# US 10,246,697 B2 · Colistin synthetases and corresponding gene cluster

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- Number: US 10,246,697 B2
- Document ID: US10246697B2
- Status: Granted
- Kind: B2
- Application number: 15/113,978
- Filed: 2015-01-26
- Published: 2016-12-01
- Granted: 2019-04-02
- Earliest claimed: 2014-01-27
- Assignees: Centre National de la Recherche Scientifique (CNRS); Universite La Rochelle
- Applicants: Centre National de la Recherche Scientifique; Universite La Rochelle
- Inventors: Romain Chevrot; Fatoumata Tambadou; Thibault Caradec; Sandrine Didelot; Cyrille Barthelemy
- Web page: https://patentgrep.com/patent/US10246697B2

## CPC

- C12N 9/93: {Ligases (6)}
- A61P 31/04: Antibacterial agents
- C07K 7/62: Polymyxins; Related peptides
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

PmxA synthetase involved in polymyxin E synthesis, comprising four adenylation sites, characterized in that the second adenylation site has at least 90% identity with the peptide sequence SEQ ID NO 1: VTEAEKADLLGRFNDTTTEFPRGKTLIQLFEEQVERIPDAAAITLNEQE LTYRELNERVNRLARTLRSHGISKGRLVAILAERSIEMVVGMLAAHKAG AAYVPIDPEYPEERIRFLIEDSGGQVMLTQSRLRERLAGSDPVILLDDE SFYHEDGTNLNTGIEATDLACVIYTSGTTGKPKGNPVSHRNIVRVVQNT NYIDITERDHVLQLSSYSFDGATFDIFGALTNGARLVLVPYETLLEIGR LADLIQRERISVMFITTAFFNILVDVNVDCLRDVRAILFGGERVSVGHV RKALAHIGPGRLNHVYGPTESTVYTTYLPVDFVDELAVTVPIGRPISNT TVYIVDSRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVP GERMYRTGDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVE GIRKTTVFARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPA YLIQLERLPLTTNGKVDRRSLPAPEESLQPGGG, the underlined amino acids DGFFLGVVYK being conserved and forming a binding pocket specific for a leucine, isoleucine or valine residue.

## Claims

### Claim 1

Method for producing polymyxin E, comprising culturing a microorganism transformed by introducing a gene encoding a PmxA synthetase in an appropriate mineral medium, and purifying, from the culture medium, the polymyxin E produced,
said PmxA synthetase comprising four adenylation sites, wherein the second adenylation site has at least 90% identity with the peptide sequence SEQ ID NO:1:
VTEAEKADLLGRFNDTTTEFPRGKTLIQLFEEQVERIPDAAAITLNEQEL
TYRELNERVNRLARTLRSHGISKGRLVAILAERSIEMVVGMLAAHKAGAA
YVPIDPEYPEERIRFLIEDSGGQVMLTQSRLRERLAGSDPVILLDDESFY
HEDGTNLNTGIEATDLACVIYTSGTTGKPKGNPVSHRNIVRVVQNTNYID
ITERDFIVLQLSSYSFDGATFDIFGALTNGARLVLVPYETLLEIGRLADL
IQRERISVMFITTAFFNILVDVNVDCLRDVRAILFGGERVSVGHVRKALA
HIGPGRLNHVYGPTESTVYTTYLPVDFVDELAVTVPIGRPISNTTVYIVD
SRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVPGERMYRT
GDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVEGIRKTTVF
ARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPAYLIQLERLP
LITNGKVDRRSLPAPEESLQPGGG,
the bold amino acids D216, G217, F220, F259, L283, G285, V309, V317, Y318, and K505 being conserved and forming a binding pocket specific for a leucine, isoleucine or valine residue.

### Claim 2

Method for producing polymyxin E, comprising culturing a microorganism transformed by introducing a gene encoding a PmxA synthetase in an appropriate mineral medium, and purifying, from the culture medium, the polymyxin E produced,
said PmxA synthetase comprising four adenylation sites, wherein the second adenylation site has at least 90% identity with the peptide sequence SEQ ID NO:1:
VTEAEKADLLGRFNDTTTEFPRGKTLIQLFEEQVERIPDAAAITLNEQEL
TYRELNERVNRLARTLRSHGISKGRLVAILAERSIEMVVGMLAAHKAGAA
YVPIDPEYPEERIRFLIEDSGGQVMLTQSRLRERLAGSDPVILLDDESFY
HEDGTNLNTGIEATDLACVIYTSGTTGKPKGNPVSHRNIVRVVQNTNYID
ITERDFIVLQLSSYSFDGATFDIFGALTNGARLVLVPYETLLEIGRLADL
IQRERISVMFITTAFFNILVDVNVDCLRDVRAILFGGERVSVGHVRKALA
HIGPGRLNHVYGPTESTVYTTYLPVDFVDELAVTVPIGRPISNTTVYIVD
SRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVPGERMYRT
GDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVEGIRKTTVF
ARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPAYLIQLERLP
LITNGKVDRRSLPAPEESLQPGGG,
the bold amino acids D216, G217, F220, F259, L283, G285, V309, V317, Y318, and K505 being conserved and forming a binding pocket specific for a leucine, isoleucine or valine residue,
wherein said microorganism belongs to the Bacillus or Paenibacillus genus.

### Claim 3 (depends on 1)

Method for producing polymyxin E according to claim 1, wherein the culturing is carried out at a temperature of 30° C., and lasts at least 25 hours.

### Claim 4

Method for producing polymyxin E variants, comprising culturing a microorganism transformed by introducing a gene encoding a PmxA synthetase in an appropriate mineral medium, and purifying, from the culture medium, the polymyxin E variant(s),
said PmxA synthetase comprising four adenylation sites, wherein the second adenylation site has at least 90% identity with the peptide sequence SEQ ID NO:1:
VTEAEKADLLGRFNDTTTEFPRGKTLIQLFEEQVERIPDAAAITLNEQEL
TYRELNERVNRLARTLRSHGISKGRLVAILAERSIEMVVGMLAAHKAGAA
YVPIDPEYPEERIRFLIEDSGGQVMLTQSRLRERLAGSDPVILLDDESFY
HEDGTNLNTGIEATDLACVIYTSGTTGKPKGNPVSHRNIVRVVQNTNYID
ITERDFIVLQLSSYSFDGATFDIFGALTNGARLVLVPYETLLEIGRLADL
IQRERISVMFITTAFFNILVDVNVDCLRDVRAILFGGERVSVGHVRKALA
HIGPGRLNHVYGPTESTVYTTYLPVDFVDELAVTVPIGRPISNTTVYIVD
SRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVPGERMYRT
GDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVEGIRKTTVF
ARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPAYLIQLERLP
LITNGKVDRRSLPAPEESLQPGGG,
the bold amino acids D216, G217, F220, F259, L283, G285, V309, V317, Y318, and K505 being conserved and forming a binding pocket specific for a leucine, isoleucine or valine residue.