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We thank Jose Maria Buesa, Marcel Veldhuizen, and Diana Coroian for yeast fermentations and for help with cloning; and Hans Roubos (Royal DSM) for providing the pDSM vector. Funding for this research was provided by Bill & Melinda Gates Foundation Grant OPP1143172 (to L.M.R). X.J. is supported by a doctoral fellowship from Universidad Politecnica de Madrid. Y.G. acknowledges financial support from Carnegie Mellon University. K.P., I.S., and D.B.G. were funded by the US Defense Advanced Research Projects Agency's Living Foundries Program Award HR0011-15-C-0084. Funding for this project was provided by the Abdul Latif Jameel Water and Food Systems Lab (J WAFS) at the Massachusetts Institute of Technology.

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Burén, StefanAuthorJiang, XiAuthorEchavarri-Erasun, CarlosAuthorRubio, Luis M.Author

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January 7, 2020
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Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae

Publicated to:Proceedings Of The National Academy Of Sciences Of The United States Of America. 116 (50): 25078-25086 - 2019-12-10 116(50), DOI: 10.1073/pnas.1904903116

Authors: Buren, Stefan; Pratt, Katelin; Jiang, Xi; Guo, Yisong; Jimenez-Vicente, Emilio; Echavarri-Erasun, Carlos; Dean, Dennis R.; Saaem, Ishtiaq; Gordon, D. Benjamin; Voigt, Christopher A.; Rubio, Luis M.;

Affiliations

Broad Inst MIT & Harvard, Cambridge, MA 02142 USA - Author
Carnegie Mellon Univ, Dept Chem, 4400 5th Ave, Pittsburgh, PA 15213 USA - Author
MIT, Dept Biol Engn, Synthet Biol Ctr, 77 Massachusetts Ave, Cambridge, MA 02139 USA - Author
Univ Politecn Madrid, Ctr Biotecnol & Genom Plantas, Inst Nacl Invest & Tecnol Agr & Alimentaria, Madrid 28223, Spain - Author
Univ Politecn Madrid, Escuela Tecn Super Ingn Agron Alimentaria & Biosi, Dept Biotecnol Biol Vegetal, E-28040 Madrid, Spain - Author
Virginia Polytech Inst & State Univ, Dept Biochem, Blacksburg, VA 24061 USA - Author
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Abstract

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.

Keywords

combinatorial designmitochondrianif genesnitrogen fixationAzotobacter vinelandiiAzotobacter-vinelandiiBacterial proteinsClusterCombinatorial designExpressionIdentificationIn-vitro synthesisIron compoundsIron-molybdenum cofactorMetabolic networks and pathwaysMethanocaldococcusMitochondriaMofe-proteinNif genesNitrogen fixationNitrogenaseProductPurificationRecombinant proteinsSaccharomyces cerevisiaeSulfurSynthetic biology

Quality index

Bibliometric impact. Analysis of the contribution and dissemination channel

The work has been published in the journal Proceedings Of The National Academy Of Sciences Of The United States Of America due to its progression and the good impact it has achieved in recent years, according to the agency WoS (JCR), it has become a reference in its field. In the year of publication of the work, 2019, it was in position 8/71, thus managing to position itself as a Q1 (Primer Cuartil), in the category Multidisciplinary Sciences.

From a relative perspective, and based on the normalized impact indicator calculated from the Field Citation Ratio (FCR) of the Dimensions source, it yields a value of: 3.93, which indicates that, compared to works in the same discipline and in the same year of publication, it ranks as a work cited above average. (source consulted: Dimensions Jul 2025)

Specifically, and according to different indexing agencies, this work has accumulated citations as of 2025-07-16, the following number of citations:

  • WoS: 13
  • Scopus: 33
  • Europe PMC: 10
  • Google Scholar: 38

Impact and social visibility

From the perspective of influence or social adoption, and based on metrics associated with mentions and interactions provided by agencies specializing in calculating the so-called "Alternative or Social Metrics," we can highlight as of 2025-07-16:

  • The use, from an academic perspective evidenced by the Altmetric agency indicator referring to aggregations made by the personal bibliographic manager Mendeley, gives us a total of: 81.
  • The use of this contribution in bookmarks, code forks, additions to favorite lists for recurrent reading, as well as general views, indicates that someone is using the publication as a basis for their current work. This may be a notable indicator of future more formal and academic citations. This claim is supported by the result of the "Capture" indicator, which yields a total of: 86 (PlumX).

With a more dissemination-oriented intent and targeting more general audiences, we can observe other more global scores such as:

  • The Total Score from Altmetric: 42.75.
  • The number of mentions on the social network X (formerly Twitter): 55 (Altmetric).

It is essential to present evidence supporting full alignment with institutional principles and guidelines on Open Science and the Conservation and Dissemination of Intellectual Heritage. A clear example of this is:

  • The work has been submitted to a journal whose editorial policy allows open Open Access publication.

Leadership analysis of institutional authors

This work has been carried out with international collaboration, specifically with researchers from: United States of America.

There is a significant leadership presence as some of the institution’s authors appear as the first or last signer, detailed as follows: First Author (BUREN, NILS STEFAN) and Last Author (RUBIO HERRERO, LUIS MANUEL).