WO2005054265A2 - Polyketides and their synthesis - Google Patents
Polyketides and their synthesis Download PDFInfo
- Publication number
- WO2005054265A2 WO2005054265A2 PCT/GB2004/005001 GB2004005001W WO2005054265A2 WO 2005054265 A2 WO2005054265 A2 WO 2005054265A2 GB 2004005001 W GB2004005001 W GB 2004005001W WO 2005054265 A2 WO2005054265 A2 WO 2005054265A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- alkyl
- plasmid
- group
- erythronolide
- isolated
- Prior art date
Links
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 33
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 15
- 229930001119 polyketide Natural products 0.000 title description 4
- 125000000830 polyketide group Chemical group 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 133
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Natural products O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 claims abstract description 131
- 101100280046 Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338) eryCIII gene Proteins 0.000 claims abstract description 20
- 101100371351 Streptomyces fradiae tylMII gene Proteins 0.000 claims abstract description 18
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 claims abstract description 16
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 claims abstract description 16
- IJUPCLYLISRDRA-ULAWRXDQSA-N mycaminose Chemical compound C[C@@H](O)[C@@H](O)[C@H](N(C)C)[C@@H](O)C=O IJUPCLYLISRDRA-ULAWRXDQSA-N 0.000 claims abstract description 15
- DIOQKPOBSJVSJS-UHFFFAOYSA-N 3,6-Dideoxy-3-dimethylamino-beta-D-glucose Natural products CC1OC(O)C(O)C(N(C)C)C1O DIOQKPOBSJVSJS-UHFFFAOYSA-N 0.000 claims abstract description 14
- IJUPCLYLISRDRA-UHFFFAOYSA-N Mycaminose Natural products CC(O)C(O)C(N(C)C)C(O)C=O IJUPCLYLISRDRA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 101100280043 Saccharopolyspora erythraea eryBV gene Proteins 0.000 claims abstract description 14
- 101100501742 Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338) eryK gene Proteins 0.000 claims abstract description 12
- -1 eryBVI Proteins 0.000 claims abstract description 10
- 101100422142 Saccharopolyspora spinosa spnO gene Proteins 0.000 claims abstract description 9
- 101100371350 Streptomyces fradiae tylM1 gene Proteins 0.000 claims abstract description 9
- 101100371352 Streptomyces fradiae tylMIII gene Proteins 0.000 claims abstract description 9
- 238000012546 transfer Methods 0.000 claims abstract description 6
- 241000187559 Saccharopolyspora erythraea Species 0.000 claims description 90
- 229960003276 erythromycin Drugs 0.000 claims description 62
- 241000588724 Escherichia coli Species 0.000 claims description 60
- 150000001875 compounds Chemical class 0.000 claims description 56
- 238000000034 method Methods 0.000 claims description 53
- 125000000217 alkyl group Chemical group 0.000 claims description 49
- ZFBRGCCVTUPRFQ-UHFFFAOYSA-N erythronolide-B Natural products CCC1OC(=O)C(C)C(O)C(C)C(O)C(C)(O)CC(C)C(=O)C(C)C(O)C1C ZFBRGCCVTUPRFQ-UHFFFAOYSA-N 0.000 claims description 49
- 125000005843 halogen group Chemical group 0.000 claims description 48
- ZFBRGCCVTUPRFQ-HWRKYNCUSA-N erythronolide B Chemical compound CC[C@H]1OC(=O)[C@H](C)[C@@H](O)[C@H](C)[C@@H](O)[C@](C)(O)C[C@@H](C)C(=O)[C@H](C)[C@@H](O)[C@H]1C ZFBRGCCVTUPRFQ-HWRKYNCUSA-N 0.000 claims description 45
- 229930006677 Erythromycin A Natural products 0.000 claims description 40
- YJSXTLYNFBFHAT-UHFFFAOYSA-N Protylonolide Natural products CCC1CC(C)C(=O)C=CC(C)=CC(C)C(CC)OC(=O)CC(O)C(C)C1O YJSXTLYNFBFHAT-UHFFFAOYSA-N 0.000 claims description 32
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 32
- YJSXTLYNFBFHAT-HJOMEYPASA-N tylactone Chemical compound CC[C@H]1C[C@@H](C)C(=O)\C=C\C(\C)=C\[C@H](C)[C@@H](CC)OC(=O)C[C@@H](O)[C@H](C)[C@H]1O YJSXTLYNFBFHAT-HJOMEYPASA-N 0.000 claims description 32
- 238000004519 manufacturing process Methods 0.000 claims description 31
- 229920006395 saturated elastomer Polymers 0.000 claims description 31
- 108700023372 Glycosyltransferases Proteins 0.000 claims description 28
- 230000008569 process Effects 0.000 claims description 28
- 125000003545 alkoxy group Chemical group 0.000 claims description 27
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims description 24
- MQTOSJVFKKJCRP-BICOPXKESA-N azithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)N(C)C[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 MQTOSJVFKKJCRP-BICOPXKESA-N 0.000 claims description 20
- CLQUUOKNEOQBSW-KEGKUKQHSA-N erythromycin D Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@H]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)C)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 CLQUUOKNEOQBSW-KEGKUKQHSA-N 0.000 claims description 19
- 229910052760 oxygen Inorganic materials 0.000 claims description 19
- 125000001424 substituent group Chemical group 0.000 claims description 19
- 229960004099 azithromycin Drugs 0.000 claims description 17
- 125000001313 C5-C10 heteroaryl group Chemical group 0.000 claims description 16
- 125000000304 alkynyl group Chemical group 0.000 claims description 16
- 125000003118 aryl group Chemical group 0.000 claims description 16
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 15
- 239000005864 Sulphur Substances 0.000 claims description 15
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 15
- 125000000623 heterocyclic group Chemical group 0.000 claims description 15
- 239000001301 oxygen Substances 0.000 claims description 15
- 239000000758 substrate Substances 0.000 claims description 15
- 125000006552 (C3-C8) cycloalkyl group Chemical group 0.000 claims description 14
- 125000000081 (C5-C8) cycloalkenyl group Chemical group 0.000 claims description 13
- WWWXDCNRNMZGEN-UPOWUTDQSA-N 3-O-alpha-mycarosylerythronolide B Chemical compound C[C@@H]1[C@@H](O)[C@](C)(O)C[C@@H](C)C(=O)[C@H](C)[C@@H](O)[C@@H](C)[C@@H](CC)OC(=O)[C@H](C)[C@H]1O[C@@H]1O[C@@H](C)[C@H](O)[C@](C)(O)C1 WWWXDCNRNMZGEN-UPOWUTDQSA-N 0.000 claims description 13
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 13
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 13
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 13
- 125000003342 alkenyl group Chemical group 0.000 claims description 12
- 229910052739 hydrogen Inorganic materials 0.000 claims description 11
- JWFRNGYBHLBCMB-UHFFFAOYSA-N D-Canaytose Natural products CC(O)C(O)C(O)CC=O JWFRNGYBHLBCMB-UHFFFAOYSA-N 0.000 claims description 10
- 125000004183 alkoxy alkyl group Chemical group 0.000 claims description 10
- 125000006350 alkyl thio alkyl group Chemical group 0.000 claims description 10
- 241001446247 uncultured actinomycete Species 0.000 claims description 10
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 9
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 claims description 9
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 claims description 9
- 125000004648 C2-C8 alkenyl group Chemical group 0.000 claims description 9
- 125000004649 C2-C8 alkynyl group Chemical group 0.000 claims description 9
- 241000868102 Saccharopolyspora spinosa Species 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 101100280049 Saccharopolyspora erythraea (strain ATCC 11635 / DSM 40517 / JCM 4748 / NBRC 13426 / NCIMB 8594 / NRRL 2338) eryG gene Proteins 0.000 claims description 8
- 241001468227 Streptomyces avermitilis Species 0.000 claims description 8
- 241000142901 Streptomyces eurythermus Species 0.000 claims description 8
- 241000531819 Streptomyces venezuelae Species 0.000 claims description 8
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 8
- 125000001072 heteroaryl group Chemical group 0.000 claims description 8
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- YQLFLCVNXSPEKQ-UHFFFAOYSA-N Mycarose Natural products CC1OC(O)CC(C)(O)C1O YQLFLCVNXSPEKQ-UHFFFAOYSA-N 0.000 claims description 7
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- 241000187398 Streptomyces lividans Species 0.000 claims description 7
- 229930185156 spinosyn Natural products 0.000 claims description 7
- DBDJCJKVEBFXHG-UHFFFAOYSA-N L-Oleandrose Natural products COC1CC(O)OC(C)C1O DBDJCJKVEBFXHG-UHFFFAOYSA-N 0.000 claims description 6
- DBDJCJKVEBFXHG-BNHYGAARSA-N Oleandrose Natural products O(C)[C@H]1[C@H](O)[C@H](C)O[C@H](O)C1 DBDJCJKVEBFXHG-BNHYGAARSA-N 0.000 claims description 6
- 241000187432 Streptomyces coelicolor Species 0.000 claims description 6
- JYAQWANEOPJVEY-LYFYHCNISA-N mycarose Chemical compound C[C@H](O)[C@H](O)[C@](C)(O)CC=O JYAQWANEOPJVEY-LYFYHCNISA-N 0.000 claims description 6
- FDWRIIDFYSUTDP-KVTDHHQDSA-N (2r,4r,5s,6r)-6-methyloxane-2,4,5-triol Chemical compound C[C@H]1O[C@@H](O)C[C@@H](O)[C@@H]1O FDWRIIDFYSUTDP-KVTDHHQDSA-N 0.000 claims description 5
- JWFRNGYBHLBCMB-PBXRRBTRSA-N (3r,4s,5r)-3,4,5-trihydroxyhexanal Chemical compound C[C@@H](O)[C@H](O)[C@H](O)CC=O JWFRNGYBHLBCMB-PBXRRBTRSA-N 0.000 claims description 5
- 125000000229 (C1-C4)alkoxy group Chemical group 0.000 claims description 5
- FDWRIIDFYSUTDP-UHFFFAOYSA-N 102850-49-7 Natural products CC1OC(O)CC(O)C1O FDWRIIDFYSUTDP-UHFFFAOYSA-N 0.000 claims description 5
- 241001468213 Amycolatopsis mediterranei Species 0.000 claims description 5
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 5
- FDWRIIDFYSUTDP-DUVQVXGLSA-N D-olivose Chemical compound C[C@H]1OC(O)C[C@@H](O)[C@@H]1O FDWRIIDFYSUTDP-DUVQVXGLSA-N 0.000 claims description 5
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 5
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 5
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- 125000004414 alkyl thio group Chemical group 0.000 claims description 5
- 125000004432 carbon atom Chemical group C* 0.000 claims description 5
- GOYBREOSJSERKM-ACZMJKKPSA-N oleandrose Chemical compound O=CC[C@H](OC)[C@@H](O)[C@H](C)O GOYBREOSJSERKM-ACZMJKKPSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 claims description 4
- 239000011203 carbon fibre reinforced carbon Substances 0.000 claims description 4
- 125000005587 carbonate group Chemical group 0.000 claims description 4
- 125000001316 cycloalkyl alkyl group Chemical group 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 125000005842 heteroatom Chemical group 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 238000007254 oxidation reaction Methods 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- HQZOLNNEQAKEHT-UHFFFAOYSA-N (3R,4S,5R,6S,7S,9R,11R,12S,13R,14R)-14-ethyl-4,6,12-trihydroxy-3,5,7,9,11,13-hexamethyloxacyclotetradecane-2,10-dione Natural products CCC1OC(=O)C(C)C(O)C(C)C(O)C(C)CC(C)C(=O)C(C)C(O)C1C HQZOLNNEQAKEHT-UHFFFAOYSA-N 0.000 claims description 3
- HQZOLNNEQAKEHT-IBBGRPSASA-N 6-deoxyerythronolide B Chemical compound CC[C@H]1OC(=O)[C@H](C)[C@@H](O)[C@H](C)[C@@H](O)[C@@H](C)C[C@@H](C)C(=O)[C@H](C)[C@@H](O)[C@H]1C HQZOLNNEQAKEHT-IBBGRPSASA-N 0.000 claims description 3
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- 125000003358 C2-C20 alkenyl group Chemical group 0.000 claims description 3
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- 125000000468 ketone group Chemical group 0.000 claims description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 3
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- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims 1
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- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
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- OYEXGNNKRQPUBW-FJYHMNRNSA-N elloramycin A Chemical compound CO[C@@H]1[C@H](OC)[C@@H](OC)[C@H](C)O[C@H]1OC(C(=C(C)C1=C2O)C(=O)OC)=CC1=CC1=C2C(=O)[C@]2(OC)C(=O)C=C(OC)[C@@H](O)[C@]2(O)C1=O OYEXGNNKRQPUBW-FJYHMNRNSA-N 0.000 description 1
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- QCAWEPFNJXQPAN-UHFFFAOYSA-N methoxyfenozide Chemical compound COC1=CC=CC(C(=O)NN(C(=O)C=2C=C(C)C=C(C)C=2)C(C)(C)C)=C1C QCAWEPFNJXQPAN-UHFFFAOYSA-N 0.000 description 1
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- UEIVQYHYALXCBD-OTUJEKPESA-N neomethymycin Chemical compound C[C@H]1C[C@@H](C)C(=O)\C=C\[C@@H](C)[C@@H]([C@H](O)C)OC(=O)[C@H](C)[C@H]1O[C@H]1[C@H](O)[C@@H](N(C)C)C[C@@H](C)O1 UEIVQYHYALXCBD-OTUJEKPESA-N 0.000 description 1
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- UZQBOFAUUTZOQE-VSLWXVDYSA-N pikromycin Chemical compound C[C@H]1C[C@@H](C)C(=O)\C=C\[C@@](O)(C)[C@@H](CC)OC(=O)[C@H](C)C(=O)[C@H](C)[C@H]1O[C@H]1[C@H](O)[C@@H](N(C)C)C[C@@H](C)O1 UZQBOFAUUTZOQE-VSLWXVDYSA-N 0.000 description 1
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- 229960002930 sirolimus Drugs 0.000 description 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 1
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- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 description 1
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- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
Definitions
- the present invention relates to processes and materials (including recombinant strains) for the preparation and isolation of macrolide compounds, particularly compounds differing from natural compounds at least in terms of glycosylation. It is particularly concerned with erythromycin and azithromycin analogues wherein the natural sugar at the 5-position has been replaced.
