Period of performance:
Plants need nitrogen to grow. Globally, the primary food crops are cereals (rice, corn, wheat). They are not able to fix their own nitrogen, but in nature, they obtain microbially derived nitrogen from soil. However, because of the density of modern agriculture, they would quickly leach the nitrogen and yields would collapse. This is avoided by adding chemically derived nitrogen. In contrast, legumes have special associations of nitrogen-fixing microbes in specialized organs, called nodules. As such, they do not need added nitrogen. The dream of biotechnology has been to move this capability into cereals so that they do not require added nitrogen. The challenge has been that nitrogen fixation requires the simultaneous transfer of many genes that must be carefully balanced in order to obtain activity. There are also biochemical constraints, including oxygen inactivation, complex metal cofactors, and high energy and redox potential requirements. This proposal will fund an international collaboration that will be the first to attempt to move the complete nitrogenase pathway into plant organelles.
The transfer of the pathway has not been previously attempted due to limitations in technology for plant genetic engineering. The DNA encoding the microbial pathway is large (20-40,000 bp) and this is technically challenging to move into a plant (albeit not impossible). The problem is that the genes are under tight regulation in their native host and this does not transfer to plants – so the transfer of the native DNA sequence would not lead to gene expression. Normally, one would replace the microbial regulation with that which would be functional in a plant. However, the model nitrogenase pathway (from Klebsiella oxytoca) requires 16 genes and even slightly misexpression causes a loss in activity. For plant chromosomes, the parts (promoters, etc.) for precision expression control do not exist.
The transfer to a plant organelle would be ideal. Chloroplasts are ancient bacterial systems and they have similar gene regulation. Chloroplasts (at night) have low oxygen tensions, they generate the required energy (ATP) and have appropriate redox capability. Genetic parts that function in bacteria with well-developed tools, such as E. coli, also function in plastid DNA. However, relatively few labs that specialize in engineering these organelles and none of the large agriculture companies are active in this area.
Our lab at MIT has overcome several critical technical hurdles. The first is that we have reengineered the 16 gene nitrogenase pathway from Klebsiella to systematically remove all of the native regulation and replace it with well-characterized synthetic genetic parts. This modularizes the system such that alternative parts can be rapidly swapped for different target organisms. This pathway is activated by the phage-derived T7 RNA polymerase. We are the first to build a nif pathway using chemical DNA synthesis. We have also established the MIT- Broad Foundry and this has become the largest DNA assembly facility in the world. We have used this capability to build hundreds of nitrogenase pathways and it enables us to take many “shots on goal.”
This proposal seeks to: 1. Develop genetic parts required for expression in these systems, 2. Design and build combinatorial nitrogenase pathways for expression in plastids and mitochondria, and 3. Screen for activity. This would represent the first transfer of nitrogenase activity to a eukaryote.