Engineering Nitrogenases for Synthetic Nitrogen Fixation: From Pathway Engineering to Directed Evolution

Abstract

Globally, agriculture depends on industrial nitrogen fertilizer to improve crop growth. Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution. A potential solution would be to harness nitrogenases-enzymes capable of converting atmospheric nitrogen N₂ to NH₃ in ambient conditions. It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants, or bacteria that form symbiotic relationships with crops, to support growth and reduce dependence on industrially produced fertilizer. This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants.

Fig. 1.

Minimum set of nif genes essential for nitrogen fixation with molybdenum-iron nitrogenase. Please note that shown stoichiometry has not been adjusted. NifB contains one catalytic cluster (shown in white) and 2 substrate [4Fe-4S] clusters that react to produce the NifB cofactor. NifEN matures the NifB cofactor producing the FeMo cofactor. The molybdenum-iron (MoFe) nitrogenase (NifHDK) contains the FeMo cofactor at its active site. Electron donors transfer single electrons to the [4Fe-4S] cluster at the interface of the NifH homodimer. Electrons are moved from the [4Fe-4S] cluster into the active site of nitrogenase using energy produced by ATP hydrolysis by NifH. A minimum of 8 electrons are used to reduce each molecule of N₂.

Fig. 2.

A hypothetical engineered nitrogen-fixing organism promoting wheat growth, highlighting current areas of focus for engineering nitrogenase in synthetic biology. (A) Engineered nitrogen-fixing organisms convert atmospheric nitrogen into ammonia, which promotes plant growth. (B) Engineered organisms exchange fixed nitrogen in the form of ammonia for carbon sources and other nutrients. They may also communicate with the plant through exchange of quorum signaling molecules (colored circles). Artificial oxygen protection systems may be employed to limit oxygen damage to nitrogenase. (C) Approaches used to optimize nitrogenase activity in heterologous contexts include inducible expression or overexpression of nitrogenase genes, tuning stoichiometries of nitrogenase proteins by altering promoters, engineering ribosome binding sites and codon usage, managing supply of ATP and electrons through incorporating additional electron donors, or supplying the organism with high concentrations of sugar, which mimics the exchange of nutrients present in a symbiotic association with plants.