US20110300584A1 - Synthesis of fucosylated compounds - Google Patents

Synthesis of fucosylated compounds Download PDF

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US20110300584A1
US20110300584A1 US13/140,548 US200913140548A US2011300584A1 US 20110300584 A1 US20110300584 A1 US 20110300584A1 US 200913140548 A US200913140548 A US 200913140548A US 2011300584 A1 US2011300584 A1 US 2011300584A1
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fucose
fucosyllactose
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Eric Hüfner
Julia Parkot
Stefan Jennewein
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Chr Hansen HMO GmbH
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    • 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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • 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)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention is related to methods of making fucosylated compounds and cells related thereto.
  • Human milk consists of a complex mixture of carbohydrates, proteins, lipids, hormones, and micronutrients, providing all necessary nutrients for infant development.
  • human milk contains several protective agents.
  • human milk contains an array of complex oligosaccharides with protective properties.
  • Human milk oligosaccha ride (HMO) fraction comprise beside the principal carbohydrate component lactose, more than 130 different complex oligosaccharides. This structural diversity of complex oligosaccharides and their occurrence at high amounts is unique to humans. In contrast, only trace amounts of much less complex oligosaccharides are found in bovine milk, and consequently commonly used infant formula lacks these oligosaccharides.
  • HMOs can drastically reduce the virulence of pathogenic Escherichia coli (Cravioto et al., 1991), Vibrio cholerae (Coppa et al., 2006), Streptococcus pneumoniae (Andersson et al., 1986) or Campylobacter jejuni (Ruiz-Palacios et al., 2003) and are also able to neutralize toxins, like the heat-stable enterotoxin of E. coli (Crane et al., 1994). Besides the mentioned local effects in the intestinal tract, HMOs are also capable of eliciting systemic effects in infants by entering the systemic circulation (Gnoth et al., 2001).
  • HMOs e.g., selectin-leukocyte binding
  • oligosaccharides represent the third largest component of human milk, after lactose and fat. They almost all have in common lactose at the reducing end, and are decorated with fucose and/or sialic acid at the non-reducing end. They are build from 3 to up to 32 monosaccharides and most of them contain fucose, with 1 to 15 fucose units. Thus, fucosylated oligosaccharides show great potential as bioactive food ingredients with anti-infective and prebiotic attributes.
  • Fucosyltransferases which catalyze the transfer of fucose residues from the donor guanosine-diphosphate activated L-fucose (GDP-L-fucose) to several acceptor molecules, are expressed in animals, plants, fungi and bacteria (Ma et al., 2006). They are categorized according to the site of fucose addition, therefore ⁇ 1,2, ⁇ 1,3/4, and ⁇ 1,6 FucTs are distinguished. Besides human FucTs, which are originally responsible for the biosynthesis of HMOs and blood group antigens, several bacterial FucTs have been described.
  • FucT activity has been best documented for the human gastric pathogen Helicobacter pylori , which decorates its lipopolysaccharide (LPS) with fucose-containing Lewis antigens (Wang et al., 2000).
  • LPS lipopolysaccharide
  • FucT activity has been best documented for the human gastric pathogen Helicobacter pylori , which decorates its lipopolysaccharide (LPS) with fucose-containing Lewis antigens (Wang et al., 2000).
  • LPS lipopolysaccharide
  • FucT activity has been best documented for the human gastric pathogen Helicobacter pylori , which decorates its lipopolysaccharide (LPS) with fucose-containing Lewis antigens (Wang et al., 2000).
  • LPS lipopolysaccharide
  • FucT activity has been best documented for the human gastric pathogen Helicobacter pylori
  • GDP- ⁇ -L-fucose is prepared by conversion of GDP-D-mannose to GDP-4-keto-6-deoxy-D-mannose. This is treated with a GDP-4-keto-6-deoxy-D-mannose 3,5 epimerase-4-reductase to produce GDP- ⁇ -L-fucose, which is purified by preparative HPLC.
