Engineering the production of a conformational variant of occidiofungin that has enhanced inhibitory activity against fungal species

Patent metadata

Field	Value
Publication number	US-12004514-B2
Application number	US-202017113764-A
Country	US
Kind code	B2
Filing date	Dec 7, 2020
Priority date	Nov 29, 2012
Publication date	Jun 11, 2024
Grant date	Jun 11, 2024

Abstract

Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity. This invention is directed to compositions enriched for particular occidiofungin diastereomers/conformers, methods of making compositions enriched for particular diastereomers/conformers and microorganisms suitable for producing enriched compositions of particular diastereomers/conformers. Methods of treating fungal infections or plants infected by fungi are also provided.

First claim

We claim:

1. A method for promoting OcfN thioesterase activity in a bacterial strain of Burkholderia contaminans MS14 comprising a step of:
contacting the bacterial strain of Burkholderia contaminans MS14 with a peptide containing more asparagine 1 than beta-hydroxy asparagine 1 to promote the OcfN thioesterase activity of the bacterial strain of Burkholderia contaminans MS14, to produce occidiofungin; wherein the bacterial strain of Burkholderia contaminans MS14 comprises one of the following features:
(A) the bacterial strain of Burkholderia contaminans MS14 comprises an ocfN gene encoding the amino acid sequence of SEQ ID NO: 3 and the activity of the ocfN gene in the bacterial strain of Burkholderia contaminans MS14 is promoted by expressing the ocfN gene in a multicopy plasmid, integrating additional copies of the ocfN gene into the chromosome, or substituting the native promoter of the ocf gene with a promoter that increases expression of the ocfN relative to the native promoter, such that the ocfN gene in the bacterial strain of Burkholderia contaminans MS14 produces an increased OcfN thioesterase activity in comparison with the ocfN gene in a wild-type bacterial strain of Burkholderia contaminans M514; or
(B) the bacterial strain of Burkholderia contaminans MS14 comprises an ocfD gene encoding the amino acid sequence of SEQ ID NO: 4 and the activity of the ocfD gene of the bacterial strain of Burkholderia contaminans M514 is decreased by a point mutation of the catalytic serine at position 2954 of the amino acid sequence of SEQ ID NO: 4, deletion, insertion or point mutations within the thioesterase motif of the amino acid sequence of SEQ ID NO: 4, deletion of the catalytic serine of the amino acid sequence of SEQ ID NO: 4, truncation of the ocfD gene, or frameshift mutation of the ocfD gene, such that the ocfD gene in the bacterial strain of Burkholderia contaminans M514 has reduced OcfD thioesterase activity in comparison with the OcfD thioesterase activity in a wild-type bacterial strain of Burkholderia contaminans M514.

2. The method of claim 1 , wherein the ocfN gene is expressed in a multicopy plasmid with a native promoter or any other promoter sequence.

3. The method of claim 1 , wherein the ocfN gene is integrated into the chromosome with additional copies of the ocfN gene using transposons.

4. The method of claim 1 , wherein the bacterial strain of Burkholderia contaminans MS14 has two or more copies of the ocfN gene.

5. The method of claim 1 , wherein the native promoter of the ocfN gene in the bacterial strain of Burkholderia contaminans MS14 is substituted with a promoter that increases expression of the ocfN relative to the native promoter.

6. The method of claim 1 , wherein the ocfD gene of the bacterial strain of Burkholderia contaminans MS14 is truncated.

7. The method of claim 1 , wherein the thioesterase motif in the amino acid sequence of SEQ ID NO: 4 is deleted in the bacterial strain of Burkholderia contaminans MS14.

8. The method of claim 1 , wherein the ocfD gene of the bacterial strain of Burkholderia contaminans MS14 has a frameshift.

Assignees

• Univ Mississippi State
• Texas A & M Univ Sys

Inventors

• Smith James Leif
• Ravichandran Akshaya
• Lu Shien
• Gu Ganyu

Classifications

• C07K7/56

  the cyclisation not occurring through 2,4-diamino-butanoic acid · CPC title

• C12Y301/02

  Thioester hydrolases (3.1.2) · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• A01N43/713

  having rings with four or more nitrogen atoms as the only ring hetero atoms · CPC title

• C07K7/54

  with at least one abnormal peptide link in the ring · CPC title