(734e) Development of a Modular, Small Molecule Responsive, Transcriptional Regulator Platform

Our ability to engineer microbial factories has accelerated as new technologies have enabled rapid mining of biological diversity and high throughput synthesis of novel ensembles of enzymatic pathways. To date, however, it remains challenging to monitor the presence of metabolites in individual cells, outside of the situations in which a natural metabolite-responsive transcriptional regulator has evolved and been identified. Such naturally occurring biosensors have proven useful in applications including both screening of high-dimensional libraries and engineering feedback control to optimize biosynthesis. To help address this need for novel biosensors, we have developed a modular and general approach for engineering novel transcriptional regulators that are actuated by the presence or absence of metabolites of interest – the Small molecule Responsive Transcriptional Regulator (SmaRTR) biosensor platform. The central goal of this approach is to develop a systematic strategy for converting proteins that bind small molecules into proteins that bind a target DNA sequence in a manner that depends on whether those small molecules are present. In our initial evaluation of this strategy, we utilized the BCR-ABL1 zinc finger protein as a model DNA-binding domain. We built and characterized a library of promoters including BCR-ABL1 binding sites at various positions to elucidate which promoter design features are most important for achieving effective zinc finger mediated-transcriptional repression. Then, we evaluated several strategies for converting the well-characterized maltose binding protein (MBP), a model of a ligand-binding protein, into a novel maltose-actuated transcription factor that regulates gene expression in a ligand-dependent manner. Our investigations using this model system resulted in several robust and functional novel biosensors, establishing the fundamental feasibility of the SmaRTR approach. We also elucidated protein and promoter engineering strategies that may ultimately be broadly applicable to developing a range of engineered biosensors. Ultimately, this technology may be harnessed to generate quantitative insights into metabolite dynamics in living cells, provide new tools for high throughput screening, and enable improved biomanufacturing through implementation of metabolic feedback control.