Transcription factor (TF)-promoter pairs have been repurposed from nature to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF-promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. Sort-seq provides a rational and less labor-intensive strategy to engineer user-defined TF-promoter pairs using FACS and deep sequencing methods. Based on the designed promoter library’s distribution characteristics, sort-seq elucidates sequence-function interactions between DNA-protein. There has been industrial interest in butanol as a renewable fuel. Biobutanol production from renewable feedstocks has been demonstrated, but the production yield remains economically unviable. High-throughput screens on non-growth-related phenotypes and dynamic butanol-dependent regulation represent powerful metabolic engineering strategies that are largely unavailable to these efforts. This capability gap is due to a lack of TF-promoter pairs with user-defined controls. A butanol-responsive TF, BmoR, and its cognate promoter, pBMO, have been previously described in the native form, but pBMO remains relatively uncharacterized and optimizing its function by sequence modification has not been attempted. In this work, we demonstrate the engineering of the pBMO promoter at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range, and to generate synthetic promoters. We use sort-seq to study the sequence-function relationship of pBMO. A mutagenized pBMO library cloned upstream of gfp in E. coli was induced with butanol and sorted into activity-based (i.e., fluorescence-based) populations. These populations were subsequently deep sequenced and their pBMO mutations correlated with changes in gfp expression, enabling construction of synthetic promoters with desirable characteristics. Identified pBMO mutations were constructed by site-directed mutagenesis and tested in E. coli. Binding characteristics of BmoR-pBMO mutants were confirmed by surface plasmon resonance. The best mutant promoter demonstrated over 4-fold increase in dynamic range over the wild-type. Sort-seq identified essential sites required for proper function of the biosensor and those that increase the sensor output. We also discuss progress toward implementing this biosensor in butanol producing gram-positive solventogenic bacteria.

Elucidation of sequence-function relationships for an improved biobutanol in vivo biosensor

ACS Fall 2021

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