Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

通过直接引入nif基因在真核细胞中工程化生物固氮需要优雅的合成生物学方法，以确保活性氮酶生物合成所需的成分稳定并以适当的化学计量表达。以前，发现来自葡萄固氮菌和产酸克雷伯菌的固氮酶MoFe蛋白的NifD亚基分别在酵母和植物线粒体中不稳定，这是真核细胞中活性MoFe蛋白装配的瓶颈。在这项研究中，我们划定了该区域，并随后确定了来自产氧假丝酵母的关键残基NifD-R98 。赋予线粒体蛋白酶介导的降解敏感性。观察到的效果是普遍的，因为在所有分析的NifD蛋白中R98是保守的。在酵母线粒体中观察到来自四个代表性重氮营养菌的NifD蛋白，但不是其R98变体，不稳定。此外，通过从重构酵母，线粒体加工肽酶（MPP的）稻，烟草，和拟南芥在大肠杆菌，我们证明了MPP负责NifD的裂解。这些结果表明由于MPPs的切割而对线粒体中NifD蛋白的稳定性具有普遍的影响。获得了具有高水平固氮酶活性的NifD-R98变体，具有将活性MoFe蛋白稳定地靶向线粒体的潜力。这种重建方法可以帮助预先评估Nif蛋白在植物中的表达稳定性，并为工程化细胞器中的活性氮酶铺平道路。