- the invention includes the use of recombinant cells in which gene cassettes are expressed to generate novel macrolide antibiotics.
- biosynthetic pathways to the macrolide antibiotics produced by actinomycete bacteria generally involve the assembly of an aglycone structure, followed by specific modifications which may include any or all of: hydroxylation or other oxidative steps, methylation and glycosylation.
- these modifications consist of the specific hydroxylation of 6-deoxyerythronolide B to erythronolide B which is catalysed by EryF, followed by the sequential attachment of dTDP-L mycarose via the hydroxyl group at C-3 catalysed by the mycarosyltransferase EryBV (Staunton and Wilkinson, 1997).
- glycosyltransferases that have been expressed in heterologous strains, including glycosyltransferases from the pathways to vancomycin (Solenberg et al, 1997), elloramycin (Wohlert et al, 1998), oleandomycin (Doumith et al, 1999; Gaisser et al, 2000), pikromycin (Tang and McDaniel, 2001), epirubicin (Madduri et al, 1998), avermectin (Wohlert et al, 2001) and spinosyn (Gaisser et al, 2002a).
- WO 99/05283 describes low but detectable levels of 5-0-dedesosaminyl-5-(3-mycarninosyl erythromycin D in the culture supernatant of an eryCIV knockout strain of S. erythraea. It also has been demonstrated that the use of the gene cassette technology described in patent WOO 1/79520 is a powerful and potentially general approach to isolate novel macrolide antibiotics by expressing combinations of genes in mutant strains of S. erythraea (Gaisser et al, 2002b). WO 01/79520 also describes the detection of 5-0-dedesosaminyl-5- ⁇ 9-mycaminosyl erythromycin A in culture supematants of the S.
- EP 1024145 refers to the isolation of azithromycin analogues carrying a mycaminosyl residue such as 5-0-dedesosaminyl-5-0-mycaminosyl azithromycin and 3"-desmethyl-5-0-dedesosaminyl-5-0- mycaminosyl azithromycin.
- the present invention provides the first demonstration of an efficient and highly effective method for making significant quantities of erythromycins and azithromycins which have non- natural sugars at the C-5 position, in particular mycaminose and angolosamine.
- the present invention provides for the synthesis of mycaminose and angolosamine using specific combinations of sugar biosynthetic genes in gene cassettes.
- the present invention relates to processes, and recombinant strains, for the preparation and isolation of erythromycins and azithromycins, which differ from the corresponding naturally occurring compound in the glycosylation of the C-5 position.
- the present invention relates to processes, and recombinant strains, for the preparation and isolation of erythromycins and azithromycins, which incorporate angolosamine or mycaminose at the C-5 position.
- the present invention relates to processes and recombinant strains for the preparation and isolation of 5- ⁇ 9-dedesosaminyl-5-0- mycaminosyl, or angolosaminyl erythromycins and azithromycins, in particular 5- ⁇ 3-dedesosaminyl-5-0- mycaminosyl erythromycins and 5-0-dedesosaminyl-5-0-mycaminosyl azithromycins, and specifically 5-(9-dedesosaminyI-5-(?-mycaminosyl erythromycin B, 5-0-dedesosaminyl-5- ⁇ 9-mycaminosyl erythromycin C, 5-0-dedesosaminyl-5-(3-mycaminosyl erythromycin D, 5- ⁇ 3-dedesosaminyl-5-0- mycaminosyl erythromycin A, and 5-0-dedesosaminyl-5- ⁇ 9-mycaminosyl,
- the present invention relates to processes, and recombinant strains, for the preparation and isolation of erythromycins and azithromycins which differ from the naturally occurring compound in the glycosylation of the C-5 position.
- compounds of the invention include a reference to 5-0-dedesosaminy 1-5-O-mycaminosyl erythromycins, 5-0-dedesosaminyl-5-0-angolosaminyl erythromycins, 5-0-dedesosaminyl-5-O- mycaminosyl azithromycins, and 5-O-dedesosaminyl-5-0-angolosaminyl azithromycins, specifically 5-0- dedesosaminyl-5-O-mycaminosyl erythromycin A, 5-0-dedesosaminyl-5-(9- ⁇ r_ycaminosyl erythromycin
- the invention relates to processes, and recombinant strains, for the preparation and isolation of compounds of the invention.
- the present invention provides a process for the production of erythromycins and azithromycins which differ from the naturally occurring compound in the glycosylation of the C-5 position, said process comprising transforming a strain with a gene cassette as described herein and culturing the strain under appropriate conditions for the production of said erythromycin or azithromycin.
- the strain is an actinomycete, a pseudomonad, a myxobacterium, or an E. coli.
- the host strain is additionally transformed with the ermE gene from S. erythraea.
- the host strain is an actinomycete.
- the host strain is selected from S. erythraea, Streptomyces griseofuscus, Streptomyces cinnamonensis, Streptomyces albus, Streptomyces lividans, Streptomyces hygroscopicus sp., Streptomyces hygroscopicus var.
- the host strain is S. erythraea. In an alternative specific embodiment the host strain is selected from the SGQ2, Q42/1 or 18A1 strains of S. erythraea.
- the present invention further relates to novel 5-0-dedesosaminyl-5- ⁇ 9-angolosaminyl erythromycins and azithromycins produced thereby ( Figure 1).
- the methodology comprises in part the expression of a gene cassette in the S. erythraea mutant strain SGQ2 (which carries genomic deletions in eryA, eryCIII, eryBV and eryCIV (WOO 1/79520)), as described in Example 3 and 6 and in S. erythraea Q42/1 (BIOT-2166) (Examples 1- 4) and S. erythraea 18A1 (BIOT-2634) (Example 6).
- S. erythraea mutant strain SGQ2 which carries genomic deletions in eryA, eryCIII, eryBV and eryCIV (WOO 1/79520)
- S. erythraea Q42/1 BIOT-2166
- the invention relates to a process involving the transformation of an actinomycete strain, including but not limited to strains of S. erythraea such as SGQ2, (see WO 01/79520) or Q42/1 or 18A1 (whose preparation is described below) with an expression plasmid containing a combination of genes which are able to direct the biosynthesis of a sugar moiety and direct its subsequent transfer to an aglycone or pseudoaglycone.
- the present invention relates to a gene cassette containing a combination of genes which are able to direct the synthesis of mycaminose or angolosamine in an appropriate strain background.
- the present invention relates to a gene cassette containing a combination of genes which are able to direct the synthesis of mycaminose in an appropriate strain background.
- the gene cassette may include genes selected from but not limited to angorfl4, tylMIII, tylMI, tylB, tylAI, tylAII, tylla, angAI, angAII, angMIII, angB, angMI, eryG, eryK and glycosyltransferase genes including but not limited to tylMII, angMII, desVII, eryCIII, eryBV, spnP, and midl.
- the gene cassette comprises tylla in combination with one or more other genes which are able to direct the synthesis of mycaminose.
- the gene cassette comprises angorfl4 in combination with one or more other genes which are able to direct the synthesis of mycaminose.
- the gene cassette comprises angAI, angAII, angorfH, angMIII, angB, angMI, in combination with one or more glycosyltransferases such as but not limited to eryCIII, tylMII, angMII,
- the gene cassette comprises tylAI, tyl ⁇ II, tylMIII, tylB, tylla, tylMI in combination with glycosyltransferases such as but not limited to eryCIII, tylMII and angMII.
- the strain is an S. erythraea strain.
- the present invention relates to a gene cassette containing combinations of genes which are able to direct the synthesis of angolosamine, including but not limited to angMIII, angMI, angB, angAI, angAII, angorfH, angorf4, tylMIII, tylMI, tylB, tylAI, tylAII, eryCVI, spnO, eryBVI, and eryK and one or more glycosyltransferase genes including but not limited to eryCIII, tylMII, angMII, des VII, eryBV, spnP and midl.
- the gene cassette contains angMIII, angMI, angB, angAI, angAII, angorfH, spnO in combination with a glycosyltransferase gene such as but not limited to angMII, tylMII or eryCIII.
- the gene cassette contains comprises angMIII, angMI, angB, angAI, angAII, angorfif, and angorfH, in combination with one or more glycosyltransferases selected from the group consisting of angMII, tylMII and eryCIII.
- the strain is an S. erythraea strain.
- the process of the present invention further involves feeding of an aglycone and/or a pseudoaglycone substrate (for definition see below), to the recombinant strain, said aglycone or pseudoaglycone is selected from the group including (but not limited to) 3-0-mycarosyl erythronolide B, erythronolide B, 6-deoxy erythronolide B, 3-0-mycarosyl-6-deoxy erythronolide B, tylactone, spinosyn pseudoaglycones, 3-0-rhamnosyl erythronolide B, 3-0-rhamnosyl-6-deoxy erythronolide B, 3-0- angolosaminyl erythronolide B, 15-hydroxy-3-0-mycarosyl erythronolide B, 15-hydroxy erythronolide B, 15-hydroxy-6-deoxy erythronolide B, 15-hydroxy-3-0-myca
- the host cell can express the desired aglycone template, either naturally or recombinantly.
- the term "pseudoaglycone” refers to a partially glycosylated intermediate of a multiply-giycosylated product.
- a preferred cell is a prokaryote or a fungal cell or a mammalian cell.
- a particularly preferred host cell is a prokaryote, more preferably host cell strains such as actinomycetes, Pseudomonas, myxobacteria, and E. coli. It will be appreciated that if the host cell does not naturally produce erythromycin, or a closely related 14- membered macrolide, it may be necessary to introduce a gene conferring self-resistance to the macrolide product, such as the ermE gene from S. erythraea. Even more preferably the host cell is an actinomycete, even more preferably strains that include but are not limited to S.
- Streptomyces griseofuscus Streptomyces cinnamonensis, Streptomyces albus, Streptomyces lividans, Streptomyces hygroscopicus sp. , Streptomyces hygroscopicus var.