  • a genetically modified cell is produced. It has been transformed to express a fucosekinase, a fucose-1-phosphate guanylyltransferase and a fucosyltransferase.
  • the genetically modified cell is a microorganism selected from the group consisting of the genera Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Lactococcus, Pichia, Saccharomyces and Kluyveromyces.
  • the fucose kinase and the fucose-1-phosphate guanylyltransferase activity are combined in a bifunctional enzyme.
  • Suitable genes for transformation, coding for a fucose kinase, a fucose-1-phosphate guanylyltransferase and/or a bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase can be obtained from the genera Bacteroides, Lentisphaera, Ruminococcus, Solibacter, Arabidopsis, Oryza, Physcomitrella, Vitis, Danio, Bos, Equus, Macaca, Pan, Homo, Rattus, Mus and Xenopus.
  • Suitable fucosyltransferase genes can be derived from organisms selected from the group of the genera Helicobacter, Escherichia, Yersinia, Enterococcus, Shigella, Klebsiella, Salmonella, Bacteroides, Dictyostelium, Arabidopsis, Drosophila, Homo, Bos, Mus, Rattus, Gallus, Canis and Sus.
  • a codon optimization may be helpful to increase the expression.
  • Some cells have a catabolic pathway for fucose. In this case, it is recommendable to inactivate this catabolic pathway.
  • Suitable methods comprise inactivating one or several genes selected from the group consisting of a fucose-1-phosphate aldolase gene, a fucose isomerase gene and a fuculose kinase gene.
  • Suitable fucose derived compounds which can be prepared by the genetically modified cells of the present invention are fucosyllactoses, preferably 2′-fucosyllactose, 3-fucosyllactose or lactodifucotetraose.
  • the present invention is a synthesis in a cell starting from fucose instead of a preparative synthesis with recombinant enzymes starting from GDP-D-mannose as described by Albermann et al. (2001).
  • a further embodiment of the invention is the genetically modified cell obtainable by the method of the invention.
  • the genetically modified cell of the invention is cultivated under suitable cultivation conditions in a medium comprising fucose and an acceptor substrate.
  • Suitable acceptor substrates are for example a mono-, di- or oligosaccharide or a peptide, for example lactose, 2′-fucosyllactose or 3-fucosyllactose.
  • the preferred fucosylated compounds obtained by the production method are fucosyllactoses, preferably 2′-fucosyllactose or 3-fucosyllactose or lactodifucotetraose.
  • fucose salvage pathway fucose is first phosphorylated to fucose-1-phosphate by the enzyme fucose kinase.
  • the fucose-1-phosphate is then converted to GDP-fucose by the action of the enzyme fucose-1-P-guanylyltransferase.
  • Fkp the first bacterial enzyme, Fkp, with both fucose kinase and L-fucose-1-P-guanylyltransferase activity was described (Coyne et al., 2005).
  • the intestinal bacterium Bacteroides fragilis uses the enzyme for the production of GDP-fucose, which serves for the decoration of capsular polysaccharides and glycoproteins with fucose residues.
  • FIG. 1 discloses the structures of the prominent complex human milk oligosaccharides (HMOs) 2′-fucosyllactose and 3-fucosyllactose.
  • HMOs human milk oligosaccharides
  • FIG. 2 shows a scheme of the photometric assay for determination of Fkp activity by coupled enzyme reactions and determination of NADH oxidation;
  • Fkp bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase
  • PK pyruvate kinase
  • LDH L-lactate dehydrogenase
  • PEP phosphoenolpyruvate.
  • FIG. 4 shows the protein formation after induction.
  • Lanes 1-4 expression of soluble Fkp (105.7 kDa) and/or FutAco (49.3 kDa) or FucT2 (35.9 kDa), in crude extracts from E. coli BW25113 ⁇ fucA (DE3) pCOLA-fkp-fucP (lane 1), E. coli BW25113 ⁇ fucA (DE3) pET-futAco (lane 2), E. coli BW25113 ⁇ fucA (DE3) pCOLA-fkp-fucP+pETfutAco (lane 3) and E.
