Theses Doctoral

PhD student:

Álvaro Eseverri Sabaté


Luis Manuel Rubio Herrero y Elena Caro Bernat

Date of presentation:


Faculty and University:

Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas. Universidad Politécnica de Madrid

Qualification grade:

Sobresaliente cum laude


Synthetic Biology efforts in engineering nitrogenase Fe protein biosynthesis in plastids


Synthetic biology (SynBio), described as the engineering of biology, aims to develop new biological systems and to modify existing ones with predictable behaviors. The generation of nitrogen-fixing crops with low nitrogen input requirements is considered a challenge that could lead to a new agricultural “green” revolution. A potential approach for their generation is the direct transfer of bacterial nif genes needed for the biogenesis of nitrogenase into the plant genome. Chloroplasts (plastids) of higher plants have been proposed as possible subcellular compartments in which to engineer active nitrogenase, as they have the necessary properties to accommodate the assembly and function of the nitrogenase enzyme (reducing power and ATP abundance), despite their oxygenic environment. Chloroplast genome engineering has been achieved in several major crops. However, cereals remain elusive to this technique, imposing the need for strategies that rely on the expression of nucleus-encoded transgenes with proteins being subsequently imported into plastids. The first part of this work reports the use of SynBio tools to optimize the production of nitrogenase Fe protein (NifH) in the chloroplasts of Nicotiana benthamiana plants. Azotobacter vinelandii nitrogen fixation genes nifH, M, U, and S were re-designed for protein accumulation in tobacco cells and targeting to the chloroplast was optimized by screening 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. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of NifU and NifS co-expression. In the second part, this thesis describes the translation of the lessons learned in tobacco to rice. The same set of A. vinelandii genes were re-designed for protein accumulation in Oryza sativa and the transit peptides selected for their performance in tobacco were tested for their efficiency in the import of a nuclear-encoded marker protein into plastids of different rice tissues, namely, callus, leaf, and root. Results showed that the A. thaliana minimal length transit peptides selected were also able to mediate Nif protein import into rice leaf chloroplasts. NifM, similar to what has been observed previously in bacteria, yeast and tobacco, proved necessary for NifH solubility in rice. However, we were unable to obtain rice transgenic plants accumulating any Nif protein in chloroplasts and thus, nitrogenase Fe protein activity in cereals remains elusive. Altogether, this thesis presents a proof of concept of the suitability of chloroplasts as organelles to host nitrogenase, and an optimization pipeline to engineer the reliable expression of any nuclear-encoded transgenes in N. benthamiana and O. sativa plastids, focusing on designing synthetic gene versions that confer higher protein accumulation and characterizing a set of chloroplast transit peptides that efficiently target their cargo proteins while minimizing scar amino acids in the mature protein after cleavage.