Designing a Nitrogen Fixation Circuit in an Oxygenic Photosynthetic Organism

Cyanobacteria are oxygenic photosynthetic prokaryotes of considerable interest given their ability to use sunlight to convert CO₂ into a number of useful chemicals including fuels. Certain cyanobacteria are also capable of fixing nitrogen through the use of either specialized cells called heterocysts or by temporally separating photosynthesis and nitrogen fixation. Cyanothece ATCC 51142 is one such strain that is capable of fixing nitrogen by performing photosynthesis and storing carbon in glycogen granules during the day and using the stored carbon for high rates of respiration to consume intracellular oxygen, followed by nitrogen fixation, during the night. While Cyanothece 51142 already up-regulates genes associated with nitrogen fixation, iron uptake, and iron-sulfur cluster biogenesis during the dark period, it is difficult to genetically manipulate. Synechocystis PCC 6803 is the most thoroughly studied cyanobacterium and a close relative of Cyanothece 51142. A detailed study found evidence for a nitrogen-fixing ancestor of Synechocystis 6803 and the subsequent loss of nitrogen-fixing ability. This close phylogenetic relationship and ability to perform targeted genetic modifications makes Synechocystis 6803 an ideal target for the incorporation of nitrogen-fixing capabilities.

The nif gene cluster in Cyanothece 51142 is the largest intact contiguous cluster of nitrogen fixation related genes compared to other nitrogen-fixing cyanobacteria. The transfer of this approximately 28 kb cluster into Synechocystis 6803 will introduce the genes necessary for nitrogen fixation. The incorporation of nitrogen fixation into Synechocystis also requires the development of other genetic elements, such as promoters that enable Synechocystis gene expression in a similar diurnal pattern as in Cyanothece 51142. Several promoters that exhibited diurnal rhythms have been characterized, and their output strengths have been fine tuned for use in nitrogen fixation. To facilitate the characterization and engineering of nitrogen fixation in Synechocystis, several ammonia biosensors have also been constructed using the glnRA operon from Lactococcus lactis. These biosensors were shown to lower targeted gene transcription given the presence of ammonia in E. coli cells. Given the high sensitivity of the nitrogenase enzyme to oxygen, the use of an oxygen sensor to control the transcription of the nif cluster would mimic the regulation found within Cyanothece.

The in silico modeling of metabolism offers a number of insights into the metabolism of an organism and genetic modifications that would optimally perturb the organism to achieve desired results. Genome-scale metabolic models have been created for both Cyanothece 51142 and Synechocystis 6803, and the Synechocystis model has been updated to incorporate recent refinements in the modeling of the organism. These advances are aimed towards implementing and optimizing nitrogen fixation in Synechocystis 6803.

Supported by funding from the NITROGEN program of the National Science Foundation.