A step closer to nitrogen-fixing plants

The study shows the power of combining synthetic biology and hard-core biochemistry to engineer a critical step of nitrogenase biosynthesis in yeast..


The study shows the power of combining synthetic biology and hard-core biochemistry to engineer a critical step of nitrogenase biosynthesis in yeast.

Nitrogen-fixing prokaryotes (bacteria and archaea) can transform the highly inert nitrogen gas (N2) present in the atmosphere into ammonia, a reduced form that can be readily assimilated by other organisms, including plants. They do so by means of nitrogenase, a complex, demanding, and exclusive enzyme. Agricultural crop production depletes soils of assimilable nitrogen forms, thus imposing the need for nitrogen fertilization. This is especially true for cereals, humanity’s food staple. Synthesizing and applying nitrogen fertilizers is not only costly but also environmentally unfriendly, making cereal production unsustainable. Endowing cereals with the ability to fix nitrogen, that is, with an active nitrogenase, would represent a major advance towards sustainably feeding the ever-increasing world population while mitigating the negative effects of agriculture production on climate change, one of the topics that will be discussed during the COP25 meeting starting this week in Madrid (https://www.cop25.cl/#/)

With sustained funding from the Bill & Melinda Gates Foundation, Luis Rubio and his team, from the CBGP (UPM-INIA), have undertaken to ambitiously modify prokaryotic nitrogenase systems so that they can be functionally expressed in plants. This is a long-range, complex endeavor, since nitrogen fixation is incompatible with oxygen and nitrogenase contains unique, extremely labile heterometallic clusters not found anywhere else. In previous work, the Rubio lab has shown that one of the nitrogenase components can be synthesized in active form in yeast mitochondria, suggesting that targeting the nitrogen fixation systems to eukaryotic mitochondria has the potential to provide the appropriate environment for nitrogenase synthesis and activity. A very recent publication in PNAS by Burén et al., resulting from a collaborative effort among the laboratories of Luis Rubio (CBGP), Chris Voigt (MIT), Dennis Dean (Virginia Tech), and Alex Guo (Carnegie Mellon), provides a major inroad into this problem by achieving synthesis of active NifB-co in yeast mitochondria. NifB-co is a complex Fe/S cofactor (see Figure), the precursor of the nitrogenase active-site cofactor where the actual reduction of N2 to ammonia takes place. NifB-co is synthesized by means of a specific enzyme, NifB, in combination with other specific enzymes (NifU, NifS, FdxN). Through a clever use of combinatorial synthetic biology and biochemistry, Burén et al tested 28 nifB genes from different organisms, in combination with nifUSfdxN genes in yeast mitochondria. For two of the NifB proteins, unlikely originating from thermophilic archaea, synthesis of active NifB-co bound to NifB was demonstrated. Further yet, for the Methanococcus thermoautotrophicus NifB, the newly synthesized NifB-co could be transferred to its natural carrier, NifX, within mitochondria, and thus proceed towards active-site cofactor synthesis.

The study by Burén et al not only shows that synthesis of nitrogenase-specific, metallic clusters is possible in aerobic yeast mitochondria, but also establishes the minimal requirements for this heterologous synthesis to occur. Furthermore, it highlights the power of combinatorial synthetic biology in providing novel gene combinations that can help overcome the anticipated and observed limitations for functional expression of nitrogenase in plants.

(Press release by Professor Juan Imperial)

About the CBGP

The mission of the CBGP (UPM-INIA) is to carry out fundamental and strategic research in plant science and in microorganisms interacting with plants. The research is focused on understanding important biological processes such as plant development, the interaction of plants with the environment and the mechanisms of plant nutrition. Additionally, CBGP is interested in developing and using computational biology tools to achieve its goals. The acquired knowledge is used to tackle major problems of agriculture and forestry, and to develop novel technological solutions. CBGP (UPM-INIA) has also an educational role and is a reference center for training scientists and Master's and Bachelor-level students in plant biotechnology and genomics. CBGP (UPM-INIA) has been recognized by the Spanish Research Agency as Centre of Excellence Severo Ochoa, the highest institutional recognition of scientific research excellence in Spain CBGP (UPM-INIA).

Saccharomyces cerevisiae strain synthesizing NifB-co in the mitochondria and paving the way for the engineering of N2-fixing eukaryotes. NifB-co is the common metallocluster precursor for the active-site cofactors of all nitrogenases. The color-coding represents the atomic composition of a NifB-co molecule (red, Fe; yellow, S; green, C). Image prepared by Stefan Burén and Xi Jiang.

Original Paper:

Burén, S., Pratt, K., Jiang, X., Guo, Y., Jimenez-Vicente, E., Echavarri-Erasun, C., Dean, D.R., Saaem, I., Gordon, D.B., Voigt, C.A., Rubio, L.M. 2019. Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences. DOI: 10.1073/pnas.1904903116