Synthesizing active nitrogenase involves about 20 specific proteins that must work individually and coordinately, and whose specific functions are only partially understood. This complexity brings about the very real possibility of the system not working because of an individual or multiple components failing. Such a scenario would become an experimental nightmare, as it would be extremely difficult to diagnose which component(s) are not working. Therefore, our proposal relied on a step-by-step approach in which every single essential component had to be tested individually, and its activity validated. We have resorted to the wide diazotroph biodiversity to look for, and find, adequate variants for each of these components by using an activity screening approach. So far, and understanding that the final goal is still distant, this strategy has proved successful and resulted in a steady progress, marked by an encouraging number of recent milestones:

• Active nitrogenase component II (NifH) isolated from transgenic rice (Baysal et al., 2022)
• Nitrogenase cofactor maturase (NifB) isolated from transgenic rice is active in FeMo-co synthesis (He et al., 2022)
• Fast colorimetric method to measure nitrogenase activity in vitro (Payá-Torno et al., 2022)
• Functional nitrogenase cofactor maturase (NifB) isolated from Nicotiana benthamiana chloroplasts and mitochondria (Jiang et al., 2022)
• Biosynthesis of cofactor-activatable iron-only nitrogenase in Saccharomyces cerevisiae (López-Torrejón et al., 2021)
• Identification of superior nitrogenase component II (NifH) variants in Saccharomyces cerevisiae and Nicotiana benthamiana mitochondria (Jiang et al., 2021)
• Structural insights into nitrogenase cofactor maturation obtained from NifB crystals (Fajardo et al., 2020)
• Synthetic tools to optimize active NifH in Nicotiana benthamiana chloroplasts (Eseverri et al., 2020)
• Biosynthesis of critical active-site cofactor precursor in yeast mitochondria (Buren et al., 2019)
• Goldenbraid molecular cloning system for heterologous nitrogenase protein expression in Saccharomyces cerevisiae (Pérez-González et al., 2017)
• Isolation of mitochondria targeted nitrogenase cofactor maturase NifB (Buren et al., 2017)
• Formation of nitrogenase component I (NifDK) tetramer in yeast mitochondria (Buren et al., 2017)
• Expression of a functional oxygen-labile nitrogenase component II (NifH) in yeast mitochondria (López-Torrejón et al., 2016)