Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast

Abstract

The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct nif gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe–S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe–S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N2-fixing cereals.

Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

“…1), such as those required for iron-sulfur cluster assembly, could also be considered. Two recent studies have shown that the [Fe-S] cluster biosynthesis components from eukaryotic organelles can substitute for NifU and NifS to incorporate the [4Fe-4S] cluster into the Fe protein of nitrogenase (19,20).…”

“…A number of studies have suggested chloroplasts, root plastids, or mitochondria as suitable locations for expression of nitrogenase in eukaryotes (18)(19)(20). These energy-conversion organelles can potentially provide reducing power and ATP required for the nitrogen-fixation process.…”

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

“…17 Yeast cells are convenient hosts to test eukaryotic expression of Nif proteins as they can be grown at varied O 2 levels, to reduce the negative effects from O 2 on Nif protein maturation. They are also easy to transform and maintain, enabling larger and more complex libraries to be constructed and analyzed.…”

Keeping the nitrogen-fixation dream alive

“…In recent studies it has been shown that an active dinitrogenase reducatase can be produced in eukaryotes, provided it is targeted to either mitochondria in yeast or plastids in tobacco (12,13). These achievements are noteworthy for two reasons.…”

“…However, neither NifU nor NifS is necessary for the production of an active nitrogenase reductase in either yeast or tobacco (12,13), indicating that counterparts involved in assembly of iron-sulfur cofactors in eukaryotic organisms-for example Isu and Nfs1 (15)-can substitute for the nitrogen-fixation-specific assembly components. It should be noted that NifU and NifS also supply the iron-sulfur building blocks necessary for formation of the complex cofactors associated with dinitrogenase (Fig.…”