Folate Synthesis, Catabolism, and Engineering in Plants

叶酸是所有生物体中一碳转移反应的重要辅因子。与植物和微生物不同，人类不能合成叶酸，因此需要在饮食中使用叶酸。叶酸缺乏是世界上最常见的营养缺乏症，其严重后果包括婴儿的神经管缺陷和成年人的血管疾病。由于植物食品是人类饮食中叶酸的单一最大来源，通过代谢工程提高植物叶酸含量是在全球范围内改善人类营养的一种有吸引力的方法。目前对植物中叶酸的合成、分解代谢和调控知之甚少，无法对叶酸水平进行合理的调控。这项研究解决了缺少的基本生化知识，并将朝着工程化本身迈出第一步。在9种与叶酸合成相关的酶中，只有3种是从植物中克隆出来的。因此，计划通过基因组方法和功能互补相结合的方法来克隆其他六种酶，并对重组蛋白进行生化特征分析。基因组序列数据表明，这些酶中的一些在结构上与其他生物体中的对应酶有如此显著的不同，以至于它们可能具有新的特性。这项工程工作将探索整个叶酸途径中的通量在多大程度上通过其蝶呤和对氨基苯甲酸分支的提交酶来调节。这些酶将单独和一起过度表达，主要依赖于预计对最终产物抑制不敏感的非植物酶。第二种工程方法将寻求通过使用反义RNA来阻止这种化合物的循环，从而使叶酸以稳定的形式(5-甲酰基四氢叶酸)过度积累。工程植物中的叶酸和途径中间体将通过高效液相色谱定量，途径通量将使用放射性标记的前体进行测量。还将使用放射性标记的底物来研究叶酸的分解代谢，以确定发生的降解反应和叶酸池翻转的速度。番茄将用于克隆和工程工作，因为：(A)水果的叶酸含量低于叶片，表明在原则上可以提高叶酸含量。(B)叶酸盐在烹调过程中会遭受巨大损失，使水果和果汁成为输送叶酸的有效工具，因为它们是新鲜食用的。(C)番茄很容易变形，是世界主要作物。番茄果实也有可能会耐受非生理性的高叶酸水平，因为它是一个程序化死亡的器官。

Folates are essential cofactors for one-carbon transfer reactions in all organisms. Unlike plants and microorganisms, humans cannot synthesize folates and so require them in the diet. A lack of folates is the world's most common nutrient deficiency and has grave consequences that include neural tube defects in infants and vascular disease in adults. Since plant foods are the single largest source of folates in human diets, enhancing plant folate content by metabolic engineering is an appealing way to improve human nutrition worldwide. At present too little is known about folate synthesis, catabolism, and regulation in plants to undertake the rational engineering of folate levels. This research addresses the missing basic biochemical knowledge and will take the first steps towards engineering itself. Of the nine enzymes specific to folate synthesis, only three have been cloned from plants. It is therefore planned to clone the other six enzymes by combining genomic approaches and functional complementation, and to biochemically characterize the recombinant proteins. Genomic sequence data indicate that some of these enzymes differ so strikingly in structure from their counterparts in other organisms that they are likely to have novel properties. The engineering work will explore the extent to which flux in the whole folate pathway is regulated via the committing enzymes of its pterin and p-aminobenzoate branches. These enzymes will be overexpressed singly and together, relying primarily upon non-plant enzymes that are expected to be insensitive to end-product inhibition. A second engineering approach will seek to divert folates towards overaccumulation in a stable form (5-formyltetrahydrofolate) by using antisense RNA to block recycling of this compound. Folates and pathway intermediates in engineered plants will be quantified by HPLC, and pathway flux will be measured using radiolabeled precursors. Folate catabolism will also be investigated using radiolabeled substrates to establish the degradative reactions that occur and the rate at which the folate pool turns over.Tomato will be used for both the cloning and the engineering work, because: (a) Fruits have lower folate contents than leaves, showing that enhancement is in principle possible. (b) Folates are subject to huge losses during cooking, making fruits and fruit juices an efficient vehicle to deliver folates because they are consumed fresh. (c) Tomato is readily transformable and is a major world crop. It is also possible that the tomato fruit will tolerate unphysiologically high folate levels because it is an organ programmed to die.