LACTOSE CONVERTING ALPHA-1,2-FUCOSYLTRANSFERASE ENZYMES

Abstract

The present invention relates to methods for producing 2′ fucosyllactose (2′-FL), as well as newly identified fucosyltransferases, more specifically newly identified lactose binding α-1,2-fucosyltransferase polypeptides, and their applications. Furthermore, the present invention provides methods for producing 2-fucosyllactose (2′FL) using the newly identified α-1,2-fucosyltransferases.

Claims

1.-35. (canceled)

36. A method of producing α-1,2-fucosyllactose, the method comprising the steps of: a) providing a polypeptide with α-1,2-fucosyltransferase activity and able to use lactose as an acceptor substrate, wherein the polypeptide i) comprises a conserved domain G-Y-[F/Y]-Q-[N/S] (SEQ ID NO: 72) and a conserved domain (X, no K/V)XXXX[I/L]H[I/L]R[R/L]GD[F/Y](X, no C/M) (SEQ ID NO: 74), wherein X can be any distinct amino acid excluding a lysine and a valine residue from the first position of the domain and excluding a cysteine and a methionine residue from the last position of the domain, and/or ii) is selected from the group consisting of: i) any one of SEQ ID NOs: 1-7, 9-17, 19, 21, 22, 25, 26, 29, 33-41, 43-48, 50-58, 61-63, 66, 67, or 76, ii) a polypeptide having 80% or more sequence identity to a full-length amino acid sequence of any one of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78, or 79, iii) a polypeptide of an allelic variant of a polypeptide of one of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78, or 79, iv) a polypeptide of an ortholog of a polypeptide of one of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78, or 79, and v) a functional fragment of a polypeptide of one of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78, or 79, b) contacting the polypeptide of step a) with a mixture comprising GDP-fucose as a donor substrate and lactose as an acceptor substrate, under conditions wherein the polypeptide catalyses a transfer of a fucose residue from the donor substrate to the acceptor substrate, thereby producing α-1,2-fucosyllactose, c) optionally separating the α-1,2-fucosyllactose, and d) optionally further comprising purifying α-1,2-fucosyllactose thus produced.

37. The method according to claim 36, wherein said polypeptide: is selected from the group consisting of: i) any one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, ii) a polypeptide having 80% or more sequence identity to a full-length amino acid sequence of any one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, iii) a polypeptide of an allelic variant of a polypeptide of one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, iv) a polypeptide of an ortholog of a polypeptide of one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, and v) a functional fragment of a polypeptide of one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, or wherein said polypeptide: i) comprises a conserved domain G-Y-[F/Y]-Q-[N/S] (SEQ ID NO: 72) and a conserved domain (X, no K/V)XXXX[I/L]H[I/L]R[R/L]GD[F/Y](X, no C/M) (SEQ ID NO: 74), wherein X can be any distinct amino acid excluding a lysine and a valine residue from the first position of the domain and excluding a cysteine and a methionine residue from the last position of the domain, and ii) is selected from the group consisting of: i) any one of SEQ ID NOs: 8, 32, 64, 68, or 69, ii) a polypeptide having 80% or more sequence identity to a full-length amino acid sequence of any one of SEQ ID NOs: 8, 32, 64, 68, or 69, iii) a polypeptide of an allelic variant of a polypeptide of one of SEQ ID NOs: 8, 32, 64, 68, or 69, iv) a polypeptide of an ortholog of a polypeptide of one of SEQ ID NOs: 8, 32, 64, 68, or 69, and v) a functional fragment of a polypeptide of one of SEQ ID NOs: 8, 32, 64, 68, or 69.

38. The method according to claim 36, which results in a diFL concentration to 2′fucosyllactose concentration ratio of less than 1:5.

39. The method according to claim 36, wherein the polypeptide is provided in a cell free system.

40. The method according to claim 36, wherein the polypeptide is produced by a host cell comprising a polynucleotide encoding the polypeptide.

41. The method according to claim 36, wherein the GDP-fucose and/or lactose is provided by a cell producing GDP-fucose and/or lactose.

42. The method according to claim 40, the method comprising: growing a host cell expressing the polypeptide under suitable nutrient conditions permissive for producing the α-1,2-fucosyllactose, and permissive for expression of the polypeptide; providing simultaneously or subsequently a donor substrate GDP-fucose and an acceptor substrate lactose, in order for the α-1,2-fucosyltransferase polypeptide to catalyze transfer of a fucose residue from GDP-fucose to lactose, thereby producing α-1,2-fucosyllactose; optionally separating the α-1,2-fucosyllactose from the host cell or medium of its growth; and optionally further comprising purifying α-1,2-fucosyllactose thus produced.

43. The method according to claim 42, wherein the host cell is transformed or transfected to express an exogenous polypeptide with alpha-1,2-fucosyltransferase activity and able to use lactose as an acceptor substrate.

44. The method according to claim 40, wherein the GDP-fucose and/or lactose is provided by an enzyme simultaneously expressed in the host cell or by the host cell's metabolism.

