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.

中文翻译：

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双低油菜籽中多种细菌类胡萝卜素基因的协调表达，导致类胡萝卜素产量的改变。

类胡萝卜素由于其对人体健康的潜在积极益处以及其在食品和动物饲料中的实用性，最近受到了广泛关注。先前在油菜籽（甘蓝型油菜）种子中过表达细菌八氢番茄红素合酶基因（crtB）的工作表明，类胡萝卜素含量发生了变化，从而使包括类胡萝卜素和下游代谢产物（如β-胡萝卜素）在内的类胡萝卜素的总含量提高了50倍。 ，其中β-胡萝卜素与α-胡萝卜素的比例为2：1。该结果增加了该途径中代谢物的组成与crtB所获得的通量增加相关的可能性。在这里，我们报告了香叶基香叶基二磷酸合酶（crtE）的其他细菌基因的表达，在转基因双低油菜籽种子中，与八氢番茄红素合酶（crtB）一起工程改造了八氢番茄红素去饱和酶（crtI）和番茄红素环化酶（crtY和植物甘蓝型油菜番茄红素β-环化酶）。通过HPLC对类胡萝卜素水平的分析显示，与crtI结合表达crtB的双构建体，其番茄红素水平降低了90％。来自所有双重构建体的转基因种子，包括表达细菌crtB和植物番茄红素β-环化酶的转基因种子，其总类胡萝卜素的水平均与以前单独表达crtB的水平相似，但对转基因种子的影响却很小。 β-胡萝卜素与原始胡萝卜素的比例。但是，当由细菌八氢番茄红素合酶组成的三重结构时，β-胡萝卜素与α-胡萝卜素的比例从2：1增加到3：1，番茄红素去饱和酶和番茄红素环化酶基因一起表达。该结果表明细菌基因可以形成聚集复合物，其允许所有三种蛋白质通过底物通道在体内活性。该发现应允许进一步加工具有增强的农艺，动物饲料和人类营养价值的下游产品的类胡萝卜素生物合成途径。