DK2970872T1 - Mikroorganismer og fremgangsmåder til fremstilling af sialylerede og N-acetylglucosamin-holdige oligosaccharider - Google Patents

Mikroorganismer og fremgangsmåder til fremstilling af sialylerede og N-acetylglucosamin-holdige oligosaccharider Download PDF

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DK2970872T1
DK2970872T1 DK14769797.3T DK14769797T DK2970872T1 DK 2970872 T1 DK2970872 T1 DK 2970872T1 DK 14769797 T DK14769797 T DK 14769797T DK 2970872 T1 DK2970872 T1 DK 2970872T1
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Massimo Merighi
Matthew Ian Heidtman
John M Mccoy
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Glycosyn LLC
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Claims (43)

1. Fremgangsmåde til fremstilling af et sialyleret oligosaccharid i en bakterie omfattende: tilvejebringelse af en bakterie, hvilken bakterie omfatter en exogen sialyltransferase, en deficient sialinsyre katabolisk proces, en sialinsyre syntetisk kapabilitet, og et funktionelt lactosepermeasegen; og dyrkning af bakterien under tilstedeværelse af lactose.
2. Fremgangsmåde ifølge krav 1, hvor den deficiente sialinsyre kataboliske proces omfatter en mutation i et vilkårligt af generne valgt fra endogen N-acetylneuraminat-lyase {nariA)-gen, endogent N-acetylmannoseaminkinasegen (nanK), endogent N-acetylmannoseamin-6-phosphatepimerasegen (nanE), og endogent N-acetylneuramin-syretransportergen (nanT), eller en vilkårlig kombination deraf.
3. Fremgangsmåde ifølge krav 1, hvor den deficiente sialinsyre kataboliske proces omfatter en mutation i endogent N-acetylneuraminat-lyase (nanA)-gen, og eventuelt, en mutation i endogent N-acetylneuraminsyretransportergen {nanT).
4. Fremgangsmåde ifølge krav 2, hvor den deficiente sialinsyre kataboliske proces yderligere omfatter et endogent N-acetylmannoseaminkinasegen (nanK) og endogent N-acetylmannoseamin-6-phosphatepimerasegen {nanE), som ikke er muterede.
5. Fremgangsmåde ifølge krav 1, hvor den deficiente sialinsyre kataboliske proces omfatter en mutation i endogent N-acetylneuraminat-lyase {nanA)-gen, en mutation i endogent N-acetylmannoseamin-6-phosphatepimerasegen {nanE), og eventuelt en mutation i endogent N-acetylneuraminsyretransportergen {nanT).
6. Fremgagnsmåde ifølge ethvert af de foregående krav, hvor mutationen omfatter en null-mutation.
7. Fremgangsmåde ifølge krav 5, hvor den deficiente sialinsyre kataboliske proces yderligere omfatter et endogent N-acetylmannoseaminkinasegen {nanK), som ikke er muteret.
8. Fremgangsmåde ifølge ethvert af de foregående krav, hvor den sialinsyresyntetiske kapabilitet omfatter et exogent CMP-Neu5Ac-syntetasegen (neuA), et exogent sialinsyresyntasegen (neuB), og en exogen UDP-GIcNac 2-epimerase (neuC).
9. Fremgangsmåde ifølge ethvert af de foregående krav, hvor det exogene sialyl-transferasegen er a(2,3)-sialyltransferase, a(2,6)-sialyltransferase eller a(2,8-sialyl-transferase.
10. Fremgangsmåde ifølge ethvert af de foregående krav, hvor det sialylerede oligosaccharid omfatter 3’-sialinlactose (3’-SL) eller 6’-sialinlactose (6’-SL).
11. Fremgangsmåde ifølge ethvert af de foregående krav, hvor bakterien omfatter et fjernet eller inaktiveret endogent β-galactosidasegen.
12. Fremgangsmåde ifølge krav 11, hvor det fjernede eller inaktiverede β-galactosidase-gen omfatter E. coli lacZ-gen.
13. Fremgangsmåde ifølge krav 1, hvor bakterien omfatter et rekombinant β-galactosidasegen, som tilvejebringer et lavt men detekterbart niveau af β-galactosidase-aktivitet.
14. Fremgangsmåde ifølge ethvert af de foregående krav, hvor bakterien yderligere omfatter et fjernet, inaktiveret, eller muteret lacA-gen.
15. Fremgangsmåde ifølge ethvert af de foregående krav, hvor bakterien omfatter en forøget UDP-GIcNAc-produktionskapabilitet.
16. Fremgangsmåde ifølge krav 15, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af et nagC-gen, et glmS-gen, et glmY-gen, et glmZ-gen eller en vilkårlig kombination deraf.
17. Fremgangsmåde ifølge krav 15, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af E. coli nagC-gen.
18. Fremgangsmåde ifølge krav 15, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af nagC og glmS.
19. Fremgangsmåde ifølge krav 15, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af nagC og glmY.
20. Fremgangsmåde ifølge krav 15, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af nagC og glmZ.
21. Fremgangsmåde ifølge ethvert af de foregående krav, hvor bakterien omfatter E. coli.
