Granted Patent B2

US 12,612,639 · App. 18/607,210 · Granted Apr 28, 2026

Inventors: Sarah Bloch (Berkeley, CA); Karsten Temme (Berkeley, CA); Alvin Tamsir (Berkeley, CA); Douglas Higgins (Berkeley, CA); Austin Davis-Richardson (Berkeley, CA); Rosemary Clark (Berkeley, CA); Shayin Gottlieb (Berkeley, CA)

Assignee: Pivot Bio, Inc.

C12N15/74C12N1/20C12Q1/6869C12Q1/689C12Q2600/156

Abstract

Claims (13)

1 . A guided microbial remodeling method for the rational improvement of the fitness of genetically modified plant-associated microbes, comprising:

a. providing a wild type microbe with nitrogen fixing activity, wherein the wild type microbe contains one or more genes for regulating nitrogen fixation or assimilation;

b. 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;

c. 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 of the genetically modified microbe, 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.

2 . The guided microbial remodeling method of claim 1 , wherein the resulting genetically modified microbe has at least the same fitness in a plant's rhizosphere as compared to the wild type microbe.

3 . The guided microbial remodeling method of claim 2 , wherein the resulting genetically modified microbe has at least the same fitness in the plant's rhizosphere during the plant's growth cycle as compared to the wild type microbe.

4 . The guided microbial remodeling method of claim 2 , wherein 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.

5 . The guided microbial remodeling method according to claim 1 , wherein the one or more genes for regulating nitrogen fixation or assimilation are selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, and combinations thereof.

6 . The guided microbial remodeling method according to claim 1 , wherein the one or more genes involved in the glutamine conversion pathway is glsA2.

7 . The guided microbial remodeling method according to claim 1 , wherein the one or more targeted genetic modifications in step (b) and step (c) are non-intergeneric.

8 . The guided microbial remodeling method according to claim 1 , wherein the one or more targeted genetic modifications in step (b) and step (c) are selected from the group consisting of: full gene deletions, partial gene deletions, promoter insertions, single base pair changes, and combinations thereof.

9 . The method of claim 1 , wherein the decreased fitness compared to the wild type microbe is evidenced by decreased colonization potential.

10 . The method of claim 1 , wherein the at least the same fitness of the wild type microbe as compared to the wild type microbe under the same growing conditions is evidenced by at least the same colonization potential of the wild type microbe as compared to the wild type microbe under the same growing conditions.

Assignments (2)

Continuity (3)

References Cited (400)