Jiang Xi, Payá-Tormo Lucía, Coroian Diana, García-Rubio Inés, Castellanos-Rueda Rocío, Eseverri Álvaro, López-Torrejón Gema, Burén Stefan, Rubio Luis Manuel
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Pozuelo de Alarcón, 28223, Madrid, Spain.
Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaría y de Biosistemas, Universidad Politécnica de Madrid, 28040, Madrid, Spain.
Commun Biol. 2021 Jan 4;4(1):4. doi: 10.1038/s42003-020-01536-6.
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [FeS] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.
在真核生物中实现固氮工程需要功能性固氮酶结构蛋白的高表达，这一目标尚未实现。在这里，我们构建了一个基于知识的文库，其中包含来自不同生态位和代谢特征的原核生物的32个固氮酶nifH序列，并与烟草中的快速筛选相结合，以鉴定用于植物线粒体表达的优质NifH变体。三种NifH变体在烟草线粒体中表现出色，并在酵母中进一步测试。嗜热氢杆菌（Aquificae）的NifH从酵母线粒体中大量分离出来，并满足高效固氮对NifH蛋白的要求，包括用于底物还原的电子转移、P簇成熟和FeMo-辅因子生物合成。在烟草叶片中表达的嗜热氢杆菌NifH的固氮酶活性低于酵母中的NifH。然而，体外将[FeS]簇从NifU转移到NifH可使烟草分离的NifH的活性提高10倍，这表明植物线粒体[Fe-S]簇的可用性是构建植物固氮酶的一个瓶颈。
