Theses Doctoral
PhD student:
Supervisors:
Luis Manuel Rubio Herrero y Stefan Burén
Date of presentation:
11-03-2022
Faculty and University:
Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas. Universidad Politécnica de Madrid
Qualification grade:
Sobresaliente cum laude
Title:
Identification of optimal NifH and NifB proteins for nitrogenase engineering in eukaryotes
Summary:
The problems associated with massive use of synthetic nitrogen fertilizers necessary to sustain high crop yields, could be alleviated by the transfer of biological nitrogen fixation (BNF) capacity into crop species. Nitrogenase, the enzyme that catalyzes BNF, is formed by NifH and NifDK and requires in addition at least the NifB and NifEN proteins, as these synthesize the nitrogenase active-site metal cofactor called FeMo-co. Two key components to engineer nitrogenase in crops are NifH, which is required at high cellular concentrations, and NifB. Prior attempts to express NifH and NifB in plants faced solubility and instability problems. In this thesis, we established a pipeline to identify NifH and NifB proteins suitable for plant engineering and use them to obtain transgenic rice lines. Two gene libraries containing 32 nifH and 30 nifB variants were expressed and targeted to tobacco mitochondria (for NifH) or tobacco mitochondria and chloroplasts (for NifB). The NifH library screening in tobacco identified a NifH variant from Hydrogenobacter thermophilus that accumulated soluble and at high levels in mitochondria of yeast, tobacco, and rice, and showed in vitro activity when purified from all of them. The NifB screening identified variants from the archaea Methanocaldococcus infernus, Methanothermobacterium thermautotrophicus and Methanosarcina acetivorans as soluble, stable, and active when purified from tobacco mitochondria and chloroplasts. Transgenic rice lines accumulating M. infernus or M. thermautotrophicus NifB in mitochondria were also generated, and NifB was shown to support in vitro FeMo-co synthesis. We demonstrate that it is possible to identify proteins with enhanced stability and solubility, by screening knowledge-based genetic libraries consisting of homologous proteins of diverse phylogenetic origin. The identification and expression of one variant of NifH, and three variants of NifB, with superior properties in tobacco and rice lays the foundation for the transfer of nitrogen fixation capacity into crops of agronomic interest.
