The marine thraustochytrids are a promising source of docosahexaenoic acid (DHA) and the ketocarotenoid astaxanthin. In this study, the biosynthetic pathways of these two important metabolites in Aurantiochytrium sp. SK4 was illustrated by the analyses of the genome, transcriptome, key enzymes, and pathway products. Two sets of genes were involved in two pathways for the biosynthesis of fatty acids. The absence of Δ-15 desaturase genes and the presence of docosapentaenoic acid (DPA), up to 12% of total fatty acids suggest that Aurantiochytrium sp. SK4 may synthesize DHA mainly via a polyketide synthase (PKS) pathway. Three enzymes, namely geranyl diphosphate synthase (GPPS), farnysyl diphosphate synthase (FPPS), and geranylgeranyle diphosphate synthase (GGPPS) were found to be involved in the formation of GGPP that was subsequently catalyzed to β-carotene by a trifunctional CrtIBY enzyme. β-Carotene might be ketolated and then hydroxylated into astaxanthin based on the carotenoid profiles. The formation of GGPP was proposed to be the limiting steps for carotenoid production. Overexpression of the Archaeoglobus GPS together with the Escherichia coli isopentenyl pyrophosphate isomerase, and Vitreoscilla hemoglobin resulted in not only 1.85- and 5.02-fold increases of total carotenoids and astaxanthin, but also 2.40- and 2.74-fold increases of total fatty acids and DHA. This study provides insights into the biosynthesis of carotenoids and fatty acids in Aurantiochytrium.

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.