Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria

Patent metadata

Field	Value
Publication number	US-10836800-B2
Application number	US-201916563086-A
Country	US
Kind code	B2
Filing date	Sep 6, 2019
Priority date	Sep 17, 2014
Publication date	Nov 17, 2020
Grant date	Nov 17, 2020

Abstract

Fusion proteins containing a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium of a Bacillus cereus family member are provided. Recombinant Bacillus cereus family members expressing such fusion proteins are also provided. Genetically inactivated Bacillus cereus family members and recombinant Bacillus cereus family members that overexpress exosporium proteins are also provided. Seeds coated with the recombinant Bacillus cereus family members and methods for using the recombinant Bacillus cereus family members (e.g., for stimulating plant growth) are also provided. Various modifications of the recombinant Bacillus cereus family members that express the fusion proteins are further provided. Fusion proteins comprising a spore coat protein and a protein or peptide of interest, recombinant bacteria that express such fusion proteins, seeds coated with such recombinant bacteria, and methods for using such recombinant bacteria (e.g., for stimulating plant growth) are also provided.

First claim

What is claimed is:

1. A recombinant Bacillus cereus family member that expresses a fusion protein comprising at least one protein or peptide of interest and a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member; wherein the recombinant Bacillus cereus family member further expresses an exosporium protein, wherein the expression of the exosporium protein is increased as compared to the expression of the exosporium protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the increased expression of the protein results in Bacillus cereus family member spores having an exosporium that is easier to remove from the spore as compared to the protein of a wild-type spore.

2. The recombinant Bacillus cereus family member of claim 1 , wherein the recombinant Bacillus cereus family member:
(i) expresses an ExsY protein, wherein the expression of the ExsY protein is increased as compared to the expression of the ExsY protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the ExsY protein comprises a carboxy-terminal tag comprising a globular protein;
(ii) expresses a BclB protein, wherein the expression of the BclB protein is increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions; or
(iii) expresses a YjcB protein, wherein the expression of the YjcB protein is increased as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions.

3. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member expresses an ExsY protein, wherein the expression of the ExsY protein is increased as compared to the expression of the ExsY protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the ExsY protein comprises a carboxy-terminal tag comprising a globular protein.

4. The recombinant Bacillus cereus family member of claim 3 , wherein:
the globular protein has a molecular weight of between 25 kDa and 100 kDa; and/or
the globular protein comprises a green fluorescent protein (GFP) or a variant thereof.

5. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member expresses a BclB protein, wherein the expression of the BclB protein is increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions.

6. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member expresses a YjcB protein, wherein the expression of the YjcB protein is increased as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions.

7. The recombinant Bacillus cereus family member of claim 1 , wherein the fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment comprising:
(1) a targeting sequence comprising 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%;
(2) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1;
(3) a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1;
(4) a targeting sequence comprising SEQ ID NO: 1;
(5) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 2;
(6) a targeting sequence comprising amino acids 2-35 of SEQ ID NO: 1;
(7) a targeting sequence comprising amino acids 5-35 of SEQ ID NO: 1;
(8) a targeting sequence comprising amino acids 8-35 of SEQ ID NO: 1;
(9) a targeting sequence comprising amino acids 10-35 of SEQ ID NO: 1;
(10) a targeting sequence comprising amino acids 15-35 of SEQ ID NO: 1;
(11) a targeting sequence comprising amino acids 1-27 of SEQ ID NO: 3;
(12) a targeting sequence comprising amino acids 12-27 of SEQ ID NO: 3;
(13) a targeting sequence comprising SEQ ID NO: 3;
(14) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 4;
(15) a targeting sequence comprising amino acids 2-27 of SEQ ID NO: 3;
(16) a targeting sequence comprising amino acids 5-27 of SEQ ID NO: 3;
(17) a targeting sequence comprising amino acids 8-27 of SEQ ID NO: 3;
(18) a targeting sequence comprising amino acids 10-27 of SEQ ID NO: 3;
(19) a targeting sequence comprising amino acids 1-38 of SEQ ID NO: 5;
(20) a targeting sequence comprising amino acids 23-38 of SEQ ID NO: 5;
(21) a targeting sequence comprising SEQ ID NO: 5;
(22) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 6;
(23) a targeting sequence comprising amino acids 2-38 of SEQ ID NO: 5;
(24) a targeting sequence comprising amino acids 5-38 of SEQ ID NO: 5;
(25) a targeting sequence comprising amino acids 8-38 of SEQ ID NO: 5;
(26) a targeting sequence comprising amino acids 10-38 of SEQ ID NO: 5;
(27) a targeting sequence comprising amino acids 15-38 of SEQ ID NO: 5;
(28) a targeting sequence comprising amino acids 20-38 of SEQ ID NO: 5;
(29) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 7;
(30) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 7;
(31) a targeting sequence comprising SEQ ID NO: 7;
(32) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 8;
(33) a targeting sequence comprising amino acids 2-28 of SEQ ID NO: 7;
(34) a targeting sequence comprising amino acids 5-28 of SEQ ID NO: 7;
(35) a targeting sequence comprising amino acids 8-28 of SEQ ID NO: 7;
(36) a targeting sequence comprising amino acids 10-28 of SEQ ID NO: 7;
(37) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 9;
(38) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 9;
(39) a targeting sequence comprising SEQ ID NO: 9;
(40) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 10;
(41) a targeting sequence comprising amino acids 2-24 of SEQ ID NO: 9;
(42) a targeting sequence comprising amino acids 5-24 of SEQ ID NO: 9;
(43) a targeting sequence comprising amino acids 8-24 of SEQ ID NO: 9;
(44) a targeting sequence comprising amino acids 1-33 of SEQ ID NO:11;
(45) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 11;
(46) a targeting sequence comprising SEQ ID NO: 11;
(47) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 12;
(48) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 11;
(49) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 11;
(50) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 11;
(51) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 11;
(52) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 11;
(53) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 13;
(54) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 13;
(55) a targeting sequence comprising SEQ ID NO:13;
(56) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:14;
(57) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 13;
(58) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 13;
(59) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 13;
(60) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 13;
(

Assignees

• Spogen Biotech Inc

Inventors

• Thompson Brian
• Siegel Ashley

Classifications

• E21B43/16

  Enhanced recovery methods for obtaining hydrocarbons · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/75

  for Bacillus · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12N3/00

  Spore forming or isolating processes · CPC title