  • FIG. 5 shows a radio thin layer chromatography (radio-TLC) of 3 H-fucose, developed with butanol:acetone:acetic acid:water (35:35:7:23) and analyzed using a radio-TLC reader.
  • radio-TLC radio thin layer chromatography
  • FIG. 6 shows a radio-TLC of a cell extract from E. coli BW25113 ⁇ fucA (DE3) pCOLADuet-1 pETDuet-1 showing fucose and fuculose and fuculose-1-phosphate, however degradation of fuculose-1-phosphate is inhibited due to the genomic knockout of the fuculose-1-phosphate aldolase gene (fucA).
  • FIG. 7 shows radio-TLC of cell extract from E. coli BW25113 ⁇ fucA (DE3) pCOLA-fkp-fucP showing accumulating GDP-fucose produced by bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase Fkp from Bacteroides fragilis as well as fucose and degradation products fuculose and fuculose-1-phosphate.
  • FIG. 8 shows a radio-TLC of a cell extract from E. coli BW25113 ⁇ fucA (DE3) pCOLA-fkp-fucP pET-futAco showing accumulating 3-fucosyllactose produced by codon optimized fucosyltransferase of Helicobacter pylori via GDP-fucose provided by bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase (Fkp). Fucose and degradation products fuculose and fuculose-1-phosphate are only minimally present; GDP-fucose amount is significantly reduced due to 3-fucosyllactose production.
  • FIG. 9 shows a HPAED analysis of cell lysate from negative control strain E. coli BW25113 ⁇ fucA (DE3) pCOLADuet-1 pETDuet-1 showing intracellular L-fucose, lactose, glycerol and L-rhamnose, but no fucosyllactose.
  • FIG. 10 shows a cell lysate of strain E. coli BW25113 ⁇ fucA (DE3) pCOLA-fkp-fucP pET-futAco producing 3-fucosyllactose (retention time of about 11 min); furthermore L-fucose, lactose, glycerol and L-rhamnose peaks can be seen.
  • FIG. 11 shows a HPAED analysis of cell lysate from strain E. coli BW25113 ⁇ fucA (DE3) pCOLA-fkp-fucP pCAW55 showed production of 2′′-fucosyllactose (retention time of about 22 min). Additionally, L-fucose, lactose, glycerol and L-rhamnose can be seen.
  • FIG. 12 a and b show HPLC-analysis with electrochemical detection of GDP-fucose expression in E. coli JM109 (DE3) ⁇ fucA ( FIG. 12 a ) and E, coli JM109 (DE3) ⁇ fucA pCOLA-fkp-fucP ( FIG. 12 b ).
  • fucA coding for the key catabolic enzyme fuculose-1-phosphate aldolase had to be deleted from the genome of E. coli strain BW25113.
  • the methodology of (Datsenko & Wanner, 2000) was applied.
  • an inducible T7 RNA polymerase was incorporated into the deletion strain E. coli BW25113 ⁇ fucA by using the ⁇ DE3 lysogenization kit (Novagen). The resulting strain was then named E. coli BW25113 ⁇ fucA (DE3).
  • the plasmids pCOLA-fkp-fucP and pET-futAco were constructed using the pCOLADuet-1 and pETDuet-1 expression vectors (Novagen). All primers used for the construction are listed in Table 2.
  • Gene fkp (GeneBank acc. no. AY849806) was amplified by PCR with primers fkp-NcoI-forward and fkp-NotI-reverse using genomic DNA of Bacteroides fragilis ATCC 25285D.
  • the fucP gene (GeneBank acc. no. CP000948) of Escherichia coli K12 was amplified from genomic DNA of E.
  • coli TOP10 (Invitrogen, USA) using primers FucP-NdeI-forward and FucP-XhoI-reverse. Both fkp and fucP were inserted into the first and second multiple cloning site (MCS) of pCOLADuet-1, respectively, using the indicated restriction sites. The resulting plasmid was designated pCOLA-fkp-fucP.