45. The method according to claim 36, wherein the method further comprises at least one of the following steps: i) adding, in a continuous manner, a lactose feed to a culture medium in a reactor having a volume, wherein the lactose feed comprises at least 50 grams of lactose per liter of initial reactor volume, wherein the total reactor volume is from 250 mL (milliliter) to 10,000 m.sup.3 (cubic meter) so that the culture medium's final volume is not more than three-fold of the volume of the culture medium before adding the lactose feed; ii) adding, in a continuous manner, a GDP-fucose feed to a culture medium, wherein the GDP-fucose feed has a concentration enabling a host cell to synthesize 2′-fucosyllactose with a diFL concentration to 2′fucosyllactose concentration ratio of less than 1:5, so that the culture medium's final volume is not more than three-fold of the volume of the culture medium before adding the GDP-fucose feed; iii) adding, in a continuous manner, a carbon-based substrate feed to a culture medium, wherein the carbon-based substrate feed is at a concentration enabling a host cell to synthesize GDP-fucose at a concentration for 2′fucosyllactose synthesis with a diFL concentration to 2′fucosyllactose concentration ratio of less than 1:5; iv) adding to a culture medium a carbon-based substrate feed at a concentration enabling a host cell to internally synthesize lactose at a concentration allowing 2′fucosyllactose synthesis; v) adding, in a continuous manner, a lactose feed, a GDP-fucose feed, and/or a carbon-based substrate feed to a culture medium over the course of 1 day, 2 days, 3 days, 4 days, or 5 days by means of at least one feeding solution; and/or vi) adding, in a continuous manner, a lactose feed to culture medium over the course of 1 day, 2 days, 3 days, 4 days, or 5 days by means of a lactose feeding solution, wherein the concentration of lactose feeding solution is at least 50 g/L, wherein the pH of the lactose feeding solution is between 3 and 7, and wherein the temperature of the lactose feeding solution is kept between 20° C. and 80° C., wherein the method results in a 2′-fucosyllactose concentration of at least 50 g/L in the culture medium's final volume.

46. A host cell genetically modified to produce alpha-1,2-fucosyllactose, the host cell comprising: at least one polynucleotide encoding an enzyme for α-1,2-fucosyllactose synthesis; wherein the host cell expresses a polypeptide with α-1,2-fucosyltransferase activity and is able to use lactose as an acceptor substrate, and wherein the polypeptide: i) comprises a conserved domain G-Y-[F/Y]-Q-[N/S] (SEQ ID NO: 72) and a conserved domain (X, no K/V)XXXX[I/L]H[I/L]R[R/L]GD[F/Y](X, no C/M) (SEQ ID NO: 74), wherein X can be any distinct amino acid excluding a lysine and a valine residue from the first position of the domain and excluding a cysteine and a methionine residue from the last position of the domain; and/or, ii) is selected from the group consisting of: a) any one of SEQ ID NOs: 1-7, 9-17, 19, 21, 22, 25, 26, 29, 33-41, 43-48, 50-58, 61-63, 66, 67, or 76; b) a polypeptide having 80% or more sequence identity to a full-length amino acid sequence of any one of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78, or 79; c) a polypeptide of an allelic variant of a polypeptide of one of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78 or 79; d) an ortholog of a polypeptide of any of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78 or 79; and e) a functional fragment of a polypeptide of any one of SEQ ID NOs: 1-31, 33-63, 65-70, 75-78 or 79.

47. The host cell of claim 46, wherein said polypeptide is selected from the group consisting of: i) any one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, ii) a polypeptide having 80% or more sequence identity to a full-length amino acid sequence of any one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, iii) a polypeptide of an allelic variant of a polypeptide of one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, iv) a polypeptide of an ortholog of a polypeptide of one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, and v) a functional fragment of a polypeptide of one of SEQ ID NOs: 1-7, 9-31, 33-63, 65-67, 70, or 75-79, or wherein said polypeptide: i) comprises a conserved domain G-Y-[F/Y]-Q-[N/S] (SEQ ID NO: 72) and a conserved domain (X, no K/V)XXXX[I/L]H[I/L]R[R/L]GD[F/Y](X, no C/M) (SEQ ID NO: 74), wherein X can be any distinct amino acid excluding a lysine and a valine residue from the first position of the domain and excluding a cysteine and a methionine residue from the last position of the domain, and ii) is selected from the group consisting of: i) any one of SEQ ID NOs: 8, 32, 64, 68, or 69, ii) a polypeptide having 80% or more sequence identity to a full-length amino acid sequence of any one of SEQ ID NOs: 8, 32, 64, 68, or 69, iii) a polypeptide of an allelic variant of a polypeptide of one of SEQ ID NOs: 8, 32, 64, 68, or 69, iv) a polypeptide of an ortholog of a polypeptide of one of SEQ ID NOs: 8, 32, 64, 68, or 69, and v) a functional fragment of a polypeptide of one of SEQ ID NOs: 8, 32, 64, 68, or 69.

48. The host cell of claim 46, wherein the host cell comprises: i) a nucleotide sequence comprising a polynucleotide encoding the polypeptide with lactose binding α-1,2-fucosyltransferase activity, wherein the nucleotide sequence is foreign to the host cell and is integrated into the host cell's genome, or ii) a vector comprising a polynucleotide encoding the polypeptide, wherein the polynucleotide is operably linked to control sequences recognized by a host cell transformed with the vector.

49. The host cell of claim 46, wherein the cell is selected from the group consisting of microorganism, plant cell and animal cell.

50. The host cell of claim 46, wherein the cell is a microorganism that heterologously expresses said α-1,2-fucosyltransferase polypeptide.

51. The host cell of claim 45, wherein the polynucleotide encoding the polypeptide with lactose binding alpha-1,2-fucosyltransferase activity is adapted to the codon usage of the respective host cell.

52. A method of using the host cell of claim 45 to produce α-1,2-fucosyllactose, the method comprising: cultivating the host cell in a medium under conditions permissive for producing α-1,2-fucosyllactose, optionally, separating the produced α-1,2-fucosyllactose from the cultivation, and optionally further comprising purifying α-1,2-fucosyllactose thus produced.

53. The method according to claim 36, wherein the separation comprises at least one of clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, and ligand exchange chromatography.

54. The method according to claim 36, further comprising purifying α-1,2-fucosyllactose thus produced, wherein purification of alpha-1,2-fucosyllactose comprises at least one of: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration or ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying, or lyophilisation.

