Induction of biosynthesis of ketocarotenoid from β-carotene in fish embryos

Abstract Ketocarotenoids are high-value molecules with powerful antioxidant properties. They are widely used as a feed supplements due to their vital functions for animals, particularly for fish and marine animals. However, fish is not able to synthesize ketocarotenoids but consume them from diet. Here, we report metabolic engineering of fish cells and fish embryos for biosynthesis of ketocarotenoid from β-carotene as a substrate. Firstly, we introduced two sets of fish-codon optimized genes (crtW and crtZ) from Brevundimonas sp., and (crtS and crtR) from Xanthophyllomyces dendrorhous, to the cultured common carp cells (Epithelioma papulosum cyprinid, EPC cells) to test the production of ketocarotenoids in vitro. We then introduced the optimized set of genes, crtW and crtZ, to zebrafish (Danio rerio) embryos and found that they could efficiently mediate the synthesis of ketocarotenoids from β-carotene in vivo. Subsequently, we constructed a bicistronic expression vector containing crtW and crtZ genes linked with a P2A peptide coding sequence, and evaluated the production of ketocarotenoids using both in vitro and in vivo systems. Overall, we unprecedentedly stimulated fish cells and zebrafish embryos to synthesize ketocarotenoids with a total content of 29.9 μg/g DW and 16.46 μg/g DW, respectively. Furthermore, we successfully optimized the ketocarotenoids biosynthesis and significantly increased the total ketocarotenoids content in fish models up to 2.5 and 2.14 fold, corresponding to 75.24 and 35.25 μg/g DW, respectively. This study is a proof of concept of engineering animals to manufacture ketocarotenoids and provides a pilot study to enable fish to synthesize ketocarotenoids in vivo.