Department of Microbiology, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Appl Microbiol Biotechnol. 2011 Nov;92(4):769-77. doi: 10.1007/s00253-011-3488-8. Epub 2011 Jul 27.
Lycopene beta-cyclase (β-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic β-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, β-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-β from Ficus carica (Lyc-β Fc), which codes for the enzyme lycopene β-cyclase (β-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. β-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to β-carotene as a result of the enzyme's action. The β-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the β-carotene. The lycopene to β-carotene conversion rate was 90%. The experiments carried out in this work showed that β-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to β-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing β-LCY in E. coli, we have obtained a new gene for β-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica.
番茄红素 β-环化酶（β-LCY）是将线性番茄红素分子修饰为环状 β-胡萝卜素的关键酶，β-胡萝卜素是光合作用装置中不可或缺的类胡萝卜素，也是人和动物营养中维生素 A 的重要来源。由于其抗氧化活性，它在化妆品和制药行业中被商业使用，并且是食品添加剂。因此，β-胡萝卜素在类胡萝卜素市场中占有很大份额。在这项研究中，我们使用逆转录-聚合酶链反应（RT-PCR）和快速扩增 cDNA 末端（RACE-PCR）获得并克隆了来自榕属（Lyc-β Fc）的 Lyc-β 的 cDNA 拷贝，该基因编码番茄红素 β-环化酶（β-LCY）。该基因在大肠杆菌中的表达产生了一个重 56 kDa 的单一多肽，含有 496 个氨基酸，能够循环番茄红素链的两端。氨基酸分析表明，该蛋白含有几个保守的植物环化酶基序。通过异源互补分析揭示了 β-LCY 的活性，结果是酶作用下将番茄红素转化为 β-胡萝卜素。通过高效液相色谱（HPLC）鉴定β-胡萝卜素证实了表达蛋白的 β-LCY 活性。表达蛋白的番茄红素转化为 β-胡萝卜素的转化率为 90%。本工作进行的实验表明，β-LYC 是将非环类胡萝卜素番茄红素转化为β-胡萝卜素的酶，β-胡萝卜素是一种双环类胡萝卜素。因此，通过在大肠杆菌中克隆和表达β-LCY，我们获得了一个新的基因，用于生产β-胡萝卜素或作为虾青素生物合成途径的一部分。到目前为止，这是在榕属中鉴定的第一个也是唯一的类胡萝卜素途径基因。