- the host cell is S. erythraea. It will readily occur to those skilled in the art that the substrate fed to the recombinant cultures of the invention need not be a natural intermediate in erythromycin biosynthesis.
- the substrate could be modified in the aglycone backbone (see Examples 8-11) or in the sugar attached at the 3-position or both.
- WO 01/79520 demonstrates that the desosaminyl transferase EryCIII exhibits relaxed specificity with respect to the pseudoaglycone substrate, converting 3-O-rhamnosyl erythronolides into the corresponding 3-O-rhamnosyl erythromycins.
- Appropriate modified substrates may also be produced by chemical semi-synthetic methods.
- erythromycin-producing polyketide synthase DEBS
- methods of engineering the erythromycin-producing polyketide synthase, DEBS, to produce modified erythromycins are well known in the art (for example WO 93/13663, WO 98/01571 , WO 98/01546, WO 98/49315, Kato, Y. et al, 2002 ).
- WO 01/79520 describes methods for obtaining erythronolides with alternative sugars attached at the 3- position. Therefore, the term "compounds of the invention” includes all such non-natural aglycone compounds as described previous additionally with alternative sugars at the C-5 position. All these documents are incorporated herein by reference.
- the compounds of the invention containing a mycaminosyl moiety at the C-5 position could be modified at the C-4 hydroxyl group of the mycaminosyl moiety, including but not limited to glycosylation (see also WO 01/79520), acylation or chemical modification.
- the present invention thus provides variants of erythromycin and related macrolides having at the 5-position a non-naturally occurring sugar, in particular an 0-mycaminosyl, or 0-angolosaminyl residue or a derivative or precursor thereof, specifically an 0-angolosaminyl residue or a derivative thereof.
- variants of erythromycin encompasses (a) erythromycins A, B, C and D; (b) semi- synthetic derivatives such as azithromycin and other derivatives as discussed in EP 1024145, which is incorporated herein by reference; (c) variants produced by genetic engineering and semi-synthetic derivatives thereof.
- Variants produced by genetic engineering include variants as taught in, or producible by, methods taught in WO 98/01571, EP 1024145, WO 93/13663, WO 98/49315 and WO 01/79520 which are incorporated herein by reference.
- the compounds of the invention include variants of erythromycin where the natural sugar at position C-5 has been replaced with mycaminose or angolosamine and also includes compounds of the following formulas (I -erythromycins and II - azithromycins) and pharmaceutically acceptable salts thereof.
- No stereochemistry is shown in Formula I or II as all possibilities are covered, including "natural" stereochemistries (as shown elsewhere in this specification) at some or all positions.
- the stereochemistry of any -CH(OH)- group is generally independently selectable.
- R H, CH 3 , C 2 H 5 or is selected from i) below;
- R 2 , R 4 , R 5 , R 6 , R 7 and R 9 are each independently H, OH, CH 3 , C 2 H 5 or OCH 3 ;
- R 3 H or OH
- R° H, , rhamnose, 2'-0-methyl rhamnose, 2',3'-bis-0-methyl rhamnose, 2',3',4'-tri-0- methyl rhamnose, oleandrose, oliose, digitoxose, olivose or angolosamine;
- R 13 H or CH 3 ;
- R 16 H or OH
- R 14 H or -C(0)NR c R d wherein each of R c and R d is independently H, d-Cio alkyl, C 2 -C 20 alkenyl, C 2 - Cio alkynyl, -(CH 2 ) m (C6 ⁇ C ⁇ 0 aryl), or -(CH 2 ) m (5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein each of the foregoing R c and R d groups, except H, may be substituted by 1 to 3 Q groups; or wherein R c and R d may be taken together to form a 4-7 membered saturated ring or a 5-10 membered heteroaryl ring, wherein said saturated and heteroaryl rings may include 1 or 2 heteroatoms selected from O, S and N, in addition to the nitrogen to which R c and R d are attached, and said saturated ring may include 1 or 2 carbon-carbon double or triple bonds, and said saturated and heteroary
- the present invention also provides compounds according to formulas I or II above in which: i) the substituent R 1 is selected from an alpha-branched C 3 -C 8 group selected from alkyl, alkenyl, alkynyl, alkoxyalkyl and alkylthioalkyl groups any of which may be optionally substituted by one or more hydroxyl groups; a C 5 - cycloalkylalkyl group wherein the alkyl group is an alpha-branched C 2 -C 5 alkyl group; a C 3 -C 8 cycloalkyl group or C 5 -C 8 cycloalkenyl group, either of which may optionally be substituted by one or more hydroxyl, or one or more C 1 -C 4 alkyl groups or halo atoms; a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more CpC 4 alky
- R 14 H or -C(0)NR c R d wherein each of R c and R d is independently H, d-Cio alkyl, C 2 -C 20 alkenyl, C 2 - C 1 0 alkynyl, -(CH 2 ) m (C6-C ⁇ 0 aryl), or -(CH 2 ) m (5-10 membered heteroaryl), wherein m is an integer ranging from 0 to 4, and wherein each of the foregoing R c and R d groups, except H, may be substituted by 1 to 3 Q groups; or wherein R c and R d may be taken together to form a 4-7 membered saturated ring or a
- saturated and heteroaryl rings may include 1 or 2 heteroatoms selected from O, S and N, in addition to the nitrogen to which R° and R d are attached, and said saturated ring may include 1 or 2 carbon-carbon double or triple bonds, and said saturated and heteroaryl rings may be substituted by 1 to 3 Q groups; or R 2 and R 17 taken together form a carbonate ring; each Q is independently selected from halo, cyano, nitro, trifluoromethyl, azido, -C(0)Q', -
- R l6 H or OH with the proviso that the compounds are not selected from the group consisting of 5-0-dedesosaminyl-5-
- the present invention provides a compound according to formula I, wherein:
- R 1 H, CH 3 , C 2 H 5 or selected from: an alpha-branched C 3 -C 8 group selected from alkyl, alkenyl, alkynyl, alkoxyalkyl and alkylthioalkyl groups any of which may be optionally substituted by one or more hydroxyl groups; a C 5 -C 8 cycloalkylalkyl group wherein the alkyl group is an alpha-branched C 2 -C 5 alkyl group; a C 3 -C 8 cycloalkyl group or C 5 -C 8 cycloalkenyl group, either of which may optionally be substituted by one or more hydroxyl, or one or more C 1 -C 4 alkyl groups or halo atoms; a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C 1 -C 4 alkyl groups, halo atom
- C 4 alkyl groups or halo atoms or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated or fully or partially unsaturated and which may optionally be substituted by one or more C 1 -C 4 alkyl groups or halo atoms; or a group of the formula SA ⁇ 6 wherein A 16 is C ⁇ -C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 8 cycloalkyl, C 5 -C 8 cycloalkenyl, phenyl or substituted phenyl wherein the substituent is C 1 -C 4 alkyl, CrC 4 alkoxy or halo, or a 3 to 6 membered oxygen or sulphur-containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C 1 -C 4 alkyl groups or halo atoms
- R 2 , R 4 , R 5 , R 6 , R 7 and R 9 are all CH 3 R 3 is H or OH
- R 16 H or OH with the proviso that the compounds are not selected from the group consisting of 5-0-dedesosaminyl-5-
- the present invention provides a compound according to formula
- R 1 C 2 H 5 optionally substituted with a hydroxyl group
- R 2 , R 4 , R 5 , R 6 , R 7 and R 9 are all CH 3
- R 3 is H or OH
- R l3 H or CH.
- R l6 H or OH with the proviso that the compounds are not selected from the group consisting of 5-0-dedesosaminyl-5-
- the present invention provides a compound according to formula I where:
- R' C 2 H 5 optionally substituted with a hydroxyl group
- R 2 , R 4 , R 5 , R ⁇ , R 7 and R 9 are all CH 3
- R 3 is H or OH
- R 10 H or CH 3
- R l3 H or CH 3
- R l5 H or A ⁇ z ° R
- R 1 H or OH
- R 2 , R 4 , R 5 , R 6 , R 7 and R 9 are all CH 3 R 3 is H or OH
- R l3 H or CH
- R l6 H or OH with the proviso that the compounds are not selected from the group consisting of 5-0-dedesosaminyl-5-
- mycaminose and angolosamine may be added to other aglycones or pseudoaglycones for example (but without limitation) a tylactone or spinosyn pseudoaglycone.
- aglycones or pseudoaglycones may be the naturally occurring structure or they may be modified in the aglycone backbone, such modified substrates may be produced by chemical semi-synthetic methods (Kaneko et al, 2000 and references cited therein), or, alternatively, via PKS engineering, such methods are well known in the art (for example WO 93/13663, WO 98/01571, WO 98/01546, WO 98/49315, Kato, Y. et l, 2002).
- the present invention provides 5-O-angolosaminyl tylactone, 5-0-mycaminosyI tylactone, 17-0-angolosaminyl spinosyn and 17-0-mycaminosyl spinosyn.
- the process of the host cell selection further comprises the optional step of deleting or inactivating or adding or manipulating genes in the host cell.
- This process comprises the improvement of recombinant host strains for the preparation and isolation of compounds of the invention, in particular 5- 0-dedesosaminyl-5-0-mycaminosyl erythromycins and 5-0-dedesosaminyl-5-O-mycaminosyl azithromycins, specifically 5-0-dedesosaminyl-5-0-mycaminosyl erythromycin A, 5-0-dedesosaminyI- 5-0-mycaminosyI erythromycin C, 5-0-dedesosaminyl-5-0-mycaminosyl erythromycin B, 5-0- dedesosaminyl-5-0-mycaminosyl erythromycin D and 5-0-dedesosaminyl-5-0-mycaminosyl azithromycin.
- Example 1 This approach is exemplified in Example 1 by introducing an eryBVI mutation into the chromosome of S. erythraea SGQ2 in order to optimise the conversion of the substrate 3-0-mycarosyl erythronolide B to 5-0-dedesosaminyl-5-0-mycaminosyl erythromycins.
- the invention relates to the construction of gene cassettes.
- the cloning method used to isolate these gene cassettes is analogous to that used in PCT/GB03/003230 and diverges significantly from the approach previously described (WO 01/79520) by assembling the gene cassette directly in an expression vector rather than pre-assembling the genes in pUCl 8/19 plasmids, thus providing a more rapid cloning procedure for the isolation of gene cassettes.
- the strategy for isolating these gene cassettes is exemplified in Example 1 to Example 11. A schematic overview of the strategy is given in Figure 2.
- Another aspect of the invention allows the enhancement of gene expression by changing the order of genes in a gene cassette, the genes including but not limited to tylMI, tylMIII, tylB, eryCVI, tylAI, tylAII, eryCIII, eryBV, angAI, angAII, angMIII, angB, angMI, angorfH, angorf4, eryBVI, eryK, eryG, angMII, tylMII, desVII equallymidI, spnO, spnN, spnP and genes with similar functions, allowing the arrangement of the genes in a multitude of permutations ( Figure 2).
- the cloning strategy outlined in this invention also allows the introduction of a histidine tag in combination with a terminator sequence 3' of the gene cassette to enhance gene expression (see Example
- Another aspect of the invention comprises the use of alternative promoters such as VtipA (Ali et al, 2002) and/or Yptr (Salah-Bey et al, 1995) to express genes and/or assembled gene cassette(s) to enhance expression.
- VtipA Ali et al, 2002
- Yptr Syah-Bey et al, 1995
- Another aspect of the invention describes the multiple uses of promoter sequences in the assembled gene cassette to enhance gene expression as exemplified in Example 6.
- Another aspect of the invention describes the addition of genes encoding for a NDP-glucose- synthase such as tylAI and a NDP-glucose-4,6-dehydratase such as tylAII to the gene cassette in order to enhance the endogenous production of the activated sugar substrate.
