Allen Robert S, Tilbrook Kimberley, Warden Andrew C, Campbell Peter C, Rolland Vivien, Singh Surinder P, Wood Craig C
CSIRO Agriculture and Food Canberra, ACT, Australia.
CSIRO Land and Water Canberra, ACT, Australia.
Front Plant Sci. 2017 Mar 3;8:287. doi: 10.3389/fpls.2017.00287. eCollection 2017.
The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in . We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.
氮肥的工业生产和使用涉及巨大的环境和经济成本。为满足世界对可持续食品、纤维和生物燃料日益增长的需求，需要采取减少对化肥依赖的策略。生物固氮是固氮细菌特有的过程，由固氮酶复合物催化，在植物细胞中重建此功能是一项旨在减少化肥使用的宏伟生物技术策略。在此，我们证实来自固氮菌的全套生物合成和催化固氮酶（Nif）蛋白可在植物中单独表达为线粒体靶向肽（MTP）-Nif融合蛋白。我们表明这些蛋白能正确靶向植物线粒体基质，这是一个具有潜在支持固氮酶功能的生化和遗传特征的亚细胞位置。虽然通过蛋白质免疫印迹分析可检测到所有Nif蛋白（B、D、E、F、H、J、K、M、N、Q、S、U、V、X、Y和Z），但NifD催化组分含量最少。为解决此问题，基于固氮酶钼铁蛋白异二聚体的晶体结构设计了NifD和NifK之间的翻译融合蛋白。该融合蛋白实现了NifD:NifK等摩尔化学计量比，并提高了植物中NifD的表达水平。最后，成功共表达了四种MTP-Nif融合蛋白（B,S,H,Y），表明固氮酶的多个组分可靶向植物线粒体。这些结果证实了在能够支持将氮气转化为氨的细胞内环境中，在植物细胞中重建固氮酶完整组件的可行性。
