Expression of chloroplastic enzymes in mitochondria of the green microalga Chlamydomonas

Chlamydomonas harbors a flexible metabolic network enabling adaptation to fluctuating environmental conditions. In anaerobic metabolism, ferredoxin 1 (FDX1) and pyruvate-ferredoxin oxidoreductase (PFR1) play central roles, mediating electron transfer from pyruvate to acetyl-CoA (1). In the frame of a synthetic biology approach, we aimed to relocate FDX1 and PFR1 to mitochondria and assess the metabolic consequences. Using a modular cloning approach (2), the sequences encoding FDX1 and PFR1 were fused to a mitochondrial targeting sequence and their expression controlled using dedicated promoters and terminators. Final constructs were transformed into a Δprf1/Δsrta strain, which lacks the endogenous PFR1 gene and the Sir2-type histone deacetylase gene (SRTA), a genetic background enhancing transgene expression (3). Western blot analysis of mitochondrial protein extracts confirmed the mitochondrial localization of PFR1. In contrast, the detection of FDX1 in mitochondrial fractions remains yet inconclusive. Functional assays are underway to determine whether the relocated enzymes possess catalytic activity and to evaluate their impact on mitochondrial redox metabolism. These findings represent a first step toward reconstituting a ferredoxin-dependent anaerobic pathway in Chlamydomonas mitochondria, providing foundation for future metabolic engineering strategies.