55. The method according to claim 36, having a lactose concentration in the culture medium of between 50 g/L and 150 g/L.

56. The method according to claim 36, having a final concentration of 2′-fucosyllactose between 70 g/L and 200 g/L.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0248] FIG. 1 shows the normalized production of 2′FL (upperpanel) and diFL (lower panel) in g product/g biomass in relative percentages of mutant strains expressing a different alpha-1,2-fucosyltransferase (with either SEQ ID NO 01, 02, 03, 04 or 05) from a transcriptional unit built with promoter-UTR combination P12U2 (De Mey et al., BMC Biotechnology, 2007) after 72 hours of cultivation in minimal medium supplemented with 20 g/L lactose as described in Example 2. The 2′FL and diFL production in each strain is normalized to the 2′FL and diFL production levels, respectively, obtained in a reference strain expressing the prior art HpFutC gene with SEQ ID NO 71 (indicated by the dashed horizontal line).

[0249] FIG. 2 shows the normalized 2′FL production (2FL) and growth speed (mu) in relative percentages (%) of mutant strains expressing a different alpha-1,2-fucosyltransferase (with either SEQ ID NO 01 or 04) from a transcriptional unit built with promoter-UTR combination P12U2 (De Mey et al., BMC Biotechnology, 2007) after 72 hours of cultivation in minimal medium supplemented with 20 g/L lactose as described in Example 2. The 2′FL production levels and the growth speed of each strain are normalized to the 2′FL production levels and the growth speed obtained in a reference strain expressing the prior art HpFutC gene with SEQ ID NO 71 (indicated by the dashed horizontal line).

[0250] FIG. 3 shows the 2′FL titers (g/L) obtained in a mutant strain expressing the alpha-1,2-fucosyltransferase with SEQ ID NO 03 from a transcriptional unit built with promoter-UTR combination P12U2 (De Mey et al., BMC Biotechnology, 2007) after 72 hours of cultivation in minimal medium supplemented with different concentrations of lactose (2.5, 5, 10, 20, 45 and 70 g/L) as described in Example 2.

[0251] FIG. 4 shows the normalized 2′FL production (2FL) and growth speed (mu) in relative percentages (%) of mutant strains expressing the alpha-1,2-fucosyltransferase with SEQ ID NO 03 from four different transcriptional units (TU) after 72 hours of cultivation in minimal medium supplemented with 20 g/L lactose as described in Example 2. The transcriptional units consist of promoter-UTR combinations P05U14 (TU 01), P05U38 (TU 02), P10U13 (TU 03) and P31U17 (TU 04) as described by De Mey et al. (BMC Biotechnology, 2007). For each TU tested, the 2′FL production levels and the growth speed of each strain are normalized to the 2′FL production levels and the growth speed respectively obtained in a reference strain expressing the prior art HpFutC gene with SEQ ID NO 71 from the same transcriptional unit (indicated by the dashed horizontal line).

[0252] FIG. 5 shows the diFL concentration to 2′FL concentration ratio obtained in whole broth samples at the end of a fed-batch fermentation process with strains expressing the alpha-1,2-fucosyltransferase with SEQ ID NO 01, 02 or 71 as described in Example 2.

[0253] FIG. 6 shows the relative 2′FL, diFL and lactose concentration (in %) at various timepoints during fermentation processes with strains expressing the alpha-1,2-fucosyltransferase with SEQ ID NO 01, 03, 04 or 71 as described in Example 2.

EXAMPLES

Example 1: Identification of Glycosyl Transferase 11 Proteins

[0254] An HMM is a probabilistic model called profile hidden Markov models. It characterizes a set of aligned proteins into a position-specific scoring system. Amino acids are given a score at each position in the sequence alignment according to the frequency by which they occur (Eddy, S. R. 1998. Profile hidden Markov models. Bioinformatics. 14: 755-63). HMMs have wide utility, as is clear from the numerous databases that use this method for protein classification, including Pfam, InterPro, SMART, TIGRFAM, PIRSF, PANTHER, SFLD, Superfamily and Gene3D. HMMsearch from the HMMER package (http://hmmer.org/) can use this HMM to search sequence databases for sequence homologs. Sequence databases that can be used are for example, but not limited to: the NCBI nr Protein Database (NR; https://www.ncbi.nlm.nih.gov/protein), UniProt Knowledgebase (UniProtKB, https://www.uniprot.org/help/uniprotkb) and the SWISS-PROT database (https://web.expasy.org/docs/swiss-prot_guideline.html). The Glycosyl transferase 11 PF01531 domain was obtained from the Pfam v 32.0 database as released in September 2018 via https://pfam.xfam.org/search#tabview=tab1 in ‘Curation & model’. HMMsearch with this model to the protein databases identified new family members (see Table 1). EMBOSS Backtranseq (https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/) can be used to translate the obtained amino acid sequence into a nucleotide sequence.

