PDF(Pubmed)
Abstract：
Lutein is a high-value carotenoid with many human health benefits. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently β-cyclize both ends of lycopene to produce β-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into β-monocyclic γ-carotene and ultimately produced α-carotene with a β-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.
摘要：
叶黄素是一种高价值的类胡萝卜素，具有许多人类健康益处。番茄红素β-和ε-环化酶（LCYB和LCYE，分别)催化番茄红素环化为不同的下游分支，其中之一是叶黄素生物合成途径，通过α-胡萝卜素。因此，LCYB和LCYE是叶黄素生物合成的关键酶。在这项研究中,富含叶黄素的海洋绿色微藻的两种番茄红素环化酶（CsLCYB和CsLCYE）的编码基因，分离并鉴定了小球藻SrookinianaFZU60。使用生物信息学进行CsLCYB和CsLCYE的序列分析和计算建模以鉴定关键结构域。Further,系统发育分析表明，CsLCYB和CsLCYE与其他绿色微藻的蛋白质均一。烟草的亚细胞定位试验表明，CsLCYB和CsLCYE定位于叶绿体中。在大肠杆菌中进行的色素互补实验表明，CsLCYB可以有效地将番茄红素的两端β环化以产生β-胡萝卜素。另一方面,CsLCYE对生产δ-胡萝卜素具有较强的ε-单环酶活性，对生产ε-胡萝卜素具有较弱的ε-双环活性。此外,CsLCYE能够将番茄红素催化成β-单环γ-胡萝卜素，并最终通过γ-胡萝卜素或δ-胡萝卜素产生具有β-环和ε-环的α-胡萝卜素。此外，CsLCYB和CsLCYE在大肠杆菌中的共表达表明，α-胡萝卜素是主要产物，这可能导致在C.sorokinianaFZU60中产生高水平的叶黄素。该研究结果为进行代谢工程以改善FZU60中叶黄素的生物合成和积累提供了理论基础。
