Genetically modified nitrogen fixing bacteria and uses thereof
Inventors
Peters, John • Mus, Florence • Ane, Jean-Michel • Khokhani, Devanshi
Assignees
Washington State University WSU • Wisconsin Alumni Research Foundation
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Publication Number
US-12391627-B2
Publication Date
2025-08-19
Expiration Date
Abstract
A genetically modified bacterium for excreting fixed nitrogen (in the form of ammonia) is disclosed. The bacterium can be made by deleting at least a portion of the nifL gene of a diazotrophic γ-proteobacterium, and inserting a promoter sequence into the diazotrophic γ-proteobacterium genome that is placed and oriented to direct transcription of the rnf1 gene complex. The resulting genetically modified bacterium excretes ammonia constitutively and at a greater rate than the wild type bacterium, and can be used to make biofertilizers to stimulate plant growth. The biofertilizers may contain a culture of the bacteria, or a co-culture of the bacteria and a mycorrhizal fungus.
Core Innovation
The invention relates to a genetically modified diazotrophic γ-proteobacterium engineered to exhibit an increased ability to fix atmospheric nitrogen. The modification comprises one or more insertions within the nifL gene of the wild type diazotrophic γ-proteobacterium, wherein the one or more insertions comprise a promoter sequence, and the insertions disrupt the nifL gene.
The promoter sequence is placed and oriented to direct transcription of at least one gene of the rnf1 gene complex selected from rnfA1, rnfB1, rnfC1, rnfD1, rnfE1, rnfG1 and rnfH1. As a result, expression of the at least one gene of the rnf1 gene complex is upregulated relative to the wild type diazotrophic γ-proteobacterium. The disclosed engineering is associated with constitutive synthesis of nitrogenase and excretion of ammonia.
The disclosure further describes biofertilizer compositions comprising the genetically modified diazotrophic γ-proteobacterium, optionally in combination with mycorrhizal fungi. In these compositions, fixed nitrogen is transferred to non-leguminous plants, and documented evidence includes transfer demonstrated by 15N enrichment across plant and fungal systems.
Claims Coverage
The coverage centers on one independent claim defining a genetically modified diazotrophic γ-proteobacterium with increased atmospheric nitrogen fixation by transcriptionally upregulating selected rnf1 gene complex genes via promoter sequence insertions within nifL. The dependent claims refine promoter orientation, promoter selection, promoter origin, additional deletions within nifL, and biofertilizer composition and use involving fixed nitrogen transfer facilitated by fungal culture.
Promoter insertion within nifL disrupting nifL
A genetically modified diazotrophic γ-proteobacterium comprising one or more insertions within the nifL gene of the wild type diazotrophic γ-proteobacterium, wherein the one or more insertions comprises a promoter sequence, and wherein the one or more insertions disrupts the nifL gene.
Promoter placement and orientation to upregulate rnf1 gene complex transcription
The promoter sequence is placed and oriented to direct transcription of at least one gene of the rnf1 gene complex selected from rnfA1, rnfB1, rnfC1, rnfD1, rnfE1, rnfG1 and rnfH1 whereby the expression of the at least one gene of the rnf1 gene complex is upregulated relative to the wild type diazotrophic γ-proteobacterium.
Overall, the independent claim requires promoter sequence insertions within nifL that disrupt nifL and are placed and oriented to direct transcription of one or more specified rnf1 gene complex genes, increasing rnf1 expression relative to the wild type diazotrophic γ-proteobacterium. The dependent claims further constrain promoter orientation, promoter selection, promoter origin as a native copy at a different genomic location, additional deletions within nifL, and use of the modified bacteria within biofertilizer compositions to facilitate fixed nitrogen transfer with fungal facilitation.
Stated Advantages
Increased ability to fix atmospheric nitrogen relative to the wild type diazotrophic γ-proteobacterium.
Upregulated expression of selected rnf1 gene complex genes relative to the wild type diazotrophic γ-proteobacterium.
Facilitation of transfer of fixed nitrogen produced by the bacterial culture to plants, including transfer facilitated by fungal culture.
Documented Applications
Biofertilizer composition containing the genetically modified diazotrophic γ-proteobacterium to facilitate transfer of fixed nitrogen to non-leguminous plants, including cereal grains such as rice.
Biofertilizer composition including mycorrhizal fungi (e.g., Laccaria bicolor and Hebeloma cylindrosporum) to facilitate fixed nitrogen transfer to plants, with documented evidence using 15N enrichment.
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