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

Patent metadata

Field	Value
Publication number	US-2018099999-A1
Application number	US-201715842062-A
Country	US
Kind code	A1
Filing date	Dec 14, 2017
Priority date	Sep 17, 2014
Publication date	Apr 12, 2018
Grant date	—

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

1 - 99 . (canceled)

100 . 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 protein or peptide of interest comprises an enzyme, a plant growth stimulating protein or peptide, a protein or peptide that protects a plant from a pathogen, a protein or peptide that enhances stress resistance in a plant, a plant binding protein or peptide, or a nucleic acid binding protein or peptide; 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.

101 . The A-recombinant Bacillus cereus family of claim 100 , 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.

102 . The recombinant Bacillus cereus family member of claim 101 , wherein the recombinant Bacillus cereus family member comprises a mutation in a CotE gene.

103 . The recombinant Bacillus cereus family member of claim 102 , wherein the mutation in the CotE gene partially or completely inhibits the ability of CotE to attach the exosporium to the spore.

104 . The recombinant Bacillus cereus family member of claim 102 , wherein the mutation in the CotE gene comprises a knock-out of the CotE gene or a dominant negative form of the CotE gene.

105 - 112 . (canceled)

113 . The recombinant Bacillus cereus family member of claim 101 , wherein the recombinant Bacillus cereus family member comprises a mutation an ExsY gene.

114 . The recombinant Bacillus cereus family member of claim 113 , 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.

115 . The recombinant Bacillus cereus family member of claim 113 , wherein the mutation in the ExsY gene comprises a knock-out of the ExsY gene.

116 . The recombinant Bacillus cereus family member of claim 101 , wherein the recombinant Bacillus cereus family member comprises a mutation in a CotY gene, the mutation in the CotY gene comprising a knock-out of the CotY gene.

117 - 118 . (canceled)

119 . The recombinant Bacillus cereus family member of claim 101 , wherein the recombinant Bacillus cereus family member comprises a mutation in an ExsA gene, the mutation in the ExsA gene comprising a knock-out of the ExsA gene.

120 - 121 . (canceled)

122 . The recombinant Bacillus cereus family member of claim 101 , wherein the recombinant Bacillus cereus family member comprises a mutation in a CotO gene, the mutation in the CotO gene comprising a knock-out of the CotO gene or a dominant negative form of the CotO gene.

123 - 129 . (canceled)

130 . The recombinant Bacillus cereus family member of claim 100 , 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) 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 com

Assignees

• Spogen Biotech Inc

Inventors

• Thompson Brian
• Siegel Ashley

Classifications

• A23K20/147

  Polymeric derivatives, e.g. peptides or proteins · CPC title

• C02F3/342

  characterised by the enzymes used · CPC title

• C07K14/32 Primary

  from Bacillus (G) · CPC title

• C07K2319/01

  containing a localisation/targetting motif · CPC title

• C02F2103/16

  from metallurgical processes, i.e. from the production, refining or treatment of metals, e.g. galvanic wastes · CPC title