- a NDP-glucose- synthase such as tylAI
- a NDP-glucose-4,6-dehydratase such as tylAII
- alternative sources include but are not limited to: TylAI- homologues: DesIII of Streptomyces venezuelae (accession no AAC68682), GrsD of Streptomyces griseus (accession no AAD31799), AveBIII of Streptomyces avermitilis (accession no BAA84594), Gtt of Saccharopolyspora spinosa (accession no AAK83289), SnogJ of Streptomyces nogalater (accession no AAF01820), AclY of Streptomyces galilaeus (accession no BAB72036), LanG of Streptomyces cyanogenus (accession no AAD13545), Graorf 16(GraD) of Streptomyces violaceoruber (accession no AAA99940), OleS of Streptomyces antibioticus (accession no AAD55453) and StrD of Streptomyces griseus (accession
- TylAII- homologues AprE of Streptomyces tenebrarius (accession no AAG18457), GdH of S. spinosa (accession no AAK83290), DesIV of S. venezuelae (accession no AAC68681), GdH of S. erythraea (accession no AAA68211), AveBII of S. avermitilis (accession no BAA84593), Scf81.08C of Streptomyces coelicolor (accession no CAB61555), LanH of S. cyanogenus (accession no AAD13546), Graorfl7 (GraE) of S.
- glycosyltransferases include but are not limited to: TylMII (Accession no CAA57472), Des VII (Accession noAAC68677), MegCIII (Accession no AAG13921), MegDI (Accession no AAG13908) or AngMII of S. eurythermus.
- the gene cassette may additionally comprise a chimeric glycosyltransferase (GT). This is particularly of benefit where the natural GT does not recognise the combination of sugar and aglycone that is required for the synthesis of the desired analogue.
- GT chimeric glycosyltransferase
- the present invention specifically contemplates the use of a chimearic GT wherein part of the GT is specific for the recognition of the sugar whose synthesis is directed by the genes in said expression cassette when expressed in an appropriate strain background and part of the GT is specific for the aglycone or pseudoaglycone template (Hu and Walker, 2002).
- aglycone or pseudoaglycone template Hu and Walker, 2002.
- plasmids containing the gene cassettes may be integrated into any neutral site on the chromosome using homologous recombination sites.
- the gene cassettes may be introduced on self-replicating plasmids (Kieser et al, 2000; WO 98/01571).
- a further aspect of the invention provides a process for the production of compounds of the invention and optionally for the isolation of said compounds.
- a further aspect of the invention is the use of different fermentation methods to optimise the production of the compounds of the invention as exemplified in Example 1.
- Another aspect of the invention is the addition of ery genes such as eryK and/or eryG into the gene cassette.
- the process can be optimised for the production of a specific erythromycin (i.e. A, B, C, D) or azithromycin by manipulation of the genes eryG (responsible for the methylation on the mycarose sugar) and/or eryK (responsible for hydroxylation at C12).
- erythromycin i.e. A, B, C, D
- azithromycin by manipulation of the genes eryG (responsible for the methylation on the mycarose sugar) and/or eryK (responsible for hydroxylation at C12).
- an extra copy of eryK may be included into the gene cassette.
- the erythromycin B analogue is required, this can be achieved by deletion of the eryK gene from the S. erythraea host strain, or by working in a heterologous host in which the gene and/or its functional homologue, is not present.
- erythromycin D analogue this can be achieved by deletion of both eryG and eryK genes from the S. erythraea host strain, or by working in a heterologous host in which both genes and/or their functional homologues are not present.
- erythromycin C analogue this can be achieved by deletion of the eryG gene from the S. erythraea host strain, or by working in a heterologous host in which the gene and/or its functional homologues are not present.
- a preferred host cell strain is a mammalian cell strain, fungal cells strain or a prokaryote.
- the host cell strain is an actinomycete, a Pseudomonad, a myxobacterium or an E. coli.
- the host cell strain is an actinomycete, still more preferably including, but not limited to Saccharopolyspora erythraea, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseofuscus, Streptomyces cinnamonensis, Streptomyces fradiae, Streptomyces eurythermus, Streptomyces longisporoflavus, Streptomyces hygroscopicus, Saccharopolyspora spinosa, Micromonospora griseorubida, Streptomyces lasaliensis, Streptomyces venezuelae, Streptomyces antibioticus, Streptomyces lividans, Streptomyces rimosus, Streptomy
- the host cell strain is selected from Saccharopolyspora erythraea, Streptomyces griseofuscus, Streptomyces cinnamonensis, Streptomyces albus, Streptomyces lividans, Streptomyces hygroscopicus sp., Streptomyces hygroscopicus var. ascomyceticus, Streptomyces longisporoflavus,
- Saccharopolyspora spinosa Streptomyces tsukubaensis, Streptomyces coelicolor, Streptomyces fradiae, Streptomyces rimosus, Streptomyces avermitilis, Streptomyces eurythermus, Streptomyces venezuelae, Amycolatopsis mediterranei.
- the host strain is Saccharopolyspora erythraea.
- the present invention provides methods for the production and isolation of compounds of the invention, in particular of erythromycin and azithromycin analogues which differ from the natural compound in the glycosylation of the C-5 position, for example but without limitation: novel 5-0- dedesosaminyI-5-O-mycaminosyI or angolosaminyl erythromycins and 5-0-dedesosaminyl-5-0- mycaminosyl, or angolosaminyl azithromycins which are useful as anti-microbial agents for use in human or animal health.
- the present invention provides novel products as obtainable by any of the processes disclosed herein. Brief description of Figures
- Figure 1A Structures of 5-0-dedesosaminyl-5-0-mycaminosyl erythromycin A, 5-0- dedesosaminyl-5-O-mycaminosyl erythromycin B and 5-0-dedesosaminyl-5-0- mycaminosyl erythromycin C.
- Figure IB Structure of 5-0-dedesosaminyl-5-0-mycaminosyl azithromycin.
- FIG. 2 Schematic overview over the gene cassette cloning strategy.
- Vector pSG144 was derived from vector pSG142 (Gaisser et al, 2000).
- dam DNA isolated from dam ' strain background
- Xbal met Xbal site sensitive to Dam methylation
- eryRHS DNA fragment of the right hand side of the ery-cluster as described previously (Gaisser et al, 2000).
- Figure 3 Amino acid comparison between the published sequence of TylAI (below, SEQ ID NO: 1) and the amino acid sequence detected from the sequencing data described in this invention (above, SEQ ID NO: 2). The changes in the amino acid sequence are underlined.
- Figure 4 Amino acid comparison between the published sequence of TylAII (below, SEQ ID NO: 3) and the amino acid sequence detected from the sequencing data described in this invention (above, SEQ ID NO: 4). The changes in the amino acid sequence are underlined.
- Figure 5 Structure of 5-0-angolosaminyl tylactone.
- FIG. 6 Shows an overview of the angolamycin polyketide synthase gene cluster.
- Figure 7 The DNA sequence which comprises orfH and or/75 (angB) from the angolamycin gene cluster (SEQ ID NO: 5).
- Figure 8 The DNA sequence which comprises or ⁇ (angAI), or ⁇ (angAII) and orf4 from the angolamycin gene cluster (SEQ ID NO: 6).
- Figure 9 The DNA sequence which comprises orfl * (angMIII), or ⁇ * (angMII), and or ⁇ * (angMI) from the angolamycin gene cluster (SEQ ID NO: 7).
- Figure 10 The amino acid sequence which corresponds to or ⁇ (angAI, SEQ ID NO: 8).
- Figure 1 1 The amino acid sequence which corresponds to or ⁇ (angAII, SEQ ID NO: 9).
- Figure 12 The amino acid sequence which corresponds to orfl (SEQ ID NO: 10)
- Figure 13 The amino acid sequence which corresponds to orfl 4 (SEQ ID NO: 11).
- Figure 14 The amino acid sequence which corresponds to orfl 5 (angB, SEQ ID NO: 12).
- Figure 15 The amino acid sequence which corresponds to orfl* (angMIII, SEQ ID NO: 13).
- Figure 16 The amino acid sequence which corresponds to or ⁇ * (angMII, SEQ ID NO: 14).
- Figure 17 The amino acid sequence which corresponds to or ⁇ * (angMI, SEQ ID NO: 15).
- Escherichia coli XLl-Blue MR (Stratagene), E. coli DH10B (GibcoBRL) and E. coli ET12567 were grown in 2xTY medium as described by Sambrook et al, (1989).
- Vector pUC18, pUC19 and Litmus 28 were obtained from New England Biolabs.
- E. coli transformants were selected with 100 ⁇ g/mL ampicilhn.
- Conditions used for growing the Saccharopolyspora erythraea NRRL 2338-red variant strain were as described previously (Gaisser et al, 1997, Gaisser etal, 1998). Expression vectors in S.
- erythraea were derived from plasmid pSG142 (Gaisser et al, 2000). Plasmid-containing S. erythraea were selected with 25-40 ⁇ g/mL thiostrepton or 50 ⁇ g/mL apramycin. To investigate the production of antibiotics, S. erythraea strains were grown in sucrose-succinate medium (Caffrey et al, 1992) as described previously (Gaisser et al, 1997) and the cells were harvested by centrifugation. Chromosomal DNA of Streptomyces rochei ATCC21250 was isolated using standard procedures (Kieser et al, 2000). Feedings of 3-O-mycarosyl erythronolide B or tylactone were carried out at concentrations between 25 to 50 mg /L.
- DNA manipulation and sequencing DNA manipulations were carried out as described in Sambrook et al, (1989). Protoplast formation and transformation procedures of S. erythraea were as described previously (Gaisser et al, 1997). Southern hybridizations were carried out with probes labelled with digoxigenin using the DIG DNA labelling kit (Boehringer Mannheim). DNA sequencing was performed as described previously (Gaisser et al, 1997), using automated DNA sequencing on double stranded DNA templates with an ABI Prism 3700 DNA Analyzer. Sequence data were analysed using standard programs.
- Extraction and mass spectrometry 1 mL of each fermentation broth was harvested and the pH was adjusted to pH 9. For extractions an equal volume of ethyl acetate, methanol or acetonitrile was added, mixed for at least 30 min and centrifuged. For extractions with ethyl acetate, the organic layer was evaporated to dryness and then re- dissolved in 0.5 mL methanol. For methanol and acetonitrile extractions, supernatant was collected after centrifugation and used for analysis. High resolution spectra were obtained on a Bruker BioApex II FT- ICR (Bruker, Bremen, FRG).
- the HPLC system comprised an Agilent HP1 100 equipped with a Luna 5 ⁇ m C18 BDS 4.6 x 250 mm column (Phenomenex, Macclesfield, UK) heated to 40 °C. The gradient elution was from 25% mobile phase B to 75% mobile phase B over 19 minutes at a flow rate of 1 mL/min.
- Mobile phase A was 10% acetonitrile: 90% water, containing 10 mM ammonium acetate and 0.15% formic acid
- mobile phase B was 90% acetonitrile: 10% water, containing 10 mM ammonium acetate and 0.15% formic acid.
- the HPLC system described was coupled to a Bruker Daltonics Esquire3000 electrospray mass spectrometer operating in positive ion mode.
- the pelleted cells were similarly extracted once more with Me 2 CO/MeOH (1:1).
- the cell extracts were combined with the Me 2 CO from the HP20 column and the solvent was removed in vacuo to give an aqueous concentrate.
- the aqueous was extracted with EtOAc (3 x) and the solvent removed in vacuo to give a crude extract.
- the residue was dissolved in CH 3 CN/MeOH and purified by repeated rounds of reverse phase (C18) high performance liquid chromatography using a Gilson HPLC, eluting a Phenomenex 21.2 x 250 mm Luna 5 ⁇ m C18 BDS column at 21 mL/min.
- c indicates that the gene is encoded by the complement DNA strand potential functions of the predicted polypeptides (SEQ ID No.8 to 15) were obtained from the NCBI database using a BLAST search.
- Example 1 Bioconversion of 3-0-mycarosyl erythronolide B to 5-0-dedesosaminy_-5-0- mycaminosyl erythromycins using gene cassette pSG144tylAItyIA ⁇ tylMIIItylBtylIatylMIeryCIII.
- Plasmid pSGl 42 (Gaisser et al, 2000) was digested with Xbal and a fill-in reaction was performed using standard protocols. The DNA was re- ligated and used to transform E. coli DH10B. Construct pSG143 was isolated and the removal of the_Yb ⁇ I site was confirmed by sequence analysis.
