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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

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Published on: August 8, 2016

Flow-cytometric cell sorting coupled with UV mutagenesis for improving pectin lyase expression.

Ke Fang^1,2, Jun Ma^1,2, Xinyu Wang^1,2

¹State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, China.

Frontiers in Bioengineering and Biotechnology

|September 18, 2023

Summary

Researchers developed a new method to create high-activity alkaline pectin lyase strains using UV mutagenesis and flow cytometry. This significantly boosts enzyme production for industrial applications.

Keywords:

Escherichia coli UV mutagenesis expression flow cytometry pectin lyase

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Area of Science:

Enzymology
Biotechnology
Molecular Biology

Background:

Alkaline pectin lyase is crucial for industries like food processing, textiles, and paper production.
Existing high-activity strains have limitations in industrial application due to insufficient enzymatic activity.
Developing cost-effective methods for higher enzyme yields is essential for industrial biotechnology.

Purpose of the Study:

To develop a rapid screening method for identifying alkaline pectin lyase variants with enhanced enzymatic activity.
To improve the industrial applicability of alkaline pectin lyase through modern breeding techniques.
To increase the cost-effectiveness of alkaline pectin lyase production.

Main Methods:

Genes for alkaline pectin lyase (PGLA-rep4) and enhanced green fluorescent protein (EGFP) were fused and cloned into a temperature-sensitive plasmid (pKD46).
The screening plasmid was transformed into an expression host and subjected to UV mutagenesis and flow-cytometric cell sorting.
Mutant strains were screened, and the highest-expressing strain (E. coli BL21/1G3) was selected for further analysis.

Main Results:

A high-expression strain, E. coli BL21/1G3, was successfully generated, exhibiting a 1.37-fold increase in pectin lyase activity compared to the parental strain (p < 0.001).
The highest observed pectin lyase activity reached 230-240 U/mL at 144 hours of fermentation.
Genome sequencing revealed mutations in genes encoding ribonuclease E (RNase E) and diadenosine tetraphosphatase (ApaH) in the mutant strain, potentially contributing to increased enzyme expression.

Conclusions:

The study presents an effective strategy for constructing E. coli strains with significantly enhanced pectin lyase expression levels.
The developed method combines genetic engineering with high-throughput screening for rapid strain improvement.
This approach holds promise for optimizing enzyme production in various industrial biotechnological processes.