DK2675899T1 - Biosyntese af humane mælkeoligosaccharider i manipulerede bakterier - Google Patents

Biosyntese af humane mælkeoligosaccharider i manipulerede bakterier Download PDF

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DK2675899T1
DK2675899T1 DK12746649.8T DK12746649T DK2675899T1 DK 2675899 T1 DK2675899 T1 DK 2675899T1 DK 12746649 T DK12746649 T DK 12746649T DK 2675899 T1 DK2675899 T1 DK 2675899T1
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gene
bacterium
exogenous
fucosyltransferase
galactosidase
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Massimo Merighi
John M Mccoy
Matthew Ian Heidtman
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Glycosyn LLC
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    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
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    • 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)
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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    • C12Y204/01065—3-Galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase (2.4.1.65), i.e. alpha-1-3 fucosyltransferase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/03—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; catalysing transmembrane movement of substances (3.6.3)
    • C12Y306/03018—Oligosaccharide-transporting ATPase (3.6.3.18)
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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Claims (38)

1. Fremgangsmåde til fremstilling af et fucosyleret oligosaccharid i en bakterie, omfattende tilvejebringelse af en bakterie, hvilken bakterie omfatter et funktionelt β-galactosidasegen, et exogent fucosyltransferasegen, en GDP-fucosesynteseproces, et funktionelt lactosepermeasegen; dyrkning af bakterien under tilstedeværelse af lactose; og udvinding af et fucosyleret oligosaccharid fra bakterien eller fra et dyrkningssupernatant fra bakterien.
2. Fremgangsmåde ifølge krav 1, hvor β-galactosidasegenet omfatter et E. coli lacZ-gen.
3. Fremgangsmåde ifølge krav 1, hvor β-galactosidasegenet er et endogent β-galactosidasegen eller et exogent β-galactosidasegen.
4. Fremgangsmåde ifølge krav 1, hvor bakterien akkumulerer en forøget intracellulær lactosepool og producerer et lavt niveau af β-galactosidase.
5. Fremgangsmåde ifølge krav 1, hvor det exogene fucosyltransferasegen koder for a(1,2)fucosyltransferase eller a(1,3)fucosyltransferase.
6. Fremgangsmåde ifølge krav 5, hvor a(1,2)fucosyltransferasegenet omfatter et Bacteroides fragilis wcfW-gen.
7. Fremgangsmåde ifølge krav 5, hvor a(1,3)fucosyltransferasegenet omfatter et Helicobacter pylori 26695 futA-gen.
8. Fremgangsmåde ifølge krav 5, hvor bakterien omfatter såvel et exogent fucosyltransferasegen, som koder for a(1,2)fucosyltransferase og et exogent fucosyltransferasegen, som koder for a(1,3)fucosyltransferase.
9. Fremgangsmåde ifølge krav 1, hvor GDP-fucosesynteseprocessen omfatter endogene enzymer eller exogene enzymer.
10. Fremgangsmåde ifølge krav 1, hvor lactosepermeasegenet er et endogent lactose-permeasegen eller et exogent lactosepermeasegen.
11. Fremgangsmåde til fremstilling af et fucosyleret oligosaccharid i en bakterie, omfattende tilvejebringelse af en enterisk bakterie, hvilken bakterie omfatter et funktionelt β-galactosidasegen, et exogent fucosyltransferasegen, en mutation i et colansyresyntesegen, og et funktionelt lactosepermeasegen; dyrkning af bakterien under tilstedeværelse af lactose; og udvinding af et fucosyleret oligosaccharid fra bakterien eller fra et dyrkningssupernatant fra bakterien.
12. Fremgangsmåde ifølge krav 11, hvor β-galactosidasegenet omfatter et E. coli lacZ-gen.
13. Fremgangsmåde ifølge krav 11, hvor det exogene fucosyltransferasegen koder for a(1,2) fucosyltransferase eller a(1,3) fucosyltransferase.
14. Fremgangsmåde ifølge krav 11, hvor den enteriske bakterie omfatter E. coli.
15. Fremgangsmåde ifølge krav 14, hvor colansyresyntesegenet omfatter et wcaJ-gen.
16. Fremgangsmåde ifølge krav 14, hvor bakterien yderligere omfatter en mutation i et Ion-gen.
17. Fremgangsmåde ifølge krav 14, hvor bakterien omfatter et funktionelt, wild-type £. coli /acZ+-gen indsat i et endogent Ion-gen.
18. Fremgangsmåde ifølge krav 14, hvor et endogent lacZ-gen i den nævnte £. coli er fjernet.
19. Fremgangsmåde ifølge krav 14, hvor bakterien yderligere omfatter et exogent rcsA-eller rcsS-gen.
20. Fremgangsmåde ifølge krav 14, hvor bakterien yderligere omfatter en mutation i et lacA-gen.
