Engineering Modular Biosensors to Confer Metabolite-Responsive Regulation of Transcription

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.