TABLE-US-00001 TABLE 1 List of alpha 1,2-fucosyltransferases together with the prior art alpha 1,2-fucosyltransferase HpFutC from H. pylori (SEQ ID NO 71), wherein SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 21, 22, 25, 26, 29, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 61, 62, 63, 66, 67 and 76 have both the conserved domain G-Y-[F/Y]-Q-[N/S] (SEQ ID NO: 72) and the conserved domain (X, no K/V)XXXX[I/L]H[I/L]R[R/L]GD[F/Y](X, no C/M) (SEQ ID NO: 74), wherein X can be any distinct amino acid excluding a lysine and a valine residue from the first position of said domain and excluding a cysteine and a methionine residue from the last position of said domain. SEQ ID NO Organism Country origin 1 Akkermansia muciniphila Human 2 Porphyromonas catoniae Unknown 3 Helicobacter sp. MIT 01-6242 USA 4 Capnocytophaga canis Unknown 5 Capnocytophaga leadbetteri Finland 6 Helicobacter sp. MIT 01-6242 USA 7 Kingella denitrificans Human 8 Pseudoalteromonas distincta Unknown 9 Porphyromonas catoniae F0037 Unknown 10 Campylobacter mucosalis UK 11 Pedobacter kyungheensis South Korea 12 Noviherbaspirillum autotrophicum Japan 13 Microbacterium trichothecenolyticum Unknown 14 Candidatus Symbiothrix dinenymphae Japan 15 Pedobacter sp. Switzerland 16 Chryseobacterium sp. Switzerland 17 Microbacterium sp. Germany 18 Ramazzottius varieornatus Japan 19 Candidatus Wolfebacteria bacterium USA 20 Candidatus Yanofskybacteria bacterium USA 21 Spirochaetes bacterium USA 22 Pedobacter soli South Korea 23 Bacteroidales bacterium KHT7 Unknown 24 Butyrivibrio sp. TB Unknown 25 Butyrivibrio fibrisolvens Unknown 26 Candidatus Magasanikbacteria bacterium USA 27 Butyrivibrio fibrisolvens Unknown 28 Chryseobacterium scophthalmum UK 29 Akkermansia sp. 54_46 Unknown 30 Lingula unguis Japan 31 Lingula unguis Japan 32 [Flexibacter] sp. ATCC 35103 USA 33 Bacteroidetes bacterium ADurb. Bin174 USA 34 Pedobacter sp. AJM USA 35 Candidatus Planktophila lacus Switzerland 36 Rhizobiales bacterium PAR1 UK 37 Helicobacter sp. 11S02629-2 Netherlands 38 Candidatus Magasanikbacteria bacterium USA 39 Candidatus Magasanikbacteria bacterium USA 40 Diaminobutyricimonas aerilata South Korea 41 Flavobacterium magnum South Korea 42 Litoreibacter ponti South Korea 43 Dysgonomonas alginatilytica Japan 44 Campylobacter hyointestinalis subsp. lawsonii USA 45 Phycisphaerales bacterium Unknown 46 Akkermansia sp. Unknown 47 Helicobacter sp. MIT 17-337 USA 48 Pedobacter sp. G11 USA 49 Empedobacter brevis Unknown 50 Akkermansia muciniphila South Korea 51 Akkermansia muciniphila Human 52 Akkermansia muciniphila USA 53 Chitinophagaceae bacterium UK 54 Chitinophagaceae bacterium UK 55 Proteobacteria bacterium UK 56 Flaviaesturariibacter sp. 17J68-12 South Korea 57 Pedobacter sp. AR-2-6 Norway 58 Helicobacter jaachi USA 59 Helicobacter japonicus USA 60 Klebsiella pneumoniae Unknown 61 Dysgonomonas capnocytophagoides USA 62 Flavobacterium sp. UK 63 Pedobacter sp. UK 64 Hoeflea phototrophica Germany 65 Subdoligranulum variabile Denmark 66 Crassostrea gigas Unknown 67 Crassostrea gigas Unknown 68 uncultured bacterium USA 69 Clostridium sp. CAG:510 Unknown 70 Helobdella robusta USA 71 Helicobacter pylori Australia 75 Akkermansia sp. KLEI798 Unknown 76 Akkermansia muciniphila Netherlands 77 Capnocytophaga gingivalis ATCC 33624 Unknown 78 Helicobacter sp. CLO-3 USA 79 Helicobacter sp. MIT 17-337 USA

Example 2: Materials & Methods Escherichia coli

[0255] Media

[0256] The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR, Leuven, Belgium). The minimal medium for the growth experiments contained 2.00 g/L NH4Cl, 5.00 g/L (NH4)2SO4, 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 14.26 g/L sucrose or another carbon source when specified in the examples, 1 ml/L vitamin solution, 100 μl/L molybdate solution, and 1 mL/L selenium solution. The medium was set to a pH of 7 with 1M KOH. Vitamin solution consisted of 3.6 g/L FeCl2.Math.4H2O, 5 g/L CaCl2.Math.2H2O, 1.3 g/L MnCl2.Math.2H2O, 0.38 g/L CuCl2.Math.2H2O, 0.5 g/L CoCl2.Math.6H2O, 0.94 g/L ZnCl2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.Math.2H2O and 1.01 g/L thiamine.Math.HCl. The molybdate solution contained 0.967 g/L NaMoO4.Math.2H2O. The selenium solution contained 42 g/L Seo2.

[0257] The minimal medium for fermentations contained 6.75 g/L NH4Cl, 1.25 g/L (NH4)2SO4, 2.93 g/L KH2PO4 and 7.31 g/L KH2PO4, 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 14.26 g/L sucrose, 1 mL/L vitamin solution, 100 μL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. Complex medium was sterilized by autoclaving (121° C., 21′) and minimal medium by filtration (0.22 μm Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g. chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L)).

[0258] Plasmids

[0259] pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium in 2007).

[0260] Plasmids were maintained in the host E. coli DH5alpha (F.sup.−, phi80dlacZΔM15, Δ(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk.sup.−, mk.sup.+), phoA, supE44, lambda.sup.−, thi-1, gyrA96, relA1) bought from Invitrogen.

[0261] Strains and Mutations

[0262] Escherichia coli K12 MG1655 [lambda.sup.−, F.sup.−, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain #: 7740, in March 2007. Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection after homologous recombination performed by lambda Red recombinase. Subsequent catalysis of a flippase recombinase ensures removal of the antibiotic selection cassette in the final production strain.

