Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Metabolic engineering of Rhodotorula toruloides IFO0880 improves C16 and C18 fatty alcohol production from synthetic media.

Microbial cell factories·2022
Same author

Selectively Lighting Up Singlet Oxygen via Aggregation-Induced Electrochemiluminescence Energy Transfer.

Analytical chemistry·2022
Same author

Directed Evolution of Replication-Competent Double-Stranded DNA Bacteriophage toward New Host Specificity.

ACS synthetic biology·2022
Same author

Metabolic engineering of threonine catabolism enables Saccharomyces cerevisiae to produce propionate under aerobic conditions.

Biotechnology journal·2022
Same author

Recent advances and perspectives of enzyme-based optical biosensing for organophosphorus pesticides detection.

Talanta·2021
Same author

Pathological diagnosis, differential diagnosis and origin investigation of easily misdiagnosed adult gastric duplication cysts.

Histology and histopathology·2021

Related Experiment Video

Updated: Dec 8, 2025

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
08:10

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

Published on: August 8, 2016

9.1K

Optically guided mass spectrometry to screen microbial colonies for directed enzyme evolution.

Pu Xue1, Tong Si2, Huimin Zhao1

  • 1Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, United States; DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois at Urbana-Champaign, Urbana, IL, United States.

Methods in Enzymology
|September 18, 2020
PubMed
Summary

Directed evolution for enzyme engineering is enhanced by a new label-free, high-throughput method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). This technique rapidly analyzes bacterial colonies for multistep enzymatic reactions, overcoming previous screening bottlenecks.

Keywords:
Directed enzyme evolutionHigh-throughput screeningMALDI-ToF MSMass spectrometry

More Related Videos

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
10:50

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Published on: April 1, 2016

11.3K
Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat
06:03

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Published on: September 20, 2016

15.0K

Related Experiment Videos

Last Updated: Dec 8, 2025

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
08:10

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

Published on: August 8, 2016

9.1K
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
10:50

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Published on: April 1, 2016

11.3K
Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat
06:03

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Published on: September 20, 2016

15.0K

Area of Science:

  • Biochemistry
  • Biotechnology
  • Analytical Chemistry

Background:

  • Directed evolution is crucial for enzyme engineering but high-throughput screening of variants remains a significant challenge.
  • Engineering multiple enzymes in a biochemical pathway exacerbates profiling difficulties.
  • Current methods often lack the speed and sensitivity required for large-scale variant analysis.

Purpose of the Study:

  • To develop and validate a label-free, high-throughput method for engineering multistep enzymatic reactions in bacterial colonies.
  • To overcome the bottleneck in characterizing enzyme variants generated through directed evolution.
  • To enable rapid profiling of microbial libraries for natural product biosynthesis.

Main Methods:

  • Utilized optically guided matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS).
  • Applied the method to bacterial colonies for analyzing multistep enzymatic reactions.
  • Developed computational algorithms for processing and visualizing mass spectral data.

Main Results:

  • Demonstrated high sensitivity and accuracy in detecting products, reactants, and byproducts.
  • Successfully applied the method to create plantazolicin analogs and profile rhamnolipid congeners.
  • Achieved improved MS acquisition efficiency (1-2.5s per colony) and obtained rich data from large populations.

Conclusions:

  • The developed MALDI-ToF MS method offers a powerful, label-free approach for high-throughput enzyme engineering.
  • This technique significantly enhances the characterization of multistep enzymatic reactions in microbial libraries.
  • The method is broadly applicable to diverse enzymatic reactions and microbial systems for rapid phenotyping.