Patent application title: SYNTHESIS OF FUCOSYLATED COMPOUNDS
Eric Hüfner (Hennef, DE)
Eric Hüfner (Hennef, DE)
Julia Parkot (Koln, DE)
Stefan Jennewein (Aachen, DE)
JENNEWEIN BIOTECHNOLOGIE GMBH
IPC8 Class: AC12P1918FI
Class name: Micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition preparing compound containing saccharide radical produced by the action of a glycosyl transferase (e.g., alpha, beta, gamma-cyclodextrins by the action of glycosyl transferase on starch, etc.)
Publication date: 2011-12-08
Patent application number: 20110300584
A method for making a genetically modified cell having the ability to
produce fucosylated compounds comprising the steps of transforming the
cell to express a fucose kinase transforming the cell to express a
fucose-1-phosphate guanylyltransferase transforming the cell to express a
1. A method for making a genetically modified cell having the ability to
produce fucosylated compounds comprising the steps of transforming the
cell to express a fucose kinase transforming the cell to express a
fucose-1-phosphate guanylyltransferase transforming the cell to express a
2. The method of claim 1, wherein the genetically modified cell is a microorganismm selected from the group consisting of the genera Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Lactococcus, Pichia, Saccharomyces and Kluyveromyces.
3. The method of claim 1, wherein the fucose kinase and the fucose-1-phosphate guanylyltransferase are combined in a bifunctional enzyme.
4. The method of claim 3, wherein the bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase is selected of bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase dervied from the group consisting of the genera Bacteroides, Lentisphaera, Ruminococcus, Solibacter, Arabidopsis, Oryza, Physcomitrella, Vitis, Danio, Bos, Equus, Macaca, Pan, Homo, Rattus, Mus and Xenopus.
5. The method of claim 1, wherein the fucosyltransferase is derived from an organism selected from the group consisting of the genera Helicobacter, Escherichia, Yersinia, Enterococcus, Shigella, Klebsiella, Salmonella, Bacteroides, Dictyosetelium, Arabidopsis, Drosophila, Homo, Bos, Mus, Rattus, Gallus, Canis and Sus.
6. The method of claim 1, wherein a catabolic pathway of said cell for fucose is inactivated.
7. The method of claim 6, wherein the catabolic pathway for fucose is inactivated by 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.
8. The method of claim 1, wherein the fucosylated compound is a fucosyllactose, preferably 2'-fucosyllactose, 3-fucosyllactose or lactodifucotetraose.
9. A genetically modified cell obtainable by the method of claim 1.
10. A method for making fucosylated compound comprising the steps of cultivating the cell of claim 9 under suitable cultivation conditions in a medium comprising fucose and an acceptor substrate.
11. The method of claim 10, wherein the acceptor substrate is a mono-, di- or oligosaccharide or a peptide.
12. The method of claim 10, wherein the acceptor substrate is lactose, 2'-fucosyllactose or 3-fucosyllactose.
13. The method of claim 10, wherein the fucosylated compound is a fucosyllactose, preferably 2'-fucosyllactose or 3-fucosyllactose, or lactodifucotetraose.
 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. In addition human milk contains several protective agents. Besides immunoglobulins 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.
 Clinical data showed that breast-fed infants have lower incidence of diarrhea, respiratory disease, and otitis media than formula-fed infants. For a long time these protective effects of human milk have been attributed to the presence of secreted immunoglobulins, however, it has now been recognized that the HMOs may be a major line of defense against pathogens for breast-fed infants.
 Many of the complex HMOs show homology to cell surface glycoconjugates such as the Lewis x (Lex) histo-blood group antigen Gal(β1-4)[Fuc-(α1-3)]GlcNAc(β1) (Newburg, 2001), which often serve as pathogen receptors. Thus, by excreting soluble decoys, mimicking cell surface glycoconjugate structures, nature developed here an efficient mechanism to prevent infections. For example it was shown that 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).
 The impact of HMOs on protein-carbohydrate interactions, e.g., selectin-leukocyte binding, can modulate immune responses and reduce inflammatory responses (Bode, 2006, Kunz & Rudloff, 2006).
