Enhancing carotenoid production in Chlorella sorokiniana through the protein-protein interaction between ORANGE and...

The green microalga Chlorella sorokiniana FZU60, capable of achieving ultra-high biomass concentrations under heterotrophic conditions, is a promising chassis for carotenoid production. Enhancing intracellular carotenoid accumulation is an effective strategy to further improve the potential of this alga. ORANGE protein is recognized as a key post-transcriptional regulator of phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis, in higher plants. However, its function in microalgae remains unclear. In this study, the genomic structures of ORANGE and PSY in C. sorokiniana FZU60 (CsORANGE and CsPSY) were characterized, revealing GC-AG splice sites within their introns. Computational analysis predicted a stable interaction between CsORANGE and CsPSY, which was subsequently confirmed by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Heterologous co-expression of CsORANGE and CsPSY in carotenoid-engineered Escherichia coli significantly increased lycopene accumulation compared with expression of CsPSY alone. Moreover, CRISPR activation (CRISPRa)-mediated upregulation of endogenous CsORANGE significantly enhanced carotenoid accumulation in the engineered transformants. These results demonstrate that CsORANGE positively regulates carotenoid biosynthesis via direct protein-protein interaction with CsPSY in C. sorokiniana FZU60.