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Updated: Mar 11, 2026
A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening
Emma Barahona1, Emilio Jiménez-Vicente1, Luis M Rubio1
1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223-Pozuelo de Alarcón, Madrid, Spain.
Researchers developed a new high-throughput screening method to detect hydrogen gas (H2) production in biological systems. This innovative technique enables the directed evolution of enzymes for improved microbial H2 fuel generation.
Area of Science:
- Biotechnology
- Bioenergy
- Enzyme Engineering
Background:
- Biological hydrogen gas (H2) production offers a clean fuel alternative.
- Current limitations in H2 enzyme discovery hinder progress.
- High-throughput screening methods for biological H2 production are lacking.
Purpose of the Study:
- To engineer a novel system for high-throughput screening of H2 production in biological systems.
- To enable the discovery and optimization of H2-producing enzymes.
- To facilitate the directed evolution of nitrogenases for enhanced H2 generation.
Main Methods:
- Re-engineered the Rhodobacter capsulatus H2 sensing system.
- Utilized a lacZ reporter gene under an H2-inducible promoter.
- Employed fluorescence-activated cell sorting flow cytometry for screening.
Main Results:
- Developed a system correlating fluorescence with H2 production levels.
- Identified a novel H2-overproducing nitrogenase Fe protein variant lacking 40% of its wild-type sequence.
- Demonstrated the effectiveness of the screening system for enzyme variant selection.
Conclusions:
- The developed screening method is effective for identifying H2-overproducing enzymes.
- This approach has significant potential for improving microbial H2 production.
- Facilitates directed evolution of nitrogenases and hydrogenases for bioenergy applications.
