Directed Evolution of the Stringency of the LuxR Vibrio fischeri Quorum Sensor without OFF-State Selection

ABSTRACT LuxR is the 3-oxohexanoyl-homoserine lactone (3OC6HSL)-dependent transcriptional activator of the prototypical acyl-homoserine lactone (AHL) quorum-sensing system of Vibrio fischeri. Wild-type LuxR exhibits no response to butanoyl-HSL (C4HSL) in quantitative bioassays at concentrations of up to 1 μM; a previously described LuxR variant (LuxR-G2E) exhibits a broadened response to diverse AHLs, including pentanoyl-HSL (C5HSL), but not to C4HSL. Here, two rounds of directed evolution of LuxR-G2E generated variants of LuxR that responded to C4HSL at concentrations as low as 10 nM. One variant, LuxR-G4E, had only one change, I45F, relative to the parent LuxR-G2E, which itself differs from the wild type at three residues. Dissection of the four mutations within LuxR-G4E demonstrated that at least three of these changes were simultaneously required to achieve any measurable C4HSL response. The four changes improved both sensitivity and specificity towards C4HSL relative to any of the other 14 possible combinations of those residues. These data confirm that LuxR is evolutionarily pliable and suggest that LuxR is not intrinsically asymmetric in its response to quorum-sensing signals with different acyl-side-chain lengths.

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Robust and flexible platform for directed evolution of yeast genetic switches

Nature Communications ◽

10.1038/s41467-021-22134-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Masahiro Tominaga ◽

Kenta Nozaki ◽

Daisuke Umeno ◽

Jun Ishii ◽

Akihiko Kondo

Keyword(s):

Synthetic Biology ◽

Directed Evolution ◽

Rational Design ◽

Signal To Noise Ratio ◽

Model Organism ◽

Carotenoid Biosynthesis ◽

Genetic Switches ◽

State Selection ◽

Yeast Genetic ◽

Selection Of

AbstractA wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system’s utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.

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Oxygen Triggering Reversible Modulation of Vibrio fischeri Strain Y1 Bioluminescence In Vivo¶

Photochemistry and Photobiology ◽

10.1562/0031-8655(2004)79<120:otrmov>2.0.co;2 ◽

2004 ◽

Vol 79 (1) ◽

pp. 120 ◽

Cited By ~ 4

Author(s):

Hajime Karatani ◽

Susumu Yoshizawa ◽

Satoshi Hirayama

Keyword(s):

Vibrio Fischeri

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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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