Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells.

The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural 'green' revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re-designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl-prolyl cis-trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe-4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co-expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.