ENZYMATIC METHOD FOR PREPARATION OF GDP-FUCOSE

Abstract

The present invention relates to an enzyme-catalyzed process for producing GDP- fucose from low-cost substrates guanosine and L-fucose or guanosine and D-Mannose in a single reaction mixture. Said process can be operated (semi)continuously or in batch mode. Further, said process can be adapted to produce fucosylated molecules and biomolecules including glycans, such as human milk oligosaccharides, proteins, peptides, glycoproteins or glycopeptides.

Claims

1. A method for producing guanosine 5′-diphospho-β-L-fucose from guanosine and L-fucose or D-mannose comprising: ##STR00010## A) providing a solution comprising (i) guanosine and L-fucose or guanosine and D-mannose represented by the following formulae ##STR00011## (ii) polyphosphate, adenosine triphosphate and a co-solvent for solubilizing guanosine; and providing a set of enzymes comprising a guanosine kinase, a polyphosphate kinase, and in case of L-fucose a L-fucokinase/L-fucose- 1-phosphate guanylyltransferase or in case of D-mannose either (a) a glucokinase, phosphomannomutase, a mannose-1-phosphate guanylyltransferase, a GDP-mannose-4,6-dehydratase and a GDP-L-fucose-synthase or (b) an N-acetylhexosamine-1-kinase, a mannose-1-phosphate guanylyltransferase, a GDP-mannose 4,6-dehydratase and a GDP-L-fucose-synthase; B) producing guanosine 5′-diphospho-β-L-fucose from guanosine and L-fucose in the presence of the set of enzymes, polyphosphate, adenosine triphosphate and the co-solvent or from guanosine and D-mannose in the presence of the set of enzymes, polyphosphate, adenosine triphosphate and the co-solvent.

2. The method according to claim 1, wherein the solution comprises guanosine and L-fucose; and the set of enzymes comprises a guanosine kinase, a polyphosphate kinase, and a L-fucokinase/L-fucose-1-phosphate guanylyltransferase.

3. The method according to claim 1, wherein the solution comprises guanosine and D-mannose; and the set of enzymes comprises a guanosine kinase, a polyphosphate kinase, and either (a) a glucokinase, phosphomannomutase, a mannose-1-phosphate guanylyltransferase, a GDP-mannose-4,6-dehydratase and a GDP-L-fucose- synthase or (b) an N-acetylhexosamine-1-kinase, a mannose-1-phosphate guanylyltransferase, a GDP-mannose 4,6-dehydratase and a GDP-L-fucose-synthase.

4. The method according to claim 1, wherein the set of enzymes further comprises a pyrophosphatase.

5. The method according to claim 1, wherein at least one enzyme of the set of enzymes is immobilized on a solid support.

6. The method according to claim 1, wherein the set of enzymes is co-immobilized on a solid support.

7. The method according to claim 5, wherein the at least one enzyme is immobilized on a solid support from cell lysate or the set of enzymes is co-immobilized on a solid support from cell lysate.

8. The method according to claim 1, wherein the concentration of guanosine and L-fucose or guanosine and D-mannose in the solution provided in A) is in the range of 0.2 mM to 5,000 mM.

9. The method according to claim 1, wherein the polyphosphate is a long-chain polyphosphate having at least 25 phosphate residues.

10. The method according to claim 1, wherein the guanosine 5’-diphospho-β-L-fucose is produced in a single reaction mixture.

11. The method according to claim 1, wherein the amount of co-solvent is from 0.01 vol % to 30 vol % based on total volume of the solution provided in A).

12. The method according to claim 1, wherein the co-solvent is dimethyl sulfoxide.

13. The method according to claim 1, wherein the method further comprises producing L-fucose from guanosine 5′-diphospho-β-L-fucose in the presence of a fucosyltransferase and in the absence of an acceptor; or producing L-fucose by heating the guanosine 5′-diphospho-β-L-fucose at the temperature in a range of 80 to 100° C.

14. The method according to claim 1, further comprising C) isolating the guanosine 5′-diphospho-β-L-fucose.

15. The method according to claim 1 further comprising D) producing a fucosylated saccharide, fucosylated glycopeptide, fucosylated glycoprotein fucosylated protein, fucosylated peptide or small molecule from guanosine 5′-diphospho-β-L-fucose and a saccharide, glycopeptide, glycoprotein, protein, peptide or small molecule by forming an O-glycosidic bond between guanosine 5′-diphospho-β-L-fucose and an available hydroxyl group of the saccharide, glycopeptide glycoprotein, protein, peptide or small molecule in the presence of a fucosyltransferase.

