[en] Ferredoxins (FDXs) are small iron-sulfur proteins that provide electrons for a variety of metabolic pathways. The green microalga Chlamydomonas reinhardtii is quite unique in having twelve FDXs. The major chloroplastic isoform, FDX1 or PETF, mediates electron transfer between photosystem I and NADPH at the level of the ferredoxin-NADP+ reductase. FDX1 also plays a central role under anoxic conditions where it couples the activity of pyruvate-ferredoxin oxidoreductase and [Fe-Fe] hydrogenases.
In the frame of a synthetic biology approach, we aimed to relocate FDX1 to mitochondria and assess the metabolic consequences. We constructed six transcription units by modular cloning. The endogenous FDX1 genomic sequence, in which the sequence encoding the predicted chloroplast targeting peptide was replaced by a mitochondrial matrix targeting sequence was combined with different promoters and terminators. The built transcription units eventually contained the sequence allowing a C-terminal fusion with nanoluciferase. The constructs were expressed in two strains: a control strain and a mutant strain in which the gene coding for Sir2-type histone deacetylase was inactivated since this mutation enhances exogenous gene expression. The expression level of FDX1 was assessed by measuring nanoluciferase activity. Transformants with low, medium and high nanoluciferase activity for each construct and each strain were retained for further analysis. The presence of the tagged protein in mitochondria is currently being analyzed by immunoblotting using nanoluciferase and FDX1 antibodies from enriched mitochondrial fractions. Comparison of these results for each expression unit and each strain will allow identifying the optimal combination for mitochondrial FDX1 expression. The next step will be to verify that the relocated FDX1 is active in mitochondria. Further characterization of the transformants will be done to investigate the effects on the mitochondrial redox metabolism.
