Engineering Modular Biosensors to Confer Metabolite-Responsive Regulation of Transcription

• Published in: ACS synthetic biology 2017-02, Vol.6 (2), p.311-325
• Main Authors: Younger, Andrew K. D, Dalvie, Neil C, Rottinghaus, Austin G, Leonard, Joshua N
• Format: Article
• Language: English
• Subjects: Biosensing Techniques - methods; Carrier Proteins - genetics; DNA-Binding Proteins - genetics; Gene Expression Regulation - genetics; Gene Library; Maltose-Binding Proteins - genetics; Metabolic Engineering - methods; Promoter Regions, Genetic - genetics; Synthetic Biology - methods; Transcription Factors - genetics; Transcription, Genetic - genetics; Zinc Fingers - genetics
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.6b00184

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.