1. Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.
- Author
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Younger, Andrew K. D., Su, Peter Y., Shepard, Andrea J., Udani, Shreya V., Cybulski, Thaddeus R., Tyo, Keith E. J., and Leonard, Joshua N.
- Subjects
CARRIER proteins ,TRANSCRIPTION factors ,TRANSPOSONS ,BIOSENSORS ,METABOLITES - Abstract
Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors–Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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