title: Engineering Modular Biosensors to Confer Metabolite-Responsive Regulation of Transcription.
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.
authors.affiliation: Interdisciplinary Biological Sciences Graduate Program, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Andrew K D
authors.initials: AK
authors.lastname: Younger
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Neil C
authors.initials: NC
authors.lastname: Dalvie
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
authors.forename: Austin G
authors.initials: AG
authors.lastname: Rottinghaus
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.|Center for Synthetic Biology, Northwestern University , Evanston, Illinois 60208, United States.|Chemistry of Life Processes Institute, Northwestern University , Evanston, Illinois 60208, United States.|Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Leonard
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D002352:Carrier Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D015723:Gene Library; D058619:Maltose-Binding Proteins / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D014157:Transcription Factors
keywords: maltose binding protein; metabolite biosensor; promoter engineering; protein engineering; zinc finger DNA binding domain