Guided microbial remodeling, a platform for the rational improvement of microbial species for agriculture
Inventors
BLOCH, Sarah • Temme, Karsten • Tamsir, Alvin • Higgins, Douglas • Davis-Richardson, Austin • CLARK, Rosemary • GOTTLIEB, Shayin
Assignees
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Publication Number
US-12612639-B2
Publication Date
2026-04-28
Expiration Date
Abstract
The present disclosure provides guided microbial remodeling (GMR) methods for the rational improvement of plant-associated microbes to perform plant-beneficial functions. The GMR methods described herein allow for non-intergeneric genetic optimization of key regulatory networks within the microbes, which improve plant-beneficial functions over wild-type microbes but don't have the risks associated with transgenic approaches (e.g., unpredictable gene function, public and regulatory concerns, etc.). The present disclosure also provides remodeled microbes and compositions thereof. The utilization of remodeled microbes and compositions thereof will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived fertilizers.
Core Innovation
The invention provides a guided microbial remodeling method for the rational improvement of the fitness of genetically modified plant-associated microbes. A wild type microbe with nitrogen fixing activity is provided, where the wild type microbe contains one or more genes for regulating nitrogen fixation or assimilation. Targeted genetic variations are introduced within one or more genes for regulating nitrogen fixation or assimilation to produce a genetically modified microbe having increased nitrogen fixation activity and decreased fitness compared to the wild type microbe.
The method further introduces targeted genetic variations into one or more genes in a glutamine conversion pathway of the genetically modified microbe to improve the fitness of the genetically modified microbe. In the resulting genetically modified microbe, nitrogen fixation activity is increased and fitness is at least the same as the wild type microbe under the same growing conditions. The approach uses non-intergeneric genetic variations in plant-associated microbes and avoids intergeneric transgenics and associated risks/regulatory concerns.
The disclosed framework emphasizes remodeling nitrogen regulation and related pathways so that nitrogen fixation persists under nitrogen excess conditions. It combines colonization and transcriptional activity under relevant conditions with genome sequencing and pathway characterization, followed by targeted non-intergeneric genetic variations, confirmation of locus integration and absence of transgenic sequences, and repetition. Nitrogen fixation and fitness assessment are documented using nitrogen-fixing validation/assessment methods.
Claims Coverage
The document provides an independent claim with two inventive phases and dependent claims that narrow the gene targets, the glutamine conversion pathway gene, the permitted types of genetic variations, and the evidentiary context for fitness comparison. The independent claim contains two key inventive features: first increasing nitrogen fixation activity via variations in nitrogen fixation or assimilation regulators, and then improving fitness via variations in a glutamine conversion pathway while maintaining increased nitrogen fixation activity.
Guided microbial remodeling for rational fitness improvement
A guided microbial remodeling method for the rational improvement of the fitness of genetically modified plant-associated microbes.
Targeted variations in nitrogen fixation or assimilation regulators
Introducing one or more targeted genetic variations within one or more of the genes for regulating nitrogen fixation or assimilation to produce a genetically modified microbe having increased nitrogen fixation activity and decreased fitness compared to the wild type microbe.
Fitness improvement via targeted glutamine conversion pathway variations
Introducing one or more targeted genetic variations into one or more genes in a glutamine conversion pathway of the genetically modified microbe to improve the fitness, wherein a resulting genetically modified microbe has increased nitrogen fixation activity and at least the same fitness of the wild type microbe as compared to the wild type microbe under the same growing conditions.
Colonization potential as fitness evidence under the same growing conditions
The at-least-the-same fitness is evidenced by at least the same colonization potential under the same growing conditions.
Fitness comparison in the plant rhizosphere in fertilized soil
The resulting genetically modified microbe has at least the same fitness in the plant's rhizosphere in fertilized soil as compared to the wild type microbe.
Overall claim coverage centers on producing a plant-associated genetically modified microbe with increased nitrogen fixation activity while remediating an initial fitness decrease by introducing targeted genetic variations in a glutamine conversion pathway so that the resulting microbe has at least the same fitness as the wild type under the same growing conditions.
Stated Advantages
Increased nitrogen fixation activity in the genetically modified microbe.
Decreased fitness after first modifications is addressed so the resulting genetically modified microbe has at least the same fitness as the wild type under the same growing conditions.
Fitness equivalence is supported by colonization potential under the same growing conditions.
Fitness comparison is achieved in the plant rhizosphere in fertilized soil.
Avoidance of intergeneric transgenics and associated risks/regulatory concerns.
Persistence of nitrogen fixation under nitrogen excess conditions.
Documented Applications
Rational improvement of the fitness of genetically modified plant-associated microbes with nitrogen fixing activity, including examples in Klebsiella variicola CI137 with stacked genetic modifications and described effects related to ammonia excretion and colonization potential.
Agricultural application of remodeled non-intergeneric strains and multi-strain microbial consortia/compositions.
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