IP Library › Granted Patent US 12,612,669
Granted Patent B2
US 12,612,669 · App. 17/604,119 · Granted Apr 28, 2026

High-throughput methods for isolating and characterizing ammonium-excreting mutant libraries generated by chemical mutagenesis

Inventors: Min-Hyung Ryu (Cambridge, MA); Bilge Ozaydin Eskiyenenturk (Berkeley, CA); Alvin Tamsir (Berkeley, CA)
Assignee: Pivot Bio, Inc.
C12Q1/6897A01N63/20C12N1/205C12Q1/689
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Quick Facts
Patent No.
US 12,612,669
App. No.
17/604,119
Granted
Apr 28, 2026
Kind
B2
Abstract

The present disclosure provides high-throughput methods for rapidly mutagenizing, screening, and targeting candidate microbes that are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The methods utilize a microbial biosensor capable of detecting the presence/absence of ammonium and/or glutamine in a composition and signaling with a fluorescent reporter. The present disclosure further utilizes rapid visual detection assays capable of processing thousands of candidate microbes. The disclosed methods and biosensor can be used to identify mutant bacteria with improved nitrogen fixing capabilities. Mutant bacteria with improved nitrogen fixing capabilities are also disclosed, as well as methods of utilizing these novel bacteria to provide fixed nitrogen to a plant.

Claims (9)

1 . A nitrogen fixing bacterium comprising a mutant GlnA protein comprising an amino acid sequence with an N339D amino acid substitution at amino acid position 339 of a Klebsiella GlnA protein with SEQ ID NO: 24, wherein the nitrogen fixing bacterium is Klebsiella variicola.

2 . The nitrogen fixing bacterium of claim 1 , wherein the mutant GlnA protein is SEQ ID NO: 36.

3 . A nitrogen fixing bacterium comprising a mutant glnA gene comprising a nucleotide substitution at nucleotide position 1015 of a Klebsiella glnA gene with SEQ ID NO: 1 that encodes a mutant GlnA protein comprising an amino acid sequence with an N339D amino acid substitution at amino acid position 339 of a Klebsiella GlnA protein with SEQ ID NO: 24, wherein the nitrogen fixing bacterium is Klebsiella variicola.

4 . The nitrogen fixing bacterium of claim 3 , wherein the mutant glnA gene is SEQ ID NO: 13.

5 . The nitrogen fixing bacterium of claim 3 , wherein the bacterium is genetically engineered.

6 . A microbial composition comprising the nitrogen fixing bacterium of claim 3 and a carrier.

7 . A method of providing fixed nitrogen to a plant comprising applying a microbial composition to a plant, a plant part or a locus in which the plant is located, or a locus in which the plant will be grown, wherein the microbial composition comprises at least one nitrogen fixing bacterium of claim 3 .

8 . A nitrogen fixing bacterium comprising a mutant glnA gene comprising a nucleotide substitution at nucleotide position 1015 of a Klebsiella glnA gene with SEQ ID NO: 1 or at a homologous nucleotide position in a homolog thereof, wherein the nitrogen fixing bacterium is Klebsiella variicola CI3938 deposited as PTA-126716.

9 . A microbial composition, comprising: one or more isolated bacteria selected from the group consisting of a bacterium deposited as PTA-126709, a bacterium deposited as PTA-126710, a bacterium deposited as PTA-126711, a bacterium deposited as PTA-126712, a bacterium deposited as PTA-126713, a bacterium deposited as PTA-126714, a bacterium deposited as PTA-126715, a bacterium deposited as PTA-126716, a bacterium deposited as PTA-126717, a bacterium deposited as PTA-126718, a bacterium deposited as PTA-126719, a bacterium deposited as PTA-126720, a bacterium deposited as PTA-126721, a bacterium deposited as PTA-126722, a bacterium deposited as PTA-126723, a bacterium deposited as PTA-126724, a bacterium deposited as PTA-126725 and a bacterium deposited as PTA-126726.

Assignments (1)
SECURITY INTEREST Recorded Dec 22, 2025
From: PIVOT BIO, INC.
To: HSBC BANK USA, N.A.
Reel/Frame 073285/0020 →
Continuity (2)
Provisional Application 62838780 · Apr 25, 2019
Related Publication 20220282340A1 · Sep 8, 2022
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