  • the futA gene (GeneBank acc. no. AE000511) of H. pylori strain 26695 was codon-optimized for expression in E. coli and prepared synthetically by GenScript Corporation (Piscataway, N.J., USA).
  • the gene was amplified using the primers FutAco-NcoI-forward and FutAco-BamHI-reverse, and inserted into the first MCS of pETDuet-1, yielding pET-futAco.
  • the correct insertion of cloned genes was checked by restriction analysis and sequencing using the recommended primers pACYCDuetUP1, pET-Upstream, DuetDOWN-1, DuetUP2 and T7-Terminator listed in the Duet Vectors Manual (Novagen). Plasmid pCAW55 containing the gene fucT2 coding for ⁇ 1,2-fucosyltransferase from Helicobacter pylori NCTC364 was donated by C.
  • E. coli strains were inoculated in 10 mL of 2 ⁇ YT broth (Sambrook & Russell, 2001), containing 100 ⁇ g mL ⁇ 1 ampicillin and/or 50 ⁇ g mL ⁇ 1 kanamycin, and incubated overnight in a rotary shaker at 37° C. The next day, 30 mL fresh 2 ⁇ YT broth supplemented with the appropriate antibiotics was inoculated 1/100 from the overnight culture, and incubated at 37° C. in a rotary shaker providing good aeration.
  • inducers isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG) and/or L-rhamnose were added in a concentration of 0.1 mM and 0.1%, respectively.
  • the cultures were further incubated at 28° C. overnight (approx. 15 h) under constant shaking.
  • For photometric activity assay an aliquot of cell culture was removed, cells were pelleted and resuspended in five times weight/volume 50 mM Tris-HCl pH 7.5. Glass beads were added four times the weight of cell pellet and the resulting suspension was vortexed two times for five minutes each and in between placed on ice for additional five minutes. Cell debris was removed by centrifugation (13200 rpm, 5 min, 4° C.) and the resulting crude extract was stored at 4° C.
  • Inducers L-rhamnose (0.1%) and IPTG (0.1 mM) were also added to all cultures regardless of which strain was cultivated to avoid different culture conditions. Again, the cultures were incubated at 28° C. overnight (approx. 15 h) under constant shaking. The cultures were centrifuged and the supernatants were decanted and stored at ⁇ 20° C. The cells were subsequently washed with PBS, resuspended in distilled water, and permeabilized by autoclaving (100° C., 5 min). To remove cell debris, the samples were centrifuged (8500 rpm, 30 min) and the clear cell lysate was stored at ⁇ 20° C.
  • heterologous proteins was checked by SDS-PAGE (Sambrook & Russell, 2001). Protein extracts were prepared in 1 ⁇ SDS gel-loading buffer, and polyacrylamide gels were stained with Coomassie Brilliant Blue.
  • fucose kinase activity of the enzyme was measured by the amount of arising ADP from ATP, used as a substrate by pyruvate kinase (PK) while dephosphorylating phosphoenolpyruvate (PEP), whereas the resulting pyruvate was then converted to L-lactate by L-lactate dehydrogenase (LDH) under NADH consumption.
  • PK pyruvate kinase
  • PEP dephosphorylating phosphoenolpyruvate
  • LDH L-lactate dehydrogenase
  • Each 1000 ⁇ L reaction was performed in 65 mM MOPS buffer (pH 7.5) containing 10 mM L-fucose, 15 mM PEP, 5 mM MgSO 4 , 0.2 mM of each ATP and NADH, and 5 U of each PK and LDH.
  • MOPS buffer pH 7.5
  • oxidation of NADH to NAD was monitored via the decrease of absorption at 340 nm using a V-630 Bio spectrophotometer (JASCO GmbH, Germany).
  • FucT activity was (as shown in FIG. 3 ) measured by arising GDP (from the donor GDP-L-fucose) which was phosphorylated to GTP by PK under conversion of PEP to pyruvate.
  • LDH catalyzed the final reaction of pyruvate reduction to L-lactate with concomitant NADH consumption.