16. The method according to claim 1, wherein the polyphosphate kinase is 2-domain polyphosphate kinase 2 or polyphosphate kinase 3.

17. The method according to claim 1, wherein the set of enzymes further comprises any one of a glucose dehydrogenase, a glucose-6-phosphate dehydrogenase and a glutamate dehydrogenase.

18. The method according to claim 17, wherein the set of enzymes further comprises a guanylate kinase.

19. The method according to claim 1, wherein the glucokinase comprises at least 85% of an amino acid sequence as set forth in SEQ ID NO: 1; the phosphomannomutase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 2; the N-acetylhexosamine-1-kinase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 3; the mannose-1-phosphate guanylyltransferase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 4; the GDP-mannose-4,6-dehydratase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 5; the GDP-L-fucose-synthase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 6; the L-fucokinase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 7; the guanosine kinase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 8; the polyphosphate kinase comprises any of at least 85% of amino acid sequences set forth in SEQ ID NO: 9, and SEQ ID NO: 14; the pyrophosphatase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 10; the guanylate kinase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 11; the glutamate dehydrogenase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 12; the glucose-6-phosphate-dehydrogenase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 13; the glucose dehydrogenase comprises at least 85% of an amino acid sequence set forth in SEQ ID NO: 15; and the fucosyltransferase comprises at least 85% of amino acid sequence set forth in SEQ ID NO: 16.

Description

DESCRIPTION OF THE FIGURES

[1226] FIG. 1: shows the reaction pathway of the inventive method for producing GDP-fucose, which consists of (a) the formation of fucose-1-phosphate (Fuc-1-P) from L-fucose and ATP, (b) the formation of guanosine triphosphate (GTP) from guanosine and polyphosphate, and (c) the reaction of fucose-1-phosphate with guanosine triphosphate to GDP-fucose.

[1227] FIG. 2: shows an exemplary reaction scheme of the inventive method for producing GDP-fucose, which consists of (a) the formation of fucose-1-phosphate (Fuc-1-P) from L-fucose and ATP catalyzed by fucokinase (9), (b) the formation of guanosine triphosphate (GTP) from guanosine and polyphosphate catalyzed by guanosine kinase/inosine kinase (7) and polyphosphate kinase (8), and (c) the reaction of fucose-1-phosphate with guanosine triphosphate to GDP-fucose catalyzed by fucose-1-phosphate guanylyltransferase (9)

[1228] FIG. 3: shows an exemplary reaction scheme of the inventive method for producing GDP-fucose, which consists of (a) the formation of mannose-1-phosphate (Man-1-P) from D-mannose and ATP catalyzed by either glucokinase (1) and phosphomannomutase (2) or N-acetylhexosamine-1-kinase (3), (b) the formation of guanosine triphosphate (GTP) from guanosine and polyphosphate catalyzed by guanosine kinase/inosine kinase (7) and polyphosphate kinase (8), and (c) the reaction of mannose-1-phosphate with guanosine triphosphate to GDP-mannose catalyzed by mannose-1-phosphate guanylyltransferase (4) and (d) formation of

[1229] GDP-4-dehydro-6-deoxy-alpha- D-mannose from GDP-mannose catalyzed by GDP-mannose-4,6-dehydratase (5) and (e) formation of GDP-fucose from GDP-4-dehydro-6-deoxy-alpha-D-mannose and NADPH catalyzed by GDP-L-fucose synthase (6).

[1230] FIG. 4A: shows concentration of GDP-fucose prepared by the inventive method at different time points (see Example 3).

[1231] FIG. 4B: shows concentration of GDP-fucose prepared by the inventive method using immobilized enzymes at different time points (see example 5).

[1232] FIG. 5A: shows a chromatogram of the starting material at t=0.

[1233] FIG. 5B: shows a chromatogram of the reaction mixture after 48 hours.

[1234] FIG. 6: shows exemplary fucosylated milk saccharides.

[1235] FIG. 7: shows amount of bound enzymes on solid support after incubation at 4° C. for 24 hours (see Example 4).

