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.

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使用植物来源的途径对虾青素和相关酮类化合物进行基于油籽的代谢工程：实验室到田间到应用

摘要酮类胡萝卜素，包括虾青素，是源自β-胡萝卜素紫罗兰酮环氧合的红色亲脂性色素。这些类胡萝卜素作为天然色素具有非凡的抗氧化能力和很高的商业价值，特别是用于水产养殖原料，赋予鲑鱼和虾红色的肉色。酮类胡萝卜素生物合成途径仅发生在选定的细菌、藻类、真菌和植物物种中，这些物种为生物技术酮类化合物生产提供遗传资源。为了实现途径优化，我们开发了一个瞬时平台，使用植物 （Adonis aestivalis） 基因、类胡萝卜素 β-环 4-脱氢酶 2 （CBFD2） 和类胡萝卜素 4-羟基-β-环 4-脱氢酶 （HBFD1） 或细菌 （Brevundimonas） 基因、β-胡萝卜素酮酶 （crtW） 和 β-胡萝卜素羟化酶 （crtZ）。在该测试系统中，植物来源的虾青素途径的异源表达赋予了更高的虾青素产量，而酮类化合物中间体比细菌途径少。我们使用油籽亚麻荠 （Camelina sativa） 作为生产平台评估了植物来源的酮类化合物生产途径。CBFD2 和 HBFD1 以及玉米八氢番茄红素合酶的基因是在种子特异性启动子的控制下引入的。与先前对细菌途径的研究相比，我们的策略导致 β-胡萝卜素几乎完全转化为酮卡罗素，主要包括虾青素。通过化学评价确定其他酮类化合物的初步身份。来自美国和英国多季节田间地点的种子最大积累了 ~135 μg/g 种子重量的酮卡罗素，包括虾青素 （~47 μg/g 种子重量）。 尽管植物没有明显的生长减少，但在虾青素生产品系中，种子大小和含油量有所降低。从酮卡罗素积累种子中提取的油显示出显着增强的氧化稳定性，可用于食品油凝胶应用。