Luis Manuel Rubio Herrero
Date of presentation:
Faculty and University:
Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas. Universidad Politécnica de Madrid
Sobresaliente cum laude
Molybdenum Metabolism in Azotobacter vinelandii and its Biotechnological Applications
Biological nitrogen fixation carried out by a group of microorganisms termed diazotrophs is key to sustainable agricultural practices. Diazotrophs use nitrogenases to reduce N2 into NH3. Depending on the metal composition of their active-site cofactors, nitrogenases are classified as Mo, V or Fe-only nitrogenases, which carry FeMo-co, FeVco or FeFe-co, respectively. Azotobacter vinelandii harbors the three types of nitrogenases but it preferentially expresses the Mo nitrogenase as the presence of molybdate in the medium represses the other two. In this thesis we have performed a genetic and biochemical analysis of the A. vinelandii unique molybdenum storage protein (MoSto) encoded in mosA and mosB genes. Because MoSto enables the accumulation of enormous amounts of Mo inside the cell, we have also investigated its application in the development of engineered N2-fixing yeast strains. We show that MoSto confers A. vinelandii a competing edge in situations of transient Mo deprivation. It also provides resistance against W, a metal that impairs the activity of Mo-enzymes such as nitrogenase. Moreover, MoSto buffers the regulatory effects of transient Mo step-down, preventing early derepression of alternative nitrogenase genes. Mo stored at MoSto is biologically active as purified MoSto replaces molybdate in the in vitro FeMoco synthesis assay. The properties of MoSto make it a desirable component for an engineered Mo pathway to nitrogenase in S. cerevisiae. Indeed, we show here that expression and targeting of MoSto to the mitochondrial matrix protected S. cerevisiae from Mo toxicity and additionally permitted the maturation of co-expressed NifQ into a fully active form. As NifQ is the physiological Mo donor to FeMo-co in A. vinelandii and other diazotrophs, achieving functional NifQ completed the Mo pathway in S. cerevisiae mitochondria. Thus, the engineered Mo pathway required, at a minimum, the incorporation of nifQ, mosA and mosB genes into the chromosome and proper targeting of their products to mitochondria. Genes encoding molybdate transporters might be required under certain yeast genetic backgrounds or growth conditions.