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Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in spp. | LitMetric

Versatile Strategy for the Construction of a Transcription Factor-Based Orthogonal Gene Expression Toolbox in spp.

ACS Synth Biol

Hubei International Scientific and Technological Cooperation Base of Traditional Fermented Foods, Huazhong Agricultural University, Wuhan, Hubei Province430070, China.

Published: January 2023

AI Article Synopsis

Article Abstract

Gene expression is needed to be conducted in an orthogonal manner and controllable independently from the host's native regulatory system. However, there is a shortage of gene expression regulatory toolboxes that function orthogonally from each other and toward the host. Herein, we developed a strategy based on the mutant library to generate orthogonal gene expression toolboxes. A transcription factor, MaR, located in the azaphilone biosynthetic gene cluster, was taken as a typical example. Nine DNA-binding residues of MaR were identified by molecular simulation and site-directed mutagenesis. We created five MaR multi-site saturation mutagenesis libraries consisting of 10743 MaR variants on the basis of five cognate promoters. A functional analysis revealed that all five tested promoters were orthogonally regulated by five different MaR variants, respectively. Furthermore, fine gene expression tunability and high signal sensitivity of this toolbox are demonstrated by introducing chemically inducible expression modules, designing synthetic promoter elements, and creating protein-protein interaction between MaRs. This study paves the way for a bottom-up approach to build orthogonal gene expression toolboxes.

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Source
http://dx.doi.org/10.1021/acssynbio.2c00500DOI Listing

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