  • Cellular extracts (25 ⁇ L) were tested in a 1000 ⁇ L reaction containing 10 mM lactose, 100 ⁇ M GDP-L-fucose, 5 mM MgSO 4 , 0.2 mM of each ATP and NADH, and 5 U of each PK and LDH in 50 mM Tris-HCl buffer (pH 7.5). The decrease of NADH was monitored at 340 nm.
  • HPAED high performance anion exchange chromatography
  • a Decade II pulsed amperometric detector from Antec Leyden (Netherlands) and a CarboPac PA20 column (Dionex, Germany) connected to a HPLC system (Shimadzu, Germany).
  • the detector sensitivity was set at 50 ⁇ A with a 0.05-V applied pulse potential.
  • Mono-, di-, and oligosaccharides eluted with 10 mM sodium hydroxide at a flow rate of 0.4 mL min ⁇ 1 . After 30 min isocratic elution with 10 mM NaOH the column was washed for 20 min with 200 mM NaOH to obtain constant retention times and thereafter regenerated with 10 mM NaOH for 20 min.
  • E. coli BW25113 ⁇ fucA (DE3) cells were transformed with the vectors pCOLADuet-1, pETDuet-1, pCOLA-fkp-fucP and pET-futAco to generate the following strains:
  • E. coli BW25113 ⁇ fucA (DE3) pCOLADuet-1 pETDuet-1 served as empty vector control in the feeding experiments. All three strains were then used for tritium labeled fucose feeding experiments.
  • cells were cultured in 3 ml of 2 ⁇ YT medium containing 20 ⁇ l L-5,6 ⁇ 3 H-Fucose (40-60 Ci/mmol and 1 mCi/mL), 50 mM lactose and 1 mM IPTG.
  • 2 ⁇ YT medium was supplemented with 100 ⁇ g mL ⁇ 1 ampicillin and/or 50 ⁇ g mL ⁇ 1 kanamycin.
  • E. coli cultures were incubated at room temperature overnight. Cells were then collected by centrifugation and separated from the culture media, the obtained cell pellets were resuspended in 200 ⁇ L of ddH 2 O and boiled for 5 min. After cooling on ice for 10 min cell debris were collected by centrifugation at 13000 rpm for 10 min. From the so obtained E. coli cell supernatants 20 ⁇ L of each culture were applied to a silica gel TLC plate (Silica gel 60). For the development of the TLC plate a solvent mixture consisting of butanol:acetone:acetic acid:water (35:35:7:23) was employed. Radio-TLC analysis was then performed with a Radio-TLC reader (Raytest).
  • the TLC plate was sprayed with anisaldehyde solution (5 mL conc. H 2 SO 4 , 100 mL ethanol, 1.5 mL acetic acid, 2 mL anisaldehyde) and heated.
  • anisaldehyde solution 5 mL conc. H 2 SO 4 , 100 mL ethanol, 1.5 mL acetic acid, 2 mL anisaldehyde
  • lacZ ⁇ ⁇ -galactosidase deficient E. coli strain BW25113 was chosen to circumvent the problem of rapid lactose degradation (Datsenko & Wanner, 2000).
  • L-Fucose can be also effectively degraded by wild type E. coli via isomerization to fuculose, phosphorylation to fuculose-1-phosphate and subsequent retro-aldol cleavage of fuculose-1-phosphate to glycerin-3-phosphate and L-lactaldehyde.
  • fucA encoding the key catabolic enzyme of the fucose degradation pathway fuculose-1-phosphate aldolase (FucA)
  • fucA fuculose-1-phosphate aldolase
  • nucleotide activation of fucose to GDP-fucose is very limited in nature and was also for a long time only known from several mammals (human, pig, mouse). Nucleotide activation of fucose is mediated here by two successive enzymatic steps, first the phosphorylation of fucose to fucose-1-phosphate, catalyzed by fucose kinase and followed by the conversion of fucose-1-phosphate to GDP-Fucose, catalyzed by guanylyltransferase, respectively. Whereas in mammals the fucose salvage pathway comprises two separate enzyme catalyzed reactions, the recently discovered bacterial and plant proteins comprise both enzymatic activities.