[0263] Transformants carrying a Red helper plasmid pKD46 were grown in 10 mL LB media with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30° C. to an OD.sub.600 nm of 0.6. The cells were made electrocompetent by washing them with 50 mL of ice-cold water, a first time, and with 1 mL ice cold water, a second time. Then, the cells were resuspended in 50 μL of ice-cold water. Electroporation was done with 50 μL of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene Pulser™ (BioRad) (600 Ω, 25 μFD, and 250 volts).

[0264] After electroporation, cells were added to 1 ml LB media incubated 1 h at 37° C., and finally spread onto LB-agar containing 25 mg/L of chloramphenicol or 50 mg/L of kanamycin to select antibiotic resistant transformants. The selected mutants were verified by PCR with primers upstream and downstream of the modified region and were grown in LB-agar at 42° C. for the loss of the helper plasmid. The mutants were tested for ampicillin sensitivity.

[0265] The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivates as template. The primers used had a part of the sequence complementary to the template and another part complementary to the side on the chromosomal DNA where the recombination must take place. For the genomic knock-out, the region of homology was designed 50-nt upstream and 50-nt downstream of the start and stop codon of the gene of interest. For the genomic knock-in, the transcriptional starting point (+1) had to be respected. PCR products were PCR-purified, digested with DpnI, repurified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).

[0266] The selected mutants (chloramphenicol or kanamycin resistant) were transformed with pCP20 plasmid, which is an ampicillin and chloramphenicol resistant plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis. The ampicillin-resistant transformants were selected at 30° C., after which a few were colony purified in LB at 42° C. and then tested for loss of all antibiotic resistance and of the FLP helper plasmid. The gene knock outs and knock ins are checked with control primers (Fw/Rv-gene-out).

[0267] For 2′FL production, the mutant strains derived from E. coli K12 MG1655 have knock-outs of the genes lacZ, lacY, lacA, glgC, agp, pfkA, pfkB, pgi, arcA, icIR, wcaJ, pgi, Ion and thyA and additionally genomic knock-ins of constitutive expression constructs containing the E. coli lacY gene, a fructose kinase gene (frk) originating from Zymomonas mobilis and a sucrose phosphorylase (SP) originating from Bifidobacterium adolescentis. These genetic modifications are also described in WO2016075243 and WO2012007481. GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for the E. coli genes manA, manB, manC, gmd and fcl. GDP-fucose production can also be obtained by genomic knock-outs of the E. coli fucK and fucI genes and genomic knock-ins of constitutive transcriptional units containing the fucose permease (fucP) from E. coli and the bifunctional fucose kinase/fucose-1-phosphate guanylyltransferase (fkp) from Bacteroides fragilis. In addition, an alpha-1,2-fucosyltransferase expression plasmid having a constitutive transcriptional unit for SEQ ID NO 01, 02, 03, 04, 05 or 71 is added to the strains.

[0268] All constitutive promoters and UTRs originate from the libraries described by De Mey et al. (BMC Biotechnology, 2007) and Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). All genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage was adapted using the tools of the supplier.

[0269] All strains are stored in cryovials at −80° C. (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).

[0270] Cultivation Conditions

[0271] A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 μL LB and was incubated overnight at 37° C. on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 μL minimal medium by diluting 400×. These final 96-well culture plates were then incubated at 37° C. on an orbital shaker at 800 rpm for 72 h, or shorter, or longer. At the end of the cultivation experiment samples were taken from each well to measure sugar concentrations in the broth supernatant (extracellular sugar concentrations, after spinning down the cells), or by boiling the culture broth for 15 min at 90° C. before spinning down the cells (=whole broth measurements, average of intra- and extracellular sugar concentrations).

[0272] A preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL of minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37° C. on an orbital shaker at 200 rpm. A 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culturing condition were set to 37° C., and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2SO4 and 20% NH40H. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.

[0273] Optical Density

[0274] Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland).

[0275] Growth Rate/Speed Measurement

[0276] The maximal growth rate (μMax) was calculated based on the observed optical densities at 600 nm using the R package grofit.

[0277] Liquid Chromatography

[0278] Standards for 2′fucosyllactose and difucosyllactose were synthetized in house. Other standards such as but not limited to lactose, sucrose, glucose, fructose, fucose were purchased from Sigma.

[0279] Carbohydrates were analyzed via a HPLC-RI (Waters, USA) method, whereby RI (Refractive Index) detects the change in the refraction index of a mobile phase when containing a sample. The sugars were separated in an isocratic flow using an X-Bridge column (Waters X-bridge HPLC column, USA) and a mobile phase containing 75 ml acetonitrile and 25 ml Ultrapure water and 0.15 ml triethylamine. The column size was 4.6×150 mm with 3.5 μm particle size. The temperature of the column was set at 35° C. and the pump flow rate was 1 mL/min.

Example 3: Materials & Methods Saccharomyces cerevisiae

[0280] Media

[0281] Strains are grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura (MP Biomedicals).

[0282] Strains

[0283] Saccharomyces cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995). Kluyveromyces marxianus lactis is available at the LMG culture collection (Ghent, Belgium).

[0284] Plasmids

[0285] Yeast expression plasmid p2a_2μ (Chan 2013 (Plasmid 70 (2013) 2-17)) was used for expression of foreign genes in Saccharomyces cerevisiae. This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli. The plasmid further contains the 2μ yeast on and the Ura3 selection marker for selection and maintenance in yeast. Next, this plasmid can be modified to p2a_2μ_fl to contain a lactose permease (for example LAC12 from Kluyveromyces lactis), a GDP-mannose 4,6-dehydratase (such as Gmd from E. coli) and a GDP-L-fucose synthase (such as fcl from E. coli).

