Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)

Bat Erdene Jugder, Jeffrey Welch, Nady Braidy, Christopher P. Marquis*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)
6 Downloads (Pure)

Abstract

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

Original languageEnglish
Article numbere2269
Pages (from-to)1-16
Number of pages16
JournalPeerJ
Volume2016
Issue number7
DOIs
Publication statusPublished - 26 Jul 2016
Externally publishedYes

Bibliographical note

Copyright the Author(s) 2016. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • Green Fluorescent Proteins
  • Cupriavidus necator
  • Promoter
  • Ralstonia eutropha
  • Soluble hydrogenase

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