NZ779656B2 - Production of human milk oligosaccharides in microbial hosts with engineered import / export - Google Patents

Production of human milk oligosaccharides in microbial hosts with engineered import / export

Info

Publication number
NZ779656B2
NZ779656B2 NZ779656A NZ77965616A NZ779656B2 NZ 779656 B2 NZ779656 B2 NZ 779656B2 NZ 779656 A NZ779656 A NZ 779656A NZ 77965616 A NZ77965616 A NZ 77965616A NZ 779656 B2 NZ779656 B2 NZ 779656B2
Authority
NZ
New Zealand
Prior art keywords
host cell
nucleic acid
acid sequence
protein
export
Prior art date
Application number
NZ779656A
Other versions
NZ779656A (en
Inventor
Stefan Jennewein
Dirk Wartenberg
Original Assignee
Chr Hansen Hmo Gmbh
Filing date
Publication date
Priority claimed from EP15184968.4A external-priority patent/EP3141610A1/en
Application filed by Chr Hansen Hmo Gmbh filed Critical Chr Hansen Hmo Gmbh
Publication of NZ779656A publication Critical patent/NZ779656A/en
Publication of NZ779656B2 publication Critical patent/NZ779656B2/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01146—Beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase (2.4.1.146)

Abstract

The present invention relates to methods for the production of oligosaccharides in genetically modified bacterial host cells, as well as to the genetically modified host cells used in the methods. The genetically modified host cell comprises at least one recombinant glycosyltransferase, and at least one nucleic acid sequence coding for a protein enabling the export of the oligosaccharide.

Claims (13)