[0286] Yeast expression plasmids p2a_2μ_fl_2ft are based on p2a_2μ_fl but modified in a way that also SEQ ID NO 01, 02, 03, 04, 05, 06, 07, 09 or 71 are expressed. Preferably but not necessarily, the fucosyltransferase proteins are N-terminally fused to a SUMOstar tag (e.g. obtained from pYSUMOstar, Life Sensors, Malvern, PA) to enhance the solubility of the fucosyltransferase enzymes.

[0287] Plasmids were maintained in the host E. coli DH5alpha (F.sup.−, phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk.sup.−, mk.sup.+), phoA, supE44, lambda.sup.−, thi-1, gyrA96, relA1) bought from Invitrogen.

[0288] Gene Expression Promoters

[0289] Genes are expressed using synthetic constitutive promoters, as described by Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

[0290] Heterologous and Homologous Expression

[0291] Genes that needed to be expressed, be it from a plasmid or from the genome, were synthetically synthetized by Twist Biosciences (San Francisco, USA). Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.

[0292] Cultivations Conditions

[0293] In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30° C.

[0294] Starting from a single colony, a preculture was grown overnight in 5 mL at 30° C., shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30° C. with an orbital shaking of 200 rpm. The use of an inducer is not required as all genes are constitutively expressed.

Example 4: Evaluation of Potential Lactose-Binding Alpha-1,2-Fucosyltransferase Enzymes Incorporated in Escherichia coli

[0295] The alpha-1,2-fucosyltransferase enzymes with SEQ ID NO 01, 02, 03, 04 and 05 identified in Example 1 were evaluated to produce 2-fucosyllactose (2′FL) from GDP-fucose and lactose in mutant E. coli strains. Mutant strains expressing said alpha-1,2-fucosyltransferases from a transcriptional unit built with promoter-UTR combination P12U2 (De Mey et al., BMC Biotechnology, 2007) were analysed in a growth experiment according to the cultivation conditions provided in Example 2. Each mutant E. coli strain was grown in multiple wells of a 96-well plate. FIG. 1 (upper panel) shows the normalized production of 2′FL (g product/g biomass in relative percentages) obtained in a growth experiment of the strains successfully expressing various alpha-1,2-fucosyltransferases from the same transcriptional unit with 20 g/L lactose in the minimal medium. All datapoints were normalized to the 2′FL production obtained with a reference strain expressing the prior art alpha-1,2-fucosyltransferase HpFutC with SEQ ID NO 71 from a transcriptional unit with the same P12U2 promoter-UTR combination, indicated by the dashed horizontal line. The experiment identified all newly identified polypeptides to have lactose binding 2-fucosyltransferase activity. Surprisingly, the polypeptides with SEQ ID NO 01, 02 and 04 were identified to have better lactose binding 2-fucosyltransferase activity than the prior art alpha-1,2-fucosyltransferase HpFutC with SEQ ID NO 71 in these conditions tested. The experiment also demonstrated that the polypeptides with SEQ ID NOs 01, 03, 04 and 05 did not produce diFL unlike the prior art enzyme with SEQ ID NO 71. The polypeptide with SEQ ID NO 02 produced diFL but this diFL production was very minimal compared to the diFL production measured with the prior art HpFutC enzyme with SEQ ID NO 71 (FIG. 1, lower panel). The diFL concentration to 2′FL concentration ratio measured in the strain expressing the polypeptide with SEQ ID NO 02 was 1:40.

[0296] The experiment also showed that the higher production of 2′FL in the mutant strains expressing the polypeptides with SEQ ID NO 01 or 04 did not affect the growth speed of these strains when compared to the reference strain expressing the prior art alpha-1,2-fucosyltransferase HpFutC with SEQ ID NO 71 (FIG. 2).

Example 5: Evaluation of an E. coli Strain Expressing a Lactose-Binding Alpha-1,2-Fucosyltransferase Enzyme when Tested on Different Lactose Concentrations

[0297] In a next experiment, the mutant E. coli strain expressing the alpha-1,2-fucosyltransferase enzyme with SEQ ID NO 03 from a transcriptional unit built with the P12U2 promoter-UTR combination (De Mey et al., BMC Biotechnology, 2007) as described in Example 4 was evaluated for 2′FL and diFL production on minimal medium containing low to high lactose concentrations. A growth experiment was performed according to the cultivation conditions provided in Example 2. For each condition, the mutant E. coli strain was grown in multiple wells of a 96-well plate.

[0298] FIG. 3 shows the 2′FL titers (g/L) obtained in this experiment. [CS21][NL-I22][23][24] This figure demonstrates that the polypeptide with SEQ ID NO 03 has lactose binding 2-fucosyltransferase activity in all conditions tested and is able to produce 2′FL from various lactose concentrations. In all tested conditions the strain did not produce diFL (Results not shown).

Example 6: Evaluation of an E. coli Strain Expressing a Lactose-Binding Alpha-1,2-Fucosyltransferase Enzyme from Different Transcriptional Units

[0299] In a next experiment, several mutant E. coli strains expressing the alpha-1,2-fucosyltransferase polypeptide with SEQ ID NO 03 from transcriptional units (TU) that were different compared to those used in previous examples were evaluated for 2′FL and diFL production. The transcriptional units used consisted of promoter-UTR combinations P05U14 (TU 01), P05U38 (TU 02), P10U13 (TU 03) and P31U17 (TU 04) respectively as described by De Mey et al. (BMC Biotechnology, 2007). A growth experiment was performed according to the cultivation conditions provided in Example 2. Each mutant E. coli strain was grown in multiple wells of a 96-well plate.

