Biology subjects

Bhattacharyya-Pakrasi, M.

Publications and source records attributed to Bhattacharyya-Pakrasi, M..

2 recordsLinked to original sources

bioRxiv2025

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

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 under explored. 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 to 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 towards a greener world. IMPORTANCEIntegrating 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.

synthetic biology↗

bioRxiv2018

Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph

Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N2 fixation is energetically highly expensive, and an energy generating process such as photosynthesis can meet the energy demand of N2 fixation. However, synthesis and expression of nitrogenase is exquisitely sensitive to oxygen. Thus, engineering nitrogen fixation activity in photosynthetic organisms that produce oxygen is challenging. Cyanobacteria are oxygenic photosynthetic prokaryotes, and some of them also fix N2. Here, we demonstrate a feasible way to engineer nitrogenase activity in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. In addition, we have identified the minimal nif cluster required for such activity in Synechocystis 6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels of nif genes. Importantly, the O2 tolerance of nitrogenase was enhanced by introduction of uptake hydrogenase genes, showing this to be a functional way to improve nitrogenase enzyme activity under micro-oxic conditions. To date, our efforts have resulted in engineered Synechocystis 6803 strains that remarkably have more than 30% N2-fixation activity compared to that in Cyanothece 51142, the highest such activity established in any non-diazotrophic oxygenic photosynthetic organism. This study establishes a baseline towards the ultimate goal of engineering nitrogen fixation ability in crop plants.\n\nIMPORTANCEApplication of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes as well as the wide spread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow crop plants the ability to fix atmospheric N2 in situ. One long-term approach is to transfer all nif genes from a prokaryote to plant cells, and express nitrogenase in an energy-producing organelle, chloroplast or mitochondrion. In this context, Synechocystis 6803, the non-diazotrophic cyanobacterium utilized in this study, provides a model chassis for rapid investigation of the necessary requirements to establish diazotrophy in an oxygenic phototroph.

synthetic biology↗