 Complex 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 (FucTs), 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 α1,2, α1,3/4, and α1,6 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). The exact role of these Lewis antigenic structures during H. pylori infection is unclear, but molecular mimicry to evade the host immune system, adhesion and colonization are discussed (Bergman et al., 2006).
 Due to the great potential of HMOs as health-promoting food supplements, there is strong interest in the cost-effective large-scale production. Biocatalytic production via bacterial fermentation processes is highly favorable over extraction of HMOs from human milk, and chemical synthesis, which is laborious and requires multiple protection and deprotection steps (Kretzschmar & Stahl, 1998). During the last decade, several attempts of HMO synthesis using either fermentation with recombinant E. coli or in vitro enzymatic conversion, have been published (Albermann et al., 2001, Dumon et al., 2006, Dumon et al., 2001, Dumon et al., 2004, Koizumi et al., 2000). The bottleneck in the production of fucosylated oligosaccharides is, however, the availability of the donor nucleotide sugar GDP-fucose. This high-energy molecule is currently neither efficiently nor cost-effectively accessible via chemical or enzymatic synthesis. Most publications reporting production systems for fucosylated compounds rely on the endogenous GDP-fucose pool of E. coli, which however is extremely limited and only used for the inducible synthesis of the fucose-containing exopolysaccharide cola nic acid (Grant et al., 1970).
 For example Albermann et al. (2001) use recombinant enzymes in an enzymatic synthesis. GDP-β-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-β-L-fucose, which is purified by preparative HPLC.
 Another approach by Koizumi and co-workers to synthesize Lex from N-acetyllactosamine (LacNAc) involved the combination of GTP production from supplemented GMP by Corynebacterium ammoniagenes, GDP-fucose synthesis via GDP-mannose, and fucosylation of LacNAc by overexpression of a H. pylori α1,3-FucT in separate E. coli strains (Koizumi et al., 2000). Since permeabilization, and thus killing the cells, had to be used for this bacterial coupling approach, a continuous and large-scale fermentation process is not possible with this chosen approach.
 There is still a need for methods for producing fucosylated compounds which overcome at least some of the drawbacks of prior art.
 One embodiment of the invention is a method for making a genetically modified cell having the ability to produce fucosylated compounds comprising the steps of  transforming the cell to express a fucose kinase  transforming the cell to express a fucose-1-phosphate guanylyltransferase  transforming the cell to express a fucosyltransferase.
 According to the method of the invention a genetically modified cell is produced. It has been transformed to express a fucosekinase, a fucose-1-phosphate guanylyltransferase and a fucosyltransferase.
 Methods of introducing genes into a cell are known to the skilled person.
 In a preferred embodiment, 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.
 In a preferred embodiment of the invention, 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.
 Depending on the source of the gene and the cell used for expression, 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. To produce fucosylated compounds, 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.
 This is the first report of efficient GDP-fucose synthesis in E. coli from externally supplied L-fucose and thus the establishment of an fucose "salvage pathway" in E. coli. However, this approach may also be transferred to other easy to culture organisms of interest to food or pharmaceutical industry (for example Lactobacillus spp.). Usage of this newly discovered pathway offers utterly new perspective for production of oligosaccharides, besides 2'-fucosyllactose and 3-fucosyllactose, without the need to rely on costly and laborious provision of GDP-fucose (in vitro) or endogenous, highly regulated, GDP-fucose biosynthetic pathways (in vivo).
 In the so called "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. Recently, 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.
BRIEF DESCRIPTION OF THE FIGURES
 FIG. 1 discloses the structures of the prominent complex human milk oligosaccharides (HMOs) 2'-fucosyllactose and 3-fucosyllactose.