- the gene eryBV was amplified by PCR using the primers cas01eG21 (WO01/79520) and 7966 5'- GGGGAATTCAGATCTGGTCTAGAGGTCAGCCGGCGTGGCGGCGCGTGAGTTCCTCCAGTCGC GGGACGATCT -3' (SEQ ID NO: 16) and pSG142 (Gaisser et al, 2000) as template.
- the PCR fragment was cloned using standard procedures and plasmid pUC 18eryBVcas was isolated with an Ndel site overlapping the start codon of eryBV a d Xb ⁇ l and BgRl sites (underlined) following the stop codon. The construct was verified by sequence analysis.
- Isolation of vector pSGLitl The isolation of this vector is described in PCT/GB03/003230. Isolation of pSGLitl eryCIII Plasmid pSGCIII (WOO 1/79520) was digested with NdellBglll and the insert fragment was isolated and ligated with the NdellBglll treated vector fragment of pSGLitl . The ligation was used to transform E. coli ET12567 and plasmid pSGLitl eryCIII was isolated using standard procedures. The construct was confirmed using restriction digests and sequence analysis. This cloning strategy allows the introduction of a his-tag C-terminal of EryCIII.
- Plasmid pSGTYLM2 (WO01/7952) was digested with NdellBglll and the insert fragment was isolated and ligated with the NdellBglll treated vector fragment of pSGLitl. The ligation was used to transform E. coli ET12567 and plasmid pSGLitl tylMII was isolated using standard procedures. The construct was confirmed using restriction digests and sequence analysis. This cloning strategy allows the introduction of a his-tag C-terminal of TylMII.
- Plasmid pSGLitl was isolated and digested with NdellBglll and an approximately 1.3 kb insert was isolated. Plasmid pSG143 was digested with NdeVBglll, the vector band was isolated and ligated with the approximately 1.3 kb band from pSGLitl followed by transformation of E. coli DH10B. Plasmid pSG144 ( Figure 2) was isolated and the construct was verified by DNA sequence analysis. This vector allows the assembly of gene cassettes directly in an expression vector ( Figure 2) without prior assembly in pUC-derived vectors (WO 01/79520) in analogy to PCT/GB03/003230 using vector pSG144 instead of pSGsetl .
- Plasmid pSG144 differs from pSG142 in that the Xbal site between the thiostrepton resistance gene and the eryRHS has been deleted and the his- tag at the end of eryBV has been removed from pSG142 and replaced in pSG144 with an Xb ⁇ l site at the end of eryBV. This is to facilitate direct cloning of genes to replace eryBV and then build up the cassette.
- Isolation ofpSGH4eryCIII EryCIII was amplified by PCR reaction using standard protocols, with primers cas01eG21 (WO 01/79520) and caseryCIII2 (WO 01/79520) and plasmid pSGCIII (Gaisser et al, 2000) as template.
- the approximately 1.3 kb PCR product was isolated and cloned into pUC18 using standard techniques. Plasmid pUCCIIIcass was isolated and the sequence was verified.
- the insert fragment of plasmid pUCCIIIcass was isolated after NdellXbal digestion and ligated with the NdellXbal digested vector fragment of pSG144. After the transformation of E. coli DH10B plasmid pSG144eryC//7was isolated using standard techniques.
- Plasmid Litmus 28 was digested with SpellXbal and the vector fragment was isolated.
- Plasmid pSGLitl (dam ) was digested with Xbal and the insert band was isolated and ligated with the SpellXbal digested vector fragment of Litmus 28 followed by the transformation of E. coli DH10B using standard techniques. Plasmid pSGLit2 was isolated and the construct was verified by restriction digest and sequence analysis.
- This plasmid can be used to add a 5' region containing n Xbal site sensitive to Dam methylation and a Shine Dalgarno region thus converting genes which were originally cloned with an Ndel site overlapping the start codon and an Xbal site 3 ' of the stop codon for the assembly of gene cassettes.
- This conversion includes the transformation of the ligations into E. coli ET12567 followed by the isolation of dam ' DNA and Xbal digests. Examples for this strategy are outlined below.
- Plasmid pSGLit2 and pUC 19tylAI were digested with Ndel lXbal and the insert band of pUCl9tylAI and the vector band of pSGLit2 were isolated, ligated and used to transform E. coli ET12567. Plasmid pSGLitltylAI (dam ' ) was isolated.
- pSGLit2 was digested with PacllXbal and the vector fragment was isolated and ligated with the PacllXbal digested insert fragment of pUC 19ty/v__7. The ligated DNA was used to transform E. coli ET12567 and plasmid was isolated.
- PCR product was cloned into Sm ⁇ l digested pUC19 using standard techniques and plasmid pGC19eryCVI was isolated and verified by sequence analysis.
- Plasmid p ⁇ JC19eryCVI was digested with NdellXbal and ligated with the NdellXbal digested vector fragment of pSGLit2 followed by transformation of E. coli ET12567. Plasmid pSGLit2eryCF_ " (dam ) was isolated.
- Plasmid pSG144 and pUC19ty _47 were digested with NdellXbal and the insert band of pUC 19tylAI and the vector band of pSG144 were isolated, ligated and used to transform E. coli DH10B. Plasmid pSG ⁇ 44tylAI was isolated using standard protocols.
- Plasmid pSGH4tylAItyl All Plasmid pSGLit2ty/_4_7 (dam ) was digested with Xbal and ligated with Xbal digested plasmid pSG144ty/_i_. The ligation was used to transform E. coli DH10B and plasmid pSG144ty/_4_ " ty/_4_7 was isolated and verified using standard protocols.
- Plasmid pUCl 8tylM3 (Isolation described in WO01/79520) was digested with NdellXbal and the insert band and the vector band of NdellXbal digested pSGLit2 were isolated, ligated and used to transform E. coli ET12567. Plasmid pSGLit2tylMIII (dam ' ) was isolated using standard protocols. The construct was verified using restriction digests and sequence analysis.
- Plasmid pSGLit2ty. /_7 (dam ) was digested with Xbal and the insert band was ligated with Xbal digested plasmid pSG 144tylAItylAII. The ligation was used to transform E. coli DH10B and plasmid pSG ⁇ 4tylAItylAIItylMIII no36 was isolated using standard protocols. The construct was verified using restriction digests and sequence analysis. Isolation of plasmid pSGLit2tylB Plasmid pUC18ty/_?
- Plasmid pSGL.t2ty._3 (dam ) was digested with_ ⁇ l and the insert band was ligated with _ ⁇ > ⁇ I digested plasmid pSGl44tylAItylAIItylMIII. The ligation was used to transform E. coli DHIOB and plasmid pSGl 44tylAItylAIItylMIIItylB no5 was isolated using standard protocols and verified by restriction digests and sequence analysis.
- Plasmid pGCtylla nol was digested with NdellXbal and the insert band and the vector band of NdellXbal digested pSGLit2 were isolated, ligated and used to transform E. coli ET12567. Plasmid pSGLit2ty// no 54 (dam ) was isolated using standard protocols. The construct was verified using sequence analysis.
- Plasmid pSGLit2ty/7 ⁇ (dam ) was digested with Xbal and the insert band was ligated with Xbal digested plasmid pSG ⁇ 44tylAItylAIItylMIIItylB.
- the ligation was used to transform E, coli DH10B and plasmid pSG ⁇ 44tylAItylAIItylMIIItylBtylIa no3 was isolated using standard protocols and verified by restriction digests and sequence analysis.
- Plasmid pUCtylMI (Isolation described in WO01/79520) was Pad digested and the insert was ligated with the Pad digested vector fragment of pSGLitl eryCIII using standard procedures. Plasmid pSGLitltv /eryC/_7no20 was isolated and the orientation was confirmed by restriction digests and sequence analysis.
- Plasmid pSGLitl tylMIeryCIII no20 was digested with Xbal/Bgl ⁇ l and the insert band was isolated and ligated with the Xbal/Bglll digested vector fragment of plasmid pSG144tylAItylAIItylMIIItylBtylIa no3.
- Plasmid pSGl 44tylAItylAIItylMIIItylBtyllatylMIeryCIII was isolated using standard procedures and the construct was confirmed using restriction digests and sequence analysis. Plasmid preparations were used to transform S. erythraea mutant strains with standard procedures.
- Plasmid pSGKCl was isolated by cloning the approximately 0.7 kb DNA fragment of the eryBVI gene by using
- Cosmid 2 containing the right hand site of the ery- cluster was isolated from an existing cosmid library (Gaisser et al, 1997) by screening with eryBV as a probe using standard techniques.
- the amplified DNA fragment was isolated and cloned into i ⁇ coRV digested pKCl 132 (Bierman et al, 1992) using standard methods.
- the ligated DNA was used to transform E. coli DH10B and plasmid pSGKCl was isolated using standard molecular biological techniques. The construct was verified by DNA sequence analysis.
- Plasmid pSGKCl was used to transform S. erythraea SGQ2 using standard techniques followed by selection with apramycin. Thiostrepton/apramycin resistant transformant S. erythraea Q42/1 was isolated.
- Bioconversion using S. erythraea Q42/lpSG144tylAItylAIItylMIIItylBtyllatylMIeryCIII Bioconversion assays using 3-O-mycarosyl erythronolide B are carried out as described in General Methods. Improved levels of mycaminosyl erythromycin A are detected in bioconversion assays using S. erythraea Q42/lpSG ⁇ 44tylAItylAIItylMIIItylBtyllatylMIeryCIII compared to bioconversion levels previously observed (WOOl/79520).
- Example 2 Isolation of mycaminosyl tylactone using gene cassette pSG 4tylAItylA ⁇ tylMIIItylBtylIatylMItylMII
- Plasmid pGCtylMI (Isolation described in WOOl/79520) was Pad digested and the insert was ligated with the Pad digested vector fragment of pSGLitl tylMII using standard procedures. Plasmid pSGLitl tylMItylMII nol6 was isolated and the construct was confirmed by restriction digests and sequence analysis.
- Plasmid pSGLitl tylMItylMII nol 6 was digested with XbaUBglll and the insert band was isolated and ligated with the Xbal/BgHl digested vector fragment of plasmid pSG144tylAItylAIItylMIIItylBtylIa no3.
- Plasmid pSGl 44tylAItylAIItylMIIItylBtyllatylMItylMII was isolated using standard procedures and the construct was confirmed using restriction digests and sequence analysis. The plasmid was isolated and used for transformation of S. erythraea mutant strains using standard protocols.
- Bioconversion using gene cassette pSG144tylAItylAIItylMIIItylBtyllatylMItylMII The conversion of fed tylactone to mycaminosyl tylactone was assessed in bioconversion assays using S. erythraea Q42l ⁇ pSG ⁇ 44tylAItylAIItylMIIItylBtyllatylMItylMII. Bioconversion assays were carried out using standard protocols. The analysis of the culture showed the major ion to be 568.8 [M+H] + consistent with the presence of mycaminosyl tylactone.
- Tylla was identified in the biosynthetic pathway of dTDP-3-acetamido- 3,6-dideoxy-alpha-D-galactose in Aneurinibacillus thermoaerophilus L420-91 ⁇ (Pfoestl et al, 2003) and the function was postulated as a novel type of isomerase capable of synthesizing dTDP-6-deoxy-D- xylohex-3-ulose from dTDP-6-deoxy-D-xylohex-4-ulose.
- Example 3 Bioconversion of 3-O-mycarosyl erythronolide B to 5-0-dedesosaminyl-5-0- mycaminosyl erythromycins using gene cassette pSG1448/27/95/21/44/193/6eryCIII (pSG144angAIangAHorfl4angMIIIangBangMIeryCIH).
- angMIII Cloning of angMIII by isolating plasmid Lit 1/4
- the gene angMIII was amplified by PCR using the primers BIOSG61 5'- GGGCATATGAGCCCCGCACCCGCCACCGAGGACCC -3' (SEQ ID NO: 27) and BIOSG62 5'- GGTCTAGAGGTCAGTTCCGCGGTGCGGTGGCGGGCAGGTCAC -3' (SEQ ID NO: 28).
- Cosmid5B2 containing a fragment of the angolamycin biosynthetic pathway was used as template.