[1236] FIG. 8: shows amount of formed GDP-fucose after reaction with the immobilized enzymes for 24 hours at 30° C. (see Example 4).

[1237] FIG. 9: shows the exemplary reaction scheme of the inventive method for producing L-fucose from guanosine and D-mannose, which consists of (a) the formation of mannose-1-phosphate (Man-1-P) from D-mannose and ATP catalyzed by either glucokinase (1) and phosphomannomutase (2) or N-acetylhexosamine-1-kinase (3), (b) the formation of guanosine triphosphate (GTP) from guanosine and polyphosphate catalyzed by guanosine kinase/inosine kinase (7) and polyphosphate kinase (8), and (c) the reaction of mannose-1-phosphate with guanosine triphosphate to GDP-mannose catalyzed by mannose-1-phosphate guanylyltransferase and (d) formation of GDP-4-dehydro-6-deoxy-alpha- D-mannose from GDP-mannose catalyzed by GDP-mannose-4,6-dehydratase and (e) formation of GDP-fucose from GDP-4-dehydro-6-deoxy-alpha-D-mannose and NADPH catalyzed by GDP-L-fucose synthase and formation of L-fucose from GDP-L-fucose catalyzed by type 1 galactoside alpha-(1,2)-fucosyltransferase in absence of an acceptor molecule.

[1238] FIG. 10: shows an exemplary reaction scheme of the inventive method for producing 2′-fucosyllactose from guanosine and D-mannose, which consists of (a) the formation of mannose-1-phosphate (Man-1-P) from D-mannose and ATP catalyzed by either glucokinase (1) and phosphomannomutase (2) or N-acetylhexosamine-1-kinase (3), (b) the formation of guanosine triphosphate (GTP) from guanosine and polyphosphate catalyzed by guanosine kinase/inosine kinase (7) and poly-phosphate kinase (8), and (c) the reaction of mannose-1-phosphate with guanosine triphosphate to GDP-mannose catalyzed by mannose-1-phosphate guanylyl-transferase and (d) formation of GDP-4-dehydro-6-deoxy-alpha- D-mannose from GDP-mannose catalyzed by GDP-mannose-4,6-dehydratase and (e) formation of GDP-fucose from GDP-4-dehydro-6-deoxy-alpha-D-mannose and NADPH catalyzed by GDP-L-fucose synthase and formation of 2′-fucosyllactose from GDP-L-fucose and lactose catalyzed by type 1 galactoside alpha-(1,2)-fucosyltransferase.

[1239] FIG. 11: shows the reaction cascade from (A1) GDP-mannose to GDP-fucose performed in Example 6-A1; (A2) GDP-mannose to GDP-fucose in presence of glucose and G6PDH as performed in Example 6-A2; and (A3) mannose and GTP to GDP-fucose in presence of glutamate and GLDH as performed in Example 6-A3. Reactions were conducted to demonstrate that NADPH recycling can drive the multi-enzyme reaction equilibrium towards the side of GDP-fucose.

[1240] FIG. 12: shows chromatogram of reaction aliquots of reactions A1, A2 and A3 after overnight incubation). The chromatogram (A1) shows that the reaction equilibrium is on the side towards GDP-mannose. When NADPH recycling is implemented the reaction equilibrium can be driven towards GDP-fucose (see A2 and A3).

[1241] FIG. 13: shows the reaction cascade from the reaction cascade from mannose and guanosine to GDP-fucose as performed in Example 7.

[1242] FIG. 14: shows reaction time course of the reaction in example 7 as measured by ion chromatography.

[1243] FIG. 15: shows the reaction cascade from mannose and guanosine to GDP-fucose as performed in Example 8.

[1244] FIG. 16: shows reaction time course of the reaction in example 8 as measured by ion chromatography.

[1245] FIG. 17: shows the reaction cascade from mannose and guanosine to 3-fucosyl-lactose as performed in Example 9.

[1246] FIG. 18: shows synthesis of 3-fucosyllactose from mannose and guanosine. Reaction chromatogram of a reaction aliquot taken after a reaction time of 48 hours (black) and of 3-fucosyllactose standard samples. The peaks were detected by amperometric detection.

[1247] FIG. 19: shows synthesis of 3-fucosyllactose from guanosine and fucose (reaction D2 of example 9). Chromatogram of a reaction aliquot taken after a reaction time of 48 hours. The peaks were detected by amperometric detection.