 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. 3 shows a scheme of the photometric assay for determination of FucT activity by coupled enzyme reactions and determination of NADH oxidation; FucT=fucosyltransferase, 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 ΔfucA (DE3) pCOLA-fkp-fucP (lane 1), E. coli BW25113 ΔfucA (DE3) pET-futAco (lane 2), E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP+pETfutAco (lane 3) and E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP+pCAW55 (lane 4); lane 5: PageRuler® Prestained Protein Ladder (Fermentas, Germany); lanes 6-9: expression of insoluble Fkp and/or FutAco or FucT2, in cell debris resuspended in 6 M urea from E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP (lane 6), E. coli BW25113 ΔfucA (DE3) pET-futAco (lane 7), E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP+pETfutAco (lane 8) and E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP+pCAW55 (lane 9).
 FIG. 5 shows a radio thin layer chromatography (radio-TLC) of 3H-fucose, developed with butanol:acetone:acetic acid:water (35:35:7:23) and analyzed using a radio-TLC reader.
 FIG. 6 shows a radio-TLC of a cell extract from E. coli BW25113 ΔfucA (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 ΔfucA (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 ΔfucA (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 ΔfucA (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 ΔfucA (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 ΔfucA (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) ΔfucA (FIG. 12a) and E, coli JM109 (DE3) ΔfucA pCOLA-fkp-fucP (FIG. 12b).
 This invention is further explained by the following, non-limiting examples:
Construction of Expression Plasmids and Development of Production Strains
 To successfully prevent the degradation of externally supplied fucose the fucA gene, coding for the key catabolic enzyme fuculose-1-phosphate aldolase had to be deleted from the genome of E. coli strain BW25113. For construction of the fucA deletion the methodology of (Datsenko & Wanner, 2000) was applied. For heterologous gene expression using the T7 promoter an inducible T7 RNA polymerase was incorporated into the deletion strain E. coli BW25113 ΔfucA by using the λDE3 lysogenization kit (Novagen). The resulting strain was then named E. coli BW25113 ΔfucA (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 α1,2-fucosyltransferase from Helicobacter pylori NCTC364 was donated by C. Albermann (Institute for Microbiology, University of Stuttgart) and is based on vector pJOE2702 (Stumpp et al., 2000). Gene fucT2 is inserted via restriction sites NdeI/PstI and controlled by L-rhamnose-inducible promoter rhaPBAD. E. coli BW25113 ΔfucA (DE3) was transformed with the expression vectors by electroporation (Dower et al., 1988). All bacterial strains used in this study are listed in Table 1.
TABLE-US-00001 TABLE 1 Bacterial strains a nd plasmids used. Name Relevant characteristic(s)* References E. coli strains BW25113 Δ(araD-araB)567, (Datsenko ΔlacZ4787(::rrnB-3), & Wanner, lambda.sup.-, rph-1, 2000) Δ(rhaD-rhaB)568, hsdR514 BW25113ΔfucA (DE3) BW25113 fucA mutant, This study carrying chromosomal copy of λDE3 T7 RNA polymerase gene BW25113ΔfucA (DE3) Negative control strain This study pCOLADuet-1 pETDuet-1 harboring empty vectors, ApR, KanR BW25113ΔfucA (DE3) KmR This study pCOLA-fkp-fucP BW25113ΔfucA (DE3) ApR, KmR This study pCOLA-fkp-fucP pET-futAco BW25113ΔfucA (DE3) ApR, KmR This study pCOLA-fkp-fucP pCAW55 Plasmids pCOLADuet-1 KmR Novagen pETDuet-1 ApR Novagen pCOLA-fkp-fucP KmR This study pET-futAco ApR This study pCAW55 ApR C. Albermann *ApR, ampicillin resistant, KmR, kanamycin resistant.
