Folate Synthesis, Turnover, and Engineering in Plants

叶酸是所有生物体中一碳转移反应的重要辅助因子，但仅由植物和微生物产生。人体所需的叶酸主要来自植物性食物。叶酸分子有三个部分-蝶啶，对氨基苯甲酸酯（pABA）和谷氨酸-通常也有一个聚谷氨酸尾巴。本研究有三个目的。(a)通过工程改造番茄果实来提高番茄果实中的叶酸水平，使它们过量产生蝶啶和pABA（从先前的工作中可以清楚地看出，仅过量产生蝶啶的果实适度富含叶酸，但已经耗尽了pABA），并分析工程改造的果实中叶酸合成基因的表达。(b)结合联合收割机和生物化学，以确定和表征参与回收叶酸分解产物的酶和基因，因为这种回收似乎是至关重要的，以维持叶酸水平在植物组织中面对高速率的化学或光化学分解。(c)探索降低番茄果实中γ-谷氨酰水解酶活性的工程方法，因为这种酶攻击多聚谷氨酰化叶酸，并可能实际上靶向它们进行破坏，因为去谷氨酰化叶酸更容易在体内分解。更广泛的影响：维生素叶酸缺乏是一个全球性的健康问题，导致可预防的出生缺陷和贫血的巨大负担，并增加血管疾病和癌症的风险。在植物或其他食品生物体中进行叶酸代谢工程（生物强化）是纠正膳食叶酸缺乏症的可行方法，并在几个国家取得进展。这项研究将推进这一国际研究和开发工作，并培养代谢生物化学和工程方面的博士生和博士后。此外，它还将包括对初中和高中的宣传，以提高对叶酸营养的认识，并提高通过生物强化改善叶酸营养的潜力。这次推广活动将包括关于测量食物中叶酸的演示和简单的实验室练习，这将表明水果和蔬菜的重要性。

Folates are vital cofactors for one-carbon transfer reactions in all organisms, but are made only by plants and microorganisms. The folate that humans need comes mainly from plant foods. Folate molecules have three parts - pteridine, p-aminobenzoate (pABA), and glutamate - and usually also have a polyglutamate tail. This research has three aims. (a) To raise folate levels in tomato fruit by engineering them overproduce both pteridine and pABA (it being clear from previous work that fruit overproducing pteridine alone are modestly enriched in folate, but have run out of pABA) and to profile the expression of folate synthesis genes in engineered fruit. (b) To combine genomics and biochemistry to identify and characterize enzymes and genes involved in recycling folate breakdown products, since this recycling appears to be crucial to maintaining folate levels in plant tissues in the face of high rates of chemical or photochemical breakdown. (c) To explore engineering lower gamma-glutamyl hydrolase activity in tomato fruit, since this enzyme attacks polyglutamylated folates and may in effect target them for destruction since deglutamylated folates are more prone to breakdown in vivo.Broader Impact: Deficiency of the vitamin folate is a global health problem that results in a huge burden of preventable birth defects and anemia, and increased risks of vascular disease and cancer. Metabolic engineering of folates in plants or other food organisms (biofortification) is a viable way to correct dietary folate deficiency and is in progress in several countries. This research will advance this international research and development effort, and train PhD students and postdoctorals in metabolic biochemistry and engineering. Furthermore, it will incorporate an outreach to middle and high schools to heighten awareness of folate nutrition and the potential for improving it by biofortification. This outreach will incorporate presentations and simple lab exercises on measuring folate in foods, which will show the importance of fruits and vegetables.