22. Fremgangsmåde til fremstilling af et N-acetylglucosamin-holdigt oligosaccharid i en bakterie, omfattende: tilvejebringelse af en bakterie, hvilken bakterie omfatter et exogent UDP-GlcNAc:Gala/3-R β 3-N-acetylglucosaminyltransferasegen og et funktionelt lactose-permeasegen; og dyrkning af bakterien under tilstedeværelse af lactose.
23. Fremgangsmåde ifølge krav 22, hvor bakterien omfatter en forøget UDP-GIcNAc-produktionskapabilitet.
24. Fremgangsmåde ifølge krav 11, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af et nagC-gen, et glmS-gen, et glmY-gen, et glmZ-gen eller en vilkårlig kombination deraf.
25. Fremgangsmåde ifølge krav 22, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af £. coli nagC-gen.
26. Fremgangsmåde ifølge krav 22, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af nagC og glmS.
27. Fremgangsmåde ifølge krav 22, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af nagC og glmY.
28. Fremgangsmåde ifølge krav 22, hvor den forøgede UDP-GIcNAc-produktions-kapabilitet omfatter overeksprimering af nagC og glmZ.
29. Fremgangsmåde ifølge ethvert af kravene 22-28, hvor det N-acetylglucosamin-holdige oligosaccharid omfatter en vilkårlig valgt fra Lacto-N-triose 2 (LNT2), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), Lacto-N-fucopentaose I (LNF I), Lacto-N-fucopentaose II (LNF II), Lacto-N-fucopentaose III (LNF III), Lacto-N-fucopentaose V (LNF V), Lacto-N-difucohexaose I (LDFH I), Lacto-N-difucohexaose II (LDFH II) og Lacto-N-neodifucohexaose II (LFNnDFH II).
30. Fremgangsmåde ifølge ethvert af kravene 22-29, hvor bakterien er E. coli.
31. Fremgangsmåde til oprensning af sialyleret oligosaccharid fremstillet ved fremgangsmåden ifølge ethvert af kravene 1-21, omfattende binding af det sialylerede oligosaccharid fra et bakteriecellelysat eller bakteriecellekultursupernatant af den nævnte bakterie til en kulstofsøjle og eluering af det sialylerede oligosaccharid fra den nævnte søjle.
32. Fremgangsmåde til oprensning af et N-acetylglucosaminholdigt oligosaccharid fremstillet ved fremgangsmåden ifølge ethvert af kravene 22-30, omfattende binding af det sialylerede oligosaccharid fra et bakterielt cellelysat eller en bakteriel cellekultur-supernatant af den nævnte bakterie til en kulstofsøjle, og eluering af det sialylerede oligosaccharid fra den nævnte søjle.
33. En isoleret E. coli bakterie omfattende et fjernet eller inaktiveret endogent β-galactosidasegen, et exogent sialyltransferasegen, en deficient sialinsyre katabolisk proces, en sialinsyre syntetisk kapabilitet, og et funktionelt lactosepermeasegen.
34. Isoleret bakterie ifølge krav 33, hvor bakterien yderligere omfatter et rekombinant β-galactosidasegen, som tilvejebringer et lavt men detekterbart niveau for β-galactosidaseaktivitet.
35. En isoleret E. coli bakterie omfattende et exogent UDP-GlcNAc:Gala^-R β 3-N-acetylglucosaminyltransferasegen og et funktionelt lactosepermeasegen.
36. Isoleret E. coli bakterie ifølge ethvert af kravene 33-35, hvor bakterien yderligere omfatter en forøget UDP-GIcNAc-produktionskapabilitet.
37. Oprenset sialyleret oligosaccharid fremstillet ved fremgangsmåden ifølge ethvert af kravene 1-21.
38. Oprenset N-acetylglucosaminholdigt oligosaccharid fremstillet ved fremgangsmåden ifølge ethvert af kravene 22-30.
39. Nukleinsyrekonstrukt omfattende et exogent sialyltransferasegen transformeret til en bakteriel værtsstamme omfattende et fjernet eller inaktiveret endogent β-galactosidasegen, en sialinsyresynteseproces, et funktionelt lactosepermeasegen, og et fjernet lactoseacetyltransferasegen.
40. Nukleinsyrekonstrukt ifølge krav 39, hvor det exogene sialyltransferasegen koder a(2,3)-sialyltransferase eller a(2,6)-sialyltransferase.
41. Nukleinsyrekonstrukt omfattende et exogent UDP-GlcNAc:Gala/p-R β 3-N-acetylglucosaminyltransferasegen transformeret til en bakterieværtsstamme omfattende et funktionelt lactosepermeasegen.
42. Fremgangsmåde ifølge ethvert af kravene 1-21, som yderligere omfatter udvinding af det sialylerede oligosaccharid fra bakterien eller fra en kultursupernatant af bakterien.
43. Fremgangsmåde ifølge ethvert af kravene 22-29, som yderligere omfatter udvinding af det N-acetylglucosaminholdige oligosaccharid fra bakterien eller fra en kultursupernatant af bakterien.
DK14769797.3T 2013-03-14 2014-03-14 Mikroorganismer og fremgangsmåder til fremstilling af sialylerede og N-acetylglucosamin-holdige oligosaccharider DK2970872T3 (da)

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