TABLE-US-00002 TABLE 2 Primers. Added restriction Name Sequence (5'→3')* site Fkp-NcoI-forward AAGGAAACCATGGGCCAAAAACTACTATCTTTACCG NcoI TCCAATCTGGTTCAGTC Fkp-NotI-reverse AAGGAAATTGCGGCCGCATTATGATCGTGATACTTG NotI GAATCCCTTATCAGATAACG FucP-NdeI-forward AAGGAATACATATGGGAAACACATCAATACAAACGC NdeI AGAGTTACCGTGCGG FucP-XhoI-reverse AAGGAAACTCGAGTCAGTTAGTTGCCGTTTGAGAAC XhoI GGAAACGGGCAAAG FutAco-NcoI-forward AAGGGAAACCATGGCTATGTTCCAGCCGCTGCTGG NcoI ACGCGTTTATCGAGTCTGC FutAco-BamHI-reverse AAGGGAAAGGATCCGGGTCCTATTACAGACCCAGT BamHI TTTTTCACCAG pACYCDuetUP1 GGATCTCGACGCTCTCCCT pET-Upstream-Primer ATGCGTCCGGCGTAGA DuetDOWN-1-Primer GATTATGCGGCCGTGTACAA DuetUP2-Primer TTGTACACGGCCGCATAATC T7-Terminator-Primer TATGCTAGTTATTGCTCAG *The restriction endonuclease recognition sites are underlined.
Cultivation Conditions and Preparation of Cell Extracts
 E. coli strains were inoculated in 10 mL of 2×YT broth (Sambrook & Russell, 2001), containing 100 μg mL-1 ampicillin and/or 50 μg 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. When the cultures reached an optical density (OD600 nm) of approximately 0.5, inducers isopropyl-1-thio-β-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.
 For in vivo production of fucosyllactose, cells were washed with one culture volume of phosphate buffered saline pH 7.4 (PBS) (Sambrook & Russell, 2001), and resuspended in 30 mL of modified M9 mineral medium; to the standard M9 recipe (Sambrook & Russell, 2001), the following substances were added: 20 mM L-fucose, 20 mM lactose, 0.5% glycerol, 0.5 mM guanosine and 1×GIBCO MEM Vitamin Solution (100×) (Invitrogen, USA). 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.
 The expression of 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.
Enzymatic Photometer Assays
 To determine Fkp activity, 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. The corresponding reactions are summarized in FIG. 2. Each 1000 μL reaction was performed in 65 mM MOPS buffer (pH 7.5) containing 10 mM L-fucose, 15 mM PEP, 5 mM MgSO4, 0.2 mM of each ATP and NADH, and 5 U of each PK and LDH. After the addition of 25 μL crude extract, the oxidation of NADH to NAD was monitored via the decrease of absorption at 340 nm using a V-630 Bio spectrophotometer (JASCO GmbH, Germany).
 Analogously, 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 μL) were tested in a 1000 μL reaction containing 10 mM lactose, 100 μM GDP-L-fucose, 5 mM MgSO4, 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.
Detection of Oligosaccharides
 Samples were analyzed by high performance anion exchange chromatography (HPAED) using 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 μA 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.
3H-Fucose Feeding Experiments
 E. coli BW25113 ΔfucA (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 ΔfucA (DE3) pCOLADuet-1 pETDuet-1
 E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP
 E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP pET-futAco.
 Strain E. coli BW25113 ΔfucA (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. For the feeding experiments cells were cultured in 3 ml of 2×YT medium containing 20 μl L-5,6-3H-Fucose (40-60 Ci/mmol and 1 mCi/mL), 50 mM lactose and 1 mM IPTG. According to the used expression vectors 2×YT medium was supplemented with 100 μg mL-1 ampicillin and/or 50 μg mL-1 kanamycin. The 3 mL 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 μL of ddH2O 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 μL 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). For the determination of Rf-values of non-radioactive reference material the TLC plate was sprayed with anisaldehyde solution (5 mL conc. H2SO4, 100 mL ethanol, 1.5 mL acetic acid, 2 mL anisaldehyde) and heated.
