Abstract
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.
Highlights
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved
The nifH sequences were codonoptimized for S. cerevisiae because codon-usage is similar to tobacco[21] and the workflow included downstream expression of tobacco-selected NifH variants in yeast for biochemical characterization
COX4-TS-NifH variants are hereafter denoted as NbNifHXx where Nb stands for the host N. benthamiana, Xx denotes variants collectively, and other superscripts indicate the species from which NifH sequence was obtained
Summary
Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. The difficulty of expressing high levels of soluble and functional NifH in yeast and tobacco poses a major problem for eukaryotic nitrogenase engineering as it is the most abundant Nif protein during N2 fixation[17]. The problem is exacerbated because, in addition to serving NifDK with electrons for substrate reduction, NifH is required to mature P-clusters onto NifDK and for the final steps of FeMo-co biosynthesis in complex with NifEN9 For these reasons it is essential to identify a NifH variant that is highly soluble and stable when expressed at very high levels in a plant cell, and that can perform all three NifH-. Mining of phylogenetically diverse nifH sources can be undertaken in order to find natural NifH proteins with superior properties, a strategy that was successful for NifB11 and for increasing carotenoid levels in “Golden Rice”[20]
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