Rapid depletion and environmental impact of fossil fuels have pushed a mandate to seek clean alternative energy sources. While ethanol is a renewable biofuel, butanol is superior due to its higher energy density, lower volatility and hygroscopicity, and fungibility to the current infrastructure. However, low production yield and high substrate costs limit butanol’s use. Furthermore, strain improvement to identify butanol hyperproducers and other biochemicals are hindered due to low-throughput screening methods. Transcription factor–based biosensors coupled with flow cytometry enable rapid screening of large genetic libraries (&gt;109 mutants) to identify desired microbial phenotypes. Therefore, a biosensor must be able to differentiate strains exhibiting different biochemical production yields to identify hyperproducing strains. A biosensor comprising of butanol-responsive transcription factor, BmoR, and its cognate promoter, pBMO, (isolated from Thauera butanivorans) was demonstrated in Escherichia coli. Here, we investigate correlation between the BmoR-pBMO operator affinity and biosensor properties such as basal activity, dynamic range, sensitivity, and linearity. GFP expression controlled by sixteen promoters containing mutant pBMO operators were designed and evaluated via flow cytometry against a range of butanol concentrations in Escherichia coli. Biophysical characteristics between the BmoR-pBMO operator were evaluated using surface plasmon resonance (SPR). This strategy was further demonstrated in which BmoR was converted into a transcriptional repressor by relocating mutant pBMO operators near or overlapping the RNA polymerase binding site. This work can potentially enable rational strategies to edit the dynamic range of transcription factor-based biosensors.

Tuning the dynamic range of 1-butanol-responsive transcription factor-based biosensor in Escherichia coli

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.