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Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

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Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system.

Irisappan Ganesh¹, Sambandam Ravikumar, Seung Hwan Lee

¹Department of Chemical Engineering, University of Ulsan, 93 Daehakro, Nam-gu, Ulsan 680-749, Republic of Korea.

Journal of Biotechnology

|September 24, 2013

Summary

Engineered a chimeric two-component system (TCS) to create a sensitive screening method for fumarate-producing microorganisms. This novel system enhances signal detection for high-throughput screening of microbial chemical production.

Keywords:

Chimeric TCS DcuS/DcuR two-component system (TCS)DcuS/EnvZ (DcuSZ) TCS Escherichia coli Fumarate

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Area of Science:

Microbiology
Biotechnology
Metabolic Engineering

Background:

Two-component systems (TCS) regulate gene expression in response to environmental stimuli.
Developing high-throughput screening (HTS) systems is crucial for identifying microorganisms that overproduce valuable chemicals like fumarate.
The native DcuS/DcuR TCS showed insufficient signal strength for effective fumarate-dependent gene expression.

Purpose of the Study:

To engineer a robust TCS for a sensitive HTS system to identify high-fumarate-producing microorganisms.
To enhance the signal output of a fumarate-responsive TCS for quantitative detection.
To assess the specificity and response of the engineered TCS to various C4-dicarboxylates.

Main Methods:

Construction of a chimeric DcuS/EnvZ (DcuSZ) TCS by fusing sensor and catalytic domains.
Utilizing the ompC gene promoter to drive reporter gene (gfp) or ompC gene expression.
Employing principal component analysis (PCA) to evaluate the specificity of the DcuSZ system to C4-dicarboxylates.

Main Results:

The chimeric DcuSZ TCS successfully mediated reporter gene expression in response to fumarate.
Expression levels of gfp and ompC were quantitatively proportional to fumarate concentration.
PCA indicated high specificity for fumarate, with cross-reactivity to other C4-dicarboxylates.

Conclusions:

The engineered DcuSZ TCS provides a sensitive and quantitative platform for fumarate detection.
This TCS-based system holds promise for developing effective HTS methods for microbial chemical production.
Further optimization could enhance specificity and broaden applications in metabolic engineering.