- Plasmid LitI/4 was isolated with an Ndel site overlapping the start codon of angMIII and an Xbal site following the stop codon. The construct was verified by sequence analysis.
- Plasmid L ⁇ ' tl/4 was digested with NdellXbal and the about 1.4 kb fragment was isolated and ligated to NdellXbal digested DNA of pSGLit2. The ligation was used to transform E. coli ET12567 and plasmid pSGL ⁇ X21/4 no7 (dam ) was isolated. This construct was digested with Xbal and used for the construction of gene cassettes.
- angMII Cloning of angMII by isolating plasmid LU2/8
- the gene angMII was amplified by PCR using the primers BIOSG63 5 '-GGGC ATATGCGTATC CTGCTGACGTCGTTCGCGCACAACAC -3' (SEQ ID NO: 29) and BIOSG64 5'-GGTCTAGAGGTCA GGCGCGGCGGTGCGCGGCGGTGAGGCGTTCG -3' (SEQ ID NO: 30) and cosmid5B2 containing a fragment of the angolamycin biosynthetic pathway was used as template.
- the 1.3 kb PCR fragment (PCR no2) was cloned using standard procedures and i ⁇ coRV digested plasmid Litmus28. Plasmid Lit2/ ⁇ was isolated with an Ndel site overlapping the start codon of angMII and anXb ⁇ l site following the stop codon. The construct was verified by sequence analysis.
- angMII Cloning of angMII by isolating plasmid pLitangMII(Bglll)
- the gene angMII was amplified by PCR using primers BIOSG63 5'-GGGCATATGCGTATCCT GCTGACGTCGTTCGCGCACAACAC -3' (SEQ ID NO: 29) and BIOSG80 5 '-GGAGATCTGGCGCG GCGGTGCGCGGCGGTGAGGCGTTCG -3' (SEQ ID NO: 31) and cosmid5B2 containing a fragment of the angolamycin biosynthetic pathway as template.
- the 1.3 kb PCR fragment was cloned using standard procedures and _ ⁇ coRV digested plasmid Litmus28.
- Plasmid LitangMII(BglII)no8 was isolated with an Ndel site overlapping the start codon of angMII and a Bgl ⁇ site instead of a stop codon thus allowing the addition of a his-tag. The construct was verified by sequence analysis.
- Plasmid L ⁇ ' tangMII(Bglll) was digested with NdellBglll and ligated with the NdellBglll digested vector fragment of pSGLitl. The ligation was used to transform E. coli ET12567 and plasmid pSGLitl angMII (dam ) was isolated using standard procedures.
- angMI Cloning of angMI by isolating plasmid Lit3/ 6
- the gene angMI was amplified by PCR using the primers BIOSG65 5'-GGGCATATGAAC CTCGAATACAGCGGCGACATCGCCCGGTTG -3' (SEQ ID NO: 32) and BIOSG66 5'- GGTCTAGAGGTCAGGCCTGGACGCCGACGAAGAGTCCGCGGTCG -3' (SEQ ID NO: 33) and cosmid5B2 containing a fragment of the angolamycin biosynthetic pathway was used as template.
- the 0.75 kb PCR fragment (PCR no3) was cloned using standard procedures and .EcoRV digested plasmid Litmus28. Plasmid Lit3/ ⁇ 5 was isolated with an Ndel site overlapping the start codon of angMI and an Xb ⁇ l site following the stop codon. The construct was verified by sequence analysis.
- Plasmid was digested with NdellXbal and the about 0.8 kb fragment was isolated and ligated to NdellXbal digested DNA of pSGLit2. The ligation was used to transform E. coli ET12567 and plasmid pSGLit23/ ⁇ 5 no8 (dam ) was isolated. This construct was digested with Xbal and the isolated about 1 kb fragment was used for the assembly of gene cassettes.
- angB Cloning of angB by isolating plasmid Lit4/ 19
- the gene angB was amplified by PCR using the primers BIOSG67 5'-GGGCATATGACTACCT ACGTCTGGGACTACCTGGCGG -3' (SEQ ID NO: 34) and BIOSG68 5 '-GGTCTAGAGGTC AG AGC GTGGCCAGTACCTCGTGCAGGGC -3' (SEQ ID NO: 35) and cosmid4H2 containing a fragment of the angolamycin biosynthetic pathway was used as template.
- the 1.2 kb PCR fragment (PCR no4) was cloned using standard procedures and EcoKY digested plasmid Litmus28. Plasmid l ⁇ t4/19 was isolated with an Ndel site overlapping the start codon of angB and an Xbal site following the stop codon. The construct was verified by sequence analysis.
- Plasmid IZA4/19 was digested with NdellXbal and the 1.2 kb fragment was isolated and ligated into NdellXbal digested DNA of pSGLit2. The ligation was used to transform E. coli ET12567 and plasmid pSGLit2 /79 no24 (dam ) was isolated. This construct was digested with_ ⁇ Z> ⁇ I and the isolated 1.2 kb fragment was used for the assembly of gene cassettes.
- Plasmid Lit5/2 was digested with NdellXbal and the approximately 1 kb fragment was isolated and ligated to NdellXbal digested DNA of pSGZ,.t2.
- the ligation was used to transform E. coli ET12567 and plasmid pSGLit25/2 no24 (dam ) was isolated.
- This construct was digested with Xbal, the about 1 kb fragment isolated and used for the assembly of gene cassettes.
- Plasmid Lit7/9 was digested with NdellXbal and the approximately 1 kb fragment was isolated and ligated to NdellXbal digested DNA of pSGLit2. The ligation was used to transform E. coli ET12567 and plasmid pSGLit27/9 no 15 (dam ) was isolated. This construct was digested with Xbal and the isolated 1 kb fragment was used for the assembly of gene cassettes.
- angAI Cloning of angAI (or ⁇ ) by isolating plasmid LU8/2
- the gene angAI was amplified by PCR using the primers BIOSG73 5'-GGGCATATGAAGGGC
- Plasmid Lit ⁇ /2 was isolated with an Ndel site overlapping the start codon of angAI and an Xbal site following the stop codon. The construct was verified by sequence analysis.
- angAII Cloning of angAII (or ⁇ ) by isolating plasmid LU7/9
- the gene angAII was amplified by PCR using the primers BIOSG71 5 '-GGGC ATATGCGGCTG
- CTGGTCACCGGAGGTGCGGGC-3' SEQ ID NO: 40
- GTGCGCCGGGCCTCCTGCG-3' (SEQ ID NO: 41) and cosmid4H2 containing a fragment of the angolamycin biosynthetic pathway was used as template.
- the 1.0 kb PCR fragment was cloned using standard procedures and i_coRV digested plasmid Litmus28. Plasmid lZ ⁇ t7/9 was isolated with an Ndel site overlapping the start codon of angAII and an Xbal site following the stop codon. The construct was verified by sequence analysis.
- Plasmid LitS/2 was digested with NdellXbal and the 1 kb fragment was isolated and ligated to
- Plasmid LitS/2 was digested with NdellXbal and the approximately 1 kb fragment was isolated and ligated with NdellXbal digested DNA of pSG144.
- the ligation was used to transform E. coli DHIOB and plasmid pSG 1448/2 (dam ' ) (pSG144 ⁇ «g_4.Z) was isolated using standard procedures. This construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit27/9 isolated from E.coli ET12567 was digested withJ ⁇ l and the 1 kb fragment was isolated and ligated with the Xbal digested vector fragment of pSGl448/2 (pSG ⁇ 44angAI). The ligation was used to transform E. coli DHIOB and plasmid pSG 1445/27/9 (pSGl 4angAIangAII) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit2_/ ⁇ isolated from E. coli ET12567 was digested with ⁇ Y ⁇ l and the 1.4 kb fragment was isolated and ligated with the Xbal digested vector fragment of pSG144 ⁇ /27/9
- pSGl 44angAIangAII The ligation was used to transform E. coli DH10B and plasmid pSG144S/27/91/4 (pSG ⁇ 44angAIangAIIangMIII) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit24/19 isolated from E. coli ET12567 was digested with Xbal and the about 1.2 kb fragment was isolated and ligated with the Xbal digested vector fragment of pSG ⁇ 448/27/91/4 (pSG ⁇ 44angAIangAIIangMIII). The ligation was used to transform E. coli DH10B and plasmid pS>G ⁇ 448/27/91/44/19 (pSGl 44angAIangAIIangMIIIangB) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit23/6 isolated from E. coli ET12567 was digested with Xbal and the about 0.8 kb fragment was isolated and ligated with theXbal digested vector fragment of pSG1448/27/91/44/19 (pSG ⁇ 44angAIangAIIangMIIIangB). The ligation was used to transform E. coli DH10B and plasmid pSG ⁇ 448/27/91/44/193/6 (pSG144angAIangAIIangMIIIangBangMI) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLitl eryCIII isolated from E. coli ET12567 was digested with Xbal/Bglll and the about 1.2 kb fragment was isolated and ligated with the-Y& ⁇ l digested and partially Bglll digested vector fragment of pSGl 448/27/91/44/ 193/6 (pSG144angAIangAIIangMIIIangBangMI).
- the Bglll partial digest was necessary due to the presence of a Bglll site in angB.
- the ligation was used to transform E.
- coli DHI OB and plasmid pSGU48/27/91/44/193/6eryCIII no9 was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis. EryCIII carries a his-tag fusion at the end.
- Plasmid pSGLit25/2 isolated from E. coli ET12567 was digested with_Y& ⁇ I and the about 1 kb fragment was isolated and ligated with the _Y& ⁇ I digested vector fragment of pSG1448/27/°
- pSGl44angAIangAII The ligation was used to transform E. coli DH10B and plasmid pSGl448/27/95/2 (pSG 44angAIangAIIorf 14) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit2_7 isolated from E. coli ET12567 was digested with _ ⁇ > ⁇ I and the 1.4 kb fragment was isolated and ligated with the Xbal digested vector fragment of pSG ⁇ 448/27/95/2 (pSGl 44angAIangAIIorfl 4). The ligation was used to transform E. coli DH10B and plasmid pSG 1448/27/95/21/4 (pSGl44angAIangAIIorfl4angMIII) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit24/19 isolated from E. coli ET12567 was digested with Xbal and the 1.2 kb fragment was isolated and ligated with the Xbal digested vector fragment of pSG1448/27/95/27/
- Plasmid pSG ⁇ 448/27/91/44/193/6eryCIII no9 was digested with Bglll and the about 2 kb fragment was isolated and ligated with the Bglll digested vector fragment of pSG ⁇ 448/27/95/21/44/19
- pSG 144angAIangAIIorfl4angMIIIangB The ligation was used to transform E. coli DHIOB and plasmid pSG ⁇ 448/27/95/21/44/193/6eryCIII(pSGl44angAIangAIIorfl4angMIIIangBangMIeryCIII) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis. EryCIII carries a his-tag fusion at the end. The construct was used to transform S. erythraea SGQ2 using standard procedures.
- Plasmid pSG 1448/27/91/44/193/6tylMII no9 was digested with Bglll and the about 2 kb fragment was isolated and ligated with the Bglll digested vector fragment of pSGl 448/27/95/21/44/19 (pSG ⁇ 44angAIangAIIorfl4angMIIIangB). The ligation was used to transform E.
- coli DH10B and plasmid pSG ⁇ 448/27/95/21/44/193/6tylMII was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis. TylMII carries a t ⁇ -tag fusion at the end.
- Example 5 Isolation of 5-0-dedesosaminyl-5-0-angolosa ⁇ r_.inyI erythromycins using gene cassette pSG1448/27/91/4spn05/2p4/193/6tylMII by bioconversion of 3-O-mycarosyl erythronolide B.
- plasmid conv nol For the multiple use of promoter sequences in act-controlled gene cassettes a 240 bp fragment was amplified by PCR using the primers BIOSG78 5'-GGGCATATGTGTCCTCCTTAATTAATCGAT GCGTTCGTCC-3' (SEQ ID NO: 42) and BIOSG79 5'-GGAGATCTGGTCTAGATCGTGTTCCCCTCC CTGCCTCGTGGTCCCTCACGC -3' (SEQ ID NO: 43) and plasmid pSG142 (Gaisser et al, 2000) as template. The 0.2 kb PCR fragment (PCR no5) was cloned using standard procedures and EcoRY digested plasmid Litmus28. Plasmid conv nol was isolated. The construct was verified by sequence analysis.
