Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles

Abstract

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction ofnifgenes 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 fromAzotobacter vinelandiiandKlebsiella oxytocawere 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, fromK. oxytocathat 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, andArabidopsis thalianainEscherichia 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.

A Research Road Map for Responsible Use of Agricultural Nitrogen

“…These features may explain why the process was never co-opted from microbes during plant evolution. Nonetheless, progress has been made toward this objective (Burén et al, 2017;Burén and Rubio, 2018;Eseverri et al, 2020;Xiang et al, 2020), and such work is testing and extending our understanding of BNF. Given the current state of knowledge and technology, the greatest impediment to solving the N problems of agriculture through N fixing plants may turn out to be public acceptance of such technology rather than our ability to develop it.…”

Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants

“…To date, functional NifH, NifU, and NifB have been purified from mitochondria of aerobically cultured Saccharomyces cerevisiae cells 10 , 11 , while active NifU and NifH were isolated from chloroplasts of Nicotiana benthamiana at the end of the dark period 12 . Also, the reported low stability of the NifD protein 13 has now been improved in two recent studies that identified key residues in the NifD sequence as susceptible to cleavage upon mitochondria import 14 , 15 . Notwithstanding these achievements, detailed analysis of yeast mitochondria-targeted Azotobacter vinelandii NifH has been hampered by low protein solubility resulting in suboptimal yields 10 .…”

Sustainable Biological Ammonia Production towards a Carbon-Free Society

“…Furthermore, the NifD protein itself contains an MTPrecognition sequence, which is subject to processing, thereby making functional expression difficult. However, R98 has been identified as a key mutation for this cleavage, and an R98P mutant that is resistant to processing and retains high levels of activity in the mitochondria has been obtained [75]. Another study found that the Y100Q mutant was also resistant to processing within the plant and yeast mitochondria [76].…”