[0300] FIG. 4 shows the normalized 2′FL production (g product/g biomass) and growth speed in relative percentages obtained in this experiment. For each strain and thus for each transcriptional unit tested, the datapoints for 2′FL production or growth speed were normalized to the 2′FL production or growth speed respectively obtained for a reference strain expressing the prior art alpha 1,2-fucosyltransferase HpFutC with SEQ ID NO 71 from the identical transcriptional unit (indicated by the dashed horizontal line). This experiment demonstrated the strains all produced 2′FL without diFL production in all tested conditions. In addition, one can increase the 2′FL production in a mutant strain depending on the transcriptional unit used, with limited effect on the strain's growth speed. The four transcriptional units used here in the mutant strains to express the polypeptide with SEQ ID NO 03 also resulted in a higher expression of said polypeptide compared to when using a transcriptional unit built of P12U2 as used in Example 4, leading to more 2′FL production.

Example 7: Production of 2′Fucosyllactose with an E. coli Strain Expressing a Combination of Lactose-Binding Alpha-1,2-Fucosyltransferase Enzymes

[0301] In another experiment, several mutant E. coli strains expressing a combination of two or more of the alpha-1,2-fucosyltransferase polypeptides with SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 09 were evaluated for 2′FL and diFL production. A growth experiment was performed according to the cultivation conditions provided in Example 2. Each mutant E. coli strain was grown in multiple wells of a 96-well plate. The experiment showed that a single strain expressing combinations of at least two alpha-1,2-fucosyltransferase polypeptides is able to produce 2′FL and in higher concentrations than when only a single polypeptide is expressed per strain.

Example 8: Production of 2′Fucosyllactose in Saccharomyces cerevisiae Using Various Lactose Binding Alpha-1,2-Fucosyltransferase Enzymes

[0302] Another example provides use of a eukaryotic organism, in the form of Saccharomyces cerevisiae, for the invention. Using the strains, plasmids and methods as described in Example 3, strains are created that express SEQ ID NO 01, 02, 03, 04, 05, 06, 07, 09 or 71. On top of that, further modifications are made in order to produce 2′fucosyllactose. These modifications comprise the addition of a lactose permease, a GDP-mannose 4,6-dehydratase and a GDP-L-fucose synthase. The preferred lactose permease is the KILAC12 gene from Kluyveromyces lactis (WO 2016/075243). The preferred GDP-mannose 4,6-dehydratase and the GDP-L-fucose synthase are respectively gmd and fcl from Escherichia coli.

[0303] These strains are capable of growing on glucose or glycerol as carbon source, converting the carbon source into GDP-L-fucose, taking up lactose, and producing 2′fucosyllactose using GDP-L-fucose and lactose as substrates for the enzymes represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07, 09 and 71 with SEQ ID NO 71 as reference. Preculture of said strains are made in 5 mL of the synthetic defined medium SD-CSM containing 22 g/L glucose and grown at 30° C. as described in Example 3. These precultures are inoculated in 25 mL medium in a shake flask with 10 g/L sucrose as sole carbon source and grown at 30° C. Regular samples are taken and the production of 2′fucosyllactose and di-fucosyllactose is measured as described in Example 2.

Example 9: Evaluation of Escherichia coli Strains Expressing Various Lactose Binding Alpha-1,2-Fucosyltransferase Enzymes in a Batch Fermentation

[0304] Batch fermentations at bioreactor scale were performed to evaluate strains, derived from the mutant E. coli K12 MG1655 strain background as described in Example 2, expressing the alpha-1,2-fucosyltransferase enzymes with SEQ ID NO NOs 01, 02, 03, 04, 05, 06, 07, 09 or 71 with SEQ ID NO 71 as reference. The bioreactor runs were performed as described in Example 2. In these examples, sucrose was used as a carbon source. Lactose was added in the batch medium at 90 g/L as a precursor for 2′FL formation. Regular samples are taken and the production of 2′fucosyllactose and di-fucosyllactose is measured as described in Example 2.

Example 10: 2′Fucosyllactose Production with Different Lactose Concentrations

[0305] A fermentation process as described in Example 2 was performed wherein the lactose concentration in the culture medium ranges from 50 to 150 g/L. Said lactose is converted during the process into 2′fucosyllactose until minor amounts of lactose is left. The final ratio lactose to 2′fucosyllactose may be manipulated during this process by stopping the process earlier (higher lactose to 2′fucosyllactose ratio) or later (lower lactose to 2′fucosyllactose ratio). The lactose concentration may be increased in the vessel by feeding high concentrations of lactose solution with or without another carbon source to the bioreactor. Said lactose feed contains lactose concentrations between 100 and 700 g/L and is kept at a temperature so that the lactose is kept soluble at a pH below or equal to 6 to avoid lactulose formation during the process, a standard method used in the dairy industry. The final concentrations of 2′fucosyllactose reached in such a production process ranges between 70 g/L when lower lactose concentrations are used and 200 g/L or higher when high lactose concentrations are used in the process as described above. The 2′FL titers that can be obtained in such a fed-batch fermentation process are much higher compared to batch growth experiments in shake flasks or multiwell plates. In a well-controlled bioreactor, additional carbon source is constantly added during the fed-batch and much higher biomass concentrations can be obtained (better aeration and less acidification compared to multiwell plate batch experiments). The final difucosyllactose concentration to 2′FL concentration ratio obtained in such a process with strains producing 2′FL from SEQ ID NO 01 or 02 e.g. is 0 or 1:55, respectively, which is much smaller than the diFL to 2′FL ratio of 1:6 (a ratio of 0,164) at a lactose concentration near 0 g/L obtained in a similar process with a reference strain producing 2′FL from SEQ ID NO 71 (FIG. 5). For this reference strain the ratios were measured at different residual lactose concentrations and this strain reaches diFL to 2′FL ratios of 1:8 at 3 g/L residual lactose, 1:9 at 5 g/L residual lactose, 1:11 at 8 g/L residual lactose, 1:12 at 14 g/L residual lactose and 1:22 at 21 g/L residual lactose. The resulting 2′FL purity ranges between 76% and 86% wherein diFL forms a substantial fraction of the remaining 24 to 14%, respectively. The obtained purities of 2′fucosyllactose on the sum of 2FL, diFL and lactose concentration for SEQ ID NO 01 or 02 ranged between 80% and 99.9% with the remaining carbohydrate (20% to 1%) mainly being lactose, which is the result of an incomplete conversion of lactose.

