Stepwise pathway engineering to the biosynthesis of zeaxanthin, astaxanthin and capsanthin in rice endosperm

Carotenoid pigments are valuable components of the human diet. A notable example is β-carotene, or provitamin A, which is converted into the derivatives astaxanthin and capsanthin, via the common intermediate zeaxanthin. To generate rice varieties producing diverse carotenoids beyond β-carotene, we specifically used a Capsicum β-carotene hydroxylase gene, B (CaBch) and a codon optimized version of the same gene, stB (stBch) to increase zeaxanthin synthesis. We also used a recombinant BAK gene (CaBch-2A-HpBkt), consisting of the CaBch sequence and a Haematococcus β-carotene ketolase gene (HpBkt) linked by a bicistronic 2 A sequence, as well as a codon optimized recombinant stBAK gene (stBch-2A-stBkt) to create astaxanthin synthesis. The four cassettes to seed-specifically express the B, stB, BAK and stBAK genes were individually combined with a PAC gene (CaPsy-2A-PaCrtI) cassette to previously impart β-carotene-enriched trait in rice endosperm. The single T-DNA vectors of B-PAC, stB-PAC, BAK-PAC and stBAK-PAC resulted in the accumulation of zeaxanthin and astaxanthin in the endosperm of the transgenic rice seeds. In addition, an extended version on the carotenoid pathway was introduced into rice to allow the production of capsanthin, by intercrossing a B-PAC rice line with a Ccs rice line, which harbors a Capsicum capsanthin-capsorubin synthase gene. Ultimately, we developed three functional rice varieties: B-PAC (0.8 μg/g zeaxanthin, deep yellow), stBAK-PAC (1.4 μg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4 μg/g of ketoxanthophylls, including capsanthin, orange-red) with the similar levels of total carotenoids to PAC rice, suggesting the capacity was dependent on β-carotene levels. Collectively, a combination of genetic engineering and conventional breeding is effective for multi-step metabolic engineering and biochemical pathway extension. •The pathways toward zeaxanthin, astaxanthin and capsanthin beyond β-carotene were constructed in rice endosperm via stepwise metabolic engineering.•To do this, multiple gene expression strategies have taken advantage of the merits of bicistronic and multi-cassette expression and conventional breeding.•Resultant colorful rice varieties generating zeaxanthin (yellow), astaxanthin (pinkish red) and capsanthin (orange-red) in addition to β-carotene (yellow) might be attractive as new cultivars with different functions.