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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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Altering the substrate specificity of RhlI by directed evolution.

Pavan Kumar Reddy Kambam¹, Dawn T Eriksen, Jason Lajoie

¹Department of Chemical Engineering, University of Massachusetts, Amherst, MA 01003, USA.

Chembiochem : a European Journal of Chemical Biology

|January 6, 2009

Summary

Researchers engineered the RhlI enzyme to enhance production of quorum sensing molecules N-butanoyl-homoserine lactone (BHL) and N-hexanoyl-homoserine lactone (HHL) in Pseudomonas aeruginosa, reducing bacterial virulence.

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Area of Science:

Microbiology
Biochemistry
Molecular Biology

Background:

Quorum sensing regulates virulence and biofilm formation in Pseudomonas aeruginosa.
The RhlI enzyme synthesizes signaling molecules N-butanoyl-homoserine lactone (BHL) and N-hexanoyl-homoserine lactone (HHL).

Purpose of the Study:

To engineer the RhlI enzyme for altered substrate specificity and enhanced signaling molecule production.
To gain insights into the molecular mechanisms of quorum sensing in P. aeruginosa.

Main Methods:

Directed evolution was employed to engineer the RhlI enzyme.
A genetic screen was used to identify mutants with improved signaling molecule production.

Main Results:

Engineered RhlI showed enhanced production of both BHL (over two-fold) and HHL (from undetectable to a level similar to BHL).
This indicates a significant shift in the enzyme's substrate specificity.
Mutations likely enhance interactions between the enzyme and acylated ACP substrates.

Conclusions:

Directed evolution and genetic screening are effective for engineering quorum sensing components.
The engineered RhlI enzyme offers a potential strategy for reducing P. aeruginosa virulence.