# US 12,004,514 B2 · Engineering the production of a conformational variant of occidiofungin that has enhanced inhibitory activity against fungal species > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,004,514 B2 - Document ID: US12004514B2 - Status: Granted - Kind: B2 - Application number: 17/113,764 - Filed: 2020-12-07 - Published: 2022-01-13 - Granted: 2024-06-11 - Earliest claimed: 2012-11-29 - Assignees: The Texas A&M University System; Mississippi State University - Applicants: Mississippi State University; The Texas A&M University System - Inventors: James Leif Smith; Akshaya Ravichandran; Shien Lu; Ganyu Gu - Web page: https://patentgrep.com/patent/US12004514B2 ## CPC - A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46) - A01N 43/713: having rings with four or more nitrogen atoms as the only ring hetero atoms - A61K 38/12: Cyclic peptides {, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C (A61K38/043 - A61K38/046 take precedence)} - A61P 31/04: Antibacterial agents - C07K 7/54: with at least one abnormal peptide link in the ring - C07K 7/56: the cyclisation not occurring through 2,4-diamino-butanoic acid - C12N 9/16: acting on ester bonds (3.1) - C12Y 301/02: Thioester hydrolases (3.1.2) - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## 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. ## Claims ### 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the ocfN gene is expressed in a multicopy plasmid with a native promoter or any other promoter sequence. ### Claim 3 (depends on 1) The method of claim 1, wherein the ocfN gene is integrated into the chromosome with additional copies of the ocfN gene using transposons. ### Claim 4 (depends on 1) The method of claim 1, wherein the bacterial strain of Burkholderia contaminans MS14 has two or more copies of the ocfN gene. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 1) The method of claim 1, wherein the ocfD gene of the bacterial strain of Burkholderia contaminans MS14 is truncated. ### Claim 7 (depends on 1) 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. ### Claim 8 (depends on 1) The method of claim 1, wherein the ocfD gene of the bacterial strain of Burkholderia contaminans MS14 has a frameshift.