Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor.
The pursuit of circuits and metabolic pathways of increasing complexity and robustness in synthetic biology will require engineering new regulatory tools. Feedback control based on relevant molecules, including toxic intermediates and environmental signals, would enable genetic circuits to react appropriately to changing conditions. In this work, variants of qacR, a tetR family repressor, were generated by computational protein design and screened in a cell-free transcription-translation (TX-TL) system for responsiveness to a new targeted effector. The modified repressors target vanillin, a growth-inhibiting small molecule found in lignocellulosic hydrolysates and other industrial processes. Promising candidates from the in vitro screen were further characterized in vitro and in vivo in a gene circuit. The screen yielded two qacR mutants that respond to vanillin both in vitro and in vivo. While the mutants exhibit some toxicity to cells, presumably due to off-target effects, they are prime starting points for directed evolution toward vanillin sensors with the specifications required for use in a dynamic control loop. We believe this process, a combination of the generation of variants coupled with in vitro screening, can serve as a framework for designing new sensors for other target compounds.
Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
Emmanuel L C
EL
de los Santos
Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
Joseph T
JT
Meyerowitz
Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
Stephen L
SL
Mayo
Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
Richard M
RM
Murray
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001547:Benzaldehydes / Q000378:metabolism*; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D015202:Protein Engineering; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D001547:Benzaldehydes; C442128:QacR protein, Staphylococcus aureus; D012097:Repressor Proteins; C100058:vanillin
TX-TL; cell-free systems; computational protein design; in vitro biological circuit prototyping; protein engineering; synthetic biology