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Developing a high-throughput screening method for threonine overproduction based on an artificial promoter.

Ya'nan Liu^1,2,3, Qinggang Li^4,5, Ping Zheng^6,7

¹College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300222, People's Republic of China. lyn8952@163.com.

Microbial Cell Factories

|August 23, 2015

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Summary

A novel biosensor enables high-throughput screening (HTS) for improved L-threonine production strains. This method identified superior mutants, enhancing industrial fermentation efficiency for this key animal feed amino acid.

Area of Science:

Biotechnology
Metabolic Engineering
Microbial Fermentation

Background:

L-Threonine is a crucial amino acid in animal feed production.
Current industrial threonine fermentation strains have potential for improvement.
High-throughput screening (HTS) is powerful but lacks suitable biosensors for many fine chemicals like threonine.

Purpose of the Study:

To develop a novel HTS method for identifying high-yielding threonine-producing microbial strains.
To establish a functional biosensor for efficient screening of threonine production.
To overcome the limitations of existing screening methods in microbial strain development.

Main Methods:

Discovered novel threonine-sensing promoters (cysJp, cysHp) using proteomic analysis of Escherichia coli.

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Constructed an HTS system utilizing fused cysJp and cysHp promoters linked to an enhanced green fluorescent protein reporter.

Employed fluorescence-activated cell sorting (FACS) to screen a library of 20 million mutants.

Main Results:

Successfully identified novel threonine-sensing promoters.
Developed and implemented a functional HTS system for threonine production.
Screened over 400 strains, with 34 exhibiting higher productivity than the original industrial strain.
Achieved a 17.95% increase in threonine production in one optimized mutant using a 5-L fermenter.

Conclusions:

The developed HTS method is crucial for the continuous improvement of the threonine industry.
The convenient construction of threonine sensors using proteomic-derived promoters can be extended to develop HTS systems for other valuable biochemicals.