Directed Evolution of Nitrogenase

Without artificial nitrogen fixation for fertilisers, the world could only support 3.5 billion of the 7.5 billion population. Cereal crops do not fix nitrogen or form intimate associations with nitrogen-fixing bacteria; consequently, global agriculture relies on application of fertilisers. These both damage the environment and are costly. This project will combine supervisory expertise on nitrogenase systems and directed evolution to develop a synthetic solution. The nitrogenase enzyme catalyses the conversion of dinitrogen to ammonia, a reaction known as biological nitrogen fixation. Synthetic biology can be used to engineer nitrogen fixation in new organisms by transferring nif genes from nitrogen-fixing species. Heterologous nif genes engineered into E. coli have lower activity than native nitrogen-fixing clusters because of non-optimal interactions of host genes with the nif operons and unbalanced nif gene expression. The MoFe nitrogenase has an ATPase component Fe protein (NifH) and a MoFe component which contains the active site of nitrogen reduction. Nitrogenase is inactivated by oxygen, which limits natural and biotechnological expression. Paenibacillus polymyxa (ATCC 15970) has a minimal nif operon of 10 kb, encoding these components and the essential assembly factors. When expressed in E. coli, this operon has 10 per cent of wild-type activity. I will re-clone the operon and evolve it for improved activity, and potentially improved oxygen tolerance. In nature, bacteria under nitrogen stress activate o54 promoters which increases expression of nitrogen fixation genes and genes involved in the nitrogen scavenging response. To evolve nitrogenase for improved activity, ammonia reporter circuits based on o54 promoters will be used to screen for improved variants, drive selection and inversely couple intracellular ammonia levels to nitrogenase expression.

The project has five parts to provide contingencies:
1. I will rationally engineer designs to improve efficiency such as faster nitrogenase ATPase component, NifH (Seefeldt & Mortenson, 1993) or to improve o2 protection on heterologous nitrogenase by adding the protective protein FeSII or NifH-SOD (superoxide dismutase) fusions.
2. I will build a library of reporter circuits using E. coli o54 promoters with different maximal expression rates and dose-response behaviour to discriminate between members of a nitrogenase variant library with different activities.
3. I will evolve E. coli for ammonia production and oxygen tolerance by screening fluorescent reporter output from the synthetic reporter circuits in (2), or through linking them directly to a gene conferring a selective advantage, such as an antibiotic resistance cassette.
4. A recently-developed evolution platform based on filamentous phage (Brodel et al, 2016) allows tuneable selection pressures and mutation rates, which is an advantage over other directed evolution systems. I will apply this platform to an important worldwide problem - how to engineer biological nitrogen fixation. I will use ammonium sensitive circuits developed in (2) for selection. We will select for phage-driven nif regulon evolution in nitrogen-poor microaerobic conditions.
5. Through use of the reporter circuits in (2) nif gene expression can be linked to host nitrogen status, thereby limiting metabolically expensive nif gene expression in non-nitrogen-fixing conditions, improving activity.