A Brief Guide to the High-Throughput Expression of Directed Evolution Libraries

2017 ◽  
pp. 131-143
Author(s):  
Ana Luísa Ribeiro ◽  
Mario Mencía ◽  
Aurelio Hidalgo
Keyword(s):  
Directed Evolution ◽  
High Throughput

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

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 <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-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 H<sub>2</sub>O<sub>2</sub> as co-substrate.

Fluorescence Activated Cell Sorting as a General Ultra-High-Throughput Screening Method for Directed Evolution of Glycosyltransferases

2010 ◽  
Vol 132 (30) ◽  
pp. 10570-10577 ◽  
Author(s):  
Guangyu Yang ◽  
Jamie R. Rich ◽  
Michel Gilbert ◽  
Warren W. Wakarchuk ◽  
Yan Feng ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Fluorescence Activated Cell Sorting

scholarly journals CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics

The Analyst ◽  
2014 ◽  
Vol 139 (13) ◽  
pp. 3314-3323 ◽  
Author(s):  
Thomas Beneyton ◽  
Faith Coldren ◽  
Jean-Christophe Baret ◽  
Andrew D. Griffiths ◽  
Valérie Taly
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Recombinant Enzyme ◽  
Cell Analysis ◽  
E Coli ◽  
Cota Laccase

A high-throughput cell analysis and sorting platform using droplet-based microfluidics is introduced for directed evolution of recombinant CotA laccase expressed in E. coli.

scholarly journals A Photoclick‐Based High‐Throughput Screening for the Directed Evolution of Decarboxylase OleT

2020 ◽  
Author(s):  
Ulrich Markel ◽  
Pia Lanvers ◽  
Daniel F. Sauer ◽  
Malte Wittwer ◽  
Gaurao V. Dhoke ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase

2013 ◽  
Vol 52 (21) ◽  
pp. 5571-5574 ◽  
Author(s):  
Ryan Lauchli ◽  
Kersten S. Rabe ◽  
Karolina Z. Kalbarczyk ◽  
Amulya Tata ◽  
Thomas Heel ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Terpene Synthase

scholarly journals A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases

1999 ◽  
Vol 6 (10) ◽  
pp. 699-706 ◽  
Author(s):  
Hyun Joo ◽  
Akira Arisawa ◽  
Zhanglin Lin ◽  
Frances H Arnold
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Digital Imaging

Development of a simple high-throughput assay for directed evolution of enantioselective sulfoxide reductases

2020 ◽  
Vol 56 (40) ◽  
pp. 5386-5388 ◽  
Author(s):  
Vincenzo Tarallo ◽  
Kasireddy Sudarshan ◽  
Vladimír Nosek ◽  
Jiří Míšek
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Assay

We report on the development of high-throughput fluorogenic assay that can streamline directed evolution of enantioselective sulfoxide reductases.

scholarly journals High‐throughput droplet‐based microfluidics for directed evolution of enzymes

2019 ◽  
Vol 40 (21) ◽  
pp. 2860-2872 ◽  
Author(s):  
Flora W. Y. Chiu ◽  
Stavros Stavrakis
Keyword(s):  
Directed Evolution ◽  
High Throughput

scholarly journals A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging

2001 ◽  
Vol 6 (4) ◽  
pp. 219-223 ◽  
Author(s):  
John M. Joern ◽  
Takeshi Sakamoto ◽  
Akira Arisawa ◽  
Frances H. Arnold
Keyword(s):  
Image Analysis ◽  
Directed Evolution ◽  
High Throughput ◽  
Digital Imaging ◽  
Solid Phase ◽  
High Throughput Screen ◽  
Microplate Reader ◽  
Dihydrodiol Dehydrogenase ◽  
Dioxygenase Activity

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-di- hydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30,uM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

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