Engineering Modular Biosensors to Confer Metabolite-Responsive Regulation of Transcription

Andrew K.D. Younger, Neil C. Dalvie, Austin G. Rottinghaus, Joshua N. Leonard*

*Corresponding author for this work

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

Efforts to engineer microbial factories have benefitted from mining biological diversity and high throughput synthesis of novel enzymatic pathways, yet screening and optimizing metabolic pathways remain rate-limiting steps. Metabolite-responsive biosensors may help to address these persistent challenges by enabling the monitoring of metabolite levels in individual cells and metabolite-responsive feedback control. We are currently limited to naturally evolved biosensors, which are insufficient for monitoring many metabolites of interest. Thus, a method for engineering novel biosensors would be powerful, yet we lack a generalizable approach that enables the construction of a wide range of biosensors. As a step toward this goal, we here explore several strategies for converting a metabolite-binding protein into a metabolite-responsive transcriptional regulator. By pairing a modular protein design approach with a library of synthetic promoters and applying robust statistical analyses, we identified strategies for engineering biosensor-regulated bacterial promoters and for achieving design-driven improvements of biosensor performance. We demonstrated the feasibility of this strategy by fusing a programmable DNA binding motif (zinc finger module) with a model ligand binding protein (maltose binding protein), to generate a novel biosensor conferring maltose-regulated gene expression. This systematic investigation provides insights that may guide the development of additional novel biosensors for diverse synthetic biology applications.

Original languageEnglish (US)
Pages (from-to)311-325
Number of pages15
JournalACS synthetic biology
Volume6
Issue number2
DOIs
StatePublished - Feb 17 2017

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Keywords

  • maltose binding protein
  • metabolite biosensor
  • promoter engineering
  • protein engineering
  • zinc finger DNA binding domain

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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