Claims
1. Method for the production of a desired oligosaccharide by a genetically modified microbial host cell, wherein the desired oligosaccharide is lacto-N-triose II, the method comprising the steps of - providing a cally modified microbial host cell that - comprises at least one recombinant ß-1,3-N- acetylglucosaminyltransferase, - has the expression or activity of at least one endogenous sugar export protein modified such, that the expression or activity of the sugar export protein is either (i) sed or (ii) decreased or inactivated as compared to an genetically unmodified host cell, so that (i) the export of a accharide into the medium is either decreased or abolished, or (ii) the transport of a desired oligosaccharide is increased , respectively, as compared to an genetically unmodified host cell, - comprises at least one gous or logous nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide into the culture medium, wherein said host cell has been modified such, that the expression of the homologous or heterologous nucleic acid sequence is overexpressed, wherein said at least one nucleic acid sequence coding for the protein enabling the export of the desired oligosaccharide is a gene ed from the group consisting of yjhB from Escherichia coli, proP from Mann- heimia succiniciproducens, setA from Cedecea neteri and functional fragments thereof, - cultivating the host cell in a medium under conditions permissive for the production of the d oligosaccharide, y the desired accharide is transported into the medium, and - obtaining the desired oligosaccharide from the medium.
2. The method of claim 1, wherein the host cell comprises: - a on, disruption, diminishment or inactivation of at least one endogenous nucleic acid sequence coding for an exporter protein that exports precursors of the desired oligosaccharide outside the host cell; and/or - an pression of at least one homologous or heterologous sequence coding for a protein mediating the import of a precursor of a desired oligosaccharide into said host cell, wherein the precursor is larger than a disaccharide.
3. The method of any of claim 1 or 2, wherein said at least one nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide is an endogenous or a inant nucleic acid.
4. The method of any one of claims 1 or 3, wherein said ß-1,3-N- acetylglucosaminyltransferase belongs to the class of lgtA of Neisseria meningiti- des and PmnagT of rella multocida.
5. The method of any of the preceding claims, wherein, where applicable, in the genetically modified host cell the endogenous ctosidase gene and the glucosaminephosphate deaminase gene are inactivated or deleted, and wherein said genetically modified host cell comprises a nucleic acid sequence coding for a functional lactose permease protein.
6. The method of any of the preceding claims, wherein the genetically modified host cell comprises an increased UDP-N-acetylglucosamine and lactose or GDP-fucose or CMP-N-acetylneuraminic acid production capability as compared to a genetically fied host cell, wherein ably said increased UDP-N- acetylglucosamine and UDP-galactose production capability comprises the overexpression of one or more genes encoding for proteins comprising the following ties for a: L-glutamine: D-fructosephosphate aminotransferase, N-acetyl glucosaminephosphate uridyltransferase/glucosaminephosphate acetyl erase , phosphoglucosamine mutase, UDP-galactoseepimerase, phosphoglucomutase , glucosephosphate uridylyltransferase.
7. The method according to claim 5 or 6, wherein said genetically modified host cell is cultivated in the presence of glucose, sucrose, glycerol or a combination thereof, but neither by addition or in the presence of N-acetylglucosamine or ose nor in a combination thereof.
8. A genetically modified microbial host cell for the production of a d oligosaccharide , the oligosaccharide being lacto-N-triose II, wherein the host cell - comprises at least one recombinant ß-1,3-N-acetylglucosaminyltransferase, - has the expression or activity of at least endogenous sugar transport protein modified such, that the expression or activity of the endogenous sugar transport n is functionally inactivated for the export of a precursor of the desired oligosaccharide, and - comprises at least one gous or heterologous nucleic acid sequence coding for a protein enabling the export of a desired oligosaccharide into the culture medium, wherein said host cell has been ed such, that the expression of the homologous or heterologous nucleic acid sequence is overexpressed, wherein said at least one nucleic acid ce coding for the protein enabling the export of the desired oligosaccharide is a gene se- lected from the group consisting of yjhB from Escherichia coli, proP from Mannheimia iciproducens, setA from Cedecea neteri and functional fragments thereof.
9. The microbial host cell of claim 8, r comprising - a on, disruption, diminishment or inactivation of at least one endogenous nucleic acid sequence coding for an exporter protein that exports precursors of the d oligosaccharide outside the host cell; , - an overexpression of at least one homologous or heterologous nucleic acid sequence coding for a protein mediating the import of a precursor of a desired oligosaccharide into said host cell, wherein the precursor is larger than a disaccharide.
10. The microbial host cell of claim 8 or 9, wherein said at least one nucleic acid sequence coding for a protein enabling the export of the desired oligosaccharide is an endogenous or a recombinant nucleic acid sequence.
11. The genetically modified microbial host cell of any one of claims 8 to 10, wherein said ß-1,3-N-acetylglucosaminyltransferase belongs to the class of lgtA of Neis- seria meningitides or PmnagT of Pasteurella multocida.
12. The genetically modified microbial host cell of any one of claims 8 to 11, wherein, where applicable, in the genetically ed host cell the endogenous ßgalactosidase gene and the glucosaminephosphate deaminase gene are vated or deleted, and wherein said cally modified host cell comprises a nucleic acid sequence coding for a functional lactose permease protein.
13. The genetically modified microbial host cell of any one of claims 8 to 12, wherein the genetically modified host cell comprises an increased UDP-N- acetylglucosamine and UDP-galactose or GDP-fucose or CMP-N-acetylneuraminic acid production capability as compared to a genetically unmodified host cell, wherein preferably said increased UDP-N-acetylglucosamine and lactose production capability comprises the overexpression of one or more genes encoding for proteins comprising the following activities for a: L-glutamine: tose phosphate aminotransferase, N-acetyl glucosaminephosphate uridyltransferase /glucosaminephosphate acetyl transferase, phosphoglucosamine mutase, UDP-galactoseepimerase, phosphoglucomutase, glucosephosphate uridylyltransferase. :25 o :0 :2: $852.28.. oxonooo 38.3.2633 0: o_._ :3 All IO IO 2.5.; $0:0o: 2.5.; __ ow%:0 __ $052.28.. O o 212 8.. o: o: O: 0?o?woioOO $203 $28.. OI OI (MMIIOHo|lao<2 :8 8:808 :56me 12 34567891011123141516171819 1 2 3 4 5 67891011121314151617
NZ779656A 2016-09-12 Production of human milk oligosaccharides in microbial hosts with engineered import / export NZ779656B2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP15184968.4A EP3141610A1 (en) 2015-09-12 2015-09-12 Production of human milk oligosaccharides in microbial hosts with engineered import / export
NZ740265A NZ740265B2 (en) 2016-09-12 Production of human milk oligosaccharides in microbial hosts with engineered import / export

Publications (2)

Publication Number Publication Date
NZ779656A NZ779656A (en) 2025-02-28
NZ779656B2 true NZ779656B2 (en) 2025-06-04

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