[1248] FIG. 20: shows the production of L-fucose from D-mannose. The reaction of sample in example 8 was heated for 1 hour at 95° C. (see, example 10). An aliquot of the heated sample was taken and measured by ion chromatography with amperometric detection (black) and compared against a fucose standard (Fuc).

[1249] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples, which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments, which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[1250] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

[1251] Abbreviations and Acronyms

[1252] ADP adenosine 5′-diphosphate

[1253] ATP adenosine 5′-triphosphate

[1254] Fuc L-fucose

[1255] GSK guanosine kinase

[1256] GDP guanosine 5′-diphosphate

[1257] GDH glucose dehydrogenase; glucose-1-dehydrogenase

[1258] GLDH glutamate dehydrogenase

[1259] G6PDH glucose-6-phosphate-dehydrogenase

[1260] GMD GDP-D-mannose-4,6-dehydratase

[1261] GMK guanylate kinase

[1262] GMP guanosine 5′-monophosphate

[1263] GLK glucokinase

[1264] GTP guanosine 5′-triphosphate

[1265] GUO guanosine

[1266] Lac D-lactose

[1267] Man D-mannose

[1268] ManB phosphomannomutase

[1269] ManC mannose-1-phosphate guanyltransferase

[1270] NADP nicotinamide adenine dinucleotide phosphate

[1271] NADPH reduced nicotinamide adenine dinucleotide phosphate

[1272] NAHK N-acetylhexosamine-1-kinase

[1273] PolyP polyphosphate

[1274] PPi pyrophosphate

[1275] Pi phosphate

[1276] PPK2 polyphosphate kinase 2

[1277] PPK3 polyphosphate kinase 3

[1278] 2D-PPK2 2-domain polyphosphate kinase 2

[1279] FKP L-fucokinase/L-fucose-1-phosphate guanylyltransferase

[1280] PmPpA Pasteurella multocida inorganic pyrophosphatase (PPA)

[1281] WCAG GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase

[1282] 3/4FT α-1-3/4-fucosyltransferase

[1283] Chemicals & Reagents

[1284] Unless otherwise stated, all chemicals and reagents were acquired from Sigma-Aldrich, and were of the highest purity available. Solid supports were obtained from Resindion, ChiralVision, ROhm GmbH & Co. KG and micromod GmbH.

Example 1

Preparation of Enzymes

[1285] The genes encoding for the enzymes GSK, PPK2, FKP and PmPpA were cloned into standard expression vectors as listed in Table 1. The expression vectors were transformed into E. coli BL21 Gold (DE3).

TABLE-US-00001 TABLE 1 Expression Enzyme Source Plasmid Inducer host guanosine kinase Exiguobacterium pET-28a(+) IPTG E. coli BL21 (GSK) acetylicum Gold (DE3) polyphosphate kinase Pseudomonas pET-28a(+) IPTG E. coli BL21 (2D-PPK2) aeruginosa Gold (DE3) L-fucokinase/L-fucose-1- Bacteroides pET-100/D- IPTG E. coli BL21 phosphate- fragilis TOPO Gold (DE3) guanylyltransferase (FKP) inorganic Pasteurella pET-28a(+) IPTG E. coli BL21 pyrophosphatase multocida Gold (DE3) (PmPpA)

[1286] Transformants were grown in 1 L shaking flasks with baffles in a volume of 500 ml of LB medium (lysogeny broth) supplemented with 50 pg/ml Kanamycin. The cultures were grown at 37° C. up to OD.sub.600=0.8. The expression was induced by addition of IPTG with a final concentration of 0.5 mM to the culture. Expression time was terminated after 12-18 hours at 20° C. Biomass was separated from the medium by centrifugation at 6,000×g for 10 min. Successful expression of the respective enzyme was analyzed by SDS-PAGE following standard operating procedures (Laemmli, Nature 1970, 227, 680-685). The wet biomass was stored at −20° C.