Establishment of an Efficient L-Fucose Salvage Pathway in E. coli
 Since lactose was used as acceptor substrate for the fucosyltransferases, the β-galactosidase deficient (lacZ.sup.-) 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. To prevent degradation of supplied fucose the gene fucA, encoding the key catabolic enzyme of the fucose degradation pathway fuculose-1-phosphate aldolase (FucA), was deleted in the genome of strain E. coli BW25113. The resulting strain E. coli BW25113 ΔfucA was unable to grow on fucose as well as lactose as sole carbon source on M9 minimal plates. Lysogenization with recombinant phage λDE3 resulted in strain E. coli BW25113 ΔfucA (DE3) compatible with the use of T7 promoter driven expression vectors. The ability of 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. Heterologous expression of human fucose kinase in E. coli resulted only in barely detectable activity (Hinderlich et al., 2002). Biochemical studies showed that mammalian fucokinase represents a highly regulated enzyme (Park et al., 1998). To examine of whether the recently discovered B. fragilis Fkp enzyme is more suitable for activation of fucose and to efficiently provide GDP-fucose for the synthesis of fucosylated oligosaccharides in E. coli we amplified the gene from B. fragilis genomic DNA and cloned it into a bacterial expression vector for heterologous expression.
 For the synthesis of 2'- and 3-fucosyllactose the following fucosyltransferases were chosen for co-expression: The futA gene of H. pylori 26695 (Appelmelk et al., 1999), encoding an α1,3-fucosyltransferase, and the α1,2 fucosyltransferase gene fucT2 of H. pylori NCTC364 (Albermann et al., 2001). Before the start of the cloning process, the codon usage of futA was optimized for expression in E. coli and the gene was then synthesized by GenScript corporation (USA). 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. Using standard induction conditions, 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. However, the gene products of futAco and fucT2 proved to be primarily located in inclusion bodies with only a small soluble fraction detectable.
Photometric Detection of Enzymatic Activity
 The crude extracts derived from induced cultures were tested for fucose kinase and fucosyltransferase activity using auxiliary enzymes in coupled enzymatic assays as described above. Apparently, there is a considerable background of either NADH oxidase and/or phosphatase activity in E. coli BW25113 ffucA (DE3), which was responsible for non-reproducible results and low measured fucose kinase and fucosyltransferase activity of the different strains. Therefore, it was decided to determine enzymatic activity by monitoring intracellular product formation (GDP-fucose and fucosyllactose).
Examination of Utilization of Externally Fed 3H-L-Fucose for GDP-Fucose and 3-Fucosyllactose Production by Recombinant E. coli
 The aim of this experiment was the verification of 3-fucosyllactose production from fucose and lactose via GDP-fucose production by the fucose salvage pathway bifunctional enzyme Fkp from Bacteroides fragilis. Negative control strain E. coli BW25113 ΔfucA (DE3) pCOLADuet-1 pETDuet-1, as well as Fkp and fucose permease expressing strain E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP and Fkp, fucose-permease and α1,3-fucosyltransferase expressing strain E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP pET-futAco were treated as described above. Cell extracts derived from these strains were applied to a TLC plate, developed as described above and analyzed by radio-TLC reader. Additionally, 3H-labelled L-fucose standard was applied to a TLC plate and developed (see FIG. 5). Non-radioactive standards for L-fucose and L-fuculose-1-phosphate, GDP-L-fucose, as well as 3-fucosyllactose were analyzed similarly by TLC and subsequent staining by anisaldehyde solution (data not shown).
 The results of the negative control experiment (see FIG. 6) 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.
 Cell extracts from strain E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP pET-futAco show production of 3-fucosyllactose and only a small amount of GDP-fucose (see FIG. 8). This result is consistent with the initial aim of the experiment, i.e. to show the production of 3-fucosyllactose via GDP-fucose supply by bifunctional salvage pathway enzyme Fkp from Bacteroides fragilis. The amount of 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.
Examination of 2'-Fucosyllactose and 3-Fucosyllactose Production by Recombinant E. coli
 Strain E. coli BW25113 ΔfucA (DE3) harboring pCOLA-fkp-fucP and either the futAco or fucT2 gene in a separate expression vector, as well as E. coli BW25113 ΔfucA (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.
 Analysis via HPAED showed retention times on the used HPLC column of approximately 3 min for the L-fucose standard, approx. 17 min for the lactose standard, approx. 11 min for the 3-fucosyllactose standard, and of approx. 22 min for the used 2'-fucosyllactose standard (data not shown). Glycerol, that is, as carbon source, part of the culture medium, was recorded with a retention time of approx. 1.5 min, and inducer L-rhamnose with a retention time of 5.5 min. Both substances are detected intraceltularly during analysis of cell lysates.
