Pyruvate Carboxylase Variants Enabling Improved Lysine Production from Glucose Identified by Biosensor-Based High-Throughput Fluorescence-Activated Cell Sorting Screening

2019 ◽  
Vol 8 (2) ◽  
pp. 274-281 ◽  
Author(s):  
Maike Kortmann ◽  
Christina Mack ◽  
Meike Baumgart ◽  
Michael Bott
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
Pyruvate Carboxylase ◽  
Fluorescence Activated Cell Sorting ◽  
Lysine Production

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.

An integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting system (μ-CFACS) for the enrichment of rare cells

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Kunpeng Cai ◽  
Shruti Mankar ◽  
Taiga Ajiri ◽  
Kentaro Shirai ◽  
Tasuku Yotoriyama
Keyword(s):  
Personalized Medicine ◽  
Regenerative Medicine ◽  
Precision Medicine ◽  
High Throughput ◽  
Cell Sorting ◽  
Next Generation ◽  
Fluorescence Activated Cell Sorting ◽  
Clinical Environments ◽  
Rare Cells ◽  
Sorting System

There is an increasing need for the enrichment of rare cells in the clinical environments of precision medicine, personalized medicine, and regenerative medicine. With the possibility of becoming the next-generation...

scholarly journals High-throughput screening of high lactic acid-producing Bacillus coagulans by droplet microfluidic based flow cytometry with fluorescence activated cell sorting

RSC Advances ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 4507-4513 ◽  
Author(s):  
Xu-Dong Zhu ◽  
Xiang Shi ◽  
Shu-Wen Wang ◽  
Ju Chu ◽  
Wei-Hong Zhu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lactic Acid ◽  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Bacillus Coagulans ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Microfluidic Sorting

A high-throughput screening system based on droplet microfluidic sorting was developed and employed for screening of high lactic acid-producing Bacillus coagulans.

scholarly journals Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting

The Analyst ◽  
2018 ◽  
Vol 143 (19) ◽  
pp. 4747-4755 ◽  
Author(s):  
Joanna C. Sadler ◽  
Andrew Currin ◽  
Douglas B. Kell
Keyword(s):  
Monoamine Oxidase ◽  
Directed Evolution ◽  
High Throughput ◽  
Cell Sorting ◽  
Oxidase Activity ◽  
Functional Enrichment ◽  
High Throughput Screen ◽  
Fluorescence Activated Cell Sorting ◽  
E Coli

A novel ultra-high throughput screen forin vivodetection of oxidase activity inE. colicells and its application to directed evolution.

scholarly journals High-throughput fluorescence-activated cell sorting for lipid hyperaccumulatingChlamydomonas reinhardtiimutants

2014 ◽  
Vol 12 (7) ◽  
pp. 872-882 ◽  
Author(s):  
Bo Xie ◽  
Dan Stessman ◽  
Jason H. Hart ◽  
Haili Dong ◽  
Yingjun Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
Fluorescence Activated Cell Sorting
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