Ravanello Monica P, Ke Dangyang, Alvarez Julie, Huang Bihua, Shewmaker Christine K
Monsanto Company, Calgene Campus, 1920 Fifth St, Davis, CA 95616, USA.
Metab Eng. 2003 Oct;5(4):255-63. doi: 10.1016/j.ymben.2003.08.001.
Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values.
类胡萝卜素最近备受关注，因为它们对人类健康具有潜在的积极益处，并且在食品和动物饲料中都有应用。先前在过量表达细菌八氢番茄红素合酶基因（crtB）的油菜籽（甘蓝型油菜）中的研究表明，类胡萝卜素含量发生了变化，包括八氢番茄红素和下游代谢产物如β-胡萝卜素在内的类胡萝卜素总水平提高了50倍，β-胡萝卜素与α-胡萝卜素的比例为2:1。这一结果增加了一种可能性，即结合crtB获得的通量增加，该途径中代谢物的组成可能会进一步改变。在此，我们报告了在转基因油菜籽中与八氢番茄红素合酶（crtB）一起构建并表达的用于香叶基香叶基二磷酸合酶（crtE）、八氢番茄红素去饱和酶（crtI）和番茄红素环化酶（crtY以及植物甘蓝型油菜番茄红素β-环化酶）的其他细菌基因的表达情况。通过高效液相色谱法对类胡萝卜素水平进行分析发现，同时表达crtB和crtI的双构建体中八氢番茄红素水平降低了90%。所有双构建体的转基因种子，包括表达细菌crtB和植物番茄红素β-环化酶的构建体，其类胡萝卜素总水平都有所增加，类似于之前单独表达crtB时观察到的情况，但与原始构建体相比，β-胡萝卜素与α-胡萝卜素的比例变化不大。然而，当由细菌八氢番茄红素合酶、八氢番茄红素去饱和酶和番茄红素环化酶基因组成的三构建体一起表达时，β-胡萝卜素与α-胡萝卜素的比例从2:1增加到了3:1。这一结果表明，细菌基因可能形成一种聚集复合物，通过底物通道化使所有三种蛋白质在体内具有活性。这一发现应该能够进一步操纵类胡萝卜素生物合成途径，以获得具有更高农艺价值、动物饲料价值和人类营养价值的下游产品。
