ISOLATION AND FUNCTIONAL IDENTIFICATION OF A NOVEL CDNA FOR ASTAXANTHIN BIOSYNTHESIS FROM HAEMATOCOCCUS-PLUVIALIS, AND ASTAXANTHIN SYNTHESIS IN ESCHERICHIA-COLI
ISOLATION AND FUNCTIONAL IDENTIFICATION OF A NOVEL CDNA FOR ASTAXANTHIN BIOSYNTHESIS FROM HAEMATOCOCCUS-PLUVIALIS, AND ASTAXANTHIN SYNTHESIS IN ESCHERICHIA-COLI
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DOI：
10.1007/bf00043657
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发表时间：
1995-10-01
影响因子：
5.1
通讯作者：
MISAWA, N
中科院分区：
文献类型：
--
作者：
KAJIWARA, S;KAKIZONO, T;MISAWA, N
我们成功地分离了一个新的cDNA参与虾青素生物合成的绿色的雨生红球藻，通过表达克隆方法使用大肠杆菌作为宿主，合成β-胡萝卜素由于欧文氏菌类胡萝卜素生物合成基因。克隆的cDNA被证明编码一种新的酶，β-胡萝卜素酮醇酶（β-胡萝卜素加氧酶），它通过海胆酮将β-胡萝卜素转化为胡萝卜黄质，通过色谱和光谱分析，在大肠杆菌中积累的色素。这表明编码的酶负责将亚甲基直接转化为酮基，这种机制通常需要通过羟基中间体进行两种不同的酶促反应。北方印迹分析表明，该mRNA仅在H.雨生植物E. coli携带H. pluvialis cDNA和E.还发现玉米黄质生物合成所需的噬夏孢子菌基因合成虾青素（3S，3 ′ S），其在通过多种光谱方法纯化后被鉴定。
We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E, coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.