Catégorie | Starter |
Période | 2025-2027 (AAP1) |
Porteur | Annie Frelet-Barrand |
Unités/Plateformes BFC | FEMTO, Agroécologie |
Collaborateurs externes | - |
Doctorants/Postdoctorants | - |
Stages Master | 2 |
Work Packages HARMI | WP4 |
Histone deacetylases (HDAs) are enzymes capable of deacetylating the lysine residues of proteins, particularly histones. Histone acetylation or deacetylation affects chromatin structure and the recruitment of regulatory proteins, thus modulating DNA replication, repair and transcription. HDAs can also target other proteins. We recently identify the HDA14 protein from the algae Klebsormidium nitens (KnHDA14) as a target of S-nitrosation, a post-translational modification induced by the signalling molecule nitric oxide (NO). KnHDA14 is orthologous to the Arabidopsis thaliana HDA14 protein (AtHDA14), whose characterization has been initiated but is far from complete. AtHDA14 is chloroplastic and, to a lesser extent, mitochondrial. Its substrates are mainly proteins involved in photosynthesis, in particular photosystem component proteins (Hartl et al., 2017). AtHDA14 is also capable of deacetylating N-acetyl-serotonin to serotonin in vitro (Lee et al., 2018).
Objectives: To functionally characterize KnHDA14, we need to produce the recombinant protein in large quantities and as purely as possible. Unfortunately, when produced using the classical Escherichia coli bacterial expression system, the protein accumulates in inclusion bodies. The aim of the present project is to produce KnHDA14 in the emerging Lactococcus lactis expression system. Indeed, in recent years, L. lactis has emerged as an alternative and efficient expression system to the well-known E. coli, as it does not produce inclusion bodies (Bernaudat et al., 2011).
How it will be achieved: Firstly, the gene encoding the protein will be cloned into the appropriate L. lactis expression vector to generate the recombinant plasmid. Protein expression will then be tested after induction of production. After bacterial lysis, the protein will be isolated and analysed by biochemical methods. We will then be able to study the structure of the protein and its activity after the in vitro S-nitrosation or not.
Relevance to HARMI: This project is in line with HARMI project Axis 4, “Developing microbial biotechnology”, since its objective is the bioproduction of a recombinant protein in the L. lactis bacterium. It will open up new ways of expressing proteins of interest from photosynthetic organisms in microbial factories.
