Flow Cytometer-Based High-Throughput Screening System for Accelerated Directed Evolution of P450 Monooxygenases

ACS Catalysis ◽  
2012 ◽  
Vol 2 (12) ◽  
pp. 2724-2728 ◽  
Author(s):  
Anna Joëlle Ruff ◽  
Alexander Dennig ◽  
Georgette Wirtz ◽  
Milan Blanusa ◽  
Ulrich Schwaneberg
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Flow Cytometer ◽  
Screening System ◽  
P450 Monooxygenases

Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase

2019 ◽  
Vol 127 (1) ◽  
pp. 30-37 ◽  
Author(s):  
Gordana Kovačević ◽  
Raluca Ostafe ◽  
Ana Marija Balaž ◽  
Rainer Fischer ◽  
Radivoje Prodanović
Keyword(s):  
Glucose Oxidase ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening System

A colourimetric high-throughput screening system for directed evolution of prodigiosin ligase PigC

2020 ◽  
Vol 56 (61) ◽  
pp. 8631-8634 ◽  
Author(s):  
Stefanie Brands ◽  
Hannah U. C. Brass ◽  
Andreas S. Klein ◽  
Jörg Pietruszka ◽  
Anna Joëlle Ruff ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening System

A colourimetric high-throughput screening system was developed for the first directed evolution campaign on PigC towards production of artificial prodiginines.

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.

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

scholarly journals Creation of Novel Protein Transduction Domain (PTD) Mutants by a Phage Display-Based High-Throughput Screening System

2006 ◽  
Vol 29 (8) ◽  
pp. 1570-1574 ◽  
Author(s):  
Yohei Mukai ◽  
Toshiki Sugita ◽  
Tomoko Yamato ◽  
Natsue Yamanada ◽  
Hiroko Shibata ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Transduction ◽  
Protein Transduction Domain ◽  
Screening System ◽  
Novel Protein

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
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