Colistin synthetases and corresponding gene cluster
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 NYIDITERDHVLQLSSYSF DG AT F DIFGALTNGARLVLVPYETLLEIGR LADLIQRERISVM F ITTAFFNILVDVNVDCLRDVRAI L F G GERVSVGHV RKALAHIGPGRLNH V YGPTEST VY TTYLPVDFVDELAVTVPIGRPISNT TVYIVDSRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVP GERMYRTGDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVE GIRKTTVFARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPA YLIQLERLPLTTNG K VDRRSLPAPEESLQPGGG, the underlined amino acids DGFFLGVVYK being conserved and forming a binding pocket specific for a leucine, isoleucine or valine residue.
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
ITERDFIVLQLSSYSF DG AT FD IFGALTNGARLVLVPYETLLEIGRLADL
IQRERISVM F ITTAFFNILVDVNVDCLRDVRAI L F G GERVSVGHVRKALA
HIGPGRLNH V YGPTEST VY TTYLPVDFVDELAVTVPIGRPISNTTVYIVD
SRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVPGERMYRT
GDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVEGIRKTTVF
ARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPAYLIQLERLP
LITNG K VDRRSLPAPEESLQPGGG,
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.
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
ITERDFIVLQLSSYSF DG AT FD IFGALTNGARLVLVPYETLLEIGRLADL
IQRERISVM F ITTAFFNILVDVNVDCLRDVRAI L F G GERVSVGHVRKALA
HIGPGRLNH V YGPTEST VY TTYLPVDFVDELAVTVPIGRPISNTTVYIVD
SRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVPGERMYRT
GDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVEGIRKTTVF
ARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPAYLIQLERLP
LITNG K VDRRSLPAPEESLQPGGG,
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.
3. 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.
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
ITERDFIVLQLSSYSF DG AT FD IFGALTNGARLVLVPYETLLEIGRLADL
IQRERISVM F ITTAFFNILVDVNVDCLRDVRAI L F G GERVSVGHVRKALA
HIGPGRLNH V YGPTEST VY TTYLPVDFVDELAVTVPIGRPISNTTVYIVD
SRNKLLPIGVAGELCVGGEGLVRGYNNRPELTAEKFVDNPFVPGERMYRT
GDLAKWLPDGTIEYVGRTDDQVKIRGFRIELGEIEAQLQKVEGIRKTTVF
ARENASGEKQLCAYYEADCELPAAELKSVLSKELPAYMIPAYLIQLERLP
LITNG K VDRRSLPAPEESLQPGGG,
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.