A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

Metabolic Engineering Communications
Ramesh K JhaTaraka Dale

Abstract

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.

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Citations

Jun 11, 2020·Biomolecules·Egle ValancieneNaglis Malys
Sep 26, 2019·Frontiers in Bioengineering and Biotechnology·Siseon LeeBong Hyun Sung
Oct 23, 2019·Biotechnology for Biofuels·Guadalupe Alvarez-Gonzalez, Neil Dixon
Apr 5, 2020·Proceedings of the National Academy of Sciences of the United States of America·Davinia SalvachúaGregg T Beckham
Feb 27, 2021·Frontiers in Microbiology·Ilenne Del ValleLauren B Stadler
May 11, 2020·Current Opinion in Chemical Biology·Kirsten Davis, Tae Seok Moon
Jan 14, 2020·Metabolic Engineering·Gayle J BentleyGregg T Beckham
Mar 13, 2019·ACS Synthetic Biology·Ramesh K JhaTaraka Dale

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Methods Mentioned

BETA
biosensing
PCA
flow cytometry
biosensors
biosensor
PCR
restriction
fluorescence-activated cell sorting
FACS
flow

Software Mentioned

GENEWIZ
Rosetta

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