Methods for thaxtomin production and modified streptomyces with increased thaxtomin production

Patent metadata

Field	Value
Publication number	US-10385372-B2
Application number	US-201515510863-A
Country	US
Kind code	B2
Filing date	Sep 17, 2015
Priority date	Sep 19, 2014
Publication date	Aug 20, 2019
Grant date	Aug 20, 2019

Abstract

In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, embodiments of the present disclosure, in some aspects, relate to genetically modified Streptomyces bacteria capable of increased thaxtomin production, genetically modified Streptomyces bacteria with reduced activity of a CebR protein encoded by a cebR gene and/or reduced activity of a β-glucosidase enzyme encoded by the bglC gene, genetically modified Streptomyces bacteria including a mutation of a native cebR gene and/or a native bglC gene, methods of increasing thaxtomin production in Streptomyces bacteria, methods of suppressing CebR and/or BglC activity, methods of producing thaxtomin, and thaxtomin produced by the methods of the present disclosure.

First claim

We claim:

1. A genetically modified Streptomyces bacterium comprising:
a mutation that reduces activity of a β-glucosidase enzyme encoded by a bglC gene, such that the genetically modified Streptomyces has increased production of a thaxtomin compound as compared to a corresponding wild type Streptomyces bacterium.

2. The genetically modified Streptomyces bacterium of claim 1 , wherein the mutation is a mutation of a native bglC gene, wherein the mutation reduces production or functionality of a β-glucosidase enzyme encoded by the bglC gene.

3. The genetically modified Streptomyces bacterium of claim 2 , wherein the mutation of bglC is a null mutation.

4. The genetically modified Streptomyces bacterium of claim 1 , wherein the bacterium does not produce functional β-glucosidase enzyme encoded by the bglC gene.

5. The genetically modified Streptomyces bacterium of claim 1 , wherein the mutation comprises an exogenous nucleic acid sequence introduced into the bacterium, wherein the exogenous nucleic acid sequence reduces activity of a β-glucosidase enzyme encoded by the bglC gene.

6. The genetically modified Streptomyces bacterium of claim 1 , wherein the bglC gene has a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having about 80% or more sequence identity with SEQ ID NO: 3.

7. The genetically modified Streptomyces bacterium of claim 1 , wherein the genetically modified Streptomyces bacterium is selected from the group of Streptomyces species consisting of: Streptomyces scabies, Streptomyces acidiscabies , and Streptomyces turgidiscabies.

8. A method of increasing production of a thaxtomin compound in a Streptomyces bacterium, the method comprising:
providing a Streptomyces bacterium from a species capable of producing one or more thaxtomin compounds under standard thaxtomin-inducing conditions;
genetically modifying the Streptomyces bacterium by creating a mutation in the bacterium, wherein the mutation reduces activity of a β-glucosidase enzyme encoded by a bglC gene, such that the genetically modified Streptomyces has increased production of a thaxtomin compound as compared to a corresponding wild type Streptomyces bacterium.

9. The method of claim 8 , wherein the mutation is a mutation of a native bglC gene, wherein the mutation reduces production or functionality of a β-glucosidase enzyme encoded by the bglC gene.

10. The method of claim 9 , wherein the mutation of bglC is a null mutation created by inserting a deletion cassette into the bglC gene to inhibit production of β-glucosidase.

11. The method of claim 8 , wherein the mutation comprises an exogenous nucleic acid sequence introduced into the bacterium, wherein the exogenous nucleic acid sequence reduces activity of a β-glucosidase enzyme encoded by the bglC gene.

12. A method of increasing production of a thaxtomin compound in a Streptomyces bacterium, the method comprising suppressing the activity of a β-glucosidase enzyme encoded by a bglC gene.

13. The method of claim 12 , wherein suppressing the activity of the β-glucosidase enzyme comprises genetically modifying the Streptomyces bacterium to inhibit expression of the bglC gene encoding the β-glucosidase enzyme.

14. The method of claim 12 , wherein suppressing the activity of the β-glucosidase enzyme comprises genetically modifying the Streptomyces bacterium to introduce an exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence reduces activity of the β-glucosidase enzyme encoded by the bglC gene.

15. The method of claim 12 , wherein the bglC gene has a nucleotide sequence of SEQ ID NO: 3 or a sequence having about 80% or more sequence identity with SEQ ID NO: 3.

16. A method of producing a thaxtomin compound, the method comprising:
culturing genetically modified Streptomyces bacteria, wherein the genetically modified Streptomyces bacteria comprise a mutation of a native bglC gene, wherein the mutation reduces production or functionality of a β-glucosidase enzyme encoded by the bglC gene, such that the modified Streptomyces bacteria have increased production of a thaxtomin compound as compared to a corresponding wild type Streptomyces bacteria.

17. The method of claim 16 , further comprising extracting the thaxtomin compound from the culture media.

18. The method of claim 16 , further comprising culturing the genetically modified Streptomyces bacteria in a culture medium that does not contain cellobiose, wherein the genetically modified Streptomyces bacteria are capable of producing a thaxtomin compound in the absence of cellobiose.

Assignees

• Univ Florida
• Univ Liege

Inventors

• Loria Rosemary
• Jourdan Samuel
• Rigali Sebastien
• Francis Isolde Maria

Classifications

• A01N43/60

  1,4-Diazines; Hydrogenated 1,4-diazines · CPC title

• C07D403/06

  linked by a carbon chain containing only aliphatic carbon atoms · CPC title

• C12N9/2445

  Beta-glucosidase (3.2.1.21) · CPC title

• C07K14/36

  from Actinomyces; from Streptomyces (G) · CPC title

• C12P17/165 Primary

  Heterorings having nitrogen atoms as the only ring heteroatoms · CPC title