Abstract
Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana. NifB proteins were targeted to the mitochondria, where O2 consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe–S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.
Highlights
Agricultural systems in developed countries are largely based on cereal crops, which provide most of the calories and proteins in the human diet (Borlaug, 2002)
Expression vectors were cointroduced into S. cerevisiae strain W303-1a, generating yeast strains SB09Y and SB10Y for the expression of mitochondria targeted NifU, NifS, and FdxN, together with NifBAv-His10 or NifBMi-His10, respectively (Table 1)
SU9-NifBMi-His10 when expressed in S. cerevisiae and N. benthamiana
Summary
Agricultural systems in developed countries are largely based on cereal crops, which provide most of the calories and proteins in the human diet (Borlaug, 2002). Nitrogen and water availability are the most important factors limiting cereal crop productivity. Over the last 100 years, cereal crop yields have been increased by addition of chemically synthesized nitrogen fertilizers (Smil, 2000; Galloway et al, 2008). The extensive use of these commercial nitrogen fertilizers in developed countries poses enormous and pressing environmental threats (Vitousek et al, 1997). The cost of chemical fertilizers is prohibitive for poor farmers, and they are scarcely used in most of Africa with consequence of poverty and hunger derived from. One way to tackle this problem could be the generation of so-called nitrogen-fixing plants, aimed to solve the nitrogen problem (Stokstad, 2016; Vicente and Dean, 2017)
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