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，因此对这种营养物质有绝对的饮食需求。植物是饮食中维生素C的主要来源，维生素C是包括胶原合成在内的几种代谢功能的关键。由于维生素C是一种很好的抗氧化剂，它也可以用作天然防腐剂。因此，含有较高水平维生素C的新鲜农产品应该会延长保质期，并吸引消费者。此外，增加植物中的维生素C会导致每部分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，我们将改造模式植物拟南芥，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，但它是一个基本的模型，因为它是唯一具有完全测序的基因组的植物。与动物不同，植物中的维生素C生物合成代谢网络目前还不完全清楚。然而，已经提出了一种植物系统的途径，一种平行的途径，可能类似于动物的途径，也可能在植物中运行。这一途径的证据来自于实验，在烟草和生菜中表达了编码动物途径末端酶L-古罗诺-g-内酯氧化酶(GLOase)的基因，导致维生素C水平增加了5-7倍。该项目的具体目标包括用代谢工程技术改造正常和维生素C缺乏的拟南芥突变体来表达GLOase，以确定是否有任何植物途径的中间产物直接供应GLOase或其前体。编码维生素C途径(S)酶的基因将通过臭氧筛选和维生素C分析在激活标记的拟南芥品系中找到，以填补途径中的剩余空白(S)。预计该项目的结果将导致：(A)拟南芥转化体的维生素C库比野生型更大，(B)鉴定与维生素C合成的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发挥作用。

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.