Compositions and methods for enhanced production of enduracidin in a genetically engineered strain of streptomyces fungicidicus

Patent metadata

Field	Value
Publication number	US-11447530-B2
Application number	US-201716465444-A
Country	US
Kind code	B2
Filing date	Dec 1, 2017
Priority date	Dec 6, 2016
Publication date	Sep 20, 2022
Grant date	Sep 20, 2022

Abstract

This disclosure describes compositions and methods for enhanced production of enduracidin in genetically engineered strains of Streptomyces fungicidicus . In particular, the present disclosure describes the genetic manipulation of regulatory genes orf24 and orf18 associated with the enduracidin (enramycin) biosynthesis gene cluster from Streptomyces fungicidicus to generate vector constructs and recombinant strains producing greater yields of enduracidin.

First claim

We claim:

1. A recombinant strain of Streptomyces fungicidicus comprising one or more modified genes selected from the group consisting of an augmented open reading frame-24 (orf24) that encodes the amino acid sequence of SEQ ID NO: 26, and a diminished open reading frame-18 (orf18), wherein an enhanced production of enduracidin is obtained with the recombinant strain of Streptomyces fungicidicus in comparison to that obtained with a control Streptomyces fungicidicus strain.

2. The recombinant strain of claim 1 , wherein the diminished orf18 is diminished because it has been nulled.

3. The recombinant strain of claim 2 , wherein the diminished orf18 has been nulled by a process selected from the group consisting of an in-frame-deletion, a frame-shift mutation, a point mutation, and any combination thereof.

4. The recombinant strain of claim 3 , wherein the diminished orf18 has been nulled by an in-frame deletion.

5. The recombinant strain of claim 4 , wherein the in-frame deletion is of nucleotides 5 through 660 of the orf18 (SEQ ID NO: 27).

6. The recombinant strain of claim 1 , wherein the augmented orf24 is operatively linked to a heterologous promoter.

7. The recombinant strain of claim 6 , wherein the heterologous promoter is a strong constitutive promoter.

8. The recombinant strain of claim 7 , wherein the strong constitutive promoter is ermE*p.

9. The recombinant strain of claim 1 , wherein the augmented ORF24 is augmented because it has been overexpressed.

10. The recombinant strain of claim 1 , wherein the Streptomyces fungicidicus is Streptomyces fungicidicus ATCC 21013.

11. The recombinant strain of claim 1 , wherein the Streptomyces fungicidicus is Streptomyces fungicidicus ATCC PTA-122342.

12. The recombinant of claim 1 , wherein the production of enduracidin by the recombinant strain is at least 1.2 fold greater than the production of enduracidin by the control Streptomyces fungicidicus.

13. The recombinant strain of claim 12 , wherein the production of enduracidin by the recombinant strain is 1.2 to 4.6 fold greater than the production of enduracidin by the control Streptomyces fungicidicus.

14. The recombinant strain of Streptomyces fungicidicus that is BM38-2.24/16 (ATCC Deposit No. PTA-124006).

15. A method of producing enduracidin, comprising culturing the recombinant strain of Streptomyces fungicidicus of claim 1 , under conditions sufficient for producing enduracidin.

16. The method of claim 15 , further comprising isolating the enduracidin from the culture medium.

17. An expression vector selected from the group consisting of pXY152-endorf24 (SEQ ID NO:3), pXY152-endorf24-camtsr (SEQ ID NO: 20), and pXY152-endorf24-blatsr (SEQ ID NO: 23).

Assignees

• Univ Oregon State

Inventors

• Zabriskie T Mark
• Yin Xihou

Classifications

• C07K14/36 Primary

  from Actinomyces; from Streptomyces (G) · CPC title

• C12N15/76

  for Actinomyces; for Streptomyces · CPC title

• C12R2001/465

  Streptomyces · CPC title

• C12N1/205

  Bacterial isolates · CPC title

• C12P21/02

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