Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion

Andrew K.D. Younger, Peter Y. Su, Andrea J. Shepard, Shreya V. Udani, Thaddeus R. Cybulski, Keith Edward Jaggard Tyo, Joshua N Leonard*

*Corresponding author for this work

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)55-63
Number of pages9
JournalProtein Engineering, Design and Selection
Volume31
Issue number2
DOIs
StatePublished - Feb 1 2018

Fingerprint

Transcription factors
Biosensing Techniques
Metabolites
Biosensors
Transcription Factors
Fusion reactions
Proteins
Carrier Proteins
Libraries
Metabolic engineering
Maltose
Metabolic Engineering
DNA
Synthetic Biology
Maltose-Binding Proteins
Biosynthetic Pathways
Zinc Fingers
Genetic Testing
Sorting
Zinc

Keywords

  • Biosensor
  • domain insertion
  • metabolic engineering
  • synthetic biology
  • transcription factor
  • transposon

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

Cite this

Younger, Andrew K.D. ; Su, Peter Y. ; Shepard, Andrea J. ; Udani, Shreya V. ; Cybulski, Thaddeus R. ; Tyo, Keith Edward Jaggard ; Leonard, Joshua N. / Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion. In: Protein Engineering, Design and Selection. 2018 ; Vol. 31, No. 2. pp. 55-63.
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Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion. / Younger, Andrew K.D.; Su, Peter Y.; Shepard, Andrea J.; Udani, Shreya V.; Cybulski, Thaddeus R.; Tyo, Keith Edward Jaggard; Leonard, Joshua N.

In: Protein Engineering, Design and Selection, Vol. 31, No. 2, 01.02.2018, p. 55-63.

Research output: Contribution to journalArticle

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