[1287] For purification, typically 30 ml of equilibration buffer were added to 3 g of frozen biomass. The equilibration buffer contains cOmplete™ protease inhibitor cocktail at pH 7.5: 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM MgCl.sub.2, 5 mM MnSO.sub.4, 300 mM NaCl and 5 vol % glycerol. Following thawing at 4° C. under stirring, cells were disrupted by four passages through a high pressure homogenizer (Emulsiflex C5, Avestin Inc., Ottawa, Canada) at 1,000 bar with intermediate cooling on ice. After centrifugation (45 min, 20,000 x g), the supernatant was applied to an equilibrated Immobilized Metal Affinity Chromatography (IMAC) column (10 ml CV) containing Ni.sup.2+ Sepharose™ High Performance chromatography material from Amersham Biosciences (Uppsala, Sweden). Unbound proteins were washed out using equilibration buffer. Immobilized protein was eluted in 1 ml fractions using elution buffer. Finally, the enzyme solutions were concentrated by Centrifugal Filter Units Amicon® Ultra-15 with a 50 kDa cut-off from Merck Millipore (Darmstadt, Germany). No enzyme loss was observed during the ultrafiltration. The enzymes were stored in 50% glycerol at −20° C. The protein concentration was determined by Bradford assay using BSA as standard (Bradford, Analytical Biochemistry 1976, 72(1), 248-254).

Example 2

Homogeneous Preparation of GDP-Fucose

[1288] The purified enzymes (Table 1) are mixed together with guanosine, L-fucose, ATP, polyphosphate, and HEPES buffer to the concentrations as listed in Table 2. Experiments conducted in low binding protein vials. Guanosine was solubilized in 6 vol % dimethyl sulfoxide (DMSO) prior addition to the mixture. The reaction was carried out at 30° C. in a thermomixer.

TABLE-US-00002 TABLE 2 Reactants Concentration [mmol/L] guanosine 2 polyphosphate (n = 14 or 25) 4 L-fucose 2 ATP 2 HEPES Buffer 50 Mg.sup.2+ 2.5 Mn.sup.2+ 2.5 Enzyme Concentration [mg/mL] GSK 0.3 2D-PPK2 0.7 FKP 0.2 PmPpA 0.2

[1289] After almost three hours 90% conversion of substrate (guanosine) to GDP-fucose was obtained (See FIG. 4A). No other end-products were detected (See FIG. 5B showing a chromatogram of the reaction mixture after 48 hours).

Example 3

Failed Synthesis of GDP-Fucose

[1290] The synthesis of GDP-fucose was carried out as described in Example 2, but without adding DMSO. The reaction was carried out at 30° C. in a thermomixer and the obtained reaction mixture remained turbid. After three hours no conversion of substrate (guanosine) to GDP-fucose was observed.

[1291] This Example demonstrates that the mere combination of the two enzymatic pathways does not provide GDP-fucose.

Example 4

Immobilization of Enzymes on Solid Support

[1292] Enzymes were immobilized on the solid supports in order to allow the multiple use of the enzymes.

[1293] Cell lysates obtained in Example 1 by high pressure homogenization were centrifuged and filtered to remove cell debris. The resins: sepabeads (Resindion): EC-EP, EP403/M, EC-HFA, EC-EA/M and EC-HA; immobeads (ChiralVision) IB-COV-1, IB-COV2, IB-COV3, IB-ANI1, IB-ANI2, IB-ANI3, IB-ANI4, IB-CAT1, IB-ADS1, IB-ADS2, IB-ADS3 and IB-ADS4; Eupergit (Rohm GmbH & Co. KG) and magnetic particles (micromod GmbH): Nano-mag, Sicastar-6 and Sicastar-1.5 wer incubated together with the enzymes for 24 hours at 4° C.

[1294] The protein assay was done by BCA assay. Results of total bound protein are shown in FIG. 7.

[1295] After immobilization the enzyme loaded resins were washed with buffer as described in Example 1. The resins were incubated with a solution of reactants as shown in Table 3 at 30° C. for 24 hours.

TABLE-US-00003 TABLE 3 Reactants Concentration [mmol/L] guanosine 3.9 (4 vol % DMSO) polyphosphate (n = 25) 12.5 L-fucose 5.3 ATP 12 HEPES Buffer 120 MgCl.sub.2 8 MnCl.sub.2 8 NaCl 200

[1296] The formation of GDP-fucose was observed for all loaded resins, as shown in FIG. 8.

Example 5

Heterogeneous Preparation of GDP-Fucose on Magnetic Particles

[1297] To this extent, fermentation broths (see Example 1) of 135 mL of FKP, 45 mL of PmPpA, 90 mL GSK and 90 mL of 2D-PPK2 were combined and lysed in 25 mL buffer (as described in Example 1) containing 10 mM imidazole, to obtain 0.5 mL of an enzyme mixture, which was incubated with 12.5 mg of Sicastar-6 magnetic resins.

