Methods and compositions for improving engineered microbes that fix nitrogen
Inventors
Tamsir, Alvin • BLOCH, Sarah • Higgins, Douglas
Assignees
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Publication Number
US-11993778-B2
Publication Date
2024-05-28
Expiration Date
Abstract
Methods and systems are provided for generating and utilizing a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.
Core Innovation
The invention relates to genetically engineered bacteria and associated agricultural compositions for increasing an amount of atmosphere derived nitrogen in a plant by contacting the plant with a genetically engineered bacterium. The genetically engineered bacterium comprises a nitrogen fixation or nitrogen assimilation coding sequence operably linked to a promoter comprising at least 90% sequence identity to the sequence set forth in SEQ ID NO: 5.
The promoter sequence identity can be increased to at least 95% sequence identity, and the disclosed nitrogen fixation or nitrogen assimilation system includes defined coding sequences such as polynucleotides encoding glutamine synthetase or glutaminase and additional nitrogenase biosynthesis-associated genes. The description also includes nitrogen fixation regulatory targets and nitrogen-cycle signaling concepts, including nifA/nifL, PII signaling via GlnD/GlnB/GlnK, AmtB, and DraT/DraG.
The disclosure further includes host-microbe evolution concepts and microbial breeding and compatibility to obtain bacteria suitable for agricultural systems across soil and geography, including association with plant roots and crops. Compositions and agricultural uses include seed and plant-based delivery formats intended to increase atmosphere-derived nitrogen, with outcomes tied to nitrogenase activity, ammonia excretion, gene expression profiling, and nitrogen uptake measurements in planta.
Claims Coverage
The independent claim is directed to a method for increasing atmosphere derived nitrogen in a plant by contacting the plant with a genetically engineered bacterium that includes a nitrogen fixation or nitrogen assimilation coding sequence operably linked to a promoter with at least 90% sequence identity to SEQ ID NO: 5. The claim set presents 7 inventive features across the independent and dependent claims.
Promoter operably linked to nitrogen fixation or nitrogen assimilation coding sequence with sequence identity threshold
Contacting the plant with a genetically engineered bacterium comprising a nitrogen fixation or nitrogen assimilation coding sequence operably linked to a promoter comprising at least 90% sequence identity to the sequence set forth in SEQ ID NO: 5.
Higher promoter sequence identity threshold
The promoter comprises at least 95% sequence identity to the sequence set forth in SEQ ID NO: 5.
Selected nitrogen fixation or nitrogen assimilation coding sequence sets
The nitrogen fixation coding sequence is selected from specified nif/ntr genes and polynucleotides encoding glutamine synthetase or glutaminase, including additional nitrogenase biosynthesis-associated genes.
Engineered diazotrophic bacterium
Using a genetically engineered diazotrophic bacterium.
Atmospheric nitrogen fixation under non-nitrogen-limiting conditions
Using a genetically engineered bacterium that fixes atmospheric nitrogen when nitrogen is not limiting.
Reduced nitrogen fertilizer requirement between planting and harvesting
Reducing the amount of nitrogen fertilizer needed between planting and harvesting of the plant crop.
Overall claim coverage centers on plant contact with a genetically engineered bacterium where nitrogen fixation or nitrogen assimilation genes are controlled by a promoter meeting a defined SEQ ID sequence identity requirement, with dependent claims further narrowing the promoter identity, specifying nitrogen-related coding sequence sets, restricting to diazotrophic engineered bacteria, qualifying atmospheric nitrogen fixation under non-nitrogen-limiting conditions, and claiming a reduced fertilizer requirement outcome.
Stated Advantages
Increases an amount of atmosphere derived nitrogen in a plant.
Reduces the amount of nitrogen fertilizer needed between planting and harvesting of the plant crop.
Documented Applications
Agricultural systems fertilized with high nitrogen are addressed by contacting plants with genetically engineered nitrogen-fixing or nitrogen-assimilating bacteria to increase atmosphere-derived nitrogen.
Plant-root and seed-based agricultural use cases are described, including contacting seeds or plants with genetically engineered bacteria for nitrogen fixation and/or nitrogen assimilation in crops.
Increasing atmosphere-derived nitrogen in plants by contacting plants with genetically engineered bacteria.
Plant-improvement workflows supported by experimental examples including identification and characterization of nitrogen-fixation-associated promoters and engineered nitrogen-fixing/assimilating bacteria.
Compatibility with different soils and field/geography observations supported by qPCR-based colonization measurements across soil types and field trial observations.
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