Methods and compositions for improving plant traits
Inventors
Temme, Karsten • Tamsir, Alvin • BLOCH, Sarah • CLARK, Rosemary • TUNG, Emily
Assignees
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Publication Number
US-12497342-B2
Publication Date
2025-12-16
Expiration Date
Abstract
Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.
Core Innovation
The invention provides methods and compositions for increasing the amount of atmospheric derived nitrogen in a plant in a field by exposing the plant to a plurality of engineered diazotrophic bacteria. The engineered bacteria increase an amount of atmospheric derived nitrogen within the exposed plant relative to a plant exposed to a same amount of a second plurality of non-engineered diazotrophic bacteria of the same species.
The engineered diazotrophic bacteria are characterized by genetic variation introduced into a nitrogen fixation genetic regulatory network that results in one or more of increased expression or activity of NifA or decreased expression or activity of NifL. At least one genetic variation includes insertion of a heterologous sequence found within a genome of a bacterium of the genus of the engineered diazotrophic bacteria.
The disclosed systems include nitrogen fixation regulation networks such as PII/GlnD–GlnB–GlnK and nitrogenase shutoff components including DraT/DraG. The document also describes engineered diazotrophic bacteria used in planta, including seed coatings and isolated deposited strains, and states measurable increases in fixed nitrogen contribution under fertilizer-related non-nitrogen-limiting conditions.
Claims Coverage
The independent claim covers a field method for increasing atmospheric-derived nitrogen in plants by exposing plants to engineered diazotrophic bacteria that outperform non-engineered diazotrophic bacteria of the same species in non-nitrogen-limiting conditions. The inventive content further specifies genetic regulatory network modifications, including heterologous sequence insertion, and greater nitrogen fixation activity in similar non-nitrogen-limiting conditions.
Field exposure to engineered diazotrophic bacteria for increased atmospheric derived nitrogen
A method of increasing the amount of atmospheric derived nitrogen in a plant in a field by exposing the plant in the field to a plurality of engineered diazotrophic bacteria, thereby increasing an amount of atmospheric derived nitrogen within the plant exposed to the plurality of engineered diazotrophic bacteria relative to a plant exposed to a same amount of a second plurality of a non-engineered diazotrophic bacteria that are the same species as the plurality of engineered diazotrophic bacteria.
Nitrogen fixation genetic regulatory network variation increasing NifA and/or decreasing NifL
The plurality of engineered diazotrophic bacteria comprises at least one genetic variation introduced into a nitrogen fixation genetic regulatory network that results in one or more of increased expression or activity of NifA or decreased expression or activity of NifL.
Heterologous sequence insertion from a bacterium of the same genus
At least one genetic variation introduced into the nitrogen fixation genetic regulatory network comprises insertion of a heterologous sequence found within a genome of a bacterium of the genus of the plurality of engineered diazotrophic bacteria.
Greater nitrogen fixation activity in non-nitrogen-limiting conditions than non-engineered comparator
Nitrogen fixation activity of the plurality of engineered diazotrophic bacteria in non-nitrogen-limiting conditions is greater than nitrogen fixation activity, in similar non-nitrogen-limiting conditions, of a same amount of the second plurality of non-engineered diazotrophic bacteria.
Overall, the claim set is centered on a field exposure method using engineered diazotrophic bacteria that modify nitrogen fixation genetic regulatory networks to increase NifA expression/activity and/or decrease NifL expression/activity, including heterologous sequence insertion from a bacterium of the same genus, and that achieve greater nitrogen fixation activity in non-nitrogen-limiting conditions than non-engineered bacteria of the same species.
Stated Advantages
Increases the amount of atmospheric derived nitrogen within a plant in a field relative to exposure to a same amount of non-engineered diazotrophic bacteria of the same species.
In non-nitrogen-limiting conditions, nitrogen fixation activity of the engineered diazotrophic bacteria is greater than that of non-engineered diazotrophic bacteria in similar conditions.
Provides improved nitrogen fixation performance under fertilizer/exogenous-nitrogen conditions as described in the disclosure.
Documented Applications
A field method for increasing atmospheric derived nitrogen in a plant by exposing the plant to engineered diazotrophic bacteria in non-nitrogen-limiting conditions.
In planta nitrogen fixation performance evaluation in plants exposed to engineered versus non-engineered diazotrophic bacteria, including measurement of fixed nitrogen contribution and colonization-related measurements.
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