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Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N2. Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the accessory proteins involved in its assembly and function are O2 sensitive and only sparingly soluble in non-native hosts. We generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH), which carries a [4Fe-4S] cluster in its active form. NifH from Hydrogenobacter thermophilus was targeted to mitochondria together with the putative peptidyl prolyl cis-trans isomerase NifM from Azotobacter vinelandii to assist in NifH polypeptide folding. The isolated NifH was partially active in electron transfer to the MoFe protein nitrogenase component (NifDK) and in the biosynthesis of the nitrogenase iron-molybdenum cofactor (FeMo-co), two fundamental roles for NifH in N2 fixation. NifH functionality was, however, limited by poor [4Fe-4S] cluster occupancy, highlighting the importance of in vivo [Fe-S] cluster insertion and stability to achieve biological N2 fixation in planta. Nevertheless, the expression and activity of a nitrogenase component in rice plants represents the first major step to engineer functional nitrogenase in cereal crops.

The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe-9S-C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) found at the nitrogenase active site. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe-S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe-4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals.

Many highly valued chemicals in the pharmaceutical, biotechnological, cosmetic, and biomedical industries belong to the terpenoid family. Biosynthesis of these chemicals relies on polymerization of Isopentenyl di-phosphate (IPP) and/or dimethylallyl diphosphate (DMAPP) monomers, which plants synthesize using two alternative pathways: a cytosolic mevalonic acid (MVA) pathway and a plastidic methyleritritol-4-phosphate (MEP) pathway. As such, developing plants for use as a platform to use IPP/DMAPP and produce high value terpenoids is an important biotechnological goal. Still, IPP/DMAPP are the precursors to many plant developmental hormones. This creates severe challenges in redirecting IPP/DMAPP towards production of non-cognate plant metabolites. A potential solution to this problem is increasing the IPP/DMAPP production flux in planta. Here, we aimed at discovering, understanding, and predicting the effects of increasing IPP/DMAPP production in plants through modelling. We used synthetic biology to create rice lines containing an additional ectopic MVA biosynthetic pathway for producing IPP/DMAPP. The rice lines express three alternative versions of the additional MVA pathway in the plastid, in addition to the normal endogenous pathways. We collected data for changes in macroscopic and molecular phenotypes, gene expression, isoprenoid content, and hormone abundance in those lines. To integrate the molecular and macroscopic data and develop a more in depth understanding of the effects of engineering the exogenous pathway in the mutant rice lines, we developed and analyzed data-centric, line-specific, multilevel mathematical models. These models connect the effects of variations in hormones and gene expression to changes in macroscopic plant phenotype and metabolite concentrations within the MVA and MEP pathways of WT and mutant rice lines. Our models allow us to predict how an exogenous IPP/DMAPP biosynthetic pathway affects the flux of terpenoid precursors. We als

Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins. Here we used TPs from four Arabidopsis thaliana proteins, three related to photosynthesis (chlorophyll a/b binding protein, Rubisco activase) and photo-protection (tocopherol cyclase) and one involved in the assimilation of ammonium into amino-acids, and whose expression is most abundant in the root (ferredoxin dependent glutamate synthase 2), to determine whether they were able to mediate import of a nuclear-encoded marker protein into plastids of different tissues of a dicot and a monocot species. In A. thaliana, import and processing efficiency was high in all cases, while TP from the rice Rubisco small chain 1, drove very low import in Arabidopsis tissues. Noteworthy, our results show that Arabidopsis photosynthesis TPs also mediate plastid import in rice callus, and in leaf and root tissues with almost a 100% efficiency, providing new biotechnological tools for crop improvement strategies based on recombinant protein accumulation in plastids by the expression of nuclear-encoded transgenes.

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism.