ME: Interagency Announcement of Opportunities in Metabolic Engineering: Metabolic Engineering of Plant Vitamin C Biosynthesis for Improved Nutrition and Health

与大多数动物不同，人类不能合成维生素C，因此对这种营养素有绝对的饮食需求。植物是膳食维生素C的主要来源，维生素C是包括胶原蛋白合成在内的几种代谢功能的关键。 因为维生素C是一种很好的抗氧化剂，它也可以用作天然防腐剂。因此，维生素C含量较高的新鲜农产品应该具有更好的保质期和消费者吸引力。此外，增加植物中的维生素C将导致每份新鲜水果或蔬菜的摄入量增加，这将对人类健康产生积极影响。为了实现这一目标，我们将设计模式植物拟南芥，以获得更高的稳态维生素C水平。拟南芥虽然不是人类饮食的一部分，但却是一个重要的模型，因为它是唯一具有完全测序基因组的植物。与动物不同，植物中维生素C的生物合成代谢网络目前仍不完全清楚。已经提出了植物系统的途径，然而，可能类似于动物途径的平行途径也可能在植物中起作用。 该途径的证据来自于实验，其中编码L-古洛糖酸-g-内酯氧化酶（GLOase）的cDNA（动物途径中的末端酶）在烟草和莴苣中表达，这导致维生素C水平增加5-7倍。该项目的具体目标包括代谢工程正常和维生素C缺乏的拟南芥突变体表达GLOase，以确定是否有任何植物途径中间体直接提供GLOase或其前体。将通过臭氧筛选和维生素C分析在活化标记的拟南芥品系中鉴定编码维生素C途径酶的基因，以填补途径中剩余的缺口。该项目的成果预计将导致（a）拟南芥转化体的维生素C比野生型更大的池，（B）维生素C合成的代谢步骤相关的植物基因的鉴定，和（c）动物途径的步骤也在植物中运作的进一步知识。

Humans, unlike most animals, cannot synthesize vitamin C and thus have an absolute dietary requirement for this nutrient. Plants are the primary source of dietary vitamin C, which is key to several metabolic functions including collagen synthesis. Because vitamin C is a good anti-oxidant it can also be used as a natural preservative. Fresh produce with higher levels of vitamin C should therefore have improved shelf-life and consumer appeal. Additionally, increasing vitamin C in plants would result in higher intake per portion of fresh fruit or vegetables that would have a positive impact on human health. Toward this goal we will engineer the model plant Arabidopsis thaliana for higher steady state levels of vitamin C. Arabidopsis, while not part of human diet, is an essential model because it is the only plant with a fully sequenced genome. Unlike animals, the vitamin C biosynthetic metabolic network in plants is at present still not completely known. A pathway for plant systems has been proposed however, a parallel pathway, possibly similar to the animal pathway, may also operate in plants. Evidence for this pathway comes from experiments in which a cDNA encoding L-gulono-g-lactone oxidase (GLOase), the terminal enzyme in the animal pathway was expressed in tobacco and lettuce, which resulted in a 5-7 fold increase in vitamin C levels. Specific aims of the project include metabolically engineering normal and vitamin C-deficient Arabidopsis mutants to express GLOase to determine if any of the plant pathway intermediates directly supply GLOase or its precursors. Genes encoding vitamin C pathway(s) enzymes will be identified in activation tagged Arabidopsis lines by ozone screening and vitamin C analysis to fill in remaining gaps in the pathway(s). The outcomes of this project are anticipated to result in (a) Arabidopsis transformants with larger pools of vitamin C than the wild type, (b) identification of plant genes related to metabolic steps of vitamin C synthesis, and (c) further knowledge of which steps of the animal pathway also operate in plants.