Molecular cloning and functional characterization of CvLCYE, a key enzyme in lutein synthesis pathway in Chlorella vulgaris
Molecular cloning and functional characterization of CvLCYE, a key enzyme in lutein synthesis pathway in Chlorella vulgaris
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小球藻叶黄素合成途径关键酶CvLCYE的分子克隆及功能表征
DOI：
10.1016/j.algal.2021.102246
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发表时间：
2021-03-01
影响因子：
5.1
通讯作者：
Hu, Zhangli
中科院分区：
文献类型：
--
作者：
Lou, Sulin;Lin, Xin;Hu, Zhangli
叶黄素是一种重要的抗氧化剂，对眼睛和心脏的健康有益。番茄红素环化酶（LCYE）是植物体内叶黄素合成途径中的关键酶，在此酶的作用下，番茄红素被转化为？胡萝卜素，？-胡萝卜素和叶黄素。在本研究中，我们首次成功克隆了富含叶黄素的绿色小球藻基因组中进化上保守的番茄红素环化酶CvLCYE。同源性分析表明，CvLCYE蛋白的氨基酸序列与其他水生绿色藻类包括小球藻、原壳小球藻CS-41、佐芬绿球藻和莱茵衣藻的氨基酸序列高度同源。同时，对CvLCYE蛋白序列进行UPGMA树分析，结果一致。CvLCYE蛋白的生物信息学分析揭示了一个典型的番茄红素环化酶结构域（Pfam 05834）之间的第64？第475个氨基酸。CvLCYE蛋白的三维结构预测显示其为β链-α螺旋-β链结构，这一结构在所有已知的番茄红素ε-环化酶中是保守的。亚细胞定位试验表明，成熟的CvLCYE-GFP融合蛋白在拟南芥原生质体中定位于细胞质。重要的是，用表达CvLCYE的大肠杆菌菌株进行番茄红素含量的靶向HPLC分析，与表达空载体的菌株相比，表达CvLCYE的大肠杆菌菌株显示出番茄红素消耗的褪色粉红色，表达空载体的菌株由于番茄红素的积累而保持粉红色。HPLC数据强烈地表明CvLCYE催化番茄红素的能力。此外，CvLCYE的酶活性也在莱茵衣藻（Chlamydiumreinhardtii）中得到了明确的验证，莱茵衣藻是藻类研究的模式生物。有趣的是，我们发现转基因C。表达CvLCYE的reinhardtii比野生型菌株增加约2.3倍。这些结果为深入了解CvLCYE在叶黄素合成中的积极作用奠定了基础，也为小球藻等藻类叶黄素代谢途径的分子育种奠定了基础。
Human completely rely on food and nutrition supply to absorb enough lutein, which is an important antioxidant beneficial to health of eyes and heart. In plants, lycopene epsilon-cyclase (LCYE) is the key enzyme in lutein synthesis pathway, where lycopene is converted into ?-carotene, ?-carotene and lutein. In this study, for the first time, we successfully cloned CvLCYE, the evolutionarily conserved lycopene cyclase in the genome of Chlorella vulgaris that is a green alga rich in lutein. Homology analysis showed that the amino acid sequence of CvLCYE protein is factually high identical with that of other aquatic green algae including Chlorella variabilis, Auxenochlorella protothecoides strain CS-41, Chromochloris zofingiensis and Chlamydomonas reinhardtii. Meanwhile, the UPGMA tree analysis of CvLCYE protein sequence showed the consistent results. Bioinformatic analysis of CvLCYE protein revealed a typical lycopene cyclase domain (Pfam05834) between the 64th?475th amino acid. The 3D structure prediction of CvLCYE protein displayed a structure of beta strand-alpha helix-beta strand, which is conserved in all known lycopene epsilon-cyclases. Subcellular localization tests demonstrated that mature CvLCYE-GFP fused protein localized to cytoplasm in Arabidopsis protoplasts. Importantly, targeted HPLC analysis on lycopene content are performed with Escherichia coli strain expressing CvLCYE, which displayed faded pink of consumption of lycopene, in comparison to strain expressing empty vector, which maintained pink because of accumulation of lycopene. HPLC data strongly suggested the capability of CvLCYE to catalyze lycopene. Furthermore, enzymatic activity of CvLCYE was also clearly verified in Chlamydomonas reinhardtii that is the model organism for algae research. Interestingly, we found that the lutein content in transgenic C. reinhardtii expressing CvLCYE increased by about 2.3 times than that of wild type strain. All these results provide insight into the positive role played by CvLCYE in lutein synthesis and lay a promising foundation for the molecular breeding of lutein metabolic pathway in Chlorella vulgaris and other algae.