Gene targets for nitrogen fixation targeting for improving plant traits
Inventors
Tamsir, Alvin • BLOCH, Sarah • Shah, Neal
Assignees
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Publication Number
US-12151988-B2
Publication Date
2024-11-26
Expiration Date
Abstract
Methods and systems are provided for generating and utilizing a genetically engineered bacterium comprising a modification in glnD, wherein said modification is selected from the group consisting of: deletion of the entire gene, deletion of substantially the entire gene, deletion of an ACT domain, deletion of more than 50% of an ACT domain, deactivation of an ACT domain, and deactivation of an UTase domain.
Core Innovation
The invention relates to genetically engineered diazotrophic bacteria for improving plant nitrogen fixation by altering nitrogen fixation and assimilation regulatory network genes. It provides genetically engineered diazotrophic bacteria capable of increasing the amount of atmospheric derived nitrogen in a plant.
A core modification is increased expression of rpoN in a genetically engineered diazotrophic bacterium. rpoN expression is increased by deleting the native rpoN promoter and replacing it with a strong intrageneric constitutive promoter.
The invention also describes genetically engineered diazotrophic bacteria with changes in the nitrogen fixation genetic network, including glnD and alterations that replace the glnA promoter and alter rpoN expression. It is described within the context of nif regulation, including NifA/NifL/NtrC and the PII-GlnD-GlnB-GlnK cascade, and includes non-limiting genetic targets such as nifA, nifL, nifH, draT, amtB, glnA, and glnE.
Claims Coverage
Two independent claims are identified. The inventive coverage centers on increased rpoN expression via promoter replacement and on the ability to increase atmospheric derived nitrogen in a plant, with one independent claim further limiting the engineered bacterium to Klebsiella variicola. Across the independent claims, the main inventive features number 3 distinct concepts.
rpoN promoter deletion and strong intrageneric constitutive promoter replacement
rpoN expression is increased by deleting the native rpoN promoter and replacing it with a strong intrageneric constitutive promoter.
Atmospheric derived nitrogen increase in a plant
the genetically engineered diazotrophic bacterium is capable of increasing the amount of atmospheric derived nitrogen in a plant.
Klebsiella variicola engineered bacterium with increased rpoN expression
the genetically engineered diazotrophic bacterium is Klebsiella variicola.
The independent claims broadly cover genetically engineered diazotrophic bacteria that increase atmospheric derived nitrogen in plants by increasing rpoN expression through deletion of the native rpoN promoter and replacement with a strong constitutive promoter. One independent claim further requires that the engineered bacterium is Klebsiella variicola.
Stated Advantages
Increases the amount of atmospheric derived nitrogen in a plant.
Documented Applications
Improving plant nitrogen fixation by using genetically engineered diazotrophic bacteria capable of increasing atmospheric derived nitrogen in a plant.
Seed coating and plant delivery context for providing genetically engineered diazotrophic bacteria to plants, with referenced applications to plant crops including corn, rice, wheat, soy, and Setaria.
In planta colonization assessment of engineered diazotrophs, including colonization tissues such as root hair, xylem, and phloem.
Nitrogen fixation assays to evaluate nitrogen fixation capability, including acetylene reduction assay and nitrogen derived from the atmosphere measurements.
Genetic compatibility screening of host-microbe combinations and geographic compatibility screening.
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