# US 2017/0283472 A1 · Compositions comprising recombinant bacillus cells and another biological control agent

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

- Number: US 2017/0283472 A1
- Document ID: US20170283472A1
- Status: Published application
- Kind: A1
- Application number: 15/511,839
- Filed: 2015-09-17
- Published: 2017-10-05
- Earliest claimed: 2014-09-17
- Assignees: Bayer CropScience AG; Spogen Biotech Inc.
- Applicants: Bayer CropScience LP; Spogen Biotech Inc.
- Inventors: Damian CURTIS; Brian THOMPSON
- Web page: https://patentgrep.com/patent/US20170283472A1

## CPC

- A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46)
- C07K 14/325: Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/82: for plant cells {, e.g. plant artificial chromosomes (PACs)}
- C12N 15/866: Baculoviral vectors
- A01N 25/00: Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application {, e.g. seed treatment or sequential application}; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates to a composition comprising a) recombinant exosporium-producing Bacillus cells that express a fusion protein comprising: (i) at least one plant growth stimulating protein or peptide and (ii) a targeting sequence that localizes the fusion protein to the exosporium of the Bacillus cells; and b) at least one further biological control agent selected from particular microorganisms disclosed herein and/or a mutant of a specific strain of a microorganism disclosed herein having all identifying characteristics of the respective strain, and/or at least one metabolite produced by the respective strain, and/or at least one metabolite produced by the respective strain that exhibits activity against insects, mites, nematodes and/or phytopathogens in a synergistically effective amount. Furthermore, the present invention relates to the use of this composition as well as a method for enhancing plant growth, promoting plant health, and/or reducing overall damage of plants and plant parts.

## Claims

### Claim 1

A composition comprising:
a) recombinant exosporium-producing Bacillus cells that express a fusion protein comprising:
at least one plant growth stimulating protein or peptide; and
(ii) a targeting sequence, exosporium protein, or exosporium protein fragment; and
b) at least one biological control agent selected from the group consisting of Bacillus thuringiensis, Bacillus megaterium, Bacillus mycoides isolate J, Bacillus methylotrophicus, Bacillus vallismortis, Chromobacterium subtsugae, Delfiia acidovorans, Streptomyces lydicus, Streptomyces colombiensis, Streptomyces galbus K61, and Penicillium bilaii
in a synergistically effective amount.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the at least one plant growth stimulating protein or peptide is selected from the group consisting of an enzyme involved in the production or activation of a plant growth stimulating compound and an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the exosporium-producing Bacillus cells are cells of a Bacillus cereus family member.

### Claim 4

(canceled)

### Claim 5 (depends on 1)

The composition according to claim 1, wherein the targeting sequence or exosporium protein comprises:
an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%;
a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 22-31 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 22-33 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 20-31 of SEQ ID NO: 1;
a targeting sequence comprising SEQ ID NO: 1; or
an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 2.

### Claim 6 (depends on 2)

The composition according to claim 2, wherein the enzyme involved in the production or activation of a plant growth stimulating compound is selected from the group consisting of an acetoin reductase, an indole-3-acetamide hydrolase, a tryptophan monooxygenase, an acetolactate synthetase, an α-acetolactate decarboxylase, a pyruvate decarboxylase, a diacetyl reductase, a butanediol dehydrogenase, an aminotransferase, a tryptophan decarboxylase, an amine oxidase, an indole-3-pyruvate decarboxylase, an indole-3-acetaldehyde dehydrogenase, a tryptophan side chain oxidase, a nitrile hydrolase, a nitril ase, a peptidase, a protease, an adenosine phosphate isopentenyltransferase, a phosphatase, an adenosine kinase, an adenine phosphoribosyltransferase, CYP735A, a 5′ribonucleotide phosphohydrolase, an adenosine nucleosidase, a zeatin cis-trans isomerase, a zeatin O-glucosyltransferase, a β-glucosidase, a cis-hydroxylase, a CK cis-hydroxylase, a CK N-glucosyltransferase, a 2,5-ribonucleotide phosphohydrolase, an adenosine nucleosidase, a purine nucleoside phosphorylase, a zeatin reductase, a hydroxylamine reductase, a 2-oxoglutarate dioxygenase, a gibberellic 2B/3B hydrolase, a gibberellin 3-oxidase, a gibberellin 20-oxidase, a chitosanase, a chitinase, a β-1,3-glucanase, a β-1,4-glucanase, a β-1,6-glucanase, an aminocyclopropane-1-carboxylic acid deaminase, and an enzyme involved in producing a nod factor.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the enzyme involved in the production or activation of a plant growth stimulating compound is a chitosanase.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the fusion protein comprises SEQ ID NO: 109.

