolean BUFFER gates ( 12 , 13 ), antirepressors function as Boolean NOT gates ( 13 , 25 , 30 ). That is to say, antirepressor-DNA affinity is increased when the ligand is present (anti-induced), inhibiting transcription.…”

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes

“…A variety of natural proteins have been used in various combinations with engineered DNA operator sequences to create transcription “circuits” that respond in desired ways to varied effector ligands 1‐6 . In addition, the transcription factors have themselves been subjected to engineering via mutagenesis and/or domain recombination to achieve novel activities 7‐13 …”

Performance Prediction of Fundamental Transcriptional Programs

“…Accordingly, several groups have demonstrated that functional chimeras can be constructed based on the given engineering principles. 29,33−37 Wilson et al, in a collection of studies, posited and demonstrated that modular design could be applied to engineered domains�i.e., alternate (engineered) DNA binding functions, 29,35 alternate (engineered) allosteric communication, 30,35,37,38 and alternate (engineered) ligand binding functions 36,37 �to create a system of transcription factors (repressors and antirepressors) that are network capable. To date, using this collection of engineered repressors and antirepressors, transcriptional programs are designed and constructed intuitively�though with an apparent rule set (see Supporting Note 4).…”