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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 55-63 |
| Number of pages | 9 |
| Journal | Protein Engineering, Design and Selection |
| Volume | 31 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 1 2018 |
Funding
This work was supported by the National Science Foundation (MCB-1341414 to J.N.L., DGE-1 324 585 to P.Y.S.); the Environmental Protection Agency (STAR Fellowship F13A30124 to A.K.D.Y.); National Institutes of Health (TRC is supported by 1R01MH103910-01, K.T supported by 5R01MH103910-02); and Northwestern University (Undergraduate Research Grant to A.J.S.). A.K.D.Y. was supported in part by the Northwestern University Graduate School Cluster in Biotechnology, Systems, and Synthetic Biology, which is affiliated with the Biotechnology Training Program. Flow cytometry experiments were conducted at the Robert H. Lurie Flow Cytometry Core Facility. Traditional sequencing services were performed at the Northwestern University Genomics Core Facility. This work was supported by the National Science Foundation (MCB-1341414 to J.N.L., DGE-1 324 585 to P.Y.S.); the Environmental Protection Agency (STAR Fellowship F13A30124 to A.K.D.Y.); National Institutes of Health (TRC is supported by 1R01MH103910-01.
Keywords
- Biosensor
- domain insertion
- metabolic engineering
- synthetic biology
- transcription factor
- transposon
ASJC Scopus subject areas
- General Medicine