- Plasmid conv nol was digested with NdellBglll and the about 0.2 kb fragment was isolated and ligated with the BamHllNdel digested vector fragment of pSGLit2. The ligation was used to transform E. coli DH10B and plasmid pSGLit3religl was isolated using standard procedures. This construct was verified using restriction digests and sequence analysis.
- Plasmid Lit4/19 was digested with NdellXbal and the 1 .2 kb fragment was isolated and ligated to
- the ligation was used to transform E. coli ET12567 and plasmid pSGLit3 /i9 no23 was isolated.
- This construct was digested with_ ⁇ l and the isolated 1.4 kb fragment was used for the assembly of gene cassettes.
- Plasmid Lit6/4 was digested with NdellXbal and the DNA was isolated and ligated to NdellXbal digested DNA of pSGLit2. The ligation was used to transform E. coli ET12567 and plasmid pSGLit2 ⁇ 5/ no9 was isolated. This construct was confirmed by restriction digests and sequence analysis.
- Plasmid pGC ⁇ 9spnO was digested with NdellXbal and the 1.5 kb fragment was isolated and ligated to NdellXbal digested DNA of pSGLit2. The ligation was used to transform E. coli ET12567 and plasmid pSGLit2_ . «0 no 4 was isolated using standard procedures. This construct was digested with Xbal and the isolated 1.5 kb fragment was used for the assembly of gene cassettes.
- Plasmid pSG ' 2spnO no4 isolated from E. coli ET12567 was digested with Xbal and the 1.5 kb fragment was isolated and ligated with theXbal digested vector fragment of pSG 1448/27/91/4 (pSG ⁇ 44angAIangAIIangMIIl). The ligation was used to transform E. coli DHIOB and plasmid pSG 1448/27/9 l/4spnO (pSG144angAIangAIIangMIIIspnO) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit25/2 no24 isolated from E. coli ET 12567 was digested with Xbal and the 1 kb fragment was isolated and ligated with the _ > ⁇ l digested vector fragment of pSG 1448/27/91/4spnO (pSG 144angAIangAIIangMIIIspnO). The ligation was used to transform E.
- coli DH10B and plasmid pSG 1448/27/9 l/4spn05/2 was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGL ⁇ ' 34/ 19 no23 was digested with_ ⁇ I and the about
- 1.4 kb fragment was isolated and ligated with the Xbal digested vector fragment of pSGl 448/27/9 l/4spn05/2 (pSGl44angAIangAIIangMIIIspnOangorfl4).
- the ligation was used to transform E. coli DHIOB and plasmid pSGU48/27/91/4spn05/2p4/19 (pSG 144angAIangAIIangMIIIspnOangorfHpangB) was isolated using standard protocols.
- the construct was verified with restriction digests and sequence analysis, 'p' indicates the presence of the promoter region in front of angB to emphasize the presence of multiple promoter sites in the construct.
- Plasmid pSG 1448/27/91/4spn05/2p4/193/6eryCIII no9 was digested with Bglll and the about 2 kb fragment was isolated and ligated with the Bglll digested vector fragment of pSG 1448/27/9 l/4spn05/2p4/19 (pSGl 4angAIangAIIangMIIIspnOorfl4pangB). The ligation was used to transform E.
- coli DHIOB and plasmid pSGl448/27/91/4spn05/2p4/193/6eryCIII (pSG ⁇ 44angAIangAIIangMIIIspnOorfl4pangBangMIeryCIII) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis. EryCIII carries a his-tag fusion at the end. 'p' indicates the presence of the promoter region in front of ⁇ ngB to emphasize the presence of multiple promoter sites in the construct. The plasmid construct was used to transform mutant strains of S. erythraea using standard procedures.
- Bioconversion of 3-O-mycarosyl erythronolide B to 5-0-dedesosaminyl-5-0-angolosaminyl erythromycins Strain S. erythraea Q42ll pSG1448/27/91/4spn05/2p4/193/6eryCIII as grown and bioconversions with fed 3-0-mycarosyl erythronolide B were performed as described in the General Methods. The cultures were analysed and peaks with m/z 704, m/z 718 and m/z 734 consistent with the presence of angolosaminyl erythromycin D, B and A, respectively, were observed.
- Plasmid pSG1448/27/91/4spn05/2p4/l 93/6tylMII (pSG144angAIangAIIangMIIIspnOorfl4pangBangMItylMII) Plasmid pSG1448/27/91/44/193/6tv/ /Jno9 was digested with Bglll and the about 2 kb fragment was isolated and ligated with the Bglll digested vector fragment of pSGl448/27/91/4spn05/2p4/19
- This construct should enable homologous recombination into the genome in both LHS and RHS regions resulting in the isolation of a strain containing a deletion between these two regions of DNA.
- the LHS fragment (2201 bp) was PCR amplified using S. erythraea chromosomal DNA as template and primers BIdelNde (5 '-CCCATATGACCGGAGTTCGAGGTACGCGGCTTG-3 ' . SEQ ID NO: 48) and BIdelSpe (5'-GATACTAGTCCGCCGACCGCACGTCGCTGAGCC-3', SEQ ID NO: 49).
- Primer BIdelNde contains an Ndel restriction site (underlined) and primer BIdelSpe contains a Spel restriction site used for subsequent cloning steps.
- the PCR product was cloned into the Smal restriction site of pUC19, and plasmid pLSB177 was isolated using standard procedures. The construct was confirmed by sequence analysis. Similarly, RHS (2158 bp) was amplified by PCR using S.
- BVIIdelSpe (5'-TGCACTAGTGGCCGGGCGCTCGACGT CATCGTCGACAT-3', SEQ ID NO: 50) and BVIIdelEco (5 '-TCGATATCGTGTCCTGCGGTTTCACC TGCAACGCTG-3', SEQ ID NO: 51).
- Primer BVIIdelSpe contains a Spel restriction site and primer BVIIdelEco contains an i ⁇ coRV restriction site.
- the PCR product was cloned into the Smal restriction site of pUC19 in the orientation with Spel positioned adjacent to Kpnl and iseoRV positioned adjacent to Xbal.
- Plasmid pLSB177 was digested with Ndel and Spel, the ⁇ 2.2kb fragment was isolated and similarly plasmid pLSB178 was digested with Ndel and Spel and the -4.6 kb fragment was isolated using standard methods. Both fragments were ligated and plasmid pLSB188 containing LHS and RHS combined together at a Spel site in pUC19 was isolated using standard protocols. An NdellXbal fragment (-4.4 kbp) from pLSB188 was isolated and ligated with Spel and Ndel treated pCJR24. The ligation was used to transform E.
- Plasmid pLSB189 was used to transform S. erythraea P2338 and transformants were selected using thiostrepton.
- iS * . erythraea Dell 8 was isolated and inoculated into 6 ml TSB medium and grown for 2 days. A 5% inoculum was used to subculture this strain 3 times. 100 ⁇ l of the final culture were used to plate onto R2T20 agar followed by incubation at 30°C to allow sporulation. Spores were harvested, filtered, diluted and plated onto R2T20 agar using standard procedures. Colonies were replica plated onto R2T20 plates with and without addition of thiostrepton. Colonies that could no longer grow on thiostrepton were selected and further grown in
- TSB medium S. erythraea 18A1 was isolated and confirmed using PCR and Southern blot analysis. The strain was designated LB-1 /BIOT-2634. For further analysis, the production of erythromycin was assessed as described in General Methods and the lack of erythromycin production was confirmed. In bioconversion assays this strain did not further process fed erythronolide B and erythromycin D was hydroxylated at C12 to give erythromycin C as expected, indicating that EryK was still functional.
- Plasmid pSGLit35/2 isolated from E. coli ET12567 was digested with Xbal and the insert fragment was isolated and ligated with the Xbal digested vector fragment of pSG 1448/27/9 l/4spnO (pSGl 44angAIangAIIangMIIIspnO). The ligation was used to transform E.
- coli DH10B and plasmid pSG 1448/27/9 l/4spnOp5/2 (pSG 144angAIangAIIangMIIIspnOpangorf 14) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit2 /79 isolated from E. coli ET12567 was digested with _YZ? ⁇ I and the insert fragment was isolated and ligated with the Xbal digested vector fragment of pSG 1448/27/9 l/4spnOp 5/2 (pSG ⁇ 44angAIangAIIangMIIIspnOpangorfl4). The ligation was used to transform E.
- coli DH10B and plasmid pSGl448/27/91/4spnOp5/24/19 (pSGl 4angAIangAIIangMIIIspnOpangorfHangB) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit23/(5 isolated from E. coli ET12567) was digested with Xbal and the insert fragment was isolated and ligated with the Xbal digested vector fragment of pSG ⁇ 448/27/91/4spnOp5/24/19 (pSGl44angAIangAIIangMIIIspnOpangorfl4angB). The ligation was used to transform E.
- coli DH10B and plasmid pSGl 448/27/91/4spnOp5/24/l 93/6 (pSGl 44angAIangAIIangMIIIspnOpangorfl4angBangMl) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis. Isolation of plasmid pSG1448/27/91/4spnOp5/24/193/6angMII (pSG144angAIangAIIangMIIIspnOpangorfl4angBangMIangMII) Plasmid pSGLitl angMII (isolated fxo E.
- Plasmid pSG1448/27/9 Plasmid pSG1448/27/9 (pSGl44angAIangAII) was digested with Xbal and treated with alkaline phosphatase using standard protocols. The vector fragment was used for ligations with Xbal treated plasmid pSGLit26/ no9 followed by transformations of ii. coli DHIOB using standard protocols. Plasmid pSG 1448/27/96/4 (pSG 44angAIangAIIangorfl) was isolated using standard procedures and the construct was confirmed by restriction digests and sequence analysis.
- Plasmid pSGLit35/2 isolated from E. coli ET12567 was digested with Xbal and the insert fragment was isolated and ligated with the -Y ⁇ l digested vector fragment of pSG ⁇ 448/27/96/4 (pSG 144angAIangAIIangorf4). The ligation was used to transform E. coli DHIOB and plasmid pSGl 448/27/96/4p5/2 (pSG 144angAIangAIIangorflpangorf 14) was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSGLit2// isolated from E. coli ET12567 was digested with Xbal and the 1.4 kb fragment was isolated and ligated with the -Y& ⁇ l digested vector fragment of pSGl 448/27/96/4p 5/2 (pSG] 44angAIangAIIangorf4pangorfl4). The ligation was used to transform E.
- coli DH10B and plasmid pSG ⁇ 448/27/96/4p5/21/4 was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSG it24/19 isolated from E. coli ET12567 was digested with Xbal and the 1.4 kb fragment was isolated and ligated with the Xbal digested vector fragment of pSG 1448/27/96/4p5/21/4 (pSGl44angAIangAIIangorflpangorfl4angMIII). The ligation was used to transform E.
- coli DHIOB and plasmid pSG ⁇ 448/27/96/4p5/21/44/19 was isolated using standard protocols. The construct was verified with restriction digests and sequence analysis.
- Plasmid pSG 1448/27/9 l/4spnOp5/24/193/6angMII was digested with _3g7II and the about 2.2 kb fragment was isolated and used to ligate with the Bglll treated vector fragment of pSG ⁇ 448/27/96/4p5/21/44/19.
- the ligation was used to transform E. coli DHIOB using standard procedures and plasmid pSGl 448/27 /96/4p5/21/44/193/6angMII
- pSGl 44angAIangAIIangorflpangorfl4angMIIIangBangMIangMII was isolated. The construct was verified using restriction digests and sequence analysis. The plasmid was used to transform mutant strains of 8. erythraea with standard protocols.
- Bioconversion of tylactone with S. erythraea Q42/1 pSG1448/27/96/4p5/21/44/193/6angMII (pSG144angAIangAIIangorflpangorfl 4angMIIIangBangMIangMII) Biotransformation experiments feeding tylactone are carried out as described in General Methods and the cultures are analysed. Angolosaminyl tylactone is detected.