[1298] After 1 hour of incubation at 10° C., resins were washed with buffer and combined in a vial with 0.25 mL of a solution of reagents (see Table 4). The mixture was incubated at 30° C. for 48 hours.

TABLE-US-00004 TABLE 4 Reactants Concentration [mmol/L] guanosine 6 (4 vol % DMSO) polyphosphate (n = 25) 9.3 L-fucose 6 ATP 9.6 HEPES Buffer 50 MgCl.sub.2 15 MnCl.sub.2 5 NaCl 30 KCl 30

[1299] After 48 hours nearly quantitative conversion (98%) of substrate (guanosine) to GDP-fucose was obtained (See FIG. 4B).

Example 6

Recycling of NADPH

[1300] A one-pot reaction was conducted (see reaction conditions of reaction Al in Table 5) to show that the equilibrium of the GDP-mannose 4,6-dehydratase (GMD, E.C.

[1301] 4.2.1.47) and GDP-L-fucose synthase (wcaG, E.C. 1.1.1.271) catalyzed reactions from GDP-mannose to GDP-fucose is on the side of GDP-mannose (see figures11-Al and 12-A1). In this experiment, first the enzymes were dispensed onto a vial up to the amounts mentioned in table 5. Buffer, co-factor, GDP-Man, NADPH were added in thus order, up to the mentioned concentration in table 5. The reaction was performed in 1.5 mL Eppendorf vials, at 37° C. and 550 rpm in the Eppendorf thermomixer comfort. Aliquots were taken at different time points and quenched by heating at 90° C. for 3 minutes and measured by ion exchange chromatography. It could be seen that the equilibrium is on the side of GDP-Man (see FIG. 12-A1).

[1302] To push the equilibrium of the reaction towards GDP-Fuc, coupling to another reaction (in a way that NADP.sup.+ is constantly removed) needs to be engineered. In reaction A2 the enzymes Glk, PPK3 and glucose-6-phosphate-dehydrogensase (purchased from Merck-10165875001) (G6PDH, E.C: 1.1.1.49) were used to recycle NAPDH and increase the GDP-fucose yield. In this experiment, first the enzymes were dispensed into a vial up to the amounts mentioned in the table 6. After addition of enzymes, buffer, co-factor, GDP-Man, NADPH, glucose, ATP and polyphosphate were added in this order, up to the mentioned concentrations in table 6. The reaction was performed in a 1.5 mL Eppendorf vial, at 37° C. and 550 rpm in the Eppendorf thermomixer Comfort. Aliquots were taken at different time points and quenched by heating at 90° C. for 3 minutes and then measured by ion exchange chromatography. For the recycling the inexpensive substrates glucose and polyphosphate are used as substrates (see FIGS. 11-A2 and 12-A2).

[1303] Another experiment was performed for the production of GDP-Fuc from Man, ATP, GTP and polyphosphate, and L-glutamate for the regeneration of NADPH and shifting the equilibrium towards GDP-Fuc. In this experiment, first enzymes were dispensed into a vial up to the amounts mentioned in the table 7. After addition of enzymes, buffer, co-factor, Man, GTP, ATP and L-glutamate, and polyphosphate were added in this order, up to the mentioned concentrations in table 7. The reaction was performed in a 1.5 mL Eppendorf vial, at 37° C. and 550 rpm in the Eppendorf thermomixer Comfort. Aliquots were taken at different time points and quenched by heating at 90° C. for 3 minutes and then measured by ion exchange chromatography. In reaction A3 (see reaction conditions of reaction A3 in Table 7) glutamate dehydrogenase (purchased from Merck - 10197734001) (GLDH, E.C. 1.4.1.4) was used to recycle NADPH from L-glutamate and water. (see FIGS. 11-A3 and 12-A3).