 Cell lysates from E. coli BW25113 ΔfucA (DE3) pCOLADuet-1 pETDuet-1 negative control strain showed intracellular L-fucose and lactose, but, as expected, no fucosyllactose (see FIG. 9). In addition to the aforementioned molecules also the medium supplied carbon source glycerol and the transcription inducer L-rhamnose are detected in the analysis.
 HPAED analysis of cell lysate from strain E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP pET-futAco, coexpressing the B. fragilis fkp gene and E. coli fucose permease gene in combination with the codon optimized Helicobacter pylori α1,3-fucosyltransferase gene, showed the intracellular production of 3-fucosyllactose (peak at about 11 min, see FIG. 10). L-fucose and lactose are also components of the cell lysate, as well as glycerol and L-rhamnose.
 Cell lysate from strain E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP pCAW55 showed intracellular production of 2'-fucosyllactose (see FIG. 11), due to the coexpression of α1,2-fucosyltransferase FucT2. Additionally, L-fucose, lactose, glycerol and L-rhamnose can be seen in cell lysate, just as in cell lysate from negative control and 3-fucosyllactose producing strain E. coli BW25113 ΔfucA (DE3) pCOLA-fkp-fucP pET-futAco.
 These results clearly show the production of 3- and 2'-fucosyllactose in recombinant E. coli cells from externally supplied L-fucose and lactose. By heterologous expression of B. fragilis Fkp protein, catalyzing the two-step reaction of fucose phosphorylation and fucose-1-phosphate guanylyl transfer, efficient production of GDP-fucose was obtained. Codon optimized α1,3-fucosyltransferase FutAco initially derived from Helicobacter pylori or α1,2-fucosyltransferase FucT2 from Helicobacter pylori, respectively, can convert the so supplied GDP-fucose into 2'- and 3-fucosyllactose.
Expression of GDP-Fucose in E. coli JM109 Cells
 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) ΔfucA was in this case used as control strain without Fkp. The strain expressing Fkp was the same strain E. coli 3M109 (DE3) ΔfucA, this time containing the plasmid pCOLA-fkp-fucP, and bearing thus the genes coding for fucose kinase/fucose-1-phosphate guanylyltransferase Fkp and fucose permease FucP. As the genes were cloned in multiple cloning sites (MCS) 1 and 2 of vector pCOLADuet-1 (Novagen, UK), expression of both genes can be induced by addition of IPTG, as both MCS are flanked by a T7 promoter/operator on the 5' side.
 Both strains were cultured in duplicate in 30 ml 2YT medium, supplemented with kanamycin for the strain with pCOLA-fkp-fucP for plasmid maintenance, at 37° C. and 220 rpm. Induction of Fkp expression was started at OD660=0.5 by addition of 1 mM IPTG and both strains were supplied with 20 mM fucose and then cultivated for additional 3 hours at 37° C. and 220 rpm. Cells were pelleted by centrifugation and pellets were resuspended in 5 v/w distilled water. These cell suspensions were incubated at 95° C. for 10 minutes to lyse the cells. Cell debris was removed by centrifugation and the supernatants were analyzed by HPLC.
 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.
TABLE-US-00003 TABLE 3 Intracellular GDP-fucose content of E. coli JM109 (DE3) ΔfucA with and without expression of fucose kinase/fucose- 1-phosphate guanylyltransferase Fkp from pCOLA-fkp-fucP. Strain GDP-fucose content [μM] E. coli JM109 (DE3) ΔfucA not detectable E. coli JM109 (DE3) ΔfucA 369 μM pCOLA-fkp-fucP
 FIG. 12a shows HPLC-analysis of E. coli JM109 (DE3) ΔfucA cells for GDP-fucose expression without expression of FKP protein.
 FIG. 12b is an analysis of E. coli JM109 (DE3) ΔfucA 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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Patent applications by Stefan Jennewein, Aachen DE
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