Elucidation of Sequence-Function Relationships for an Improved Biobutanol Biosensor in

Overview

Journal Front Bioeng Biotechnol

Date 2022 Mar 10

PMID 35265600

Authors

Nancy M Kim

Riley W Sinnott

Lily N Rothschild

Nicholas R Sandoval

Affiliations

Soon will be listed here.

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 σ-dependent, butanol-responsive TF-promoter pair, BmoR-P derived from , at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P library were sorted based on 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 P 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 σ-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.

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