TABLE-US-00005 TABLE 5 Reactions conditions of examples A1. Enzymes Concentration WCAG 1.03 μg/μL GMD 1.75 μg/μL Substrates GDP-Man 2 mM NADPH 2 mM Buffer and co-factor Tris-HCl (pH = 8) 100 mM MgCl.sub.2 10 mM Volume 40 μL

TABLE-US-00006 TABLE 6 Reaction conditions for reaction A2. Enzymes Concentration WCAG 0.8 μg/μL GMD 1.4 μg/μL GLK 0.129 μg/μL PPK3 0.07 μg/μL G6PDH 0.28 μg/μL Substrates GDP-Man 4 mM NADPH 0.5 mM Glucose 10 mM ATP 1 mM PolyP.sub.25 2 mM Buffer and co-factor Tris-HCl (pH = 8) 100 mM MgCl.sub.2 10 mM Volume 50 μL

TABLE-US-00007 TABLE 7 Reaction conditions of reaction A3. Enzymes Concentration WCAG 0.3 μg/μL GMD 0.52 μg/μL GLK 0.09 μg/μL PPK3 0.27 μg/μL GLDH 2.99 μg/μL ManB/C 0.046 μg/μL PPA 0.047 μg/μL Substrates Man 20 mM GTP 20 mM ATP 2 mM PolyP.sub.25 5 mM L-glutamate 50 mM NADPH 1 mM Buffer and co-factor Tris-HCl (pH = 9) 150 mM MgCl.sub.2 50 mM Volume 200 μL

Example 7

Synthesis of GDP-Fucose from D-Mannose using Glk

[1304] A one-pot enzymatic reaction was conducted to validate GDP-fucose synthesis from mannose through the cascade shown in FIG. 13. In this experiment, first enzymes were dispensed into a vial up to amounts mentioned in the table 8. After addition of enzymes, buffer, co-factor, mannose, ATP, L-glutamate, NADPH, guanosine (from a stock containing DMSO) and polyphosphate were added in this order, up to the mentioned concentrations in table 8. The reaction was performed in a 1.5 mL Eppendorf vial, at 37° C. and 550 rpm in a Eppendorf thermomixer Comfort. Aliquots were taken at different time points and quenched by heating at 90° C. for 3 minutes and then measured by ion exchange chromatography. Reaction time course of the reaction in example 7 is measured by ion chromatography as shown in FIG. 14.

TABLE-US-00008 TABLE 8 Reaction conditions as used in example 7. Enzymes Concentrations GSK 0.128 μg/μL GMK 0.034 μg/μL PPK3 0.2 μg/μL GLK 0.11 μg/μL MANB/C 0.071 μg/μL WCAG 0.24 μg/μL GMD 0.4 μg/μL GLDH 3.45 μg/μL PPA 0.04 μg/μL Substrates Man 11.5 mM Guanosine (in DMSO) 11.5 mM ATP 2.9 mM PolyP.sub.25 5.17 mM L-glutamate 86 mM NADPH 2.8 mM Buffer and co-factor Tris-HCl (pH = 8) 115 mM MgCl.sub.2 29 mM Volume 175 μL

Example 8

Synthesis of GDP-Fucose from D-Mannose using NahK

[1305] A one-pot enzymatic reaction was conducted to validate GDP-fucose synthesis from mannose through the cascade shown in FIG. 15. In this experiment, first enzymes were dispensed into a vial up to amounts mentioned in the table 9. After addition of enzymes, buffer, co-factor, mannose, ATP, L-glutamate, NADPH, guanosine (stock was prepared in DMSO) and polyphosphate were added in the order, up to the mentioned concentrations in table 9. The reaction was performed in 1.5 mL Eppendorf vials, at 37° C. and 550 rpm in a Eppendorf thermomixer comfort. Aliquots were taken at different time points and quenched by heating at 90° C. for 3 minutes and then measured by ion exchange chromatography. Reaction time course of the reaction in example 8 is measured by ion chromatography as shown in FIG. 16.

TABLE-US-00009 TABLE 9 Reaction conditions for example 8. Enzymes Concentration GSK 0.12 μg/μL GMK 0.03 μg/μL PPK3 0.19 μg/μL NAHK 0.14 μg/μL MANB/C 0.06 μg/μL WCAG 0.22 μg/μL GMD 0.38 μg/μL GLDH 3.26 μg/μL PPA 0.03 μg/μL Substrates Man 10.9 mM Guanosine (in DMSO) 10.9 mM ATP 2.75 mM PolyP.sub.25 4.9 mM L-glutamate 81 mM NADPH 2.6 mM Buffer and co-factor Tris-HCl (pH = 8) 108 mM MgCl.sub.2 27 mM Volume 185 μL