Example 11: Enzymatic Production of 2′ Fucosyllactose

[0306] Another example provides the use of an enzyme with SEQ ID NO 01 and 02 of the present invention. These enzymes are produced in a cell-free expression system such as but not limited to the PURExpress system (NEB), or in a host organism such as but not limited to Escherichia coli or Saccharomyces cerevisiae, after which the above listed enzymes can be isolated and optionally further purified.

[0307] Each of the above enzyme extracts or purified enzymes are added to a reaction mixture together with GDP-fucose and a buffering component such as Tris-HCl or HEPES and either lactose or 2′FL. Said reaction mixture is then incubated at a certain temperature (for example 37° C.) for a certain amount of time (for example 24 hours), during which the lactose or 2′FL will be converted by the enzyme using GDP-fucose to 2′fucosyllactose and difucosyllactose, respectively, the latter reaction only when the 2-fucosyltransferase has side activity on 2′FL. The 2′fucosyllactose and di-fucosyllactose are then separated from the reaction mixture by methods known in the art. Further purification of the 2′FL and/or diFL can be performed if preferred. At the end of the reaction or after separation and/or purification, the production of 2′fucosyllactose and di-fucosyllactose is measured as described in Example 2.

Example 12: 2′Fucosyllactose Production in a Bioreactor

[0308] A fed-batch fermentation process as described in Example 2 was performed wherein the lactose concentration in the culture medium was set at 120 g/L. The same process was performed with different 2′fucosyllactose production strains, as described in Example 2, containing an expression plasmid with the alpha-1,2-fucosyltransferase enzymes with SEQ ID NOs 01, 03, 04 or 71. Said lactose is converted during the process into 2′fucosyllactose until minor amounts of lactose is left. The final ratio lactose to 2′fucosyllactose may be manipulated during this process by stopping the process earlier (higher lactose to 2′fucosyllactose ratio) or later (lower lactose to 2′fucosyllactose ratio).[CS25] Also the final ratio diFL to 2′fucosyllactose may be manipulated similarly for strains expressing a 1,2-fucosyltransferase enzymes producing 2′FL and having side-activity forming diFL by stopping the process earlier (lower diFL to 2′fucosyllactose ratio) or later (higher diFL to 2′fucosyllactose ratio).[NL-I26][27]

[0309] FIG. 6 shows the conversion of lactose into 2′fucosyllactose and diFL during these fermentations. The x-axis depicts various timepoints during the fermentation from start (100% lactose present) to finish (when no or almost no lactose is detected anymore). The y-axis depicts the percentages of lactose, 2′FL and diFL present in the fermentation broth, determined by dividing each sugar by the sum of the three sugars. At the end of the fermentations with the strains with SEQ ID NO 01, 03 or 04, a very high 2′FL purity (near 100%) could be reached. No lactose could be detected anymore and no diFL was formed, even when the lactose was completely depleted. In contrast, in a similar fermentation with a strain containing SEQ ID NO 71, small amounts of lactose remained at the end of the fermentation. Simultaneously, when the lactose concentration dropped below 5%, diFL started to appear in the fermentation broth (up to 19% of the total sugar concentration in this example).

[0310] It is thus clear that much higher 2′fucosyllactose purities can be obtained using the alpha-1,2-fucosyltransferase enzymes with SEQ ID NO 01, 03 or 04 compared to the enzyme with SEQ ID NO 71.

Example 13: Enzymatic Production of 2′ Fucosyllactose and Di-Fucosyllactose

[0311] Another example provides the use of an enzyme with SEQ ID NO 01, 02, 03, 04 or 71 of the present invention. These enzymes are produced in Escherichia coli, after which the cells are harvested and lysed in Tris-HCl buffer using sonication. The soluble protein fraction is separated from the cell debris and used for downstream enzymatic reaction. Each of the above enzyme extracts are added to a reaction mixture together with GDP-L-fucose as the donor substrate (5 mM), a buffering component (Tris-HCl) and either lactose or 2′FL as the acceptor substrate (5 mM). Note that both the donor and acceptor substrates are present in excess, in concentrations higher than physiologically possible. Said reaction mixture is then incubated at 37° C. for 24 hours. The lactose, 2′fucosyllactose and di-fucosyllactose are then separated and detected using liquid chromatography as described in Example 2.

[0312] The experiment showed that using lactose as a substrate, the enzyme with SEQ ID NO 71 (HpFutC) was able to produce both 2′FL and diFL, while the enzymes with SEQ ID NO 01, 02, 03 and 04 only produced 2′FL and no diFL. Similarly, using 2′FL as a substrate, only the enzyme with SEQ ID NO 71 (HpFutC) was able to produce diFL, while no further conversion of 2′FL to diFL was observed for the enzymes with SEQ ID NOs 01, 02, 03 and 04. Thus, the enzyme with SEQ ID NO 71 (HpFutC) produced diFL as a side-product, while the other tested enzymes did not (or at least to a much lesser extent, below detection limit). While for strains with HpFutC, the in vivo produced diFL to 2′FL ratio could be kept low by limiting the GDP-L-fucose donor supply, this low GDP-fucose donor supply simultaneously limited the 2′FL productivity. For strains containing the enzymes with SEQ ID NOs 01, 02, 03 or 04 the in vivo GDP-L-fucose concentration supplied was irrelevant with respect to the diFL to 2′FL ratio and thus a much higher 2′FL productivity could be achieved.