# US 11,993,778 B2 · Methods and compositions for improving engineered microbes that fix nitrogen

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- Number: US 11,993,778 B2
- Document ID: US11993778B2
- Status: Granted
- Kind: B2
- Application number: 16/759,212
- Filed: 2018-10-23
- Published: 2020-10-01
- Granted: 2024-05-28
- Earliest claimed: 2017-10-25
- Assignees: Pivot Bio, Inc.
- Applicants: Pivot Bio, Inc.
- Inventors: Alvin Tamsir; Sarah Bloch; Douglas Higgins
- Web page: https://patentgrep.com/patent/US11993778B2

## CPC

- C12N 15/8202: {by biological means, e.g. cell mediated or natural vector}
- C12Q 1/689: for bacteria
- C07K 14/195: from bacteria
- C12N 9/0095: {acting on iron-sulfur proteins as donor (1.18)}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/8262: {involving plant development}
- C12Y 118/06001: Nitrogenase (1.18.6.1)
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/13: Plant traits
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## 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.

## Claims

### Claim 1

A method of increasing an amount of atmosphere derived nitrogen in a plant, comprising contacting said plant with a genetically engineered bacterium, wherein said genetically engineered bacterium comprises (a) a nitrogen fixation or nitrogen assimilation coding sequence, operably linked to (b) a promoter comprising at least 90% sequence identity to the sequence set forth in SEQ ID NO: 5.

### Claim 2 (depends on 1)

The method of claim 1, wherein said promoter comprising at least 90% sequence identity to the sequence set forth in SEQ ID No. 5 replaces a native promoter sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein said nitrogen fixation coding sequence is 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, and a gene associated with biosynthesis of a nitrogenase enzyme.

### Claim 4 (depends on 1)

The method of claim 1, wherein said engineered bacterium is a genetically engineered diazotrophic bacterium.

### Claim 5 (depends on 1)

The method of claim 1, wherein said genetically engineered bacterium is non-intergeneric.

### Claim 6 (depends on 1)

The method of claim 1, wherein said genetically engineered bacterium fixes atmospheric nitrogen under non nitrogen limiting conditions.

### Claim 7 (depends on 1)

The method of claim 1, wherein said genetically engineered bacterium fixes more atmospheric nitrogen than a non-engineered bacterium of the same species.

### Claim 8 (depends on 1)

The method of claim 1, wherein said genetically engineered bacterium is of the genus Kosakonia.

### Claim 9 (depends on 1)

The method of claim 1, wherein said genetically engineered bacterium is of the species Kosakonia sacchari.

### Claim 10 (depends on 1)

The method of claim 1, wherein said inserted sequence is a native sequence inserted in a non-native context.

### Claim 11 (depends on 1)

The method of claim 1, wherein said promoter comprises at least 95% sequence identity to the sequence set forth in SEQ ID No. 5.

### Claim 12 (depends on 1)

The method of claim 1, wherein said promoter comprises at least 98% sequence identity to the sequence set forth in SEQ ID No. 5.

### Claim 13 (depends on 1)

The method of claim 1, wherein the method decreases the amount of nitrogen fertilizer required between planting and harvesting of a crop of said plant.