Oilseed-based metabolic engineering of astaxanthin and related ketocarotenoids using a plant-derived pathway: Lab-to-field-to-application.

Ketocarotenoids, including astaxanthin, are red lipophilic pigments derived from the oxygenation of β-carotene ionone rings. These carotenoids have exceptional antioxidant capacity and high commercial value as natural pigments, especially for aquaculture feedstocks to confer red flesh colour to salmon and shrimp. Ketocarotenoid biosynthetic pathways occur only in selected bacterial, algal, fungal and plant species, which provide genetic resources for biotechnological ketocarotenoid production. Toward pathway optimization, we developed a transient platform for ketocarotenoid production using Agrobacterium infiltration of Nicotiana benthamiana leaves with plant (Adonis aestivalis) genes, carotenoid β-ring 4-dehydrogenase 2 (CBFD2) and carotenoid 4-hydroxy-β-ring 4-dehydrogenase (HBFD1), or bacterial (Brevundimonas) genes, β-carotene ketolase (crtW) and β-carotene hydroxylase (crtZ). In this test system, heterologous expression of the plant-derived astaxanthin pathway conferred higher astaxanthin production with fewer ketocarotenoid intermediates than the bacterial pathway. We evaluated the plant-derived pathway for ketocarotenoid production using the oilseed camelina (Camelina sativa) as a production platform. Genes for CBFD2 and HBFD1 and maize phytoene synthase were introduced under the control of seed-specific promoters. In contrast to prior research with bacterial pathways, our strategy resulted in nearly complete conversion of β-carotene to ketocarotenoids, including primarily astaxanthin. Tentative identities of other ketocarotenoids were established by chemical evaluation. Seeds from multi-season US and UK field sites maximally accumulated ~135 μg/g seed weight of ketocarotenoids, including astaxanthin (~47 μg/g seed weight). Although plants had no observable growth reduction, seed size and oil content were reduced in astaxanthin-producing lines. Oil extracted from ketocarotenoid-accumulating seeds showed significantly enhanced oxidative stability and was useful for food oleogel applications.