Heterologous expression of a mutant Orange gene from Brassica oleracea increases carotenoids and induces phenotypic changes in the microalga Chlamydomonas reinhardtii

In plants, a mutation in the Orange (Or) gene is known to enhance the accumulation of carotenoids in comparison to the wild-type (WT) allele. We investigated the effect of a point mutation in the Or gene of Brassica oleracea on its protein structure via in silico analysis. Subsequently we studied the effect of the Or mutation on carotenoid regulation in Chlamydomonas reinhardtii. The Or mutation in Brassica oleracea—mBoOr (R88H)—results in a small β-sheet in place of the α-helix that is present in the WT protein, and was found to be associated with enhanced carotenoid content when screened in Escherichia coli. The expression of the equivalent mutant from C. reinhardtii—mCrOr (R106H)—did not result in any change in protein structure and exhibited lower levels of carotenoid in E. coli. An in silico analysis of microalgal genomes revealed the presence of Or homologues in several microalgae from the class Trebouxiphyceae, while C. reinhardtii was the only representative of the Chlorophyceae. The heterologous expression of mBoOr in C. reinhardtii resulted in a 1.7-fold increase in carotenoid content, as well as an increase in cell size and the appearance of multicellularity in C. reinhardtii, which was evidenced by the presence of both orange and green transgenic cells grouping together as a population. A palmelloid formation of the orange and green cells impaired the motility of the transgenic population, resulting in flocculation. This study establishes mBoOr expression as a promising strategy for enhancing carotenoid content in microalgae.