Phylloquinone Biosynthesis in Plants: Enzyme Discovery and Pathway Flux Control

科学价值：叶绿醌（维生素K1）是一种脂溶性分子，在光合作用中起着电子载体的关键作用。它只由植物和某些细菌合成。人类需要它作为血液凝固和骨骼钙固定的膳食供应。虽然叶绿醌的生物合成已经被默认为已经定义了十多年，细菌和植物基因组的比较分析表明，参与叶绿醌生物合成的几种酶缺失。此外，植物已经进化出一种非凡的代谢结构来合成这种维生素，包括基因融合的多个事件，高度分化的酶，以及叶绿体和过氧化物酶体中的分离区室化。植物叶绿醌的生物合成也通过共同的中间体与生育酚（维生素E）、叶绿素和水杨酸盐（一种植物激素）的生物合成有关。结合比较基因组学、反向遗传学、代谢分析、代谢工程和蛋白质生物化学，该项目旨在表征叶绿醌生物合成中缺失的关键步骤，并确定植物如何调节该途径与其他代谢分支之间的通量分配。由于植物性食物是人类膳食维生素K的主要来源，因此本项目将提高人们对叶绿醌和其他植物微量营养素的认识（维生素，矿物质，抗氧化剂）营养的内布拉斯加州4-H计划（头部，手，心脏和健康）的儿童。为此，该项目将开发两个演示和一个研讨会，在此期间，中学生年龄的儿童将学习计算他们每天在家里和学校用餐时获得的每种维生素的量，以及这些值与推荐的膳食摄入量的比较。该项目还将有助于在PI实验室培训两名本科生研究助理，两名博士生和一名博士后。

Scientific merit: Phylloquinone (vitamin K1) is a fat-soluble molecule that plays a crucial role as electron carrier in photosynthesis. It is synthesized exclusively by plants and certain bacteria. Humans require it as a dietary supply for blood coagulation and for calcium fixation in bones. Although the biosynthesis of phylloquinone has been tacitly assumed to have been defined for more than a decade, the comparative analysis of bacterial and plant genomes reveals that several enzymes involved in phylloquinone biosynthesis are missing. Moreover, plants have evolved an extraordinary metabolic architecture to synthesize this vitamin, including multiple events of gene fusion, highly diverged enzymes, and likely, a separated compartmentalization in chloroplasts and peroxisomes. Plant phylloquinone biosynthesis is also connected via shared intermediates to that of tocopherols (vitamin E), chlorophylls, and salicylate (a plant hormone). Combining comparative genomics, reverse genetics, metabolic profiling, metabolic engineering, and protein biochemistry, this project aims to characterize the missing key steps of phylloquinone biosynthesis, and to establish how plants regulate the flux splits between this pathway and other metabolic branches.Broader impacts: As plant-based foods represent the main source of dietary vitamin K for humans, this project will raise the awareness of phylloquinone and other plant micronutrient (vitamins, minerals, antioxidants) nutrition for children of the Nebraska 4-H program (Head, Hands, Heart, and Health). For that, the project will develop two presentations and a workshop during which children of middle-school age will learn to calculate how much of each vitamin they get per day during their meals at home and at school, and how these values compare to the recommended dietary intakes. This project will also contribute to the training of two undergraduate research assistants, two PhD students, and one postdoctoral associate in the laboratory of the PI.