High-Throughput Screening or Selection Methods for Evolutionary Enzyme Engineering

2016 ◽  
pp. 707-744
Author(s):  
Shuobo Shi ◽  
Hongfang Zhang ◽  
Ee Ang ◽  
Huimin Zhao
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Engineering ◽  
Selection Methods

scholarly journals Synchronized Reagent Delivery in Double Emulsions for Triggering Chemical Reactions and Gene Expression

2021 ◽  
Author(s):  
Ariane Stucki ◽  
Petra Jusková ◽  
Nicola Nuti ◽  
Steven Schmitt ◽  
Petra Dittrich
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Double Emulsion ◽  
Lipid Vesicles ◽  
Enzyme Engineering ◽  
Droplet Microfluidics ◽  
Enzymatic Reactions ◽  
High Throughput Analysis ◽  
Double Emulsions

Microfluidic methods to form single emulsion and double emulsion (DE) droplets have greatly enhanced the toolbox for high throughput screening for cell or enzyme engineering and drug discovery. However, remaining challenges in the supply of reagents into these enclosed nanoliter compartments limit the applicability of droplet microfluidics. Here, we introduce a strategy for on-demand delivery of reactants in DEs. We use lipid vesicles as transport carriers, which are co-encapsulated in double emulsions and release their cargo upon addition of an external trigger, here the anionic surfactant SDS. The reagent present inside the lipid vesicles stays isolated from the remaining content of the DE vessel until SDS enters the DE lumen and solubilizes the lipid bilayer. We demonstrate the versatility of the method with two critical applications, chosen as representative assays for high throughput screening. First, we trigger enzymatic reactions after releasing a reactant and second, we encapsulate bacteria and induce gene expression at a delayed time. The presented technique is compatible with the high throughput analysis of individual DE droplets using conventional flow cytometry as well as with microfluidic time-resolved studies. The possibility of delaying and controlling reagent delivery in current high throughput compartmentalization systems will significantly extend their range of applications e.g. for directed evolution, and further improve their compatibility with biological systems.

scholarly journals Synchronized Reagent Delivery in Double Emulsions for Triggering Chemical Reactions and Gene Expression

2021 ◽  
Author(s):  
Ariane Stucki ◽  
Petra Jusková ◽  
Nicola Nuti ◽  
Steven Schmitt ◽  
Petra Dittrich
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Double Emulsion ◽  
Lipid Vesicles ◽  
Enzyme Engineering ◽  
Droplet Microfluidics ◽  
Enzymatic Reactions ◽  
High Throughput Analysis ◽  
Double Emulsions

Microfluidic methods to form single emulsion and double emulsion (DE) droplets have greatly enhanced the toolbox for high throughput screening for cell or enzyme engineering and drug discovery. However, remaining challenges in the supply of reagents into these enclosed nanoliter compartments limit the applicability of droplet microfluidics. Here, we introduce a strategy for on-demand delivery of reactants in DEs. We use lipid vesicles as transport carriers, which are co-encapsulated in double emulsions and release their cargo upon addition of an external trigger, here the anionic surfactant SDS. The reagent present inside the lipid vesicles stays isolated from the remaining content of the DE vessel until SDS enters the DE lumen and solubilizes the lipid bilayer. We demonstrate the versatility of the method with two critical applications, chosen as representative assays for high throughput screening. First, we trigger enzymatic reactions after releasing a reactant and second, we encapsulate bacteria and induce gene expression at a delayed time. The presented technique is compatible with the high throughput analysis of individual DE droplets using conventional flow cytometry as well as with microfluidic time-resolved studies. The possibility of delaying and controlling reagent delivery in current high throughput compartmentalization systems will significantly extend their range of applications e.g. for directed evolution, and further improve their compatibility with biological systems.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR).Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS.Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals Enzyme Functional Screening, Discovery and Engineering; Automation, Metagenomics and High-throughput Approaches

2020 ◽  
pp. 1-12
Author(s):  
Sikander Ali ◽  
Syed Shahid Hussain
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Immense Potential

Concerned with the construction and design of novel biocatalysts, the enzyme engineering served to overcome the limitations of native enzymes, in order to create biocatalysts with tailored functions, to facilitate industrial applications. The enzymes, being recognized by screening and discovery workflows and further tailored by engineering platforms, are of immense potential as improved biocatalysts. Functional metagenomics is a powerful tool to identify novel enzymes followed by the construction of metagenome-based enzyme libraries. And the subsequent screening of these enzyme libraries is in turn facilitated by ultra-high-throughput-based, for example FACS or microfluidics, enzyme engineering technologies. Relies on the compartmentalization of reaction components, in order to detect and measure assay signal within the reaction compartments, the enzyme engineering platforms are designed which include cell-as-compartment platforms, droplet-based platforms and micro-chamber-based platforms. The metagenomics approach and high-throughput screening by these three prime enzyme engineer platforms are the focus of this review.

scholarly journals An Automated Workflow to Screen Alkene Reductases Using High-throughput Thin Layer Chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening

PLoS ONE ◽  
2017 ◽  
Vol 12 (2) ◽  
pp. e0171741 ◽  
Author(s):  
Daniela Quaglia ◽  
Maximilian C. C. J. C. Ebert ◽  
Paul F. Mugford ◽  
Joelle N. Pelletier
Keyword(s):  
Synthetic Biology ◽  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Engineering ◽  
Synthetic Biology Approach

scholarly journals Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1

2020 ◽  
Vol 33 ◽  
Author(s):  
P L Herzog ◽  
E Borghi ◽  
M W Traxlmayr ◽  
C Obinger ◽  
H D Sikes ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Single Cell Analysis ◽  
Detection System ◽  
Surface Display ◽  
Enzyme Engineering ◽  
H2o2 Formation ◽  
A Cell ◽  
The Cost ◽  
Time Frames

Abstract Accurate yet efficient high-throughput screenings have emerged as essential technology for enzyme engineering via directed evolution. Modern high-throughput screening platforms for oxidoreductases are commonly assisted by technologies such as surface display and rely on emulsification techniques to facilitate single-cell analysis via fluorescence-activated cell sorting. Empowered by the dramatically increased throughput, the screening of significantly larger sequence spaces in acceptable time frames is achieved but usually comes at the cost of restricted applicability. In this work, we tackle this problem by utilizing roGFP2-Orp1 as a fluorescent one-component detection system for enzymatic H2O2 formation. We determined the kinetic parameters of the roGFP2-Orp1 reaction with H2O2 and established an efficient immobilization technique for the sensor on Saccharomyces cerevisiae cells employing the lectin Concanavalin A. This allowed to realize a peroxide-sensing shell on enzyme-displaying cells, a system that was successfully employed to screen for H2O2 formation of enzyme variants in a whole-cell setting.

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