Abstract
Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.
Highlights
Nitrogen fixation, that is, the reduction of N2 to NH3, is a prokaryotic process catalyzed by a family of enzymes called nitrogenases, the most ecologically relevant and abundant one being the Mo-nitrogenase.[1]
The nif B, nif E, nif N, and nif H gene products have been shown to carry out all essential biochemical reactions in FeMo-co biosynthesis in vitro,[8] the exact number of nif genes required for the genetic transfer of nitrogen fixation among bacterial species depends on the nature of both the nitrogenase source and the engineered host.[9−11] To add more complexity, Nif protein stoichiometry has been shown to be dynamic and strongly regulated.[12]
Gene products were targeted to the yeast mitochondria using three different mitochondria leading sequences
Summary
That is, the reduction of N2 to NH3, is a prokaryotic process catalyzed by a family of enzymes called nitrogenases, the most ecologically relevant and abundant one being the Mo-nitrogenase.[1] The Mo-nitrogenase is a two-component metalloenzyme consisting of a molybdenum−iron (MoFe) protein that catalyzes N2 reduction and an iron (Fe) protein that acts as specific electron donor to the MoFe protein.[2] The Fe protein is a homodimer of the nif H gene product carrying a single [Fe4S4] cluster between its subunits, whereas the MoFe protein is a heterotetramer of the nif D and nif K gene products carrying two pairs of a [Fe8S7] cluster and a [Fe7S9MoC-homocitrate], designated as the Pcluster and FeMo-co, respectively.[3,4] In addition to the nitrogenase structural genes, a number of accessory nitrogen fixation (nif) gene products are required for the maturation of the structural components to their catalytically active forms These include proteins that interact in complex biosynthetic pathways for the assembly of nitrogenase metal clusters and their insertion into target apo-proteins.[5]. ATP is required for electron transfer from the NifH [Fe4S4] cluster to the P-cluster of the NifDK component during N2 reduction, and possibly for P-cluster maturation and FeMo-co synthesis.[2,5] While plastid proteins can be expressed either via transformation of the plastid genome, or imported post-translationally following nuclear expression, transformation of the mitochondrial genome is more difficult.[16]
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