Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily

ABSTRACTAcinetobacter baumanniiis a major human pathogen associated with multidrug-resistant nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm formation, and disruption of biofilm formation is an attractive antivirulence strategy. Here, we report the first successful demonstration of biofilm disruption in a clinical isolate ofA. baumanniiS1, using a quorum-quenching lactonase obtained by directed evolution; this engineered lactonase significantly reduced the biomass ofA. baumannii-associated biofilms, demonstrating the utility of this antivirulence strategy.

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Directed Evolution of Quorum-Quenching Enzymes: A Method for the Construction of a Directed Evolution Platform and Characterization of a Quorum-Quenching Lactonase from Geobacillus kaustophilus

Methods in Molecular Biology - Quorum Sensing ◽

10.1007/978-1-4939-7309-5_24 ◽

2017 ◽

pp. 311-323

Author(s):

Maybelle Kho Go ◽

Jeng Yeong Chow ◽

Wen Shan Yew

Keyword(s):

Directed Evolution ◽

Quorum Quenching ◽

Geobacillus Kaustophilus

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Antisense RNA-based High-Throughput Screen System for Directed Evolution of Quorum Quenching Enzymes

ACS Chemical Biology ◽

10.1021/acschembio.5b00714 ◽

2015 ◽

Vol 10 (11) ◽

pp. 2598-2605 ◽

Cited By ~ 2

Author(s):

Sang-Soo Han ◽

Won-Ji Park ◽

Hak-Sung Kim ◽

Geun-Joong Kim

Keyword(s):

Directed Evolution ◽

High Throughput ◽

Antisense Rna ◽

Quorum Quenching ◽

High Throughput Screen ◽

Screen System

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Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

10.26434/chemrxiv.9791162 ◽

2019 ◽

Author(s):

Huifang Xu ◽

Weinan Liang ◽

Linlin Ning ◽

Yuanyuan Jiang ◽

Wenxia Yang ◽

...

Keyword(s):

Catalytic Activity ◽

Fatty Acid ◽

Directed Evolution ◽

High Throughput ◽

High Throughput Screening ◽

Colorimetric Detection ◽

Screening Method ◽

Random Mutagenesis ◽

Direct Production ◽

Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

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