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

Patent metadata

Field	Value
Publication number	US-9845342-B2
Application number	US-201514857606-A
Country	US
Kind code	B2
Filing date	Sep 17, 2015
Priority date	Sep 17, 2014
Publication date	Dec 19, 2017
Grant date	Dec 19, 2017

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;
a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member; and
an amino acid linker between the targeting sequence, the exosporium protein, or the exosporium protein fragment and the protein or peptide of interest, wherein the linker comprises a polyalanine linker, a polyglycine linker, or a linker comprising a mixture of both alanine and glycine residues;
and wherein the recombinant Bacillus cereus family member comprises a mutation that results in Bacillus cereus family member spores having an exosporium that is easier to remove from the spore as compared to the exosporium of a wild-type spore.

2. The recombinant Bacillus cereus family member of claim 1 , wherein the recombinant Bacillus cereus family member:
(i) comprises a mutation in a CotE gene;
(ii) comprises a mutation in an ExsY gene;
(iii) comprises a mutation in a CotY gene;
(iv) comprises a mutation in an ExsA gene; or
(v) comprises a mutation in a CotO gene.

3. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member comprises a mutation in a CotE gene.

4. The recombinant Bacillus cereus family member of claim 3 , wherein the mutation in the CotE gene partially or completely inhibits the ability of CotE to attach the exosporium to the spore.

5. The recombinant Bacillus cereus family member of claim 3 , wherein the mutation in the CotE gene comprises a knock-out of the CotE gene or a dominant negative form of the CotE gene.

6. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member comprises a mutation in an ExsY gene.

7. The recombinant Bacillus cereus family member of claim 6 , wherein the mutation in the ExsY gene partially or completely inhibits the ability of ExsY to complete the formation of the exosporium or attach the exosporium to the spore.

8. The recombinant Bacillus cereus family member of claim 6 , wherein the mutation in the ExsY gene comprises a knock-out of the ExsY gene.

9. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member comprises a mutation in a CotY gene.

10. The recombinant Bacillus cereus family member of claim 9 , wherein the mutation in the CotY gene comprises a knock-out of the CotY gene.

11. The recombinant Bacillus cereus family member of claim 9 , wherein the mutation in the CotY gene results in the formation of a fragile exosporium.

12. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member comprises a mutation in an ExsA gene.

13. The recombinant Bacillus cereus family member of claim 12 , wherein the mutation in the ExsA gene comprises a knock-out of the ExsA gene.

14. The recombinant Bacillus cereus family member of claim 12 , wherein the mutation in the ExsA gene results in the formation of a fragile exosporium.

15. The recombinant Bacillus cereus family member of claim 2 , wherein the recombinant Bacillus cereus family member comprises a mutation in a CotO gene.

16. The recombinant Bacillus cereus family member of claim 15 , wherein the mutation in the CotO gene comprises a knock-out of the CotO gene or a dominant negative form of the CotO gene.

17. The recombinant Bacillus cereus family member of claim 15 , wherein the mutation in the CotO gene causes the exosporium to form in strips.

18. The recombinant Bacillus cereus family member of claim 1 , wherein the targeting sequence, exosporium protein, or exosporium protein fragment comprises:
(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

Assignees

• Spogen Biotech Inc

Inventors

• Thompson Brian
• Siegel Ashley

Classifications

• A01N37/44

  containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids · CPC title

• C07K14/32 Primary

  from Bacillus (G) · CPC title

• C12N3/00

  Spore forming or isolating processes · CPC title

• C12P21/02

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

• C02F2103/10

  from quarries or from mining activities · CPC title