Example 9

Synthesis of 3-Fucosyllactose

[1306] The cascades for GDP-fucose production can be coupled to fucosyltransferases to fucosylate molecules or biomolecules, e.g. human milk oligosaccharides and therapeutic proteins. The coupling is performed in one-pot reactions (see FIG. 17). Reactions were conducted where the GDP-fucose cascade was coupled to 3/4-fucosyltransferase to produce 3-fucosyllactose starting from D-mannose (reaction Dl; see FIG. 18). In this experiment, first enzymes were dispensed into a vial up to amounts mentioned in the table 10. After addition of enzymes, buffer, co-factor, mannose, ATP, L-glutamate, NADPH, lactose, guanosine (stock was prepared in DMSO) and polyphosphate were added in this order, up to the mentioned concentrations in table 10. The reaction was performed in 1.5 mL Eppendorf vials, at 37° C. and 550 rpm in a Eppendorf thermomixer comfort. Aliquot was taken and quenched by heating at 90° C. for 3 minutes and then measured by ion exchange chromatography. Another experiment was performed to produce 3-fucosyllactosein which GDP-Fuc was produced from guanosine, L-fucose (reaction D2; see FIG. 19). In this experiment, first enzymes added up to amounts mentioned in the table 11. After addition of enzymes, buffer, co-factor, L-fucose, ATP, lactose, guanosine (stock was prepared in DMSO) and polyphosphate were added in the order, up to the mentioned concentration in table 11. The reaction was performed in 1.5 mL Eppendorf vials, at 37° C. and 550 rpm in a Eppendorf thermomixer comfort. Aliquot was taken and quenched by heating at 90° C. for 3 minutes and then measured by ion exchange chromatography.

##STR00009##

TABLE-US-00010 TABLE 10 Reaction conditions for reaction D1. Enzymes Concentration GSK 0.128 μg/μL GMK 0.034 μg/μL PPK3 0.2 μg/μL GLK 0.11 μg/μL MANB/C 0.071 μg/μL WCAG 0.24 μg/μL GMD 0.4 μg/μL GLDH 3.45 μg/μL PPA 0.04 μg/μL 3/4FT 0.07 μg/μL Substrates Man 8.7 mM Guanosine (in DMSO) 2.9 mM ATP 2.9 mM PolyP.sub.25 5.2 mM L-glutamate 29 mM NADPH 1.45 mM Lactose 11.6 mM Buffer and co-factor Tris-HCl (pH = 8) 116 mM MgCl.sub.2 43 mM Volume 172 μL

TABLE-US-00011 TABLE 11 Reaction conditions for reaction D2. Enzymes Concentration GSK 0.16 μg/μL GMK 0.042 μg/μL PPK3 0.25 μg/μL FKP 0.42 μg/μL PPA 0.05 μg/μL 3/4FT 0.07 μg/μL Substrates Fucose 14.1 mM Guanosine (in DMSO) 3.5 mM ATP 3.6 mM PolyP.sub.25 6.3 mM Lactose 14.1 mM Buffer and co-factor Tris-HCl (pH = 8) 141 mM MgCl.sub.2 35 mM Volume 141 μL

Example 10

Production of L-Fucose from D-Mannose

[1307] The cascade described in examples 7 and 8 can be used to produce L-fucose from D-mannose and guanosine. The reaction in example 8 was heated at 95° C. for 1 hour. An aliquot taken after heating was measured by ion chromatography (see FIG. 20).

[1308] A sequence listing is attached to this application comprising the sequences of the following table:

TABLE-US-00012 SEQ ID description 1 Glucokinase 2 Phosphomannomutase 3 N-acetylhexosamine-1-kinase 4 Mannose-1-phosphate guanylyltransferase 5 GDP-mannose 4,6-dehydratase 6 GDP-L-fucose synthase 7 L-fucokinase 8 Guanosine kinase 9 2-domain polyphosphate kinase 2 10 inorganic pyrophosphatase 11 Guanylate kinase 12 Glutamate dehydrogenase 1 (GLDH) 13 Glucose-6-phosphate 1-dehydrogenase (G6PDH) 14 phosphotransferase 3 (PPK3) 15 Glucose/galactose 1-dehydrogenase (GDH) 16 alpha-1,3/4-fucosyltransferase (3/4FT)