Elucidation of Sequence–Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli

Nancy M Kim,Riley W Sinnott,Nicholas R Sandoval,Lily N Rothschild

doi:10.3389/fbioe.2022.821152

Nancy M Kim, Riley W Sinnott + Show 2 more

Open Access

https://doi.org/10.3389/fbioe.2022.821152

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Abstract

Transcription factor (TF)–promoter pairs have been repurposed from native hosts 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. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ54-dependent, butanol-responsive TF–promoter pair, BmoR-PBMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized PBMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in PBMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ54-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.

Highlights

Cells can monitor and respond to a wide range of fluctuations in their environment and the ability to coordinate rapid and finely tuned responses
The DNA binding domain (DBD) of transcription factor (TF) interact with its respective promoters by recognizing specific operator sequences that often form hairpin structures affected by having inverted repeat sequences (Stanton et al, 2014; Rohlhill et al, 2017; Kim et al, 2020)
We engineered a butanol responsive transcription factor (TF)–promoter to increase its dynamic range through sequence–function elucidation of the promoter region that interacts with the TF with sort-seq

Summary

Introduction

Cells can monitor and respond to a wide range of fluctuations in their environment and the ability to coordinate rapid and finely tuned responses Due to this ability, these systems have been engineered to be used as information processing circuits in vivo for industrial applications. Ligand-responsive transcriptional regulators have been valuable tools in constructing complex metabolic pathways to increase metabolite concentrations (Kim et al, 2020). They can allow microorganisms to monitor levels of internal and/or external metabolites and adjust gene expression levels to balance pathway function (Zhang et al, 2012; Rohlhill et al, 2017; Tan and Prather, 2017). Coupling transcription factor (TF)–based biosensors with fluorescent gene reporters such as GFP screened via fluorescenceactivated cell sorting (FACS) has proven to be an efficient high-throughput strategy to accelerate the identification of high producers and fill in gaps of knowledge in metabolic pathways (Zeng et al, 2020).

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