### Claim 9 (depends on 2)

The composition according to claim 2, wherein the enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source is selected from the group consisting of a cellulase, a lipase, a lignin oxidase, a protease, a glycoside hydrolase, a phosphatase, a nitrogenase, a nuclease, an amidase, a nitrate reductase, a nitrite reductase, an amylase, an ammonia oxidase, a ligninase, a glucosidase, a phospholipase, a phytase, a pectinase, a glucanase, a sulfatase, a urease, a xylanase, and a siderophore.

### Claim 10 (depends on 9)

The composition of claim 9, wherein the enzyme is a cellulase selected from the group consisting of an endocellulase, an exocellulase, and a β-glucosidase.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the fusion protein comprises a Bacillus subtilis endoglucanase.

### Claim 12 (depends on 11)

The composition of claim 11, wherein the fusion protein comprises SEQ ID NO: 107.

### Claim 13 (depends on 12)

The composition of claim 12, wherein the recombinant Bacillus cells are derived from Bacillus thuringiensis BT013A.

### Claim 14 (depends on 9)

The composition of claim 9, wherein the enzyme is a phospholipase.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the fusion protein comprises SEQ ID NO: 108.

### Claim at position 16

16-19. (canceled)

### Claim 20 (depends on 1)

The composition according to claim 1, wherein the at least one biological control agent is a Bacillus thuringiensis strain.

### Claim 21 (depends on 20)

The composition according to claim 20, wherein the Bacillus thuringiensis strain is selected from the group consisting of B. thuringiensis var. israelensis, B. thuringiensis subsp. aizawai strain ABTS-1857, B. thuringiensis subsp. kurstaki strain HD-1, B. thuringiensis subsp. tenebrionis strain NB 176, B. th. var. aegyptii, B. th. var. cohneri, B. th. var. darmstadiensis, B. th. var. dendrolimus, B. th. var. galleriae, B. th. var.japonensis, B.th. subsp. Morrisoni, B. th. var. san diego, B. th subsp. thuringiensis strain MPPL002, B. th. var. thuringiensis, or B. th. var 7216, B. th. strain BD#32 (NRRL Accession No. B-21530), or B. th. var T36, or mutants of any thereof having all the identifying characteristics of the respective strain.

### Claim 22 (depends on 1)

The composition according to claim 1, wherein the at least one biological control agent is Penicillium bilaii ATCC22348 or mutants thereof having all the identifying characteristics of the respective strain.

### Claim 23 (depends on 1)

The composition according to claim 1, wherein the at least one biological control agent is Streptomyces lydicus WYCD108 or Streptomyces lydicus WYCE108 or mutants of any thereof having all the identifying characteristics of the respective strain.

### Claim 24 (depends on 1)

The composition according to claim 1, wherein the at least one biological control agent is Delftia acidovorans RAY209 or mutants thereof having all the identifying characteristics of the respective strain.

### Claim 25 (depends on 1)

The composition according to claim 1, wherein the at least one biological control agent is Chromobacterium subtsugae strain PRAA4-1T or mutants thereof having all of the identifying characteristics of the respective strain.

### Claim 26 (depends on 1)

A seed treated with the composition according to claim 1.

### Claim at position 24

27-28. (canceled)

### Claim 29

A method of treating a plant, a plant part, or the locus surrounding the plant to enhance plant growth and/or promote plant health comprising the step of simultaneously or sequentially applying:
a) recombinant exosporium-producing Bacillus cells that express a fusion protein comprising:
(i) at least one plant growth stimulating protein or peptide; and
(ii) a targeting sequence, exosporium protein, or exosporium protein fragment; and
b) at least one biological control agent selected from the group consisting of a Bacillus thuringiensis, Bacillus megaterium, Bacillus mycoides isolate J, Bacillus methylotrophicus, Bacillus vallismortis, Chromobacterium subtsugae, Delftia acidovorans, Streptomyces lydicus, Streptomyces colombiensis, Streptomyces galbus K61, and Penicillium bilaii,
in a synergistically effective amount.

### Claim 30 (depends on 29)

The method according to claim 29, wherein the targeting sequence or exosporium protein comprises:
an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%;
a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 22-31 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 22-33 of SEQ ID NO: 1;
a targeting sequence comprising amino acids 20-31 of SEQ ID NO: 1;
a targeting sequence comprising SEQ ID NO: 1; or
an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 2.

### Claim at position 27

31-34. (canceled)