Guo Wenjing, Liu Yuxuan, Yan Xin, Liu Mingyi, Tang Hui, Liu Zheng, Zhang Liping
The Key Laboratory of Microbial Diversity Research and Application of Hebei Province; Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education; College of Life Sciences, Hebei University, Baoding, 071002, China.
Antonie Van Leeuwenhoek. 2015 Apr;107(4):1017-27. doi: 10.1007/s10482-015-0394-6. Epub 2015 Jan 28.
Carotenoids are a naturally occurring and widely distributed group of pigments that provide protection against photooxidation and inactivate free radicals due to their highly conjugated double-bond systems. Phytoene dehydrogenation is the first rate-limiting step in the carotenoid biosynthetic pathway. Phytoene dehydrogenase is the key enzyme in the transformation of carotenoid from colorless to colored; therefore it is the first target of gene manipulation. The present study describes the identification and functional characterization of a carontenoid synthesis gene from Rhodosporidium diobovatum, designated as crtI, which catalyzes the dehydrogenation of phytoene. We obtained a full-length cDNA clone of crtI, encoding phytoene dehydrogenase (EC Number: 1.3.99.31), from R. diobovatum ATCC 2527 by rapid amplification of cDNA ends. Complementation mapping of the crtI gene in Escherichia coli allowed us to localize the regions responsible for phytoene dehydrogenase function within the protein. Enzyme activity of the expressed protein in E. coli was verified using high performance liquid chromatography analysis. We were able to determine the nucleotide sequence of crtI from R. diobovatum. The publicly available sequence will be useful in future studies on phytoene dehydrogenase.
类胡萝卜素是一类天然存在且分布广泛的色素，由于其高度共轭的双键系统，它们能够提供抗光氧化保护并使自由基失活。八氢番茄红素脱氢是类胡萝卜素生物合成途径中的第一个限速步骤。八氢番茄红素脱氢酶是类胡萝卜素从无色转变为有色过程中的关键酶；因此，它是基因操作的首要靶点。本研究描述了来自双卵圆红冬孢酵母（Rhodosporidium diobovatum）的一个类胡萝卜素合成基因的鉴定和功能表征，该基因命名为crtI，它催化八氢番茄红素的脱氢反应。我们通过cDNA末端快速扩增从双卵圆红冬孢酵母ATCC 2527中获得了crtI的全长cDNA克隆，其编码八氢番茄红素脱氢酶（酶编号：1.3.99.31）。crtI基因在大肠杆菌中的互补定位使我们能够在该蛋白质中定位负责八氢番茄红素脱氢酶功能的区域。使用高效液相色谱分析验证了大肠杆菌中表达蛋白的酶活性。我们能够确定双卵圆红冬孢酵母crtI的核苷酸序列。公开可用的序列将对未来八氢番茄红素脱氢酶的研究有用。
