J.W. Goethe University Frankfurt, Institute of Molecular Bioscience, Max von Laue Str. 9, D-60438 Frankfurt, Germany; Department of Food and Nutrition, Japan Women's University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan.
J.W. Goethe University Frankfurt, Institute of Molecular Bioscience, Max von Laue Str. 9, D-60438 Frankfurt, Germany; Department of Food and Nutrition, Japan Women's University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan.
Arch Biochem Biophys. 2014 Mar 1;545:141-7. doi: 10.1016/j.abb.2014.01.014. Epub 2014 Jan 30.
Xanthophyllomces dendrorhous (in asexual state named as Phaffia rhodozyma) is a fungus which produces astaxanthin, a high value carotenoid used in aquafarming. Genetic pathway engineering is one of several steps to increase the astaxanthin yield. The limiting enzyme of the carotenoid pathway is phytoene synthase. Integration plasmids were constructed for transformation with up to three copies of the crtYB gene. Upon stepwise transformation, the copy numbers of crtYB was continuously increased leading to an almost saturated level of phytoene synthase as indicated by total carotenoid content. Several carotenoid intermediates accumulated which were absent in the wild type. Some of them are substrates and intermediates of astaxanthin synthase. They could be further converted into astaxanthin by additional transformation with the astaxanthin synthase gene. However, three intermediates exhibited an unusual optical absorbance spectrum not found before. These novel keto carotenoid were identified by HPLC co-chromatography with reference compounds generated in Escherichia coli and one of them 3-HO-4-keto-7',8'-dihydro-β-carotene additionally by NMR spectroscopy. The others were 4-keto-β-zeacarotene and 4-keto-7',8'-dihydro-β-carotene. A biosynthesis pathway with their origin from neurosporene and the reason for their synthesis especially in our transformants has been discussed.
球孢藻（无性态命名为红发夫酵母）是一种真菌，能够产生虾青素，这是一种高附加值的类胡萝卜素，用于水产养殖。基因途径工程是提高虾青素产量的几个步骤之一。类胡萝卜素途径的限速酶是八氢番茄红素合酶。构建了整合质粒，可进行多达三个 crtYB 基因的转化。逐步转化后，crtYB 的拷贝数不断增加，总类胡萝卜素含量表明八氢番茄红素合酶几乎达到饱和水平。积累了几种类胡萝卜素中间产物，这些中间产物在野生型中不存在。其中一些是虾青素合酶的底物和中间产物。通过进一步转化虾青素合酶基因，它们可以进一步转化为虾青素。然而，三种中间产物表现出以前从未发现过的异常光吸收光谱。这些新的酮类胡萝卜素通过与大肠杆菌中生成的参考化合物的 HPLC 共色谱分析得到鉴定，其中一种 3-HO-4-酮-7'，8'-二氢-β-胡萝卜素还通过 NMR 光谱鉴定。其他两种是 4-酮-β-玉米黄质和 4-酮-7'，8'-二氢-β-胡萝卜素。讨论了它们的生物合成途径及其起源于新茄红素以及它们在我们的转化体中合成的原因。
