Nitrogen Fixation in Salmonella using Refactored Nif Clusters

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Nitrogen fixation in three-different Salmonella strains. (A) acetylene reduction assay (B) nitrogen fixation efficiency compared to that of Kiebsiella oxytoca M5a1 wild-type.
Professor Christopher Voigt
Department of Biological Engineering, MIT
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Mi Ryoung Song
Department of Biological Engineering, MIT
Min-Hyung Ryu
Department of Biological Engineering, MIT
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Jon Gilbert
MIT Technology Licensing Officer
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Nitrogen Fixation in Salmonella Using Refactored Nif Clusters

PCT Patent Application WO 2017-062412


This invention promotes crop growth by supplying nitrogen to plants without the use of fertilizers.

Problem Addressed

Available nitrogen is a main limiting factor in crop production. Excessive use of synthetic fertilizers can cause harmful algae booms or disrupt beneficial bacterial communities, but a lack of nitrogen damages agricultural land and makes farmers suffer small crop yields. This invention is a method for providing fixed nitrogen from atmospheric nitrogen using endophytic bacteria.


Endophytic bacteria that do not damage host plants have the potential to promote plant growth by providing fixed nitrogen. Biological nitrogen fixation is carried out by the nif cluster, a complex of three proteins (nitrogenase), encoded by nifH, nifD and nifK, which are assembled and activated by an additional 17 genes. Since this pathway is very sensitive to small changes in gene expression, a significant obstacle to transferring the nif cluster to a new host, the inventors refactored the gene cluster to modularize it into a set of well-characterized genetic parts.  This process has an additional benefit of eliminating the response to signals that repress the native nif cluster, including ammonia and oxygen. The inventors were able to transfer both the Klebsiella wild-type nif clusters and refactored nif clusters into three different Salmonella strains using electro-transformation and conjugation. Nitrogen fixation by Salmonella in free-living conditions was confirmed with an acetylene reduction assay. This method may be used on enteric bacteria to allow them to deliver nitrogen or plant growth-stimulating peptides directly to plants.


  • Provides plants with nitrogen or growth-stimulating peptides without the use of fertilizer
  • Environmentally safe alternative; no disruption of beneficial microbial communities or promotion of algae blooms
  • Refactoring protects bacteria from nif cluster suppression signals