Bacillus based delivery system and methods of use

Patent metadata

Field	Value
Publication number	US-10081790-B2
Application number	US-201514849295-A
Country	US
Kind code	B2
Filing date	Sep 9, 2015
Priority date	Feb 22, 2008
Publication date	Sep 25, 2018
Grant date	Sep 25, 2018

Abstract

Herein a Bacillus exosporium molecule delivery (BEMD) system that provides a means to deliver molecules of interest (MOIs) to an environment is disclosed. The system results in the display of MOIs on the exosporium surface of Bacillus family members such that they can be delivered to an environment in a stable and active form. In addition, methods of making and using the system are described.

First claim

What is claimed is:

1. A method of producing an energy source comprising distributing to a biomass material a recombinant Bacillus cereus family member expressing a fusion construct, wherein the fusion construct comprises at least one molecule of interest (MOI) and:
(a) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 10;
(b) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 10;
(c) a targeting sequence comprising SEQ ID NO: 10;
(d) a targeting sequence having at least 50% identity with amino acids 13-28 of SEQ ID NO: 10, wherein the identity with amino acids 18-28 of SEQ ID NO: 10 is at least 63%; or
(e) a targeting sequence comprising at least the first 35 amino acids of a Bacillus BclB protein;
wherein expression of the fusion construct results in display of the MOI on the exosporium of the recombinant Bacillus cereus family member; and wherein the MOI comprises an enzyme, the enzyme comprising an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, a β-galactosidase, a lipase, a hydrogenase, a laccase, a redox enzyme, or an enzyme capable of hydrolyzing starch, sucrose, lactose, cellulose, or hemicellulose into fermentable sugars.

2. The method of claim 1 , wherein the recombinant Bacillus cereus family member is selected from the group consisting of strains of B. anthracis, B. cereus, B. thuringiensis , and combinations thereof.

3. The method of claim 2 , wherein the recombinant Bacillus cereus family member comprises B. thuringiensis.

4. The method of claim 1 , wherein the enzyme comprises a lipase.

5. The method of claim 1 , wherein the MOI is physically attached to the exosporium of the recombinant Bacillus cereus family member.

6. The method of claim 1 , wherein the fusion construct is expressed under the control of a BclA promoter.

7. The method of claim 1 , further comprising inactivating the Bacillus cereus family member prior to distribution to the biomass material.

8. The method of claim 1 , wherein the enzyme comprises an enzyme capable of hydrolyzing cellulose into fermentable sugars.

9. The method of claim 1 , wherein the targeting sequence comprises amino acids 1-28 of SEQ ID NO: 10.

10. The method of claim 1 , wherein the targeting sequence comprises amino acids 13-28 of SEQ ID NO: 10.

11. The method of claim 1 , wherein the targeting sequence comprises SEQ ID NO: 10.

12. The method of claim 1 , wherein the targeting sequence has at least 50% identity with amino acids 13-28 of SEQ ID NO: 10, wherein the identity with amino acids 18-28 of SEQ ID NO: 10 is at least 63%.

13. The method of claim 1 , wherein the targeting sequence comprises at least the first 35 amino acids of a Bacillus BclB protein.

14. The method of claim 1 , wherein the enzyme comprises an oxidoreductase.

15. The method of claim 1 , wherein the enzyme comprises a transferase.

16. The method of claim 1 , wherein the enzyme comprises a hydrolase.

17. The method of claim 1 , wherein the enzyme comprises a lyase.

18. The method of claim 1 , wherein the enzyme comprises an isomerase.

19. The method of claim 1 , wherein the enzyme comprises a ligase.

20. The method of claim 1 , wherein the enzyme comprises a β-galactosidase.

21. The method of claim 1 , wherein the enzyme comprises a hydrogenase.

22. The method of claim 1 , wherein the enzyme comprises a laccase.

23. The method of claim 1 , wherein the enzyme comprises a redox enzyme.

24. The method of claim 1 , wherein the enzyme comprises an enzyme capable of hydrolyzing starch into fermentable sugars.

25. The method of claim 1 , wherein the enzyme comprises an enzyme capable of hydrolyzing sucrose or lactose into fermentable sugars.

26. The method of claim 1 , wherein the enzyme comprises an enzyme capable of hydrolyzing hemicellulose into fermentable sugars.

Assignees

• Univ Missouri

Inventors

• Stewart George C
• Thompson Brian Matthew
• Lin Chung-Ho

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C12P7/10

  substrate containing cellulosic material · CPC title

• C02F3/342

  characterised by the enzymes used · CPC title

• A61K39/07

  Bacillus · CPC title

• C12N15/75

  for Bacillus · CPC title