# US 7,611,897 B2 · AP1 amine oxidase variants

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,611,897 B2
- Document ID: US7611897B2
- Status: Granted
- Kind: B2
- Application number: 10/872,750
- Filed: 2004-06-21
- Published: 2005-03-03
- Granted: 2009-11-03
- Earliest claimed: 2001-02-06
- Assignees: Pioneer Hi-Bred International, Inc.
- Inventors: Ranjini Chatterjee; Jonathan P. Duvick; James English
- Web page: https://patentgrep.com/patent/US7611897B2

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/18: Carboxylic ester hydrolases {(3.1.1)}
- C12N 15/8209: {Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers}
- C12N 15/8242: {with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits}
- C12N 15/8282: {for fungal resistance}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

New fumonisin detoxifying or fumonisin-derivative detoxifying homologues (both nucleic acids and proteins) are provided. Compositions which include these new proteins, recombinant cells, antibodies to the new homologues, and methods of using the homologues are also provided.

## Claims

### Claim 1

An isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide that is at least 97% identical to the entire length of SEQ ID NO:50 or a fully complementary polynucleotide sequence thereof, wherein at pH 5.5 said polypeptide has a fumonisin detoxification activity or a fumonisin derivative detoxification activity that is at least 1.5-fold greater than any of the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:5 land SEQ ID NO:52.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein said polypeptide has a fumonisin detoxification activity that is at least 1.5-fold greater than any of the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:51 and SEQ ID NO:52.

### Claim 3 (depends on 2)

The nucleic acid of claim 2, wherein said polypeptide has a fumonisin detoxification activity that is at least 20-fold greater than any of the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:51 and SEQ ID NO:52.

### Claim 4 (depends on 2)

The nucleic acid of claim 2, wherein said nucleic acid is selected from the group consisting of:
(a) a nucleic acid encoding a polypeptide, wherein said polypeptide is at least 97% identical to the entire length of SEQ ID NO:50;
(b) a nucleic acid encoding a polypeptide, wherein said polypeptide is at least 97% identical to the entire length of a sequence selected from the group consisting of SEQ ID NOs: and 47 and 47;
(c) a nucleic acid encoding a polypeptide wherein said polypeptide is at least 97% identical to the entire length of a sequence selected from the group consisting of SEQ ID NOs: 48-49, 71, and SEQ ID NOs: 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127; and
(d) a nucleic acid that is fully complementary to the nucleic acid of (a), (b) or (c);
wherein the nucleic acid in (b) and (c) encodes a polypeptide having at least 97% identical to SEQ ID NO: 50.

### Claim 5

An isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 21-25, SEQ ID NOs: 67-68, and SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, and 126;
(b) a polynucleotide sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 46-50, SEQ ID NO: 71, and SEQ ID NOs: 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127,
wherein at pH 5.5 said polypeptide has a fumonisin detoxification activity or a fumonisin derivative detoxification activity that is at least 1.5-fold greater than any of the polypeptides selected from the group consisting of: SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 51 and SEQ ID NO: 52; and
(c) a polynucleotide sequence fully complementary to the polynucleotide of (a) or (b).

### Claim 6 (depends on 1)

The nucleic acid of claim 1, wherein the polynucleotide encodes a fumonisin amine oxidase.

### Claim 7 (depends on 1)

The nucleic acid of claim 1, wherein said nucleic acid comprises a characteristic selected from the group consisting of:
(a) wherein the optimum pH of said fumonisin detoxification activity is lower for said polypeptide than for any of the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:51 and SEQ ID NO:52; and
(b) wherein the thermostability of said fumonisin detoxification activity is higher for said polypeptide than for any of the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:51 and SEQ ID NO:52.

### Claim 8 (depends on 1)

The nucleic acid of claim 1, wherein said polypeptide comprises a leader sequence that directs the secretion of the polypeptide from a plant cell.

### Claim 9 (depends on 1)

The nucleic acid of claim 1, wherein said nucleic acid comprises a characteristic selected from the group consisting of:
(a) wherein at pH 5.5, the kcat of the fumonisin detoxification reaction catalyzed by the polypeptide is higher than the kcat of the fumonisin detoxification reaction catalyzed by any of the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:51 and SEQ ID NO:52;
(b) wherein at pH 5.5, the fumonisin KM for the fumonisin detoxification reaction catalyzed by the polypeptide is lower than the fumonisin KM for the fumonisin detoxification reaction catalyzed by the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:51 and SEQ ID NO:52; and
(c) wherein at pH 5.5, the fumonisin kcat/KM of the fumonisin detoxification reaction catalyzed by the polypeptide is higher than the fumonisin kcat/KM of the fumonisin detoxification reaction catalyzed by any of the polypeptides selected from the group consisting of: SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:51 and SEQ ID NO:52.

### Claim 10 (depends on 1)

The nucleic acid of claim 1, operably linked to a promoter.

### Claim 11 (depends on 1)

A nucleic acid construct comprising a promoter operably linked to the polynucleotide of claim 1.

### Claim 12 (depends on 11)

The nucleic acid construct of claim 11, wherein the promoter is heterologous with respect to the polynucleotide and effective to cause sufficient expression of the encoded polypeptide to cause the detoxification of fumonisin.

### Claim 13 (depends on 12)

The nucleic acid construct of claim 12, wherein the polynucleotide functions as a selectable marker.

### Claim 14 (depends on 12)

The nucleic acid construct of claim 12, wherein a parental codon of the polynucleotide has been replaced by a synonymous codon that is preferentially used in a plant relative to the parental codon.

### Claim 15 (depends on 11)

The nucleic acid construct of claim 11, wherein the construct is a vector.

### Claim 16 (depends on 1)

A vector comprising a first polynucleotide sequence comprising a promoter operably linked to the polynucleotide of claim 1 and a second polynucleotide sequence encoding a second polypeptide that confers a detectable phenotypic trait upon a cell or organism expressing the second polypeptide at an effective level.

### Claim 17 (depends on 16)

The vector of claim 16, wherein the detectable phenotypic trait consists of one or more traits selected from the group consisting of: herbicide resistance; pesticide resistance; and a visible marker.

### Claim 18 (depends on 15)

The vector of claim 15, wherein the vector is a plant transformation vector.

### Claim 19 (depends on 15)

An isolated host cell comprising the vector of claim 15, wherein the vector has been stably incorporated into the genome of the host cell.

### Claim 20 (depends on 19)

The cell of claim 19, wherein the polynucleotide is operably linked to a regulatory sequence.

### Claim 21 (depends on 19)

The cell of claim 19, wherein the cell is a transgenic plant cell.

### Claim 22 (depends on 21)

The transgenic plant cell of claim 21, wherein the plant cell expresses an exogenous polypeptide with fumonisin detoxification activity.

### Claim 23 (depends on 1)

An isolated transformed cell comprising the nucleic acid of claim 1.

### Claim 24 (depends on 1)

A composition comprising at least two different nucleic acids of claim 1.

### Claim 25 (depends on 24)

The composition of claim 24 comprising at least ten different nucleic acids.