Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of .

Biological nitrogen fixation, the conversion of inert N to metabolically tractable NH, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo--homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as , represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation.

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation.

Biological nitrogen fixation (BNF) is the reduction of N into NH in a group of prokaryotes by an extremely O-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph.

Biosynthesis of cofactor-activatable iron-only nitrogenase in Saccharomyces cerevisiae.

Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe-only nitrogenase component proteins in the low-oxygen mitochondria matrix of S.

Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria.

Background: There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions.

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle's high O 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.