  • the resulting gene futAco was inserted in the expression vector pETDuet-1, and expression was tested with and without co-expression of Fkp and FucP.
  • Fkp, FucP and FutAco or FucT2 were co-expressed.
  • Protein formation was examined after induction with IPTG and/or L-rhamnose with SDS-PAGE (see FIG. 4 ), documenting pronounced soluble production of Fkp protein, whereas induction of membrane localized fucose permease protein (FucP) could, as expected, not be detected in cell cytoplasm by SDS-PAGE.
  • the gene products of futAco and fucT2 proved to be primarily located in inclusion bodies with only a small soluble fraction detectable.
  • the results of the negative control experiment showed products of the first and second catabolic steps from the fucose metabolism, i.e. L-fuculose (produced from fucose by fucose isomerase) and L-fuculose-1-phosphate (produced from fuculose by fuculose kinase). Further degradation of fucose is effectively inhibited by the knock-out of the gene fucA, which encodes the enzyme fuculose-1-phosphate aldolase, which catalyzes the retro-aldol cleavage reaction of fuculose-1-phosphate to L-lactaldehyde and dihydroxyacetone phosphate.
  • E. coli cells coexpressing bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase Fkp from Bacteroides fragilis show the production of GDP-fucose (see FIG. 7 ) which is apparently accumulating in the cells and may only minimally divert into other metabolic pathways whose products would otherwise appear on the radio-TLC.
  • fucose degradation products fuculose and fuculose-1-phosphate is also greatly diminished, due to the consumption of GDP-fucose in fucosyllactose production and the deriving drift of the reaction equilibrium from fuculose-1-phosphate and fuculose to fucose, which is constantly drawn from the reaction by GDP-fucose production.
  • E. coli BW25113 ⁇ fucA (DE3) harboring pCOLA-fkp-fucP and either the futAco or fucT2 gene in a separate expression vector, as well as E. coli BW25113 ⁇ fucA (DE3) harboring the empty vectors pCOLADuet-1 and pETDuet-1 (negative control) were grown in 2 ⁇ YT broth, and protein expression was induced with IPTG and/or L-rhamnose for 15 h at 28° C. The cells were subsequently washed with PBS and resuspended in modified M9 medium supplemented with L-fucose, lactose and guanosine, IPTG and L-rhamnose. After a fermentation phase (28° C., 15 h), the cells were harvested, supernatants collected and cell lysates prepared as described above.
  • L-fucose and lactose are also components of the cell lysate, as well as glycerol and L-rhamnose.
  • Codon optimized ⁇ 1,3-fucosyltransferase FutAco initially derived from Helicobacter pylori or ⁇ 1,2-fucosyltransferase FucT2 from Helicobacter pylori , respectively, can convert the so supplied GDP-fucose into 2′- and 3-fucosyllactose.
  • Elevation of intracellular GDP-fucose content due to expression of Fkp was shown by parallel cultivation of an E. coli strain expressing Fkp from a plasmid and an E. coli strain not containing a copy of Fkp.
  • Strain E. coli 3M109 (DE3) ⁇ fucA was in this case used as control strain without Fkp.
  • the strain expressing Fkp was the same strain E.
  • HPLC analysis was carried out by electrochemical detection with a Decade II pulsed amperometric detector (Antec Leyden, Netherlands). 20 mM sodium hydroxide+825 mM sodium acetate was used as eluent on a CarboPac PA20 column (Dionex, USA). GDP-fucose eluted with a retention time of 16.0 minutes.
  • FIG. 12 a shows HPLC-analysis of E. coli JM109 (DE3) ⁇ fucA cells for GDP-fucose expression without expression of FKP protein.
  • FIG. 12 b is an analysis of E. coli JM109 (DE3) ⁇ fucA pCOLA-fkp-fucP cells co-expressing the Fkp protein together with the fucose importer FucP.
  • the peak at 16.0 min corresponds to GDP-fucose, as verified with an authentic standard.

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US20190382737A1 (en) 2019-12-19
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