- Plasmid pUCl 9eryK was digested with NdellXbal and the insert band was ligated with NdellXbal digested pCJR24. Plasmid pLSBl 11 (pCJR24eryK) was isolated and the construct was verified with restriction digests.
- Plasmid pLSBl 1 1 (pCJR24eryi ⁇ ) was digested with NdellXbal and the insert fragment was isolated and ligated with the NdellXbal digested vector fragment of plasmid pSGLit2 and plasmid pLSBl 15 was isolated using standard protocols. The plasmid was verified using restriction digestion and DNA sequence analysis.
- Plasmid pLSB1 15 from s. coli ET12567 was digested with Xbal and the insert fragment was isolated and ligated with the Xbal treated vector fragment of pSGl 448/27/9 /21/4 (pSG ⁇ 44angAIangAIIangorfl4angMIIT). The ligation was used to transform E. coli DHIOB with standard procedures and plasmid pSGl448/27/95/21/4eryK(pSGl44angAIangAIIangorfl4angMIIIeryK) is isolated. The construct is confirmed with restriction digests.
- Plasmid pSGLit24/19 from E. coli ET12567 is digested with Xbal and the insert fragment is isolated and ligated with the Xbal treated vector fragment of plasmid pSGl448/27/95/21/4eryK
- the ligation is used to transform E. coli DH10B with standard procedures and plasmid pSG 1448/27/95/2 l/4eryK4/19 (pSGl 4angAIangAIIangorfl 4 ' angMIIIeryKangB) is isolated.
- the construct is confirmed with restriction digests.
- Plasmid pSG 1448/27/95/21/44/193/6eryCIII is digested with Bglll and the about 2.1 kb fragment is isolated and ligated with the Bglll treated vector fragment of pSG 1448/27/95/2 l/4eryK4/19. Plasmid pSG1448/27/95/21/4eryK4/193/6eryCIII is isolated using standard procedures and the construct is confirmed using restriction digests. The plasmid is used to transform mutant strains of S. erythraea with standard methods.
- Bioconversion of 3-O-mycarosyl erythronolide B to 5-0-dedesosaminyl-5-0-mycaminosyl erythromycin A The S. erythraea strain Q42/lpSG1448/27/95/2/// ery ⁇ /i93/deryCZ/7 is grown and bioconversions with fed 3-O-mycarosyl erythronolide B are performed as described in the General Methods. The cultures are analysed and a compound with m/z 750 is detected consistent with the presence of 5-0-dedesosaminyl-5-0-mycaminosyl erythromycin A.
- Example 8 Production of 13-desethyl-13-methyl-5-0-mycaminosyl erythromycins A and B; 13- desethyI-13-isopropyl-5-0-mycaminosyl erythromycin A and B; 13-desethyl-13-secbutyl-5-0- mycaminosyl erythromycin A and B
- DEBS3 genes in which the loading module of DEBS1 has been replaced by the loading module of the avermectin biosynthetic cluster, was used to transform S. erythraea JC2 ⁇ eryCIII (isolated using techniques and plasmids described previously (Rowe et al, 1998; Gaisser et al, 2000)) using standard techniques.
- the transformant JC2 ⁇ eryCIIIpLS025 was isolated and cultures were grown using standard protocols. Cultures of S. erythraea JC2 ⁇ eryCIIIpLS025 are extracted using methods described in the
- Example 9 13-desethy_-13-methyl-5-0-dedesosaminyI-5-0-mycaminosyl erythromycin A and 13- desethyI-13-methyl-5-0-dedesosaminyI-5-0- mycaminosyl erythromycin B
- Azithromycin aglycones were prepared using methods described in EP 1024145 A2 (Pfizer Products Inc. Groton, Connecticut).
- the S. erythraea strain SGT2pSG142 was isolated using techniques and plasmid constructs described earlier (Gaisser et al, 2000). Feeding experiments are carried out using methods described previously (Gaisser et al, 2000) with the S. erythraea mutant SGT2pSG142 thus converting azithromycin aglycone to 3-O-mycarosyl azithronolide. Biotransformation experiments are carried out using S.
- erythraea SGQ2pSG 1448/27/95/21/44/193/6eryCIII and crude extracts containing 3- 0-mycarosyl azithronolide are added using standard microbiological techniques.
- the bioconversion of 3- 0-mycarosyl azithronolide to 5-0-dedesosaminyl-5-0-mycaminosyl azithromycin is verified by LCMS analysis.
- construct pSG ⁇ G3 was isolated as follows:
- Fragment 1 was amplified using primers BIOSG53 5'-
- Plasmid pSG ⁇ Gl is isolated using standard methods and digested with SphllXbal followed by a ligation with the Sphl/Xbal digested insert fragment of pUCPCR2. Plasmid pSG ⁇ G2 is isolated using standard procedures, digested with SphllHindlll and ligated with the SphllHindlll fragment of pCJR24 (Rowe et al, 1998) containing the gene encoding for thiostrepton resistance. Plasmid pSG ⁇ G3 is isolated and used to delete eryG in the genome of S. erythraea strain SGQ2 using methods described previously (Gaisser et al, 1997; Gaisser et al, 1998) and the S. erythraea mutant SGP1 (SGQ2 ⁇ eryG) is created.
- S. erythraea strain SGP1 (S. erythraea SGQ2 ⁇ eryG) is isolated using standard techniques and consequently used to transform the cassette construct pSGl448/27/95/21/44/193/6eryCIII as formerly described.
- S. erythraea strain SGP1 S. erythraea SGQ2 ⁇ eryG
- erythraea strain SGPlpSG 1448/27/95/21/44/193/6eryCIII is isolated and used for biotransformation as described in Example 2 and assays are carried out as described above to verify the conversion of 3-0-mycarosyl-erythronolide B to 5-0-dedesosaminyl-5-0-mycaminosyi erythromycin C by LCMS analysis.
- the _re gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome. JBact. 178: 3374-3376. Mendez, C. and Salas, J.A. (2001) Altering the glycosylation pattern of bioactive compounds.
Abstract
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MXPA06006022A MXPA06006022A (en) | 2003-11-28 | 2004-11-29 | Polyketides and their synthesis. |
CA002547560A CA2547560A1 (en) | 2003-11-28 | 2004-11-29 | Polyketides and their synthesis |
BRPI0417026-1A BRPI0417026A (en) | 2003-11-28 | 2004-11-29 | gene cassette, process for producing erythromycin and azithromycin, and, compound |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010034243A1 (en) | 2008-09-24 | 2010-04-01 | Shanghai Institute Of Organic Chemistry, Chinese Academy Of Sciences | Novel gene cluster |
WO2018153960A1 (en) | 2017-02-22 | 2018-08-30 | Immune System Regulation Holding Ab | Novel immune stimulating macrolides |
WO2018153959A1 (en) | 2017-02-22 | 2018-08-30 | Immune System Regulation Holding Ab | Novel immune stimulating macrolides |
WO2018153957A1 (en) | 2017-02-22 | 2018-08-30 | Immune System Regulation Holding Ab | Novel immune stimulating compound |
WO2018153954A1 (en) | 2017-02-22 | 2018-08-30 | Immune System Regulation Holding Ab | Novel immune stimulating macrolide |
WO2019180265A1 (en) * | 2018-03-23 | 2019-09-26 | Immune System Regulation Holding Ab | Combinations of macrolide compounds and immune checkpoint inhibitors |
EP3716274A1 (en) | 2019-03-29 | 2020-09-30 | Ajinomoto Co., Inc. | Control device, control method, computer program, and method for producing organic compound |
WO2022049260A1 (en) | 2020-09-03 | 2022-03-10 | ISR Immune System Regulation Holding AB (publ) | Vaccine comprising an antigen and a tlr2 agonist |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3598805A (en) * | 1970-02-26 | 1971-08-10 | Abbott Lab | Erythromycin ester derivatives |
WO1999005283A2 (en) * | 1997-07-25 | 1999-02-04 | Hoechst Marion Roussel | Biosynthesis genes and transfer of 6-desoxy-hexoses in saccharopolyspora erythraea and in streptomyces antibioticus and their use |
EP1024145A2 (en) * | 1999-01-28 | 2000-08-02 | Pfizer Products Inc. | Novel azalides and methods of making same |
WO2001079520A1 (en) * | 2000-04-13 | 2001-10-25 | Biotica Technology Limited | Hybrid glycosylated products and their production and use |
US6521406B1 (en) * | 1998-03-09 | 2003-02-18 | Dow Agrosciences Llc | SpnG, a gene for spinosyn insecticide biosynthesis |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5998194A (en) * | 1995-12-21 | 1999-12-07 | Abbott Laboratories | Polyketide-associated sugar biosynthesis genes |
GB9814006D0 (en) * | 1998-06-29 | 1998-08-26 | Biotica Tech Ltd | Polyketides and their synthesis |
GB0019986D0 (en) * | 2000-08-14 | 2000-10-04 | Biotica Tech Ltd | Polyketides and their synthesis |
GB0125043D0 (en) * | 2001-10-17 | 2001-12-12 | Biotica Tech Ltd | Production, detection and use of transformant cells |
GB0128680D0 (en) * | 2001-11-29 | 2002-01-23 | Biotica Tech Ltd | Hybrid glycosylated products and their production and use |
-
2003
- 2003-11-28 GB GBGB0327721.7A patent/GB0327721D0/en not_active Ceased
-
2004
- 2004-11-29 MX MXPA06006022A patent/MXPA06006022A/en unknown
- 2004-11-29 CA CA002547560A patent/CA2547560A1/en not_active Abandoned
- 2004-11-29 US US10/580,872 patent/US20080044860A1/en not_active Abandoned
- 2004-11-29 JP JP2006540616A patent/JP2007512013A/en active Pending
- 2004-11-29 WO PCT/GB2004/005001 patent/WO2005054265A2/en active Application Filing
- 2004-11-29 EP EP04798692A patent/EP1749101A2/en not_active Withdrawn
-
2011
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3598805A (en) * | 1970-02-26 | 1971-08-10 | Abbott Lab | Erythromycin ester derivatives |
WO1999005283A2 (en) * | 1997-07-25 | 1999-02-04 | Hoechst Marion Roussel | Biosynthesis genes and transfer of 6-desoxy-hexoses in saccharopolyspora erythraea and in streptomyces antibioticus and their use |
US6521406B1 (en) * | 1998-03-09 | 2003-02-18 | Dow Agrosciences Llc | SpnG, a gene for spinosyn insecticide biosynthesis |
EP1024145A2 (en) * | 1999-01-28 | 2000-08-02 | Pfizer Products Inc. | Novel azalides and methods of making same |
WO2001079520A1 (en) * | 2000-04-13 | 2001-10-25 | Biotica Technology Limited | Hybrid glycosylated products and their production and use |
Non-Patent Citations (5)
Title |
---|
L. ZHAO ET AL.: "Engineering a methymycin/ pikromycin-calicheamicin hybrid: two new macrolides carrying a designed sugar moiety" J. AM. CHEM. SOC., vol. 121, 1999, pages 9881-9882, XP002210927 * |
M. SUZUKI ET AL.: "Biological glycosidation of macrolide aglycones I. Isolation and characterization of 5-O-mycaminosyl narbonolide and 9-dihydro-5-O-mycaminosyl narbonolide" J. ANTIBIOT., vol. XXIX, 1976, pages 1203-1208, XP009045970 * |
N. BATE ET AL.: "Thy mycarose biosynthetic genes of Streptomyces fradiae, producer of tylosin" MICROBIOLOGY, vol. 146, 2000, pages 139-146, XP002284343 * |
P.H. JONES ET AL.: "Chemical modifications of erythromycin antibiotics II. Synthesis of 4'-hydroxyerythromycin A" ANTIMICROB. AGENTS CHEMOTHER., 1969, pages 123-130, XP009045968 * |
S. RENGARAJU ET AL.: SCI. REPORTS OF MEIJI SEIKA KAISHA, vol. 24, 1985, pages 52-54, XP009045973 * |
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US20080044860A1 (en) | 2008-02-21 |
JP2011147446A (en) | 2011-08-04 |
GB0327721D0 (en) | 2003-12-31 |
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CA2547560A1 (en) | 2005-06-16 |
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