[Cloning of Enterobacter aerogenes fh1A gene and overexpression of hydrogen production]
Related Experiment Video
Updated: Jun 10, 2026
Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Jinfang Zhao1, Wenlu Song, Jun Cheng
1Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Zhejiang University, Hangzhou 310029, China). jinfangzhao@126.com
Objective:
We amplified and overexpressed the FHL activator (fh1A) in E. aerogenes ATCC13408 to enhance hydrogen production.
Methods:
By using universal primers and genome walking, we cloned the full open reading frame (ORF) of fh1A gene. We inserted it into the glutathion S-transferase (GST) fusion expression vector pGEX4T-2-Cat, and transformed the recombinant plasmid into E. aerogenes ATCC13408 via electroporation for expression. Then we measured the hydrogen production of the recombinant strain in a batch culture.
Results:
We found that the ORF of fh1A was 2073 base pair in length, potential to encode a 690 amino acid peptide (GenBank accession GU188474). The Fh1A protein from E. aerogenes ATCC13408 shared high amino acid identities with those from other bacterial species. By using SDS-PAGE and Western blot analysis, we confirmed that the fh1A gene had successfully expressed in the strain. The hydrogen yield of the recombinant strain was increased from 1.23 to 1.48 mol H2/mol glucose. [ Conclusion ] Enhancement of hydrogen productivity was attained under anaerobic conditions with the recombinant strain.
