Defining the biosynthesis of ketocarotenoids in Chromochloris zofingiensis

Carotenoids are important pigments in photosynthetic organisms where they play essential roles in photoreception and photoprotection. Chromochloris zofingiensis is a unicellular green alga that is able to accumulate high amounts of ketocarotenoids including astaxanthin, canthaxanthin and ketolutein when growing heterotrophically or mixotrophically with glucose as a carbon source. Here we elucidate the ketocarotenoid biosynthesis pathway in C. zofingiensis by analyzing five algal mutants. The mutants were shown to have a single nucleotide insertion or substitution in β-carotene ketolase (BKT) gene 1, which resulted in a lack of ketocarotenoid production in Cz-bkt1-1, and decreased ketocarotenoid content in the other four mutants. These mutants accumulated much higher amounts of non-ketocarotenoids (β-carotene, zeaxanthin and lutein). Interestingly, the Cz-bkt1-5 mutant synthesized 2-fold the ketolutein and only 1/30 of the canthaxanthin and astaxanthin as its parent strain, suggesting that the mutated BKT1 exhibits much higher activity in catalyzing lutein to ketolutein but lower activity in ketolating β-carotene and zeaxanthin. Mutant and WT BKT2 gene sequences did not differ. Taken together, we conclude that BKT1 is the key gene involved in ketocarotenoid biosynthesis in C. zofingiensis. Our study provides insight into the biosynthesis of ketocarotenoids in green algae. Furthermore, Cz-bkt1 mutants may serve as a natural source for the production of zeaxanthin, lutein, and β-carotene.