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。植物是膳食中维生素C的主要来源，而维生素C对包括胶原蛋白合成在内的几种代谢功能至关重要。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长的保质期和对消费者的吸引力。此外，增加植物中的维生素C会导致每一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，以获得更高的维生素c稳态水平。拟南芥虽然不是人类饮食的一部分，但它是一个必不可少的模型，因为它是唯一具有完整基因组测序的植物。与动物不同，植物体内维生素C的生物合成代谢网络目前还不完全清楚。植物系统的途径已被提出，然而，一个平行的途径，可能类似于动物途径，也可能在植物中起作用。该途径的证据来自于实验，在烟草和生菜中表达了一种编码l -古洛诺-g-内酯氧化酶（GLOase）的cDNA，该酶是动物途径的末端酶，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。通过臭氧筛选和维生素C分析，将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，以填补该途径的空白。该项目的结果预计将导致(a)拟南芥转化体比野生型具有更大的维生素C库，(b)鉴定与维生素C合成代谢步骤相关的植物基因，以及(C)进一步了解动物途径的哪些步骤也在植物中起作用。

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.