21. Fremgangsmåde til fremstilling af en 3'-sialyl-3-fucosyllactose (3-S3FL) i en bakterie, hvilken bakterie omfatter et funktionelt β-galactosidasegen, et exogent sialyl-transferasegen, et exogent fucosyltransferasegen, en GDP-fucosesynteseproces, en deficient sialinsyrekatabolisk proces, en sialinsyresyntesekapabilitet, og et funktionelt lactosepermeasegen; dyrkning af bakterien under tilstedeværelse af lactose; og udvinding af det nævnte 3'-S3FL fra bakterien eller fra et dyrkningssupernatant fra bakterien.
22. Fremgangsmåde ifølge krav 21, hvor det exogene sialyltransferasegen koder for a(2,3)sialyltransferase.
23. Fremgangsmåde ifølge krav 21, hvor det exogene fucosyltransferasegen koder for a(1,3)fucosyltransferase.
24. Fremgangsmåde ifølge krav 21, hvor den deficiente sialinsyrekataboliske proces omfatter en nulmutation i endogene N-acetylneuraminatlyase- eller N-acetyl-mannosaminkinase-gener.
25. Fremgangsmåde ifølge krav 21, hvor sialinsyresyntesekapabiliteten omfatter et exogent UDP-GIcNAc 2-epimerasegen, et exogent Neu5Ac-syntasegen eller et exogent CMP-Neu5Ac-syntetasegen.
26. Fremgangsmåde til fremstilling afen 3'-sialyl-3-fucosyllactose (3'-S3FL) i en enterisk bakterie, hvilken enterisk bakterie omfatter et funktionelt lacZ-gen, et exogent fucosyltransferasegen, et exogent sialyltransferasegen, en mutation i et endogent colansyre-syntesegen, et funktionelt lactosepermeasegen, en deficient sialinsyrekatabolisk proces, og en sialinsyresyntesekapabilitet; dyrkning af bakterien under tilstedeværelse af lactose; og udvinding af det nævnte 3-S3FL fra bakterien eller fra et dyrkningssupernatant fra bakterien.
27. Fremgangsmåde ifølge krav 26, hvor det exogene fucosyltransferasegen koder for a(1,3)fucosyltransferase.
28. Fremgangsmåde ifølge krav 26, hvor det exogene sialyltransferasegen koder for en a(2,3)sialyltransferase.
29. Fremgangsmåde ifølge krav 26, hvor den deficiente sialinsyrekataboliske proces omfatter en nulmutation i endogene N-acetylneuraminatlyase- eller N-acetyl-mannosaminkinase-gener.
30. Fremgangsmåde ifølge krav 26, hvor sialinsyresyntesekapabiliteten omfatter et exogent UDP-GIcNAc 2-epimerasegen, et exogent Neu5Ac-syntasegen eller et exogent CMP-Neu5Ac-syntetasegen.
31. Fremgangsmåde til phenotypisk markering af en genposition i en værtscelle, hvis oprindelige β-galactosidasegen er fjernet eller inaktiveret, ved anvendelse af et indsat rekombinant β-galactosidasegen manipuleret til at producere et lavt, men detekterbart niveau af β-galactosidaseaktivitet.
32. Fremgangsmåde til udtømning af en bakteriel kultur for restlactose i en β-galactosidasenegativ værtscelle, hvis oprindelige β-galactosidasegen er fjernet eller inaktiveret ved anvendelse af et indsat rekombinant β-galactosidasegen, som er manipuleret til at producere et lavt, men detekterbart niveau af β-galactosidaseaktivitet.
33. Fremgangsmåde til detektering af bakteriel cellelyse i en kultur af en β-galactosidasenegativ værtscelle, hvis oprindelige β-galactosidasegen er fjernet eller inaktiveret, ved anvendelse af et indsat rekombinant β-galactosidasegen manipuleret til at producere et lavt, men detekterbart niveau af β-galactosidaseaktivitet.
34. Fremgangsmåde til rensning af et fucosyleret oligosaccharid fremstillet ved fremgangsmåden ifølge krav 1, omfattende binding af det fucosylerede oligosaccharid fra et bakterielt cellelysat eller bakteriel cellekultursupernatant fra bakterien til en kulstofsøjle, og eluering af det fucosylerede oligosaccharid fra søjlen.
35. Isoleret E. coli bakterie omfattende en defekt colansyresynteseproces, reduceret niveau af β-galactosidaseaktivitet, og et exogent fucosyltransferasegen.
36. Renset fucosyleret oligosaccharid fremstillet ved fremgangsmåden ifølge krav 1.
37. Nukleinsyrekonstrukt omfattende et exogent fucosyltransferasegen transformere til en bakteriel værtsstamme omfattende et fjernet endogent β-galactosidasegen, et erstatningsfunktionelt β-galactosidasegen med lav aktivitet, en GDP-fucose-synteseproces, et funktionelt lactosepermeasegen, og et fjernet lactoseacetyl-transferasegen.
38. Nukleinsyrekonstrukt ifølge krav 37, hvor det exogene fucosyltransferasegen koder for a(1,2)fucosyltransferase eller a(1,3)fucosyltransferase.
DK12746649.8T 2011-02-16 2012-02-16 Biosyntese af humane mælkeoligosaccharider i manipulerede bakterier DK2675899T1 (da)

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PCT/US2012/025450 WO2012112777A2 (en) 2011-02-16 2012-02-16 Biosynthesis of human milk oligosaccharides in engineered bacteria

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