# Investigation of the Cyanothece nitrogenase cluster in Synechocystis: a blueprint for engineering nitrogen-fixing photoautotrophs

## TL;DR

This study identifies key genes from Cyanothece that enable nitrogen fixation in photosynthetic organisms, offering a blueprint for engineering green, solar-powered nitrogen-fixing systems.

## Contribution

The study defines a minimal gene set from Cyanothece's nitrogenase cluster essential for nitrogen fixation in phototrophic systems.

## Key findings

- The structural genes nifHDK, nifBSUENPVZTXW, and hesAB are crucial for optimal nitrogenase function in phototrophs.
- Optimal gene expression is essential for efficient nitrogen fixation in engineered systems.
- A refactored gene cluster in Synechocystis demonstrates the feasibility of synthetic nitrogen-fixing phototrophs.

## Abstract

The nitrogenase gene cluster of unicellular diazotrophic cyanobacteria, such as Cyanothece, is frequently selected by nature for nitrogen-fixing partnerships with eukaryotic phototrophs. The essential cluster components that confer an advantage in such partnerships remain underexplored. To use this cluster for the development of synthetic, phototrophic nitrogen-fixing systems, a thorough and systematic analysis of its constituent genes is necessary. An initial effort to assess the possibility of engineering this cluster into non-diazotrophic phototrophs led to the generation of a Synechocystis 6803 strain with significant nitrogenase activity. In the current study, a refactoring approach was taken to determine the dispensability of the non-structural genes in the cluster and define a minimal gene set for constructing a functional nitrogenase for phototrophs. Using a bottom-up strategy, the nif genes from Cyanothece 51142 were re-organized to form new operons. The genes were then seamlessly removed to determine their essentiality in the nitrogen fixation process. We demonstrate that besides the structural genes nifHDK, nifBSUENPVZTXW, as well as hesAB, are important for optimal nitrogenase function in a phototroph. We also show that optimal expression of these genes is crucial for efficient nitrogenase activity. Our findings provide a solid foundation for generating synthetic systems that will facilitate solar-powered conversion of atmospheric nitrogen into nitrogen-rich compounds, a stride toward a greener world.

Integrating nitrogen fixation genes into various photosynthetic organisms is an exciting strategy for converting atmospheric nitrogen into nitrogen-rich products in a green and energy-efficient way. In order to facilitate this process, it is essential that we understand the fundamentals of the functioning of a prokaryotic nitrogen-fixing machinery in a non-diazotrophic, photoautotrophic cell. This study examines a nitrogenase gene cluster that has been naturally selected on multiple occasions for a nitrogen-fixing partnership by eukaryotic photoautotrophs and provides a basic blueprint for designing a photosynthetic organism with nitrogen-fixing ability.